Induced IVM: A New Approach to Oocyte Maturation in vitro2.3.4 Oocyte nuclear maturation assessment...

Induced IVM: A New Approach to Oocyte Maturation in vitro

Firas Albuz, MSc.

The Robinson Institute The Research Centre for Reproductive Health,

Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health,

University of Adelaide, Australia. A thesis submitted to the University of Adelaide in total fulfilment of the requirements for the

degree of Doctor of Philosophy in Medicine.

October 2009

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Table of Contents

Abstract vii

Declaration ix

Acknowledgements x

Glossary/Abbreviations xii

Publications and Conference Proceedings xiii

Provisional Patent xvi

Visits to Overseas Laboratories xvi

Invited Guest Seminar - International xvi

Awards, Scholarship & Prizes xvii

CHAPTER 1

LITERATURE REVIEW 18

1.1 Introduction 19

1.2 Infertility Treatment and Current Limitation 21

1.3 In vitro Maturation of Oocytes 21

1.3.1 Rationale for the Clinical Use of IVM 22

1.3.2 Potential Applications of IVM (Patient Groups) 23

1.3.3 Rescue IVM (GV Oocytes from Stimulated Cycles) 25

1.3.4 IVM Limitations 25

1.3.4.1 Fertilization Rates 26

1.3.4.2 Embryo Development Rates 26

1.3.4.3 Embryo Transfer Outcomes 28

1.3.4.4 Long-Term Health (Offspring) Outcomes 28

1.3.5 Conclusion 30

1.4 Biological Aspects 31

1.4.1 Oocyte Development 31

1.4.2 Oocyte Meiotic Arrest 34

1.4.3 Cumulus Cell-Oocyte Gap Junctional Communication 34

1.4.4 cAMP 35

1.4.5 Oocyte Maturation (in vivo) 39

1.4.6 Prophase I & Chromatin Configurations 39

1.4.7 Cytoplasmic Maturation 40

1.4.8 Meiotic Resumption 42

1.4.8.1 Nuclear Maturation 42

1.4.8.2 Role of Gonadotrophins/EGF on Meiotic Resumption 43

1.4.8.3 cAMP/PDEs 43

1.4.8.4 Loss of Gap Junctional Communication (GJC) 45

1.4.8.5 MPF 48

1.4.8.6 FF-MAS 49

1.5 Oocyte Maturation (in vitro) 49

1.6 Prospective Concepts for Improving IVM Culture System 50

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1.6.1 IVM Collection and Handling Media 52

1.6.2 IVM Culture Media 53

1.6.3 Prematuration Culture (PMC) 54

1.6.3.1 Physiological Conditions & Meiotic Arrest in vitro 54

1.6.3.2 Inorganic Additives & Meiotic Arrest in vitro 55 1.6.3.2.1 Purines 55 1.6.3.2.2 Protein Kinase Inhibitors 55 1. 6.3.2.3 cAMP Modulators (AC, PDEs) 57 1.6.3.2.4 AMPK Activators 60

1.7 Conclusion 63

1.8 Hypothesis and Aims for PhD Project 64

1.8.1 Hypothesis 64

1.8.2 Aims 64

CHAPTER 2

EFFECT OF INHIBITION OF PHOSPHODIESTERASE TYPE 8 DURING IN

VITRO MATURATION OF BOVINE OOCYTE ON CAMP LEVELS, MEIOTIC

AND DEVELOPMENTAL CAPACITY 65

2.1 Abstract 66

2.2 Introduction 67

2.3 Materials & methods 69

2.3.1 Oocytes recovery and culture 69

2.3.2 cAMP modulators 69

2.3.3 Measurement of intracellular cAMP 70

2.3.4 Oocyte nuclear maturation assessment 70

2.3.5 In vitro maturation, fertilization and embryo culture 71

2.3.6 Statistical analyses 72

2.4 Results 73

2.4.1 The role of PDE8 on cAMP degradation in the cumulus-oocyte complex 73

2.4.2 The effect of PDE8 inhibition on oocyte meiotic maturation 73

2.4.3 The effect of PDE8 inhibition on cleavage and development to the blastocyst stage 74

2.5 Discussion 75

2.6 Figures 79

2.7 References 83

CHAPTER 3

SUBSTANTIAL IMPROVEMENTS IN BOVINE EMBRYO YIELD USING A

NOVEL SYSTEM OF INDUCED IVM BY EXPLOITING CAMP MODULATORS

IN PRE-IVM AND IVM 86

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3.1 Abstract 87

3.2 Introduction 89

3.3 Materials and Methods 91

3.3.1 Oocyte collection and selection (pre-IVM phase) 91

3.3.2 Oocyte in vitro maturation (IVM phase) 91

3.3.3 In vitro fertilization and embryo culture 92

3.3.4 Blastocyst differential staining 93


3.3.6 Oocyte nuclear morphology assessment 94

3.3.7 Oocyte-cumulus cells gap junctional communication assay 95

3.3.8 Statistical analysis 96

3.4 Results 97

3.4.1 cAMP content of COC and DO during pre-IVM phase 97

3.4.2 Effect of cAMP modulators in pre-IVM phase and type-3 PDE inhibition in IVM phase on spontaneous oocyte maturation (GV/GVBD) 98

3.4.3 Effect of cAMP modulators in pre-IVM phase and type-3 PDE inhibition in IVM phase on oocyte germinal vesicle configurations 98

3.4.4 Effect of cAMP modulators during pre-IVM and IVM on oocyte-cumulus cell gap junctional communication 100

3.4.5 Effect of cAMP modulators in pre-IVM phase and type-3 PDE inhibition in IVM phase on oocyte meiotic progression to MII stage 100

3.4.6 Intracellular concentration of cAMP in oocyte after pre-IVM and IVM phase 101

3.4.7 Effect of (EGFR) kinase inhibitor on FSH-induced oocyte maturation in the presence of the type-3 PDE inhibitor (Cilostamide) 101

3.4.8 Effect of cAMP modulators during pre-IVM and IVM phase on oocyte developmental competence 102

3.4.9 Effect of cAMP modulators during pre-IVM and IVM on blastocyst cell numbers 103

3.5 Discussion 104

3.6 Figures 109

3.7 Tables 118

3.8 References 120

CHAPTER 4

SPECIFIC PRE-IVM CONDITIONS THAT IMPROVE BOVINE OOCYTE

DEVELOPMENTAL COMPETENCE 123

4.1 Abstract 124

4.2 Introduction 126

4.3 Materials & methods 130

4.3.1 Oocyte recovery, selection and incubation (pre-IVM phase) 130

4.3.2 In vitro oocyte maturation, fertilization and embryo culture 130


4.3.4 Experimental design 132


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4.4 Results 135

4.4.1 Experiment 1. The effect of increasing concentrations of forskolin and the presence/absence of IBMX in collection medium during the pre-IVM phase on COC cAMP levels 135

4.4.2 Experiment 2. The effect of pre-IVM duration on COC cAMP when incubated with increasing concentrations of forskolin and IBMX 135

4.4.3 Experiment 3. The effect of pre-IVM duration on intra-oocyte cAMP when incubated with increasing concentrations of forskolin and IBMX 136

4.4.4 Expeirment 4. The effect of increasing cAMP levels prior to IVM on oocyte developmental competence 137

4.5 Discussion 138

4.6 Figures 142

4.7 References 146

CHAPTER 5

INDUCED OOCYTE IVM SUBSTANTIALLY IMPROVES MOUSE EMBRYO

YIELD AND PREGNANCY OUTCOMES 150

5.1 Abstract 151

5.2 Introduction 153

5.3 Materials and Methods 156

5.3.1 Source of oocytes 156

5.3.2 Oocyte collection (pre-IVM phase) 156

5.3.3 Oocytes in vitro maturation (IVM phase) 157

5.3.4 In vitro fertilization 157

5.3.5 Differential nuclear staining 158

5.3.6 Embryo transfer 158


5.4 Experimental Design 159

5.4.1 COC cAMP levels and initiation of maturation 159

5.4.2 The effect of increasing doses of FSH to induce meiotic maturation of COCs matured in the presence of the type 3 PDE inhibitor (cilostamide) 159

5.4.3 Effect of pre-IVM phase on oocyte meiotic resumption that matured by induced IVM (22 hours IVM) 160

5.4.4 The effect of standard or induced IVM on oocyte meiotic maturation 160

5.4.5 The effect of time of maturation (18 or 22 hours) on oocytes matured by standard or induced IVM on oocyte developmental capacity 161

5.4.6 The effect of different cAMP modulators during the pre-IVM & IVM phases on oocyte developmental capacity 161

5.4.7 Embryo developmental capacity, pregnancy outcomes & fetal parameters of oocytes derived from induced and standard IVM and from in vivo matured oocytes 161


5.5 Results 163

5.5.1 COC cAMP measurements in vitro versus in vivo 163

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5.5.2 Increasing doses of FSH to induce meiotic maturation of COCs matured in the presence of the type 3 PDE inhibitor (cilostamide) 163

5.5.3 Effect of pre-IVM phase on oocyte meiotic resumption that matured by induced IVM 163

5.5.4 The effect of Induced IVM on GV/GVBD (0-14 hours of IVM) 164

5.5.5 The effect of Induced IVM on oocyte maturation (18 and 22 hours of IVM) 164

5.5.6 The effect of induced IVM vs. standard IVM on oocyte developmental capacity & blastocyst quality 164

5.5.7 The effect of maturing oocytes by induced or standard IVM compared to in vivo matured oocytes on developmental capacity, pregnancy outcomes & fetal parameters 165

5.6 Discussion 167

5.7 Figures 172

5.8 References 183

CHAPTER 6

FINAL DISCUSSION 187

FUTURE DIRECTIONS 198

THESIS REFERENCES 220

CHAPTER 7

APPENDICES 220

Appendix 1: Additional Experiments 221

Appendix 1: Figures 226

Appendix 2: Culture Media 233

Appendix 3: Reagents 239

Appendix 4: Bovine Blastocyst Scoring System 241

Appendix 5: Published Version of Chapter II 243

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Abstract Oocyte in vitro maturation (IVM) is a technique that would alter the management of human

infertility if success rates were notably higher. Oocyte maturation in vivo is a highly

orchestrated, induced process, whereby 3'-5'-cyclic adenosine monophosphate (cAMP)-

mediated meiotic arrest is overridden by the gonadotrophin surge. However, using standard

IVM, oocytes resume maturation spontaneously hence compromising developmental

competence. The aim of this thesis was to establish an improved system for mammalian

oocyte IVM by studying the inclusion of various forms of cAMP modulators during IVM and

examine oocyte quality and developmental capacity.

Firstly, this thesis includes a series of experiments designed to examine the effect of specific

inhibition of phosphodiesterase type 8 (PDE8) during IVM of bovine oocytes on cAMP

levels, meiotic and developmental capacity. The inhibition of PDE8 degradation resulted in a

dose-dependent increase in cAMP levels and delayed oocyte meiotic resumption. However,

the inhibition of PDE8 degradation failed to enhance oocyte developmental competence.

This thesis includes an extensive series of studies designed to establish a novel induced-IVM

system. Firstly, a pre-IVM phase was developed where immature bovine or mouse oocytes

were briefly treated with the adenylate cyclase activator, forskolin and a non-specific PDE

inhibitor, IBMX, which substantially increased intra-oocyte cAMP to in vivo physiological

levels. Secondly, to maintain oocyte cAMP levels and prevent precocious oocyte maturation,

oocytes were then matured with an oocyte-specific PDE 3 inhibitor, cilostamide and

simultaneously induced to mature by FSH. The net effect of this system was an increase in

oocyte-somatic cell gap-junctional communication and a delay in meiotic progression through

prophase I to metaphase II, extending the standard IVM interval. Moreover FSH-induced

maturation was prevented by an epidermal growth factor receptor inhibitor, AG1478,

demonstrating that induced oocyte maturation functions via secondary autocrine signalling

within the cumulus cell compartment.

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Results from the present thesis also demonstrated that induced-IVM leads to a substantial

improvement in oocyte quality, which in turn had long-term developmental consequences

improving embryo/fetal yield and pregnancy outcomes. The work presented in this thesis

validates this technology using two mammalian models. In the bovine, induced-IVM more

than doubled embryo yield (27% to 69%), relative to standard-IVM. Similarly in the mouse,

induced-IVM substantially increased fertilization rate (55% vs. 82%), embryo yield (55% vs.

86%), embryo quality, implantation rate (28% vs. 53%), fetal yield (8% vs. 26%) and fetal

weights (0.5g vs. 0.9g). All these embryo and fetal readouts using induced-IVM in mice were

equivalent to those using in vivo matured oocytes (conventional IVF).

In conclusion, induced-IVM mimics some of the characteristics of oocyte maturation in vivo

and substantially improves oocyte developmental outcomes in two disparate mammalian

species. The outcomes of the research presented in this thesis have provided a new

perspective to our understanding of the mechanisms regulating oocyte maturation and the

acquisition of developmental competence. The novel IVM system will provide new options

for a wide range of reproductive biotechnologies including livestock breeding and

conservation applications. Application of induced-IVM to human infertility will bring

substantial cost and health benefits by simplifying ART protocols.

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Declaration This work contains no material which has been accepted for the award of any other degree or

diploma in any university or other tertiary institution to Firas Albuz and, to the best of my

knowledge and belief, contains no material previously published or written by another person,

except where due reference is made in the text.

I give consent to this copy of my thesis when deposited in the University Library, being made

available for loan and photocopying, subject to the provisions of the Copyright Act 1968.

The author acknowledges that copyright of published works contained within this thesis (as

listed below*) resides with the copyright holder(s) of those works.

I also give permission for the digital version of my thesis to be available on the web, via the

university`s digital research repository, the Library catalogue, the Australasian Digital Theses

Program (ADTP) and also through web search engines, unless permission has been granted

by the University to restrict access for a period of time.

* List of Publications

1. Maxime Sasseville, Firas K Albuz, Nancy Côté N, Christine Guillemette, Robert B Gilchrist and Francois J Richard. (2009). Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle. Biology of Reproduction.81: 415-425.

2. Firas K Albuz, Maxime Sasseville, Michelle Lane, David T Armstrong, Jeremy G Thompson

and Robert B Gilchrist. (2009). Induced oocyte IVM substantially improves embryo yield and pregnancy outcomes. Nature Biotechnology. Manuscript No. NBT-TR21146A (submitted 01/06/2009).

October 2009

Firas Albuz

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Acknowledgements

Achieving my infinite dream of pursuing a PhD degree was a painful and enjoyable

experience. It was just like climbing a high peak, step by step, accompanied by bitterness,

hardship, frustration, encouragement, trust and with so many people’s kind help. When I

found myself at the top enjoying the beautiful scenery, I realized that it was, in fact,

teamwork that got me there and a group of awesome people to whom I owe a huge debt of

gratitude.

I would like to express my most sincere gratitude to my two supervisors, Dr Robert Gilchrist

and A/Prof. Jeremy Thompson, for allowing me the opportunity to undertake a PhD in the

field of research that I was always dreaming to achieve immensely. Many thanks for the

continual support, advice and encouragement throughout my project. Your enthusiasm for the

subject and never ending expertise in this area of research has been of tremendous help.

My PhD project could not have occurred without the support of Cook Australia, in particular

Kim Giliam and Aidan McMahon. Thank you for having faith in my project and work.

My very special thanks go to Prof. David Armstrong & Dr. Maxime Sasseville who have

helped me in every aspect of my study and who have provided numerous valuable comments.

Exceptional thanks goes to Dr. Michelle Lane, who helped me with embryo transfer

experiments and gave me the great opportunity to work in the clinical embryology field at

Repromed.

An enormous thanks goes out to past and present staff and students of the Robinson Institute/

The Research Centre for Reproductive Health. In particular, I would like to thank Lesley

Ritter for too many things to name so I will not even try. I would also like to thank Megan

Mitchell, Melanie McDowall, Deanne Feil & Annie Whitty for all the support over the years.

Fellow students: Kim, Sarah, Laura, Kathryn and Hannah over the past 3 years please know

that all your friendship and support have been valuable to my sanity.

I must also give gargantuan thanks to Dave Froiland and Ashley Gauld. I will miss the

Thursday lunch sessions with you guys, thanks for the great support throughout my residency

in Adelaide. I will miss our chats about almost everything and working with you in the lab.

To me, you have been more than just work colleagues; you are great friends.

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Friends sustain you on life`s journey and without them this PhD would not have been

possible. I must thank my incredible network of friends who are members of the “Olive

circle” for all the love and support they have provided throughout the course of my PhD,

Raeleen, George, Raik, Dimi, Tamer & Sarah. Moreover I would like to thank my real friend

and flatmate “the famous wine maker” Marcos for our long chats and real friendship. I will

always remember these beautiful days and your neighbourhood in Henley beach, we were

living in such a beautiful unforgettable place, I will remember this place always and forever.

Raeleen thanks for the food and the hospitality, you guys were my family in Adelaide. I am

so fortunate to have such wonderful, supportive friends and I appreciate everything that you

have done to help me.

My deep love and appreciation goes to my family with whom I shared my childhood and

whose love and support still sustain me today. My sisters and brother: Einas, Eiman and

Mohm`d who are all so much part of me and my memories of childhood. My beloved parents

have given me more than I can even realize or mention here. My mother: Samiha whose

ambition, love and sacrifice is boundless, you are always a constant source of pride, support

and encouragement and of course not to mention the untold number of sacrifices for the entire

family, and specifically for me to continue my schooling. I know that you are always proud of

me. My father: Kamal who is my role model in tolerance, patience and sacrifice; he is a man

who dedicated his whole life to see his children happy and successful. Last but not least, in

this world there are always facts, proved and unproved hypotheses. However, there is only

one hypothesis which will never ever fail: the hypothesis of love. I am finishing up my degree

and writing this thesis because of you my lovely wife Reem. You are the first, the last and

everything in my life, I love you, you put up with a lot of things, but I will never forget your

support, patience and love. Thanks for your understanding and your smile which keep me

persistent day by day. I would therefore like to dedicate this thesis to you and to my family

and to those whose names are not included, but who assisted me in one form or another. I

sincerely thank them all.

These studies were financially supported by a linkage grant (LP0562536) awarded by the

Australian Research Council (ARC) in partnership with COOK Australia and through

Australian Postgraduate Award/Industry.

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Glossary/Abbreviations AC adenylate cyclase AM acetoxy-methyl ester 5’-AMP adenosine 5’-monophosphate AMPK AMP-activated protein kinase ATP adenosine triphosphate BSA bovine serum albumin B-TCM bicarbonate-buffered tissue culture medium CAM calcein acetoxy-methyl ester cAMP cyclic adenosine monophosphate cAMP-PKA cAMP-dependant protein kinase A cGMP cyclic guanosine monophosphate CM cilostamide COC cumulus-oocyte complex dbcAMP dibutyryl cyclic adenosine monophosphate DMSO dimethyl-sulphoxide DO cumulus-oocyte complex derived oocyte ET embryo transfer FAF fatty acid-free FF-MAS follicular fluid meiosis activating sterol FSK forskolin FSH follicle stimulating hormone GC granulosa cell GJC gap junctional communication GV germinal vesicle GVBD germinal vesicle breakdown hCG human chorionic gonadotrophin H-TCM hepes-buffered tissue culture medium iAC invasive adenylate cyclase IBMX 3-isobutyl-1-methylxanthine ICM inner cell mass IVF in vitro fertilization IVM in vitro maturation IVP in vitro production (of embryos) LH luteinising hormone M meiosis phase of the cell cycle MGC mural granulosa cell MI metaphase I MII metaphase II MR milrinone PDE phosphodiesterase PDE3 phosphodiesterase subtype 3 PDE4 phosphodiesterase subtype 4 PDE8 phosphodiesterase subtype 8 PVA polyvinyl alcohol rhFSH recombinant human FSH RIA radioimmunoassay TE trophoectoderm

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Publications and Conference Proceedings Scientific Publications

1. Maxime Sasseville, Firas K Albuz, Nancy Côté N, Christine Guillemette, Robert B Gilchrist

and Francois J Richard. (2009). Characterization of Novel Phosphodiesterases in the Bovine

Ovarian Follicle. Biology of Reproduction.81: 415-425.

[Impact Factor: 3.6]

2. Firas K Albuz, Maxime Sasseville, Michelle Lane, David T Armstrong, Jeremy G

Thompson and Robert B Gilchrist. (2009). Induced oocyte IVM substantially improves

embryo yield and pregnancy outcomes. Nature Biotechnology. Manuscript No. NBT-

TR21146A (submitted 01/06/2009).

[Impact Factor: 22.3]

Other Journal Contributions

1. Firas K Albuz, Maxime Sasseville, David T Armstrong, Jeremy G Thompson and Robert B

Gilchrist. (2008). Synergistic effects of cAMP modulating agents in pre-IVM and in IVM on

bovine cumulus and oocyte functions. Reproduction, Fertility and Development; 20 (Suppl);

Abstract 260.

2. Maxime Sasseville, Firas K Albuz, Francois J Richard, Robert B Gilchrist. (2008). Evidence

for a novel cAMP-phosphodiesterase expressed in the bovine ovarian follicle. Reproduction,

Fertility and Development; 20 (Suppl); Abstract 254.

3. Firas K Albuz, Maxime Sasseville, David T Armstrong, Michelle Lane, Jeremy G

Thompson and Robert B Gilchrist. (2009). Induced oocyte in vitro maturation (IVM)

substantially improves embryo yield and pregnancy outcomes. Reproduction, Fertility and

Development; 21 (Suppl); Abstract 131.

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Conference Proceedings

1. International


Gilchrist. (2009). Substantial improvements in embryo yield using a novel system of induced

IVM by exploiting cAMP modulators in pre-IVM and IVM. Proceedings of the twenty-fifth

annual conference of the European Society of Human Reproduction and Embryology,

Amsterdam, The Netherlands. Abstract 167. (Oral presentation).

2. Robert B Gilchrist, Firas K Albuz and Jeremy G Thompson. (2010). A new approach to

IVM and embryo IVP: Induced-IVM substantially improves embryo yield and pregnancy

outcomes. Proceedings of the thirty-sixth annual conference of International Embryo Transfer

Society Conference, Cordoba, Argentina.

2. National


Gilchrist. (2008). Synergistic effects of cAMP modulating agents in pre-IVM and in IVM on

bovine cumulus and oocyte functions. Proceedings of the thirty-ninth annual conference of

The Society for Reproductive Biology, Melbourne, Australia. (Abstract 260)

2. Maxime Sasseville, Firas K Albuz, Francois J Richard, Robert B Gilchrist. (2008.)

Evidences for a novel cAMP-phosphodiesterase expressed in the bovine ovarian follicle.

Proceedings of the thirty-ninth annual conference of The Society for Reproductive Biology,

Melbourne, Australia. (Abstract 254)


Thompson and Robert B Gilchrist. (2009). Induced oocyte in vitro maturation (IVM)

substantially improves embryo yield and pregnancy outcomes. Proceedings of the fortieth

annual conference of The Society for Reproductive Biology, Adelaide, Australia. (Abstract

131)

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3. Local

1. Firas K Albuz. (2008). Improving the efficiency of oocyte maturation in vitro. The 2008

AusBiotech-GSK Student Excellence Awards National Lauch: Research to Revenue,

Adelaide, Australia.


Thompson and Robert B Gilchrist. (2009). Regulation of cAMP during oocyte maturation

substantially improves developmental outcomes. Proceedings of the Australian Society for

Medical Research SA conference, Adelaide, Australia. (Abstract RW1.)

3. Firas K Albuz (2009). IVF without hormones. The 2009 Women and Children`s Hospital

Young Investigator Award (YIA) Semi-finals-Scientific Presentation, Adelaide, Australia.

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Provisional Patent Firas K Albuz, Robert B Gilchrist, Jeremy G Thompson. “Methods for the collection and

maturation of oocytes” US Provisional Patent Application No. IP10922/473 (14/05/2009).

Visits to Overseas Laboratories

2009 Prof. Johan Smitz, Follicle Biology lab, Vrije Universitiet Brussel (VUB),

Brussels, Belgium.

Invited Guest Seminar - International

2009 Induced oocyte in vitro maturation (IVM) substantially improves

developmental outcomes. Vrije Universitiet Brussel (VUB), Brussels, Belgium (24/06/2009).

xvii

Awards, Scholarship & Prizes

2006-2009 Australian Postgraduate Award Industry (APAI) under the Australian

Research Council (ARC) Linkage Project (PhD scholarship)

2008 AusBiotech-GSK Student Excellence Award Finalist

2008 The Society for Reproductive Biology (SRB) Meat and Livestock

Australia New Investigator Award Finalist

2008 Society for Reproductive Biology Travel Scholarship

2009 Ross Wishart Memorial Award Finalist

2009 AUGU/RC Heddle Award Finalist

2009 The Society for Reproductive Biology (SRB) Meat and Livestock

Australia New Investigator Award Finalist

2009 Research Centre for Reproductive Health Research Scholarship

The University of Adelaide

2009 Faculty of Health Science Travelling Fellowship

The University of Adelaide

2009 Network in Genes and Environment in Development

Conference Participation Award

2009 The Women & Children`s Hospital Young Investigator Award Semi-

Finalist

CHAPTER 1

LITERATURE REVIEW

Chapter 1. Literature review

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1.1 Introduction

Although superovulation protocols used in assisted reproductive technologies (ART) result in

a high number of mature oocytes, major side effects can threaten the patient’s health. Some

patients who undergo exogenous gonadotrophin treatment, such as those suffering from

polycystic ovarian syndrome (PCOS), are at high risk of developing ovarian hyperstimulation

syndrome (OHSS) during the luteal phase or during early pregnancy. The prevalence of

severe OHSS is reported to range from 0.5-5%, and the syndrome may cause liver

dysfunction and sometimes fatal thromboembolism (Dumesic et al. 2007).

Hence, an important goal in ART is to reduce the need for ovarian hyperstimulation; this goal

may be achieved through in vitro maturation (IVM) of oocytes prior to fertilization. IVM is a

reproductive technology by which immature oocytes are retrieved and subsequently matured

in vitro without gonadotrophins. For this reason, IVM is now being used by some fertility

clinics. Owning to the fact that a vast supply of oocytes is captured from an ovary, IVM is

also a feasible tool for livestock breeding and commercial in vitro embryo production (IVP).

Despite its number of potential and important applications, IVM is currently under utilized.

This is because oocytes that undergo IVM demonstrate a reduced level of developmental

competence compared to in vivo matured oocytes; developmental competence is defined as

the ability of an oocyte to produce normal, viable and fertile offspring after fertilization. The

main challenge in improving IVM techniques is to understand the mechanisms involved in

developmental competence.

There are multiple differences between in vitro and in vivo matured oocytes. Unlike in vivo

matured oocytes, in vitro derived cumulus oocyte complexes (COCs) are generally retrieved

from mid-sized antral follicles that may not have achieved complete “oocyte capacitation;


20

which describes the modifications that take place in the oocyte of dominant follicles before

the LH peak (Gilchrist and Thompson 2007). These modifications permit the oocyte to attain

full developmental competence, and therefore, oocytes from smaller follicles may not possess

the complete molecular and cellular pathways needed to support early embryogenesis.

Factors affecting oocyte developmental competence in vitro include culture media additives

such as serum, hormones and growth factors, as well as the companion somatic cells which

surround the oocyte. Considering that current IVM uses media designed for tissue culture

there is a necessity for media formulations tailored specifically for oocytes (Gilchrist and

Thompson 2007).

Overall, IVM is a valuable technique; however, oocytes are inevitably collected from follicles

at various stages of development, ranging from dominant (antral) follicles to atretic ones and

cultured in suboptimal conditions. By understanding the mechanism of meiotic resumption, as

well as developing improved IVM conditions, we can endeavour to develop better IVM

techniques. This will not only improve oocyte developmental competence but could also

improve pregnancy outcomes.

This literature review will firstly cover current clinical aspects of IVM. The review will then

discuss the biological aspects of oocyte maturation in vivo vs. in vitro. Moreover, the review

will focus on current oocyte maturation culture conditions and finally discuss prospective

technologies for improving the developmental competence of oocyte maturation in vitro.


21

1.2 Infertility Treatment and Current Limitation

Assisted reproductive technologies (ART) have become a common practice in human

reproduction. The most common routine form of ART is in vitro fertilization (IVF), a

technology that utilizes ovarian stimulation with gonadotrophins to provide multiple growing

antral follicles (figure 1). This controlled ovarian hyperstimulation results in supernumerary

oocytes being ready for insemination by IVF. Despite the world wide success of IVF, the use

of ovarian stimulation increases the patients` cost and discomfort, and is associated with side

effects including ovarian hyperstimulation syndrome (OHSS) and potential cancer risk

(Duckitt and Templeton 1998). The recovery of immature oocytes followed by in vitro

maturation (IVM) and fertilization is an alternative approach for generating mature oocytes

that eliminates or significantly reduces the need for hormonal stimulation of the ovary.

1.3 In vitro Maturation of Oocytes

In IVM, immature germinal vesicle oocytes (GV) are retrieved transvaginally under

ultrasound guidance from antral follicles before the emergence of a dominant follicle in

unstimulated or minimally stimulated ovaries (Chian et al. 2004b). Oocytes are then matured

in the laboratory under controlled laboratory conditions for 24-48 hours (figure 1), signified

by the extrusion of a first polar body before standard IVF or intracytoplasmic sperm injection

(ICSI) procedures are used to generate a viable embryo (Chian et al. 2004c).


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Conventional IVFOvarian hyperstimulation (FSH + hCG)

In Vitro Maturation(No or minimal ovarian

stimulation)

IVF or ICSI

IVF or ICSI

24 to 48 hours in vitro

Figure L1. Schematic of the major differences between IVM and conventional IVF. Adapted from (Gilchrist

and Thompson 2009).

1.3.1 Rationale for the Clinical Use of IVM

One of the greatest advantages of IVM is the use of a simpler and less stressful protocol,

leading to the reduction of physical hardship to patients, as well as the reduction of time and

costs involved for frequent monitoring of follicular development. Women undergoing IVM

either completely forgo ovarian hormonal stimulation prior to egg collection or receive

minimal stimulation either by minimal follicle stimulating hormone (FSH) stimulation, 3–6

days of low-dose FSH prior ovum pick-up, or either by one bolus dose of 10,000 IU human


23

chorionic gonadotrophin (hCG) stimulation 36 hours prior to ovum pick-up or in combination

of FSH and hCG. This allows a reduced treatment time and avoids common side effects. In

addition the cost of the treatment is significantly reduced compared to conventional IVF, as

the amount of gonadotrophins is low.

Another application of IVM is preservation of women`s fertility; for patients undergoing

cancer treatment, they usually looses their fertility potential due to the devastating effects of

radiation and chemotherapy. With the advances of anti-cancer treatment, many of these

women survive and are desirous of child bearing. With the possibility of oocyte and ovarian

tissue cryopreservation, coupled with IVM, is a potential way to preserve fertility (Tucker et

al. 1998). Designing protocols for immature oocyte cryopreservation may increase the chance

of creating oocyte banking and its application for donation programmes. Pregnancies

resulting from immature oocyte donation have been reported from oophrectomy specimen

(Cha et al. 1991), during caesarean section (Hwang et al. 1997) and from a woman with

polycystic ovaries (Hwang et al. 2002).

Other potential benefits of IVM include the provision of a source of oocytes for research in

the fields of stem cells and cloning, and to test the effects of toxic substances on oocyte

development and maturation.

1.3.2 Potential Applications of IVM (Patient Groups)

The best candidates for IVM are women with PCOS or polycystic appearing ovaries (PCO),

who are at high risk of OHSS, which occurs in approximately 5-10% of women enrolled in

IVF cycles, because of the high antral follicle numbers (Table 1). Pregnancy in PCOS


24

patients can be obtained in unstimulated cycles (Barnes et al. 1996) and mildly stimulated

cycles (Jaroudi et al. 1997; Jaroudi et al. 1999; Mikkelsen and Lindenberg 2001). (Tan et al.

2002) demonstrated that the pregnancy rates after IVM correlated with the number of

immature oocytes retrieved, with the highest pregnancy rate of 26.8% in those with > 10

immature oocytes. Moreover, (Child et al. 2001b) demonstrated that the number of immature

oocytes and pregnancy rates were higher in women with PCOS or PCO than in women with

normal ovaries, and in PCOS women, IVM was comparable to IVF (Child et al. 2002).

Table L1: Incidence of OHSS in IVM vs. IVF in women with PCOS

IVM (n = 107) IVF (n = 107) P

Total units FSH injected 0 2355 ± 833 < 0.01

Moderate or severe ovarian

hyperstimulation syndrome

0% 11.2% < 0.01

Source: Adapted from (Child et al. 2002)

Other potential candidates of IVM are poor responders. Although several stimulation

protocols and strategies have been proposed, some women may still respond poorly to

ovarian stimulation. IVM may be an option for these women. Although the outcomes are still

poor for these women because only a few immature oocytes can be obtained, (Liu et al. 2003)

reported three pregnancies out of eight patients (37.5%), suggesting that IVM may be an

alternative for poor responders.

IVM can also be applied for patients who choose less drug administration. There is an

increasing interest in ‘natural cycle IVF’ or minimal ovarian stimulation, thus IVM represents

a potential application in this area (Chian et al. 2004a; Edwards 2007; Kadoch et al. 2007).

Moreover, previous studies showed a link between ovarian stimulation and increased health

risks: ovarian, breast and endometrial cancer (Brinton 2007; Pappo et al. 2008) and increased


25

incidence of stroke (Demirol et al. 2007). Hence, avoiding the use of gonadotrophins in

applications such as IVM may reduce long-term adverse health outcomes risks, relative to

conventional IVF.

A major advantage of IVM is the substantial reduction in cost. Hence, in low-income

families, in countries where infertility treatment is not publicly subsidized, IVM can be an

alternative and more socially accepted than conventional IVF.

1.3.3 Rescue IVM (GV Oocytes from Stimulated Cycles)

In a conventional IVF cycle after ovarian stimulation with gonadotrophins, about 15% of

oocytes are found in the GV stage or metaphase I stage at the time of oocyte retrieval.

Attempts have been made to mature these oocytes in vitro but the results are poor (Veeck et

al. 1983; Nagy et al. 1996; Jaroudi et al. 1997; Jaroudi et al. 1999). Low fertilization rates

were noticed and those embryos generated were with high incidence of non cleavage (embryo

arrest) (21%) and chromosomal abnormalities (Nogueira et al. 2000). Thus further studies are

necessary to examine the safety of IVM of GV oocytes from stimulated cycles, since these

oocytes are of inferior quality or have intrinsic defects.

1.3.4 IVM Limitations

Although there have been more than 300 births of babies with IVM procedures (Chian et al.

2004c). Up to this date, IVM has not become mainstream in ART, with ovarian stimulation

protocols that generate mature (MII) oocytes followed by IVF still the highly favored

approach. Although some clinics are reporting no differences in pregnancy rates between

IVM and conventional IVF (Cha et al. 2005b). However, most studies reported that embryo


26

development rates, pregnancy and live births from IVM is significantly lower than IVF cases

using ovarian stimulation with triggered maturation in vivo.

1.3.4.1 Fertilization Rates

Insemination by IVF of in vitro matured oocytes has been performed with lower success

compared with in vivo matured oocytes (45% vs. 73% respectively) (Cha et al. 1991).

Hardening of the zona pellucida of the oocytes caused by a prolonged culture period could be

responsible for the decreased fertilization (De Vos and Van Steirteghem 2000). Thus, ICSI is

used to overcome the problem of zona hardening and could in these cases lead to results

comparable to those with IVF. ICSI is now the preferred insemination method for IVM

oocytes. Table 2, show similar fertilization rate between IVM and IVF (in vivo matured

oocytes) when ICSI is applied.

Table L2: Fertilization rate from IVM vs. IVF in women with PCOS

IVM (n = 860) IVF (n = 1244) P

Fertilization rate 78% 78% n.s.


1.3.4.2 Embryo Development Rates

Subsequent embryo development is significantly lower when oocytes are matured by IVM

compared to in vivo matured oocytes (Chian et al. 2004b); with a high incidence of cleavage

arrest after IVM and increased multinucleation owning to cell division arrest (Nogueira et al.

2000). Possible causes of cleavage arrest include inadequate culture conditions (Menezo et al.

1990), inherent or induced oocyte abnormalities (Pickering et al. 1990) and failure of


27

embryonic gene expression (Macas et al. 1990). Consequently, the incidence of polyploidy

and mosaicism is also increased, due to cell division arrest (Munne et al. 1995).

The best measure of embryo developmental rates generated from IVM oocytes is

implantation rate, which is defined as the number of gestational sacs observed at 6 weeks of

pregnancy relative to number of embryo transferred. Implantation rates from conventional

IVF are usually double those matured by IVM as demonstrated in the table below (Table 3)

from (Child et al. 2002). Additionally, Table 4 summarises some of the major studies of

modern IVM, and implantations are consistently just above 10%: ~13% in non-PCOS patients

and 11.6% in PCOS patients (Chian et al. 2003; Jurema and Nogueira 2006).

Table L3: Implantation rate from IVM vs. IVF in women with PCOS

IVM IVF P

Implantation rate 9.5% 17.1% < 0.01


Table L4: Embryo development and implantation rates after clinical IVM

Cycles (n) Cleavage

rate (%)

Embryos transferred

(mean)

Implantation rate

(%)

Reference

94 88 4.9 6.9 (Cha et al. 2000)

24 95 2.7 15.7 (Chian et al. 2000)

36 60 1.8 15.0 (Mikkelsen and Lindenberg

2001)

121 93 3.2 9.3 (Child et al. 2001a)

68 88 3.8 10.5 (Lin et al. 2003)

203 N/A 5.0 5.5 (Cha et al. 2005a)

45 96 2.5 10.9 (Le Du et al. 2005)

48 75 1.6 18.5 (Soderstrom-Anttila et al.

2005)

140 90 3.2 15.4 (Zhao et al. 2008)


28

1.3.4.3 Embryo Transfer Outcomes

Clinical pregnancy is usually evidenced by clinical or ultrasound parameters (ultrasound

visualization of gestational sac). Pregnancy rates are confounded by the number of clinical

pregnancies expressed per 100 initiated cycles, aspiration cycles, or embryo transfer cycles.

Since IVM oocytes have a lower developmental capacity, evidenced by reduced

developmental rates, more embryos are transferred compared to IVF cycles. (Child et al.

2002) transferred (3.2) embryos in IVM cycles, significantly more than (2.7) in IVF. Despite

this, the pregnancy rate from IVM cycles was 21.5% compared to 33.7% from conventional

IVF (Child et al. 2002).

Live birth rate is defined as number of live-birth deliveries expressed per 100 initiated cycles,

aspiration cycles or embryo transfer cycle. (Child et al. 2002) reported that live birth from

IVM oocytes is 15.9% and significantly lower than IVF oocytes (26.2%).

1.3.4.4 Long-Term Health (Offspring) Outcomes

A growing body of evidence has raised concerns about the long-term health outcomes of

IVM. The development of embryos under suboptimal culture conditions clearly produces

offspring that display either immediate or long-term health concerns using animal models

(Ecker et al. 2004)

Since there are approximately 300 reported IVM conceived children worldwide, reports on

follow up data and analysis of offspring by human IVM are recent and still few in the

literature. Based on these very preliminary studies, analysis of offspring produced by IVM


29

detected no adverse health consequences and appear to have normal developmental rates. In a

study that investigated growth parameters including, body weight, body height and head

circumference from 0 to 2 years of age, IVM children were within normal limits (Cha et al.

2005b).

In a separate follow-up study of 46 IVM children (Soderstrom-Anttila et al. 2006), obstetrics

and perinatal outcomes including birth weight were within the normal range. At 6 months of

age, none were assessed as having Considerable Development Disorder and three of them

(7%) had Minor Development Disorder, with a delay in 1-2 fields of development. At the age

of 12 months, 8 children (19%) had Minor Development Disorder and one child demonstrated

Considerable Development Disorder. By 24 months of age, neuropsychological development

of the cohort was within the normal range; 97% of children with an Bayley Mental

Development Index between 92-114 and 3% with an index between 70 – 84 (Soderstrom-

Anttila et al. 2006).

Moreover, there are few studies in animal models investigating long-term outcomes of IVM.

In sheep, following embryo transfer, (Thompson et al. 1995) demonstrated that regardless of

whether embryos were matured in vivo or in vitro, there were no differences in pregnancy rate

between the two groups. This was despite the differing developmental rates and morphology

of embryos.

Although the previously mentioned studies showed no adverse health consequences, these

studies did not report health parameters beyond early childhood and it will be many years

before potential long-term outcomes can be evaluated. Nevertheless, studies with animal

models revealed no potential long-term health consequences to offspring produced by IVM of


30

animal oocytes. Recently, (Eppig et al. 2009) compared the long-term health status and

lifespan of offspring generated from mouse oocytes matured in vitro versus matured in vivo.

There were no differences between the two groups in lifespan or in a wide range of

physiological and behavioural analyses, except the pulse rate and cardiac output were slightly

and significantly lower in IVM offspring.

1.3.5 Conclusion

In vitro maturation of immature oocytes is a promising developing assisted reproductive

technology. In comparison with conventional IVF, the major benefits of IVM include

avoiding potential risks of OHSS, lower costs and simpler treatments. However, the clinical

results of IVM treatment have been far from satisfactory; the rates of implantation and

clinical pregnancy remain disappointingly low. The suboptimal environment for maturation in

vitro is considered to be one of the factors that could account for the poor developmental

outcomes.

The early stages of development are critical in determining fetal development and longer-

term adult health (Barker 1998). Moreover, in vitro embryo production is a multi-step

process, in which IVM is an additional step prior to carrying out IVF for individuals who

respond inappropriately to gonadotrophin stimulation or as an alternative to stimulation for all

patients. Thus studies to optimize culture conditions for immature oocytes using animal

models will be valuable. Understanding the biological aspect of oocyte maturation and trying

to mimic the in vivo environment and adapt it to in vitro, is an important emerging concept

for the development of oocyte culture systems that improving developmental outcomes.


31

1.4 Biological Aspects

1.4.1 Oocyte Development

Oogenesis involves the development of oogonium into a primary oocyte, secondary oocyte,

and finally to an ovum that is capable of fertilization, embryonic development and finally live

birth support. The process of oogenesis begins as primordial germ cells migrate from the yolk

sac to the gonads, where they begin to proliferate mitotically. These oogonia multiply from

only a few thousand cells to millions, and then develop into primary oocytes once they enter

meiosis. However, primary oocytes progress only through part of meiosis and arrest at the

dictyate stage of first meiotic prophase (Picton 2001).

When the oocyte enters meiosis, a single layer of flattened pregranulosa cells encloses it

(figure 2), thus forming the primordial follicle. This is the first step of folliculogenesis: the

process that leads to the formation and development of the ovarian follicle. The follicle

consists of several layers of stromal cells, which will differentiate into theca layers after

follicle growth commences. Oocytes which are not incorporated into primordial follicle will

degenerate (Fortune 2003).

Unlike male germ cells, which are generated continuously from puberty onward, the store of

primordial follicles serves the entire reproductive life span of the adult. The size of the oocyte

in primordial follicles in cattle is approximately 30 µm (Picton 2001). As soon as primordial

follicle stores are established, the process of recruiting follicles into the growing pool begins.

Follicle recruitment continues in a wave-like pattern during the oestrous cycle (Fortune 2003;

Fortune et al. 2004). Moreover, follicle development becomes gonadotrophin dependent once

the follicle forms an antrum (antral follicle) and reaches a certain size (i.e. 4 mm-6 mm in


32

cattle and 2 mm-4 mm in sheep). At this stage oocytes are fully grown having reached a

diameter of 120 µm (Picton 2001).

Oocyte growth commences at the beginning of follicular growth and it is almost completed

by the antrum formation. The oocyte undergoes modifications that confer the oocyte with its

developmental competence. Oocyte maturation which takes place after the LH surge allows

the oocyte to reach the metaphase II stage and to express its developmental competence

following fertilization.


33

Figure L2. Preovulatory follicles consist of a central fluid-filled cavity called the antrum. Layers of cumulus cells surround the oocyte inside the follicle, while the mural granulosa cells surround the central antrum (Thomas 2004).


34

1.4.2 Oocyte Meiotic Arrest

Oocytes are arrested in prophase I of meiosis during prenatal life, and the oocyte remains

under meiotic arrest for many years until follicular growth is completed by gonadotrophins

induction (Tsafriri and Pomerantz 1986). However; oocytes resume meiosis spontaneously

when removed from the follicle (Pincus and Enzmann 1935). This demonstrates the presence

of inhibitory molecules in the follicle which maintain oocytes in meiotic arrest. Moreover,

several possible inhibitory molecules such as intracellular 3`-5`-cyclic adenosine

monophosphate ([cAMP]i), hypoxanthine, steroid hormones and several factors derived from

granulosa cells (Cho et al. 1974; Thomas et al. 2002). Overall, a complex of several

intercellular and paracrine interactions including growth factors, cAMP and gap junctions

between oocytes and somatic cells plays a role in keeping oocytes meiotically arrested in

vivo.

1.4.3 Cumulus Cell-Oocyte Gap Junctional Communication

Oocyte and follicular cells are intimately associated and communicate by an extensive

network of gap junctions (GJ) (Gilula et al. 1978). Gap junction channels are composed of

double membrane protein structures called connexons, each connexon is made up of

transmembrane protein subunits called connexins. Many different connexin types and

combinations have been localised in the ovarian follicle (Kidder and Mhawi 2002).

Homologous gap junctions are found between adjacent cumulus cells while heterologous gap

junctions are found between cumulus cells and the oocyte (Albertini et al. 1975).


35

Gap junctions play an important role in maintaining the oocyte in meiotic arrest by

transferring inhibitory molecules, such as cAMP, which are generated by somatic follicular

cells (Dekel et al. 1981). Recently, it has been hypothesized that heterologous gap junctions

regulate oocyte (prophase I) chromatin configurations (Lodde et al. 2007).

1.4.4 cAMP

Cyclic AMP is the second messenger for gonadotrophin signal transduction. FSH and

luteinising hormone (LH) exert their functions by activating membrane receptors of target

cells, consequently, activating adenylate cyclase (AC) leading to cAMP production. cAMP is

one of the important intracellular signalling molecules which is responsible for maintaining

meiotic arrest in oocytes. Reinitiation of meiosis occurs after a drop in oocyte cAMP levels;

moreover, cAMP acts as a regulator of gap junctional communication. (Dekel and Beers

1978; Dekel and Beers 1980). There are two hypothesized mechanisms by which cAMP

maintains meiotic arrest. Firstly, cAMP diffuses from the somatic cells (namely granulosa

and cumulus cells) to the oocyte and increased intra-oocyte cAMP levels prevent oocyte

maturation (Anderson and Albertini 1976). Secondly, phosphodiesterases (PDEs) degrade

cAMP and the specific type 3 phosphodiesterase (PDE3A) in the oocyte (Tsafriri et al. 1996)

is inhibited by cyclic guanine monophosphate (cGMP) (Hambleton et al. 2005; Norris et al.

2009). Somatic cell derived cGMP enters the oocyte through the gap junctions, thus inhibiting

PDE3A and maintaining meiotic arrest (Tornell et al. 1991; Norris et al. 2009).

It has been suggested that the oocyte is also capable of generating cAMP on its own, due to

the presence of adenylate cyclase (Horner et al. 2003), heterotrimeric G protein (Kalinowski

et al. 2004; Mehlmann et al. 2004) and an orphan member of the G protein-coupled receptor


36

family (GPR3) (Mehlmann et al. 2004; Ledent et al. 2005). Moreover, recent reports suggest

that all these component molecules are involved in maintaining the prophase I arrest (Richard

2007).

In general, intracellular cAMP is regulated by two groups of enzyme: the adenylate cyclases

which generate cAMP; and the PDEs hydrolyse cAMP. cAMP is synthesized from adenosine

triphosphate (ATP) by the membrane bound AC enzyme, cAMP then phosphorylates cAMP-

dependent protein kinase A (PKA) producing its active form, the active PKA then causes a

cascade of protein kinase A pathway protein phosphorylation, and the phosphoproteins

products enable the maintenance of oocyte meiotic arrest (Masciarelli et al. 2004).

Phosphdieseterases are enzymes that hydrolyse cAMP to adenosine 5'-monophosphate

(5'AMP); which is an allosteric activator of an important stress response kinase AMP-

activated protein kinase (AMPK). AMPK and inactivate PKA are associated with the

resumption of oocyte meiosis in mice (Conti et al. 2002; Bilodeau-Goeseels 2003; Masciarelli

et al. 2004). In contrast, recent studies in non-rodent species showed that activation of AMPK

leads to oocyte meiotic arrest (figure 3) (Bilodeau-Goeseels et al. 2007; Mayes et al. 2007).

The role of the PDE isoenzyme in oocyte maturation is being studied using isoenzyme-

specific PDE inhibitors in vitro. These inhibitors act mainly as active site competitors and are

considerably more potent in blocking oocyte maturation than non-selective PDEs (Atienza et

al. 1999). Isoenzyme-specific PDE inhibitors have enabled distinct PDE families presented in

the ovary to be differentiated. In vitro, ovarian follicles treated with a PDE 4 inhibitor

(rolipram) induce oocyte maturation and hence ovulation, and thus have the same effect as

LH (Tsafriri et al. 1996). However, PDE 4 inhibitors have no effect on cAMP elevation in

denuded oocytes (DO) but enhance somatic cell cAMP production in the COC. Treatment


37

with PDE 4 inhibitors doesn’t prevent bovine oocyte maturation (Mayes and Sirard 2002;

Thomas et al. 2002). Moreover, the PDE 3 inhibitors, such as cilostazol, cilostamide and

milirinone play also an important role in regulating oocyte maturation in vitro. PDE 3

inhibitors completely inhibit PDE activity and cause a complete maturation block in murine

oocytes (Richard et al. 2001). Moreover, in other species, such as monkeys (Jensen et al.

2002) and bovine (Mayes and Sirard 2002; Thomas et al. 2002), PDE 3 inhibitors have been

used to block oocyte meiotic resumption in vitro.

The precise mechanism by which intracellular concentrations of cAMP produce a stimulatory

or inhibitory response in oocyte during meiosis is not fully understood. As discussed

previously, high levels of cAMP maintain the oocyte in meiotic arrest and this is supported by

in vitro experiments. Culturing oocytes with agents that maintain high intracellular cAMP

levels or agents that prevent cAMP degradation will maintain oocyte meiotic arrest. However,

transient elevation of cAMP by using cAMP analogues can induce oocyte maturation in vitro.

Consequently, the stimulatory or inhibitory effect of cAMP is presumably dependent on the

levels of cAMP in different compartments of the follicle.


38

Figure L3. Proposed mechanism of cAMP-mediated meiotic arrest. Adapted from (Bornslaeger and Schultz 1985) . Based on the proposition that high intra-oocyte cAMP maintains meiotic arrest, cAMP is synthesised from adenosine triphosphate (ATP) by the membrane bound adenylate cyclase (AC) enzyme (reaction 1). cAMP then phosphorylates cAMP-dependant protein kinase A (cAMP-PKA), producing its active form (reaction 2). The active cAMP-PKA then causes a cascade of protein kinase A pathway protein phosphorylation (reaction 3). The phosphoprotein(s) (x-P) enable the maintenance of oocyte meiotic arrest (ie. the germinal vesicle stage is maintained) (reaction 4). cAMP-phosphodiesterases (PDEs) cause the degradation of cAMP to adenosine 5’-monophosphate (5’AMP) (reaction 5). The consequent decrease in intra-oocyte cAMP levels due to increased activity of or increased expression of the PDEs leads to a shift in the equilibrium of reaction 2, causing active cAMP-PKA to be de-phosphorylated to its inactive form (reaction 6). This reaction is associated with GVBD and the resumption of oocyte meiosis.


39

1.4.5 Oocyte Maturation (in vivo)

Oocyte developmental competence is gradually acquired during the prolonged period of

oocyte growth. Oocyte maturation is a complex process involving nuclear maturation (the

progression of the meiotic cycle) and cytoplasmic maturation. In vitro studies have provided

insight into the importance of substances affecting oocyte maturation and its inhibition, such

as cAMP, growth factors, gonadotrophins, purines, and steroids.

1.4.6 Prophase I & Chromatin Configurations

Mammalian oocyte acquires a series of competencies during follicular development that play

critical roles at fertilization and subsequent stages of preimplantation embryonic

development. Recent studies indicate that these competencies involve remodelling of

chromatin occurring in the germinal vesicle (GV) (Albertini and Barrett 2003). Configuration

of GV chromatin has been studied and correlated with developmental competence of oocytes

in several mammalian species. A common feature in the configuration of GV chromatin in

most species is that the diffuse chromatin (the so-called non-surrounded nucleolar [NSN]

pattern) condenses into a perinuclear ring (the so-called surrounded nucleolar [SN]

configuration) during follicular growth (mice: (Mattson and Albertini 1990; Oviedo-Orta et

al. 2000), pig (Motlik and Fulka 1976; Guthrie and Garrett 2000; Sun et al. 2004), horse

(Hinrichs and Williams 1997; Hinrichs et al. 2005), monkey (Schramm et al. 1993) and

human (Parfenov et al. 1989; Miyara et al. 2003). However, studies are few and the results

are conflicting on the configuration of GV chromatin in bovine oocyte. Liu et al. (2006)

showed that during follicular growth, bovine GV chromatin is condensed into a perinucleolar

ring and that oocytes were synchronized at the floccular stage (floccular chromatin near the

nucleoli and nuclear envelope before GVBD) (Liu et al. 2006). Whereas Lodde et al. (2007)


40

characterized the morphological transitions in the GV during the later phases of oocyte

growth, in which chromatin becomes progressively condensed (Lodde et al. 2007). In

particular, chromatin remodelling , can be timely related with the morphological changes that

occur in both nuclear and cytoplasmic compartment, as well as to the decrease of the

transcriptional activity, which are essential processes to improve oocyte developmental

competence (Lodde et al. 2007; Lodde et al. 2008).

1.4.7 Cytoplasmic Maturation

A number of events take place in the oocyte cytoplasm that plays a role in fertilization and

early embryonic development. Oocyte cytoplasmic maturation involves the accumulation of

mRNA, protein reprogramming and post-translational modifications that are required to

achieve oocyte developmental competence. Moreover, a number of cytoplasmic organelles

(golgi complexes, mitochondria, endoplasmic reticulum) proliferate in the ooplasm followed

by their peripheral dislocation which is regulated by microtubules (Brevini-Gandolfi and

Gandolfi 2001; Krisher 2004; Sirard et al. 2006).

Proper cytoplasmic maturation of the developing oocytes increases the developmental

potential of the oocyte (Bell et al. 1997). While it is true that the proportion of oocytes

competent to complete nuclear and cytoplasmic maturation increases during follicular

development, some oocytes that are able to undergo nuclear maturation to M II are of low

developmental potential and fail to develop to the blastocyst stage. The process utilised for

the mass harvesting of oocytes for bovine IVF does not discriminate between oocytes

retrieved from different follicle sizes or between oocytes retrieved from partially atretic

follicles. Khatir et al. demonstrated that 90% of bovine oocyte progress through meiosis to

the MII stage under routine in vitro culture conditions (Khatir et al. 1996), however most


41

laboratories can only produce blastocysts from 30-40% of these inseminated oocyte (Khatir et

al. 1996). It is well known not all GV stage oocytes, or all oocytes which have matured in

vitro to MII for that matter, posses equivalent developmental competencies and that the

nuclear maturity of an oocyte can be used as an indicator of its development competence

(Gilchrist and Thompson 2007). The fact that in vivo matured oocytes fertilized and cultured

in vitro yield twice as many blastocyst as those matured in vitro, suggest that suboptimal in

vitro oocyte maturation conditions lead to incomplete oocyte cytoplasmic maturation

(Banwell and Thompson 2008).


42

1.4.8 Meiotic Resumption

1.4.8.1 Nuclear Maturation

Meiosis consists of two successive cell divisions following one round of DNA replication.

Meiosis gives rise to four haploid cells from a single diploid cell. This type of cell division is

characteristic of germ cells. Meiosis up to the diplotene stage occurs in the fetal ovary.

During the first meiotic division, maternal and paternal genes are exchanged before the pairs

of chromosomes are divided into two daughter cells, each containing 1n chromosome and 2c

DNA. The second meiotic division occurs without being preceded by DNA synthesis and

nuclear reformation. Haploid germ cells are formed with a 1n set of chromosomes and 1c

DNA. The two meiotic divisions of the oocyte are asymmetrical, resulting in expulsion of

polar bodies. Meiosis in each female germ cell results in a single egg and two polar bodies.

Meiotic stages of oocyte maturation can be classified according to chromosome

configurations (Motlik and Fulka 1976). Immature bovine oocytes show a germinal vesicle

containing dispersed chromatin (GV stage. Meiotic resumption is initiated by germinal

vesicle breakdown (GVBD), leading to diakenesis, where the chromatin is organized into

bivalents. Contraction of chromatin takes place during late diakenesis to metaphase I. At

metaphase I, the contracted bivalents are closely associated at the spindle equator. In

anaphase I, some chromatid pairs are at the spindle equator and others at the pole. In early

telophase, after separation the oocyte and polar body chromosomes are similar in appearance.

Morphologically, the polar body chromosomes appear to degenerate and are enclosed by the

polar body at metaphase II.


43

1.4.8.2 Role of Gonadotrophins/EGF on Meiotic Resumption

The resumption of oocyte maturation is initiated by the preovulatory surge of gonadotrophins

in the follicle. LH is the key hormone regulating oocyte maturation and ovulation. In vitro

studies in cattle have shown that LH receptor mRNA could not be detected in cumulus cells

nor cumulus oocyte complexes either before or after the initiation of oocyte maturation in

vitro or in vivo (Nuttinck et al. 2004). However, theca and mural granulosa cells do express

LH receptors. This suggests that LH is not acting directly on oocytes or COCs to promote

maturation, but that paracrine factors are released by surrounding granulosa cells in response

to LH and then promote the process of oocyte maturation (Peng et al. 1991). Recently, it has

been elucidated that these paracrine factors are members of the epidermal growth factor

(EGF) family (Park et al. 2004).

Amphiregulin, epiregulin and betacellulin are growth factors belonging to the EGF-like

growth factor family and appear to stimulate the process of oocyte maturation (Park et al.

2004). It has been suggested that LH binds to receptors on mural granulosa cells stimulating

cAMP signalling, which in turn, induces the expression of the EGF-like growth factors

epiregulin, amphiregulin, and betacellulin. These growth factors can act as paracrine

mediators and stimulate maturation of follicle-enclosed rodent oocytes and isolated COCs

(Park et al. 2004).

1.4.8.3 cAMP/PDEs

cAMP plays an important role in oocyte maturation not only by controlling meiotic arrest but

in meiotic resumption as well (Dekel and Beers 1978; Dekel et al. 1988; Eppig 1989; Eppig

1991). When ovulation takes place, the LH surge leads to PDE activation, which in turn,


44

hydrolyses cAMP and promotes the reassociation of active catalytic subunits of PKA with its

regulatory subunits, which in turn prevents PKA from further phosphorylating target proteins.

Oocyte proteins become dephosphorylated and meiotic maturation will be initiated (Conti et

al. 2002; Mehlmann 2005b). Other studies showed that a drop in cAMP also takes place in

the early stage of oocyte maturation (Schultz et al. 1983; Tornell et al. 1990; Conti et al.

2002).

Recent studies have revealed that signalling molecules may play a role in the synthesis or

degradation of cAMP. Some of these signalling molecules (GPR3, Gs, Gi) belong to

heterotrimeric G protein family (Mehlmann 2005b). The LH surge may influence the activity

of a ligand which activates GPR3 and turns it off. Consequently, lowering the level of GPR3

diminishes and this is accompanied by a drop in cAMP, and hence, meiotic resumption takes

place. Oocytes of GPR3-null mice oocyte resumes meiosis within antral follicles (Mehlmann

et al. 2004), and mouse oocytes injected with small interfering double-stranded RNA targeted

GPR3 also resume meiosis (Mehlmann 2005a). Overall, in prophase-arrested mouse oocytes,

high levels of cAMP maintain active GPR3 which is coupled to Gs proteins, this in turn

stimulates adenylate cyclase to generate more cAMP (Mehlmann 2005a). However, recent

studies in other cell types have shown that regulators of G-protein signalling (RGS2) protein

can inhibit Gs-mediated cAMP production (Sinnarajah et al. 2001; Roy et al. 2003),

nevertheless, the role of RGS2 protein in the oocyte is still unclear.

As mentioned above, PDEs are important regulators and play a critical role in oocyte

maturation and meiotic resumption. In mammals, PDEs constitute a large family of

isoenzymes and are classified into 11 families, PDE1-PDE11. Their regulation is cell and

tissue specific. (Conti and Jin 2000). The current dogma, established principally in rodents,


45

presents PDE3 as the “oocyte PDE”, while PDE4 is the “granulosa/cumulus PDE” (Tsafriri et

al. 1996). Recently, it has been shown that within the bovine ovarian follicle there is an

alternative distribution of PDE families (Sasseville et al. 2009), in which PDE3A is expressed

and functional in the oocyte and PDE8A and PDE8B are highly expressed in the cumulus

cells with low levels of PDE4D. Furthermore, PDE8A, PDE8B and PDE4D proteins are

present in mural granulosa cells and cumulus-oocyte complexes (COCs) (Sasseville et al.

2009).

PDE inhibitors have been used to understand the role of cAMP in oocyte maturation and

meiotic resumption. In vitro studies showed that PDE3 specific inhibitors (e.g. cilostamide,

milrinone) result in meiotic resumption block in murine (Shitsukawa et al. 2001), bovine

(Mayes and Sirard 2002; Thomas et al. 2002), porcine (Laforest et al. 2005; Grupen et al.

2006) and human (Nogueira et al. 2003a) oocytes. Moreover, PDE3A- and PDE4D-null

female mice show impaired fertility (Jin et al. 1999; Masciarelli et al. 2004). In porcine

showed that PDE3A is the major isoform responsible for cAMP degradation and regulates

meiotic resumption (Sasseville et al. 2006).

1.4.8.4 Loss of Gap Junctional Communication (GJC)

Previous studies suggested that oocyte meiotic resumption in vivo takes place due to the

termination in transferring meiotic inhibitory substances such as cAMP, purines and other

molecules originated from the follicular somatic cells to the oocyte which has an effect on

gap junctional communication (GJC) loss (Motlik et al. 1986; Larsen et al. 1987).

GJC plays an important role in oocyte maturation; during the LH surge, the number of gap

junctions decreases and meiotic resumption takes place in the oocyte (Eppig 1982; Larsen et


46

al. 1987). Moreover, these events are accompanied by progressive cumulus cells expansion

and hyaluronic acid synthesis which is induced by FSH (Salustri et al. 1990) via cAMP

mediated mechanisms (Ball et al. 1983).

As mentioned previously, one of the theories hypothesized is that cAMP is generated by

surrounding granulosa cells and is transmitted through gap junctions and hence increase the

intracellular cAMP levels in the oocyte (Dekel and Beers 1978; Dekel et al. 1981). Moreover,

this hypothesis further suggests that the LH surge accompanied with oocyte maturation occurs

leading to interruption of GJC (Gilula et al. 1978; Edry et al. 2006). Furthermore, according

to this hypothesis, the release of the oocyte from the ovarian follicle actually mimics the effect

of LH, thus the supply of the inhibitory cAMP from the granulosa cells to the oocyte is

terminated and oocyte resumes maturation. Webb et al. (2002) demonstrated that cAMP

transport from somatic cells into the oocyte through gap junctions is subjected to negative

regulation by gonadotrophins (Webb et al. 2002). Recently a conflicting study showed that

GJC controls meiotic arrest in mammalian oocytes, providing physiological channels that

transmit inhibitory cAMP from the granulosa cell into the oocyte (Edry et al. 2006). The

exact relationship between GJC loss and oocyte meiotic resumption still remains

controversial.

In bovine, in vivo and in vitro studies have suggested that the breakdown of gap junctions

between cumulus cells takes place in parallel to GVBD incidence. In vivo, gap junctional

breakdown occurs 9-12 hours after the LH peak in super-ovulated cattle while in vitro was

reduced after 3 hours (Hyttel 1987). However, studies in porcine (Motlik et al. 1986) and

murine (Eppig and Downs 1984) oocytes suggest GVBD precedes gap junctional breakdown.

Other studies suggested that gap junctions between the cumulus cells and the oocyte are


47

maintained for hours after the loss of gap junctional coupling within the cumulus vestment

itself (Motlik et al. 1986; Buccione et al. 1990).


48

1.4.8.5 MPF

Maturation promoting factor or M-phase promoting factor (MPF) is a protein kinase presents

in dividing cells. MPF regulates the G2/M transition in both germ and somatic cells (Nurse

1990). Thus, in all studied species, it has been shown that MPF is active during oocyte

maturation and triggers a series of reactions leading to nuclear breakdown, chromosome

condensation, spindle formation and therefore entry into meiosis and second meiotic arrest.

MPF is composed of two subunits; cyclin B regulatory protein and p34cdc2 kinase, also

known as CDK1 (Gautier et al. 1988). The activation of MPF refers to the dephosphorylation

of CDK1, and synthesis of cyclin B (Nurse 1990; Levesque and Sirard 1996). In mouse

meiotic competent oocytes, pre-MPF is already present and de novo protein synthesis is not

required to undergo GVBD (Wickramasinghe and Albertini 1993). In human and other

domestic species, GVBD requires protein synthesis, because pattern of proteins is different

before and after maturation (Schultz and Wassarman 1977).

During oocyte maturation an increase in MPF takes place and stimulates meiotic resumption

and oocytes enter metaphase I stage of the first meiotic division. After metaphase I, it

decreases during the anaphase to telophase transition, but remains elevated above a certain

baseline. This causes the extension of the condensed state of chromatin and prevention of

DNA replication. The activity of MPF increases again, due to neo-synthesis of cyclin B, until

the oocyte proceeds to meiosis II, where it arrests with high levels of MPF activity that are

stabilized by cytostatic factor which is activated by the gene product of the proto-oncogene c-

mos (Ledan et al. 2001; Vigneron et al. 2004).


49

1.4.8.6 FF-MAS

In vitro studies on murine oocytes cultured with inhibitors of spontaneous maturation, such as

hypoxanthine, suggest that a positive regulator produced by somatic cells, is transferred to the

oocyte and induce meiotic resumption (Eppig and Downs 1987; Downs et al. 1988). Byscov

et al. 1995 identified this activity as a sterol now known as ‘meiosis activating sterol’. The

sterol in human follicular fluid (FF-MAS) is 4,4-dimethyl-5 -cholest-8,14,24-triene-3ß-ol, an

intermediate in the sterol biosynthetic pathway produced by demethylation of lanosterol by

the cytochrome P450 enzyme, 14 -demethylase. The sterol from bull testicular tissue (T-

MAS) is 4,4-dimethyl-5 -cholest-8,24-diene-3ß-ol. Both FF-MAS and T-MAS are involved

in increasing the percentage of matured murine oocyte cultured with hypoxanthine (Byskov et

al. 1995). FF-MAS was also capable of initiating meiosis in denuded oocytes and COCs from

several mammalian species when meiotic arrest was maintained with a variety of inhibitors,

including hypoxanthine, isobutylmethylxanthine (IBMX) and dibutyryl cAMP (dbcAMP)

(Byskov et al. 1995; Hegele-Hartung et al. 1999). Recent studies showed that FF-MAS also

promotes the meiotic progression from MI to MII; and may play a role in stabilizing oocytes

in metaphase II arrest (Marin Bivens et al. 2004a). Additionally, FF-MAS production is

increased in response to gonadotrophins; however, which of the gonadotrophins

physiologically mediate FF-MAS production in vivo remains to be clarified (Downs et al.

2001; Tsafriri et al. 2002; Grondahl et al. 2003).

1.5 Oocyte Maturation (in vitro)

In vitro, oocytes undergo spontaneous maturation when removed from the follicle without the

prerequisite for gonadotrophin stimulation (Pincus and Enzmann 1935). It was this

observation, along with other experimental results which demonstrated that oocytes cultured


50

inside their own follicles in vitro are maintained in meiotic arrest, which implicates the

follicular environment in maintaining the oocyte at meiotic arrest (Sirard et al. 2001).

The previous studies also showed that oocytes matured in vitro are compromised in their

developmental capacity compared to oocytes matured in vivo, and a low percentage of in vitro

matured oocytes have full developmental potential (Thompson et al. 1995; Hyttel et al. 1997;

Farin et al. 2001; Gilchrist and Thompson 2007).

There are two fundamental differences between in vivo and in vitro matured oocytes. Firstly,

oocytes matured in vitro bypass oocyte capacitation. Important factors which allow the oocyte

to attain full developmental competence such as proteins or mRNAs which are stored during

oocyte growth are bypassed during in vitro spontaneous maturation. This is considered a

defect in the molecular and cellular machinery for supporting early embryo development

(Hyttel et al. 1997). Secondly, oocytes undergo spontaneous maturation when removed from

the follicle, due to the loss of the in vivo inhibiting environment (Thompson et al. 1995;

Hyttel et al. 1997; Farin et al. 2001; Gilchrist and Thompson 2007).

Overall, current efforts are focused to understand the complex interaction between the oocyte

and the cumulus cells, in attempt to overcome the artefact of oocyte maturation in vitro and

develop a system to support developmental competence.

1.6 Prospective Concepts for Improving IVM Culture System

The challenge of oocyte maturation in vitro is firstly, to provide a milieu that reflects the

naturally changing environment to which the oocyte is exposed; and secondly, support is

needed for the complex cellular changes taking place in the follicular cells and the oocyte.

Culture systems need to support a lengthy period of maturation and the ultimate goal in the


51

process of maturing the oocyte in vitro is to produce developmentally competent oocytes. The

development of an efficient oocyte collection, handling and maturation system is, therefore,

very important.


52

1.6.1 IVM Collection and Handling Media

Media used for the collection and handling of oocytes has received little attention and has

been largely ignored in oocyte biology research. Collection of oocytes usually occurs in

buffered tissue culture medium, in the case of oocytes aspirated from abattoir collected

ovaries, or even simple phosphate buffered saline (PBS) when oocytes are retrieved from

living animals using Trans-Vaginal Oocyte Recovery (TVOR). In either case, the follicular

fluid in which the oocyte was bathed in in vivo is rapidly diluted and eliminated, and the

oocyte is then exposed to a non-physiological environment. These oocyte collection

procedures, together with the media commonly used, actually promote a form of precocious

oocyte meiotic maturation. Although this is the means by which oocyte maturation is

spontaneously initiated in vitro, in this sense, oocyte IVM is an artefact. Triggering oocyte

maturation during collection and handling by this means has a deleterious effect on

subsequent oocyte developmental competence. Designing a novel oocyte collection base

medium supplemented with additives will actively prevent precocious oocyte maturation

during the collection and the handling phase.

Within the follicle, the level of cAMP within the oocyte is high, but removal from the follicle

leads to a precipitous fall in intra-oocyte cAMP levels within 15-30 minutes (Luciano et al.

1999), leading to immediate meiotic resumption. According to different studies, attenuating

this very rapid drop in cAMP and the associated precocious oocyte maturation has a positive

effect on cattle oocyte quality, as measured by developmental competence (Luciano et al.

1999; Thomas et al. 2004b).

Moreover, it has recently been shown that this precocious oocyte maturation is exacerbated

when using buffered tissue culture media for collection and handling procedures, specifically


53

due to high glucose concentrations (5.6 mM) and absence of gonadotrophins (Sutton-

McDowall et al. 2005). Finally, designing a novel collection base medium containing low

concentrations of glucose and additives which delay the process of meiotic progression

significantly improve the efficiency of the IVM process.

1.6.2 IVM Culture Media

Several commercially supplied media are commonly used for the base IVM culture system;

such as TCM-199, Waymouth MB 752/1, Ham`s F-12, Minimal Essential Medium (MEM)

and Dulbecco`s modification of Eagle`s medium (DMEM). In general, culture media consists

of a balanced salt solution, bicarbonate ions, amino acids, an energy source and various

additives; allowing approximately 80% of immature oocytes to reach metaphase of the

second meiotic division. However, up to this date, commonly only 15%-40% of the matured

oocytes reach the blastocyst stage by fertilization in vitro (Ward et al. 2002). The low

blastocyst outcome can be attributed to several factors. Firstly, current culture media, such as

TCM-199 is a complex media designed specifically for somatic cell culture and consequently,

doesn’t reflect the follicular milieu environment (Gilchrist and Thompson 2007). Secondly,

diverse fetal calf serum batch variations generate great variability in the efficiency of embryo

production (McKiernan and Bavister 1992).

Moreover, more studies in in vitro embryo production systems in murine (Khosla et al. 2001),

bovine (van Wagtendonk-de Leeuw et al. 2000) and ovine (Young et al. 2001) suggest that

using serum in culture leads to epigenetic modifications and fetal overgrowth consistent with

large offspring syndrome (LOS). Serum is thought to be containing a variety of growth

factors and endotoxins that can interfere with the process of oocyte maturation and embryo

development (Young and Fairburn 2000).


54

1.6.3 Prematuration Culture (PMC)

In vitro, resumption of meiosis occurs spontaneously once the oocyte is removed from its

follicular environment. This is accompanied by a rapid drop in cAMP transcriptional activity

and results in reduced developmental incompetence (Eppig et al. 1994b). Interestingly,

recent studies have shown that oocytes develop greater developmental competence when

cultured under conditions that arrest meiosis at the GV stage before in vitro maturation

occurs. This is known as prematuration culture (PMC) (Luciano et al. 1999; Thomas et al.

2004b).

It has been recognized that the deficiency in IVM outcomes is due to the asynchrony between

nuclear and cytoplasmic maturation. It is hypothesized that during this prematuration period

there is a build up of mRNA stores, morphological changes and ultrastructural remodelling

(e.g. changes in shape, number and migration of ooplasmic organelles and changes in

chromatin distribution), which might positively reflect on the oocytes` capacity for embryonic

development (Luciano et al. 1999; Thomas et al. 2004b). Meiotic arrest in vitro has been

studied using either physiological conditions by using follicular fractions or chemicals to

block spontaneous maturation.

1.6.3.1 Physiological Conditions & Meiotic Arrest in vitro

Prematuration culture conducted by culturing COCs with follicular fluid (Sirard and First

1988), co-culture with granulosa or theca monolayers (Richard et al. 1997), follicular

hemisections or by culturing antral follicle (Sirard and Coenen 1993).

Follicular fluid provides nourishment to the oocyte and is involved in regulating oocyte

maturation. Follicular fluid contains high concentrations of inhibitory substances such as

linoleic acid in bovine and purines in porcine, murine and primates (Sirard and First 1988;


55

Downs et al. 1989). However, the effect of follicular fluid on oocyte meiotic inhibition

depends on the size of the follicle, for example, follicular fluid from small follicles have the

greatest ability to inhibit meiosis in bovine COCs (Sirard and First 1988).

Bovine COCs can also be kept arrested when co-cultured with granulosa or theca cell

monolayers (Richard and Sirard 1996). The inhibitory effects of granulosa cells may depend

on the number of cell layers surrounding the COC. Theca cells monolayers enhance the

proportion of bovine oocytes kept in meiotic arrest even when FSH is supplied; however, the

presence of cumulus cells is essential to maintain meiotic arrest (Richard and Sirard 1996).

1.6.3.2 Inorganic Additives & Meiotic Arrest in vitro

1.6.3.2.1 Purines

Purines such as adenosine and hypoxanthine maintain murine oocytes in meiotic arrest

(Downs and Eppig 1987; Downs 1993; Downs 1999), whereas in bovine oocytes, the

inhibitory effect is transient (Sirard and First 1988). Moreover, analysis of bovine follicular

fluid revealed the presence of sufficient amounts of these purines to maintain murine oocytes

in meiotic arrest (Downs et al. 1986b). However, bovine oocytes treated with adenosine

resume meiosis (Sirard and First 1988) suggesting that purines play a different role in these

two species.

1.6.3.2.2 Protein Kinase Inhibitors

Changes in protein phosphorylation are correlated with the activation of two major M-phase

kinases: p34cdc2 component of MPF and MAPK. Phosphorylation inhibitors prevent meiotic


56

resumption by acting on regulatory proteins that feature kinase activity (Lonergan et al.

1997). Protein kinase inhibitors are used to control the phosphorylation status of specific

residues of p34cdc2; MPF activity is therefore blocked and hence maintains oocytes in

meiotic arrest.

Oocyte prematuration culture has been applied by using various inhibitors of specific cyclin-

dependent kinases (e.g. roscovitine and butyrolactone I) which inhibit oocyte maturation MPF

activity. The effect of these inhibitors on embryo development are summarised in Table 5.

These studies showed that delaying spontaneous maturation by manipulating MPF activity, in

general either adversely affect or have no positive effect on developmental competence.

Hence, using these kinase inhibitors in IVM culture systems to improve developmental

competence is probably not realistic.

Table L5: Effect of kinase inhibitors used during oocyte meiotic arrest on embryo development

Inhibitor type Site of action Species studied Embryonic development Authors Butyrolactone I MPF activity bovin unchanged Lonergan et. al, 1997; 2000 Butyrolactone I MPF activity bovine improved Hashimoto et. al, 2002 Butyrolactone I MPF activity bovine unchanged Wu et. al, 2002 Butyrolactone I MPF activity bovine unchanged Adona et. al, 2004 roscovitine + MPF activity bovine unchanged Ponderato et. al, 2002 Butyrolactone I roscovitine MPF activity bovine unchanged Mermillod et. al, 2000 roscovitine MPF activity bovine decreased Adona et. al, 2004 6-DMAP MPF/MAPK activity bovine decreased Avery et. al, 1998 6-DMAP MPF/MAPK activity rodents/human unchanged Anderiesz et. al, 2000


57

1. 6.3.2.3 cAMP Modulators (AC, PDEs)

Cho et al., 1974 were the first to suggest that mammalian oocytes are regulated by cAMP

(Cho et al. 1974); moreover, as mentioned previously increasing the intracellular cAMP

levels in oocytes delays meiotic maturation. Recently, the modulation of cAMP levels in vitro

by using membrane-permeable cAMP analogues or PDE inhibitors has become an area of

interest in the field of IVM.

Treatment with dbcAMP or 8-bromo-3`-5`-cAMP (8-Br-cAMP) transiently delays meiotic

resumption in bovine oocytes (Homa 1988; Sirard and First 1988). The efficiency of 8-Br-

cAMP in delaying meiotic progression is more effective than dibutyryl cAMP (Homa 1988).

This is due to the fact that dbcAMP is easily degraded whereas 8-Br-cAMP is only partially

degradable by PDEs (Beebe et al. 1988).

Non-selective inhibitors of PDEs that degrade cAMP, such as hypoxanthine and IBMX, have

also been studied in mammalian species in vitro and used to block spontaneous oocyte

maturation (Dekel and Beers 1978; Eppig et al. 1985). However, in bovine oocytes the effect

is transient (approximately 45% of bovine COCs treated with IBMX are meiotically arrested)

(Sirard and First 1988; Downs 1999).

Additionally, stimulation of adenylate cyclase results in increasing cAMP levels in oocytes.

Forskolin, a direct stimulator of the catalytic subunit of adenylate cyclase, increases the

synthesis of cAMP in both bovine COC and DO (Bilodeau et al. 1993). Forskolin treatment

significantly increases the number of bovine oocyte in meiotic arrest (Homa 1988; Kuyt et al.

1988; Richard et al. 1997). The inhibitory effect of forskolin on oocyte maturation is

increased in combination with IBMX (Bilodeau et al. 1993).


58

The enzyme invasive adenylate cyclase (iAC), which is purified from Bordetella pertussis,

plays an important role in modulating ([cAMP]i). However, high concentrations of iAC can

reversibly inhibit meiotic resumption (Aktas et al. 1995a), whereas low concentrations can

promote oocyte maturation and improve oocyte developmental competence in bovine species

(Luciano et al. 1999; Luciano et al. 2004). The presence of 0.01 µg/ml of iAC in bovine

maturation medium results in an increase in blastocysts numbers compared to control

standard maturation medium supplemented with FCS and gonadotrophins only. Moreover, the

positive effect was also associated with prolonged gap junctional permeability that

presumably enhances the communication between oocyte and cumulus cells during IVM

(Luciano et al. 1999; Luciano et al. 2004).

The presence of specific PDE3 inhibitors during IVM blocked meiotic resumption of mouse

(Eppig 1989; Nogueira et al. 2003b; Vanhoutte et al. 2008; Vanhoutte et al. 2009b) and

human oocytes (Nogueira et al. 2003a; Vanhoutte et al. 2007; Shu et al. 2008; Vanhoutte et

al. 2009a; Vanhoutte et al. 2009b), but temporarily attenuate meiotic resumption in bovine

oocytes (Sirard and First 1988; Aktas et al. 1995b; Thomas et al. 2004a; Thomas et al.

2004b). Exposure of mouse immature oocytes to Org9935 (Nogueira et al. 2003b) or

cilostamide (Vanhoutte et al. 2008; Vanhoutte et al. 2009b) improves their quality and

developmental potential. In bovine, the addition of milrinone delays also the rupture of gap-

junctional communication between the oocyte and the surrounding cumulus cells, resulting in

increased blastocyst yield and quality (Thomas et al. 2004a; Thomas et al. 2004b). In

addition, enhancement of nuclear maturation rates occur when human oocytes are matured in

the presence of Org9935 (Nogueira et al. 2006). Furthermore, combined exposure of human


59

oocytes to cilostamide and forskolin has a positive effect on fertilization rate following IVM

(Shu et al. 2008).

Overall, in contrast to the MPF inhibitors, adding these cAMP-modulating agents to IVM

culture systems often improve or has no detrimental effect on subsequent oocyte

developmental competence. The effect of these inhibitors on embryo development are

summarised in Table L6.

Although considerable evidence supports cAMP-dependent meiotic arrest, conditions exist

whereby mammalian oocyte maturation can apparently occur without a preceding drop in

oocyte cAMP. For instance, whole follicles or COCs maintained in meiotic arrest with cAMP

analogues or PDE inhibitors can resume meiotic maturation when stimulated with

gonadotrophins, despite the continued presence of an arresting factor (Dekel and Beers 1978;

Downs et al. 1988).

These studies show that additional or alternative mechanisms exist other than lowered cAMP

levels to complete the meiotic resumption process. An important key for this is the product of

PDE activity, 5’AMP; a compound that has not been studied in oocyte biology, presumed to

be an inactive product of the enzyme in oocytes. However, 5’AMP is a potent regulator of

AMP-activated protein kinase (AMPK), which is allosterically activated by the binding of

5’AMP (Hardie 2003).


60

Table L6: Effect of cAMP modulating agents used during oocyte meiotic arrest on embryo development

cAMP modulating agent Species studied Embryonic development Authors

cAMP analogues Porcine Improved Funahashi et. al, 1997 Improved Bagg et. al, 2006 PDE inhibitors Bovine Improved Thomas et. al, 2004 Porcine Unchanged Grupen et. al, 2006 Murine Improved Nogueira et. al, 2003 Improved Vanhoutte et. al, 2008 Improved Vanhoutte et. al, 2009 Human Unchanged Nogueira et. al, 2006 Improved Shu et. al 2008 Improved Vanhoutte et. al, 2009 iAC Bovine Unchanged Aktas et. a l, 1995 Bovine Improved Luciano et. al, 1999

1.6.3.2.4 AMPK Activators

AMPK is a stress activated kinase present in many different cell types, whose activity is

stimulated by a myriad of different cellular stresses that increase the intracellular AMP:ATP

ratio. Minute increases in AMP level or significant decreases in ATP levels considerably

increase the AMP/ATP ratio and hence stimulate AMPK activation (Hardie 2003). In vitro,

the compound 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) stimulates

AMPK activation in murine oocytes (Downs et al. 2002); moreover, phosphorylation of

AICAR by adenosine kinase results in the production of an AMP analog AICA ribotide

(ZMP).


61

Treatment of murine meiotically arrested oocytes with AICAR, stimulates both AMPK

activity and germinal vesicle breakdown (GVBD), suggesting a potential meiosis-inducing

function of AMPK in rodent oocytes (Downs et al. 2002). By using a non spontaneous oocyte

maturation model, oocytes were cultured in medium containing dbcAMP or hypoxanthine to

maintain meiotic arrest, then meiotic resumption was induced by AMPK (Downs et al. 2002).

Moreover, isoforms of the catalytic subunit of AMPK were detected in both cumulus cells

and oocytes, and it was concluded that PDE-generated AMP may act as an activator of

meiotic progression through activation of AMPK (Downs et al. 2002).

Recently, studies conducted in bovine and porcine oocytes shown that AMPK is also present

in cumulus cells and oocytes as well (Bilodeau-Goeseels et al. 2007; Mayes et al. 2007).

However, the effect of AICAR on bovine and porcine oocyte maturation is opposite to that of

murine. AICAR totally blocks meiotic resumption and keeps oocytes in meiotic arrest in a

dose-dependent manner in non-rodents species, suggesting that AMP, the activator of AMPK,

has an inhibitory effect on meiosis. Moreover, due to the presence of AMPK in cumulus cells,

AMPK activators are more potent in COCs than in denuded oocytes. This suggests a role for

AMPK in gap junctional communication and inhibiting meiotic progression in non-rodent

oocytes. Furthermore, the combination of forskolin and low doses of AICAR in culture

medium results in non-rodent oocyte meiotic progression (Bilodeau-Goeseels et al. 2007;

Mayes et al. 2007).

Extensive in vitro studies should be performed to investigate the effect of AMPK on oocyte

developmental competence in the presence of AMPK activators. Given the fact that there are

no studies that demonstrate the relative importance of AMPK activators used as an additive in


62

IVM media, addition of AMPK activators in IVM medium would potentially improve the

culture condition for in vitro matured oocytes.


63

1.7 Conclusion

Oocyte maturation is a complex process involving oocyte-follicular cell interaction in which

many of the mechanisms involved remain unclear. Many questions remain about the

mechanisms governing the final process of oocyte development. Due to the lack of substantial

knowledge, the quality of oocytes matured in vitro is far from optimal. Although oocyte

nuclear maturation can be successfully achieved in vitro, the ooplasm is insufficiently mature

to promote normal pre- and post-implantation embryonic development.

It is becoming increasingly clear that oocytes ascribe some of the causes of failed embryonic

development to faulty microtubular and chromatin organization and stability as well as the

abnormal onset of the embryonic genome. It has been recognized that the deficiency in in

vitro matured oocytes is mostly a result of the asynchrony between nuclear and cytoplasmic

maturation. Manipulating the pace of oocyte maturation in vitro (i.e., PMC) might alleviate

this asynchrony by allowing time for ooplasmic maturation to catch up and, therefore, better

synchronize the nuclear and cytoplasmic compartments. This can be achieved by adding

modulators of biochemical pathways to IVM medium with the interaction of improving the

developmental competence of maturing oocyte and thereby the efficiency of IVP.

The successful characterization of fully defined optimized culture conditions for

preimplantation development in vitro is a priority. It is of particular importance as assisted

reproductive technology clinics prepare to adopt human in vitro oocyte maturation and

embryo culture as a routine procedure.


64

1.8 Hypothesis and Aims for PhD Project

1.8.1 Hypothesis

Regulating the pace of oocyte maturation by adding cAMP modulators to IVM culture

systems synchronizes oocyte nuclear and cytoplasmic compartments, leading to improved

developmental competence of maturing oocytes.

1.8.2 Aims

1. To examine the effect of inhibition of PDE8 during IVM of bovine oocytes on cAMP levels, meiotic and developmental capacity.

2. To establish an induced IVM model in bovine, by manipulating oocyte cAMP immediately following collection and during the maturation process, and to examine the effect of induced IVM on oocyte/cumulus cells functions and hence oocyte developmental competence.

3. To optimize the pre-IVM phase in bovine induced IVM: the duration of exposure and

effect of maintaining or increasing intra-oocyte cAMP before IVM on the acquisition of embryonic developmental competence.

4. To devise a murine induced IVM system and examine the effects of induced IVM on

oocyte maturation, developmental competence, pregnancy outcomes and fetal parameters.

CHAPTER 2

EFFECT OF INHIBITION OF PHOSPHODIESTERASE TYPE 8

DURING IN VITRO MATURATION OF BOVINE OOCYTE ON cAMP LEVELS,

MEIOTIC AND DEVELOPMENTAL CAPACITY

Chapter 2. Effect of inhibition of phosphodiesterase type 8 during in vitro maturation of

bovine oocytes on cAMP levels, meiotic and developmental capacity

66

2.1 Abstract

Cyclic adenosine mono-phosphate (cAMP) is an important second messenger that controls

numerous functions in the ovarian follicle, such as steroidogenesis, gene expression and

oocyte meiotic maturation. The phosphodiesterase (PDE) protein family is a group of

enzymes that catalyses the transformation of cAMP into its inactive form 5`- adenosine

monophosphate (5`AMP). In rodents, the current dogma of PDE distribution in the ovarian

follicle predicts functional expression of the PDE type 3 (PDE3) in the oocyte and of PDE4 in

cumulus and granulosa cells. Our recent results show that PDE8 is a prominent PDE in

bovine cumulus/granulosa cells. The present study was conducted to examine the effect of

inhibition of PDE8 during in vitro maturation (IVM) of bovine oocytes on cAMP levels,

meiotic and developmental capacity. Inhibition of PDE8 degradation using dipyridamole

resulted in a dose-dependent increase in cAMP levels within the COC (P < 0.05), whereas

PDE4 inhibition with rolipram did not. Moreover, in combination of forskolin during IVM,

dipyridamole significantly delayed oocytes progression to the M II stage (10 %) compared to

forskolin-treated control (42%) or forskolin-rolipram treatment (40%) (P < 0.05). Whereas, in

absence of forskolin, dipyridamole had no further effect in meiotic maturation; regardless of

increasing concentration. However, dipyridamole supplementation during in vitro maturation

(IVM) failed to enhance oocyte developmental potential. These results are the first to

demonstrate the functional presence of PDE8 in the mammalian ovarian follicle and explain

why IBMX is inefficient in maintaining bovine oocyte meiotic arrest. In conclusion, this

challenges the recently described compartmentalization of PDEs in the follicle and the notion

that PDE4 is the predominant granulosa/cumulus cell PDE. These findings have implications

for our understanding of mechanisms underpinning hormonal regulation of folliculogenesis

and the potential application of PDE inhibitors as novel contraceptives.



67

2.2 Introduction

The mechanisms that regulate mammalian oocyte meiotic maturation have not been fully

elucidated. In vivo, oocytes remain arrested at the germinal vesicle (GV) stage unless exposed

to the pre-ovulatory surge of gonadotrophins. Alternately, in vitro, isolation of the oocyte

from an antral follicle initiates spontaneous meiotic resumption independent of gonadotrophic

hormonal stimulation (Pincus and Enzmann 1935).

It is generally accepted that the second messenger cyclic adenosine mono-phosphate (cAMP)

plays an important role in regulating and maintaining oocytes arrested at the GV stage. Cyclic

AMP is synthesized by adenylate cyclase and degraded to its inactive form into 5`- adenosine

monophosphate (5`AMP) by phosphodiesterases (PDEs). Mammalian PDEs currently

constitute 11 distinct families of isoenzymes (PDE1-11), which consist of at least 23 gene

types (Conti and Beavo 2007). Compartmentalisation of two PDE types has been

demonstrated in rat follicles, where PDE3A has been localized and restricted to the oocyte,

while PDE4D and 4B is detected in the granulosa/cumulus cell compartment (Tsafriri et al.

1996) .

Spontaneous resumption of meiosis after liberation from the follicular environment is thought

to result in compromised oocyte developmental potential compared to in vivo matured

oocytes, as demonstrated by reduced blastocyst development post-fertilization (Greve et al.

1987; Leibfried-Rutledge et al. 1987; Lonergan et al. 2003). Previous studies showed that

supplementing bovine oocyte maturation medium with specific PDE inhibitors, delays

spontaneous nuclear maturation and the rupture of gap-junctional communication between the

oocyte and the surrounding cumulus cells, resulting in increased blastocyst yield and quality

(Thomas et al. 2004a; Thomas et al. 2004b).



68

In the ovarian follicle, the current PDE distribution model is based mainly on observations in

rodents (Tsafriri et al. 1996). Broad spectrum PDE inhibitors and PDE3-specific inhibitors

can maintain meiotic arrest when used during in vitro maturation (IVM) in pig, mouse and rat

oocytes (Tsafriri et al. 1996; Nogueira et al. 2003b; Laforest et al. 2005). However, these

inhibitors are only partially effective in preventing bovine oocyte meiotic resumption from

occurring (Sirard and First 1988; Aktas et al. 1995b; Thomas et al. 2002; Barretto et al.

2007).

The current dogma, established principally in rodents, presents PDE3 as the “oocyte PDE”,

while PDE4 is the “granulosa/cumulus PDE” (Tsafriri et al. 1996). Our recent results show

that within the bovine ovarian follicle there is an alternative distribution of PDE families

(Sasseville et al. 2009), Appendix 5, paper I), in which PDE3A is expressed and functional in

the oocyte and PDE8A and PDE8B are highly expressed in the cumulus cells with low levels

of PDE4D. Furthermore, PDE8A, PDE8B and PDE4D proteins are present in mural

granulosa cells and cumulus-oocyte complexes (COCs) (Sasseville et al. 2009), Appendix 5,

paper I).

Based on this information, the aim of this present study was to determine how inhibition of

PDE8 degradation during IVM affects cAMP levels and hence oocyte meiotic resumption.

PDE8 could be a novel pharmacological target to improve bovine oocyte IVM conditions and

to increase oocyte developmental competence.



69

2.3 Materials & methods

Chemicals

Unless otherwise noted, all chemicals were bought from Sigma Chemical Co. (St. Louis, MO,

USA).

2.3.1 Oocytes recovery and culture

Bovine ovaries were collected from local abattoirs and transported to the laboratory in warm

saline (~37 ºC). The contents of antral follicles (2 to 8mm diameter) were aspirated using an

18-gauge needle and a 10-ml syringe. COCs with intact cumulus vestments were selected

under a dissecting microscope and transferred to 35mm Petri dishes (Falcon) containing

undiluted freshly aspirated follicular fluid. COCs were washed three times with H-TCM (16

mM Hepes-buffered tissue culture medium 199) supplemented with 100 IU/ml penicillin, 100

µg/mL streptomycin sulfate and 0.03% (w/v) polyvinyl alcohol (PVA). The base medium for

oocyte maturation (for oocyte meiotic maturation assessment and cAMP measurement

experiments) was synthetic oviduct fluid (SOF) medium (Ali and Sirard 2002) with 8 mg/ml

bovine serum albumin (BSA fraction V), modified Eagle medium (MEM) nonessential amino

acids (Gibco BRL, Burlington, ON, Canada), MEM essential amino acids (Gibco), 0.33 mM

pyruvate, 50 μg/mL gentamicin, 1 mM glutamine and 0.1 IU/ml FSH. Where indicated,

cAMP modulators were added from a millimolar stock solution stored -20�C dissolved in

DMSO. Groups of 30 COCs were cultured in 4-well multidishes in pre-equilibrated 500 µl

drops overlaid with mineral oil and incubated at 38.5 ºC with 6% CO2 in humidified air.

2.3.2 cAMP modulators

In this study, different cAMP modulators were used. The 3-Isobutyl-methylxanthine (IBMX)

was used as a broad-spectrum PDE inhibitor. It inhibits all PDE families, with the important

exception of PDE8 and PDE9 (Sasseville et al. 2009), Appendix 5, paper I). For specific PDE



70

inhibition, cilostamide and rolipram were used as specific PDE3 and PDE4 inhibitors,

respectively. Dipyridamole (Calbiochem, La Jolla, CA) was used as specific PDE8 inhibitor,

which is currently the best PDE8 inhibitor commercially available. In addition to PDE

inhibitors, forskolin was used which is an adenylate cyclase activator that increase cAMP

levels. All pharmacological modulators were stored in aliquots at -20�C until use.

2.3.3 Measurement of intracellular cAMP

Cyclic AMP quantification was performed using a radioimmunoassay method described and

validated (Reddoch et al. 1986). After 4 hours of IVM, 10 COC were washed twice with H-

TCM with 2 mM IBMX, transferred to 0.5ml 100% ethanol and stored at –20�C. Prior to

quantification, samples were vortexed for 30 seconds and centrifuged at 3000g for 15 min at

4�C. Supernatant was evaporated, resuspended in assay buffer (50mM sodium acetate, pH

5.5) acetylated in 2:1 triethylamine (AJAX Chemicals, Sydney, Australia) : acetic anhydride

(BDH Laboratory Supplies, Poole, England) solution. 125I-labelled cAMP (specific activity of

2175 Ci/mM) and cAMP antibody as prepared by (Reddoch et al. 1986) were added to

samples and left overnight at 4�C. The following day, 1ml cold 100% ethanol was added to

samples and were centrifuged at 3000g. The supernatant was removed and the pellet dried

and counted using a gamma counter. Samples were performed in duplicates and compared to

a standard curve of known cAMP concentration (0-1024 fmol cAMP).

2.3.4 Oocyte nuclear maturation assessment

Oocyte nuclear status was assessed by aceto-orcein staining. At the end of the incubation,

COCs were denuded by gentle pipetting and transferred to a fixing solution (3:1 ethanol:

acetic acid) for > 24hrs before staining with nuclear dye (1% orcein dissolved in 45% acetic

acid). Fixed oocytes were then mounted on slides and compressed beneath a cover slip



71

supported by petroleum jelly and retained with glue. Oocytes were examined with a phase

contrast microscope at 400x and classified as being at either germinal vesicle (GV),

diakinesis I, metaphase I (M I), anaphase I, telophase I or metaphase II (M II) stages (Motlik

et al. 1978; Homa 1988). For graph simplicity, those oocytes at diakinesis I were pooled with

and classified as MI, and those at anaphase I and telophase I were pooled with and classified

as MII. Approximately 10% of oocytes that were denuded prior to culture were at the GV

stage and of a degenerative appearance upon meiotic assessment and were consequently not

included in the final assessment.

2.3.5 In vitro maturation, fertilization and embryo culture

COCs were recovered as described above. For this experiment, the basic medium for oocyte

maturation was Bovine VitroMat (IVF Vet solutions, Adelaide, Australia), a medium based

on the ionic composition of bovine follicular fluid (Sutton-McDowall et al. 2005). All IVM

treatments were supplemented with 0.1 IU/ml FSH (Puregon, Organon, Oss, Netherlands). 30

COCs were cultured in pre-equilibrated 300 μl drops overlaid with mineral oil and incubated

at 38.5 ºC with 6% CO2 in humidified air for 24 or 30 hours.

After 24 or 30 hours of culture, COCs were removed from the IVM wells and washed twice

using Bovine VitroWash (IVF Vet solutions), and transferred to insemination dishes

containing Bovine VitroFert (IVF Vet solutions) supplemented with penicillamine (0.2 mM),

hypotaurine (0.1 mM), and heparin (2 mg/ml). Frozen semen from a single bull of proven

fertility was used for inseminating all treated oocytes in all experiments. Briefly, thawed

semen was layered over a discontinuous (45%: 90%) Percoll gradient (Amersham

Bioscience) and centrifuged (RT) for 20-25 mins at 700 g. The supernatant was removed and

the sperm pellet was washed with 500 μl Bovine VitroWash and centrifuged for a further 5

minutes at 200 g. Spermatozoa were resuspended with VitroFert and added to the fertilization



72

media drops (Bovine VitroFert, supplemented with 0.01 mM heparin, 0.2 mM penicillamine

and 0.1 mM hypotaurine) at a final concentration of 1 x 106 spermatozoa/ml. COCs were

inseminated at a density of 10 μl of IVF medium per COC for 24 h, at 39�C in 6% CO2 in

humidified air (day 0).

Cumulus Cells were removed by gentle pipetting 23–24 h post insemination and five

presumptive zygotes were transferred into 20µl drops of pre-equilibrated Cook Bovine

VitroCleave (IVF Vet solutions) and cultured under mineral oil at 38.5°C in 7% O2, 6% CO2,

balance N2, for five days (day 1 to day 5). On day 5, embryos were transferred in groups of 5-

6 to 20 μl drops of pre-equilibrated Bovine VitroBlast (IVF Vet solutions) at 38.5°C overlaid

with mineral oil and cultured to Day 8. Embryos were assessed for quality at Day 8

according to the definitions presented in the Manual of the International Embryo Transfer

Society (Stingfellow 1998) and were performed independently and blinded by an experienced

bovine embryologist.

2.3.6 Statistical analyses

Statistical analyses were conducted using Prism 5.00 GraphPad for Windows (GraphPad

Software, San Diego, CA, USA, www.graphpad.com). Statistical significance were assessed

by analysis of variances (ANOVA), followed by either Dunnett's (figures 1 and 2) or

Bonferroni’s (figure 3) multiple-comparison post-hoc tests to identify individual differences

between means. Probabilities of P < 0.05 were considered statistically significant. All values

are presented with their corresponding standard error of the mean (SEM).



73

2.4 Results

2.4.1 The role of PDE8 on cAMP degradation in the cumulus-oocyte complex

Currently the only known inhibitor of the PDE8 family is dipyridamole (Fisher et al. 1998;

Soderling et al. 1998). To assess the role of PDE4 and PDE8 in the management of ovarian

follicle cAMP, COC were matured for 4 hours in the presence of 100 µM forskolin (an

adenylate cyclase activator) in combination with 10 or 100 µM rolipram (a specific PDE4

inhibitor) or 10, 50 and 250 µM dipyridamole. The presence of forskolin substantially

increased (P < 0.05) cAMP in the COCs after 4 hours (figure 1). PDE4 inhibition by co-

culturing COCs with rolipram did not lead to a further increase in cAMP within the COC

(figure 1). However, the addition of 50 µM of dipyridamole or higher doses led to a further

significant increase in cAMP in the COCs (figure 1). This supports the hypothesis that PDE8

is a regulator of intracellular concentrations of cAMP in the COCs and is more efficient than

PDE4.

2.4.2 The effect of PDE8 inhibition on oocyte meiotic maturation

It has been demonstrated that the spontaneous maturation of isolated bovine COCs is delayed

by elevating or maintaining intra-oocyte cAMP levels, or by inducing a massive increase in

cAMP in the cumulus cells that surround the oocyte (Thomas et al. 2002; Luciano et al.

2004). In order to examine the role of dipyridamole in delaying meiotic resumption, COCs

were matured for 16 hours in the presence of either forskolin or various PDE inhibitors.

Figure 2 shows that 87% of untreated control oocytes reached metaphase II (M II) stage by 16

hours of IVM; which is not significantly different from oocytes matured in the presence of

dipyridamole, regardless of concentration. However, only 42% of oocytes matured in the

presence of forskolin progressed to the M II stage (figure 2B). Interestingly, maturing COCs

in the presence of rolipram and forskolin had no further effect on meiotic maturation, while

COCs matured in the presence of forskolin and 50 µM of dipyridamole and above



74

significantly, delayed meiotic completion compared to forskolin-treated controls (forskolin +

dipyridamole = 10% M II vs. forskolin = 42% M II, P < 0.05). This experiment further

demonstrates the importance of PDE8, in contrast to PDE4, in modulating a cAMP-regulated

function of the COC.

2.4.3 The effect of PDE8 inhibition on cleavage and development to the blastocyst

stage

Inhibition of PDE3 and PDE4 during IVM improved oocyte developmental competence of

bovine oocytes, demonstrated by increased blastocyst yield post-fertilization (Thomas et al.

2004b). Thus, we elucidated a possible role of PDE8 inhibition in oocyte developmental

potential, since our results show that PDE8 is also an important functional PDE in bovine

cumulus cells and oocytes. As treatment with forskolin and dipyridamole prevented or

delayed polar body extrusion, the optimum time for fertilization was determined by maturing

COCs for 24 or 30 hours in FSH-stimulated IVM in the presence of 50 µM dipyridamole.

Following IVM, oocyte meiotic maturation was assessed (figure 3A) or COCs were fertilized

and cultured for 5 days to determine the rate of embryos that reached the blastocyst stage

(figures 3B & C). Figure 3A shows that dipyridamole supplementation during IVM doesn’t

show any further delay in COCs meiotic progression. Moreover, the results in figure 3B show

that dipyridamole treatment significantly reduced the cleavage rate of oocytes fertilized after

30 hours of IVM (figure 3B, P < 0.05). Figure 3C shows a decreased blastocyst formation rate

in treated COCs after 30 hours of IVM compared to controls (P < 0.01). These results suggest

that the continuous inhibition of PDE8 during IVM using dipyridamole, does not promote

oocyte cytoplasmic compartments and developmental competence.



75

2.5 Discussion

Compared to in vivo matured oocytes, oocytes isolated from follicles and matured in vitro

have significantly lower capacity to support embryo development (Greve et al. 1987;

Leibfried-Rutledge et al. 1987; Trounson et al. 2001; Gilchrist and Thompson 2007). Unlike

nuclear maturation, which can easily be measured, the phenomenon of cytoplasmic

maturation is largely uncharacterized, with embryonic development post-fertilization being

the best indicator of completed maturation (Greve et al. 1987). Decreased oocyte

developmental competence is associated with deficiencies in the mRNA storage during

oocyte growth in non-rodents (Greve et al. 1987). Premature removal of oocytes from mid-

sized antral follicles induces precocious spontaneous maturation, that occurs in the absence of

crucial events and environments that are required for complete cytoplasmic maturation of the

oocyte (Gilchrist and Thompson 2007).

The use of subtype-specific PDE inhibitors provides a new opportunity for more extensive

examination of oocyte maturation mechanisms and represents new and powerful experimental

tools for investigating oocyte-follicular cell interactions during oocyte maturation (Thomas et

al. 2002). Moreover, the addition of PDE inhibitors during IVM results in improved

developmental outcomes in human (Nogueira et al. 2006; Shu et al. 2008), bovine (Thomas et

al. 2004b) and mouse oocytes (Nogueira et al. 2003b).

The present study investigated the role of PDE8 inhibition during IVM on bovine oocyte and

cumulus cells functions and subsequent oocyte developmental competence. Although PDE8

inhibition has no effect on oocyte meiotic maturation and oocyte developmental competence,

the current results results put a new perspective to the current model of PDE distribution in

the mammalian ovarian follicle as we showed that PDE8 is the predominant PDE expressed

in the cumulus cells along with low levels of PDE4. Inhibiting PDE8 degradation in cumulus



76

cells using dipyridamole, increased cAMP levels in the COCs, however didn’t show any

further delay in oocyte nuclear maturation.

Together with previous experiments conducted in this study, by measuring cAMP-PDE

activity in the ovarian follicle; various PDEs expression and detection by RT-PCR and

immunoblot detection in the various compartments of the ovarian follicle and western

blotting (Sasseville et al. 2009), Appendix 5, paper I), these results put a new perspective to

the accepted distribution of PDE families in the ovarian follicle and enlighten new avenues of

hormonal and pharmacological manipulation of follicle growth and ovulation (McKenna et

al. 2005).

Levels of cAMP were significantly higher when COCs were matured in the presence of

forskolin and dipyridamole; compared to when oocytes are matured and treated in presence of

forskolin and rolipram (figure 1).

Treating COCs with dipyridamole alone (the PDE8 inhibition) did not affect the proportion of

oocytes reaching M II after 16 hours of IVM (figure 2A). However, combined treatment of

dipyridamole and forskolin significantly impaired meiosis compared to combined treatment

of dipyridamole and rolipram (PDE4 inhibition) or forskolin alone, with less COCs reaching

M II (figure 2B). This experiment demonstrates the importance of PDE8, in contrast to PDE4,

in regulating bovine oocyte nuclear maturation and these results are not consistent with

previously published results in rodents (Reinhardt et al. 1995; Tsafriri et al. 1996).

The present results also demonstrate that dipyridamole treatment does not improve oocyte

developmental capacity when COCs are matured for either 24 or 30 hours, despite the

treatment leading to increased COC cAMP levels and delaying spontaneous meiotic

resumption. Moreover, it seems that dipyridamole is harmful to oocytes matured for long



77

periods such as 30 hours (figures 3 B & C), evidenced by significantly reduced cleavage and

blastocyst rates. The surrounding cumulus cells were not successfully expanded and were

fragile after maturing COCs in the presence of dipyridamole (figure 4B). Furthermore,

several oocytes had atypical ooplasmic contour. Fragile cumulus cells and abnormal

ooplasmic features were not observed in the control group (figure 4A).

Thomas and collaborators showed that PDE3- or PDE4 specific inhibition of FSH-treated

bovine COCs significantly increased blastocyst development rate and cell number compared

to COCs treated with FSH alone (Thomas et al. 2004b). These results suggest that subtle

PDE4 inhibition (<5% of total PDE activity in the cumulus) (Sasseville et al. 2009),

Appendix 5, paper I), rather than broad inhibition using IBMX or more specific inhibition

using dipyridamole, is a better way to improve oocyte quality and developmental capacity.

Bearing in mind that the only known PDE8 inhibitor is the one used in this study

(dipyridamole), it might be that this specific inhibitor negatively affects essential cumulus

cells functions such as gap-junctional communication, cell signalling or supply of metabolites

to the oocyte. To dissect these possibilities, the development of alternative specific PDE8

inhibitors would be required.

In conclusion, this study is the first to characterize novel functions of PDE8 in the

mammalian ovary. During IVM, PDE8 inhibition using dipyridamole increased COC cAMP

and did not delay oocyte spontaneous maturation (meiotic resumption), although oocyte

developmental capacity was not improved. Further studies should provide better

understanding into the role of PDE8 inhibition during IVM on fundamental mechanisms such

as cumulus cells-oocyte gap junctional communications. Moreover, investigations are also



78

needed for applying specific PDE8 inhibitor during IVM other than dipyridamole which can

explain the exact role of PDE8 inhibition on oocyte developmental outcomes.



79

2.6 Figures

Figure 1. The effect of an adenylate cyclase activator (forskolin) in combination with PDE inhibitors (rolipram or dipyridamole) on the cAMP content of bovine oocytes cultured and assayed with their vestments intact (COCs) after 4 hours of IVM. Values are expressed as the mean concentration of cAMP/COC ± SEM of three replicates using 6-10 COCs per treatment replicate. Means with different letters indicate significantly different amounts of cAMP between treatments (one-way ANOVA, P<0.05).

0

500

1000

1500

Forskolin (�M)Rolipram (�M)

Dipyridamole (�M)

000

10000

100

1000

010

050

0250

100 100 100 100 100

a

bb

b b

c

ccA

MP

(fMol

/CO

C)



80

Figure 2. The effect of an adenylate cyclase activator (forskolin) and PDE inhibitors (rolipram or dipyridamole) on bovine COC-cultured oocyte nuclear maturation after 16 hours of IVM. The effect of dipyridamole treatment alone (A) and of PDE inhibitors in the presence of forskolin (B) was investigated. The data represent the mean metaphase II percentage ± SEM of 3 replicates. Each replicate had at least 25 COCs per treatment. Means with different letters indicate significant differences between treatments (one-way ANOVA, P<0.05).

0

20

40

60

80

100

Forskolin (100 �M)Dipyridamole (�M)

-

-

-

10

-

250

-

50

% M

II

0

20

40

60

80

100

Forskolin (100 �M)

Dipyridamole (�M)Rolipram (�M)

+

--

-

--

+

10-

+

250-

+

50-

+

-100

+

-10

a

b bbb

c c

% M

II

A

B



81

Figure 3. The effect of dipyridamole treatment of COC during in vitro maturation on oocyte maturation and developmental capacity. (A) Bovine COC were cultured with FSH for 24 or 30 hours alone or together with dipyridamole (50 μM) where nuclear status was assessed and classified as GV arrested (GV), metaphase I (MI) or metaphase II (MII). In subsequent experiments, oocytes were submitted to in vitro fertilization and embryo development. Oocyte developmental capacity was assessed by the cleavage rate (B) and the blastocyst rate on day 8 (C). The data represent a mean ± SEM of 3 replicates. (one-way ANOVA).

0

25

50

75

100GVMIMII

Time of IVM (Hours)

Dipyridamole (50 �M)

24-

24+

28-

28+

Perc

enta

ge o

f ooc

yte

atde

fined

sta

ge

0

20

40

60

80

100 P<0.05

Time of IVM (Hours)Dipyridamole (50�M) -

24

+

24

-

30

+

30

Perc

enta

ge o

f cle

aved

embr

yo p

er fe

rtiliz

ed o

ocyt

e

0

20

40

60

80

100

P<0.01

Time of IVM (Hours)Dipyridamole (50�M) -

24+

24-

30+

30

Perc

enta

ge o

f bla

stoc

yst

per c

leav

ed e

mbr

yo

A

B

C



82

A

B Figure 4. COCs post 24 hours of IVM matured either in control group with 100 mIU/ml FSH (A) or matured in the presence of 100 mIU/ml FSH and 20 µM dipyridamole (B). All micrographs were originally taken using a 40x objective.



83

2.7 References Aktas H, Wheeler MB, Rosenkrans CF, Jr., First NL, Leibfried-Rutledge ML (1995) Maintenance of bovine oocytes in prophase of meiosis I by high [cAMP]i. J Reprod Fertil 105, 227-35. Ali A, Sirard MA (2002) Effect of the absence or presence of various protein supplements on further development of bovine oocytes during in vitro maturation. Biol Reprod 66, 901-5. Barretto LS, Caiado Castro VS, Garcia JM, Mingoti GZ (2007) Role of roscovitine and IBMX on kinetics of nuclear and cytoplasmic maturation of bovine oocytes in vitro. Anim Reprod Sci 99, 202-7. Conti M, Beavo J (2007) Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem 76, 481-511. Fisher DA, Smith JF, Pillar JS, St Denis SH, Cheng JB (1998) Isolation and characterization of PDE8A, a novel human cAMP-specific phosphodiesterase. Biochem Biophys Res Commun 246, 570-7. Gilchrist RB, Thompson JG (2007) Oocyte maturation: emerging concepts and technologies to improve developmental potential in vitro. Theriogenology 67, 6-15. Greve T, Xu KP, Callesen H, Hyttel P (1987) In vivo development of in vitro fertilized bovine oocytes matured in vivo versus in vitro. J In vitro Fert Embryo Transf 4, 281-5. Homa ST (1988) Effects of cyclic AMP on the spontaneous meiotic maturation of cumulus- free bovine oocytes cultured in chemically defined medium. J Exp Zool 248, 222-31. Laforest MF, Pouliot E, Gueguen L, Richard FJ (2005) Fundamental significance of specific phosphodiesterases in the control of spontaneous meiotic resumption in porcine oocytes. Mol Reprod Dev 70, 361-72. Leibfried-Rutledge ML, Critser ES, Eyestone WH, Northey DL, First NL (1987) Development potential of bovine oocytes matured in vitro or in vivo. Biol Reprod 36, 376-83. Lonergan P, Rizos D, Gutierrez-Adan A, Fair T, Boland MP (2003) Oocyte and embryo quality: effect of origin, culture conditions and gene expression patterns. Reprod Domest Anim 38, 259-67. Luciano AM, Modina S, Vassena R, Milanesi E, Lauria A, Gandolfi F (2004) Role of intracellular cyclic adenosine 3',5'-monophosphate concentration and oocyte-cumulus cells communications on the acquisition of the developmental competence during in vitro maturation of bovine oocyte. Biol Reprod 70, 465-72. McKenna SD, Pietropaolo M, Tos EG, Clark A, Fischer D, Kagan D, Bao B, Chedrese PJ, Palmer S (2005) Pharmacological inhibition of phosphodiesterase 4 triggers ovulation in follicle-stimulating hormone-primed rats. Endocrinology 146, 208-14.



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Motlik J, Koefoed-Johnsen HH, Fulka J (1978) Breakdown of the germinal vesicle in bovine oocytes cultivated in vitro. J Exp Zool 205, 377-83. Nogueira D, Cortvrindt R, De Matos DG, Vanhoutte L, Smitz J (2003a) Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro. Biol Reprod 69, 2045-52. Nogueira D, Ron-El R, Friedler S, Schachter M, Raziel A, Cortvrindt R, Smitz J (2006) Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development. Biol Reprod 74, 177-84. Pincus G, Enzmann E (1935) The comparative behaviour of mammalian eggs in vivo and in vitro. J. Exp. Med. 62, 665-675. Reddoch RB, Pelletier RM, Barbe GJ, Armstrong DT (1986) Lack of ovarian responsiveness to gonadotropic hormones in infantile rats sterilized with busulfan. Endocrinology 119, 879-86. Reinhardt RR, Chin E, Zhou J, Taira M, Murata T, Manganiello VC, Bondy CA (1995) Distinctive anatomical patterns of gene expression for cGMP-inhibited cyclic nucleotide phosphodiesterases. J Clin Invest 95, 1528-38. Sasseville M, Albuz FK, Cote N, Guillemette C, Gilchrist RB, Richard FJ (2009) Characterization of novel phosphodiesterases in the bovine ovarian follicle. Biol Reprod 81, 415-25. Shu YM, Zeng HT, Ren Z, Zhuang GL, Liang XY, Shen HW, Yao SZ, Ke PQ, Wang NN (2008) Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes. Hum Reprod 23, 504-13. Sirard MA, First NL (1988) In vitro inhibition of oocyte nuclear maturation in the bovine. Biol Reprod 39, 229-34. Soderling SH, Bayuga SJ, Beavo JA (1998) Cloning and characterization of a cAMP-specific cyclic nucleotide phosphodiesterase. Proc Natl Acad Sci U S A 95, 8991-6. Stingfellow DA, and Seidel, S. M (1998) Manual of the International Embryo Transfer Society. In. (IETS: Savoy, IL, USA) Sutton-McDowall ML, Gilchrist RB, Thompson JG (2005) Effect of hexoses and gonadotrophin supplementation on bovine oocyte nuclear maturation during in vitro maturation in a synthetic follicle fluid medium. Reprod Fertil Dev 17, 407-15. Thomas RE, Armstrong DT, Gilchrist RB (2002) Differential effects of specific phosphodiesterase isoenzyme inhibitors on bovine oocyte meiotic maturation. Dev Biol 244, 215-25. Thomas RE, Armstrong DT, Gilchrist RB (2004a) Bovine cumulus cell-oocyte gap junctional communication during in vitro maturation in response to manipulation of cell-specific cyclic adenosine 3',5'-monophosophate levels. Biol Reprod 70, 548-56.



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Thomas RE, Thompson JG, Armstrong DT, Gilchrist RB (2004b) Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity. Biol Reprod 71, 1142-9. Trounson A, Anderiesz C, Jones G (2001) Maturation of human oocytes in vitro and their developmental competence. Reproduction 121, 51-75. Tsafriri A, Chun SY, Zhang R, Hsueh AJ, Conti M (1996) Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors. Dev Biol 178, 393-402.

CHAPTER 3

SUBSTANTIAL IMPROVEMENTS IN BOVINE EMBRYO YIELD USING A

NOVEL SYSTEM OF INDUCED IVM BY EXPLOITING cAMP MODULATORS IN

PRE-IVM AND IVM

Chapter 3. Substantial improvements in bovine embryo yield using a novel system of induced IVM by exploiting cAMP modulators in pre-IVM and IVM

87

3.1 Abstract

Cellular and molecular events in oocyte maturation vary markedly in vivo and in vitro (IVM).

In vivo, maturation occurs in a synchronized, induced manner when meiotic inhibitory effects

found in the follicular environment are overridden by an LH surge. In contrast, maturation in

vitro occurs spontaneously when oocytes are extracted from the follicle. Accordingly, it has

been hypothesized that prolonging spontaneous maturation during oocyte maturation will

promote molecular events that more closely resemble in vivo events, thereby improving post-

maturation outcomes via improved cytoplasmic maturation. We hypothesized that

establishing an induced system in vitro, by manipulating oocyte cAMP immediately

following collection and during the maturation process, synchronizes nuclear and cytoplasmic

compartments leading to improved oocyte developmental competence.

Cumulus-oocyte complexes (COCs) were obtained by aspiration of >3mm follicles from

abattoir-derived bovine ovaries. Within 5 min of retrieval, COCs were precultured for 5-120

min in medium supplemented with/without cAMP modulators: non-specific

phosphodiesterase inhibitor (IBMX, 500 µM) and adenylate cyclase activator (forskolin,

FSK, 100 µM). After pre-IVM, COCs were cultured +/- FSH and +/- type-3

phosphodiesterase inhibitor (cilostamide, 20 µM). At multiple time points during pre-IVM

and IVM, cAMP levels, cumulus-oocyte gap junctional communication (GJC), germinal

vesicle (GV) configurations and meiotic maturation were measured. Following either 24 or 30

hours IVM, in vitro production of embryos was performed.

Within 30 min of aspiration, cAMP in control COCs dropped significantly from 15±4 to 2±1

fmol/COC, whereas FSK+IBMX during pre-IVM increased levels to 180±19 fmol/COC, and

this had substantial persistent effects on a number of COC functions throughout IVM and

embryo development. After 2 hours of pre-IVM, FSK+IBMX delayed meiotic resumption


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(GV3-4: control, 80%; treated, 25%) and significantly increased oocyte-cumulus gap

junctional communication (GJC) (340±73 vs. 1000±148 units; control vs. treated, P < 0.05).

This increased GJC persisted at 5 hours regardless of +/- cilostamide in IVM. After 9 hours of

IVM, 92% of pre-IVM treated COCs were arrested at the GV stage compared to 28% in

controls. Moreover, progression to metaphase II (MII) was delayed by modulators in pre-

IVM and IVM (24±1, 75±2, 92±1% MII at 20, 24 and 28 hours, respectively), compared to

absence in the pre-IVM alone (78±1, 95±1, 98±2% MII). Progression to MII with modulators

in pre-IVM + IVM required FSH suggesting a form of induced maturation. Confirming this,

FSH-induced maturation of cAMP modulator-treated COCs was prevented by the EGF-

receptor kinase inhibitor AG1478, demonstrating autocrine ligand-induced oocyte maturation.

After 24 hours of IVM, intra-oocyte cAMP levels remained 12-fold higher with modulators in

both phases compared to in IVM alone. Notably, embryo development was only substantially

enhanced by cAMP modulators in pre-IVM and in IVM and when IVF was delayed. cAMP

modulators in either pre-IVM or IVM alone did not improve blastocyst rates. Blastocyst rates

were higher in pre-IVM + IVM treated COCs (48%) than IVM alone (22%) or pre-IVM alone

(32%) with IVF at 24 hours. However, substantial improvements in blastocyst yield (69%)

and blastocyst cell number were achieved by delaying IVF to 30 hours in pre-IVM + IVM

treated COCs.

These results show that intracellular levels of cAMP prior to IVM have a profound effect on

oocyte and cumulus cell functions and hence on blastocyst yield and quality. Moreover, the

efficacy of cAMP modulators during IVM is substantially improved by management of COC

cAMP levels during the pre-IVM phase. Careful modulation of intracellular cAMP levels

within a physiological range in the oocyte and cumulus cells during collection and

maturation, along with specific FSH concentrations during IVM, generate a form of induced

maturation in vitro that leads to acquisition of high oocyte developmental competence.


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3.2 Introduction

Oocyte developmental competence is gradually acquired during the growth and development

of the follicle. The process of oocyte maturation is a complex, poorly defined orchestration of

cytoplasmic, molecular and nuclear events that need to occur in a synchronized fashion

(Gilchrist and Thompson 2007; Richard 2007). Immature germinal vesicle (GV)-stage

oocytes, when retrieved from antral follicles, are capable of resuming meiosis spontaneously

and progress to metaphase II (MII) (Pincus and Enzmann 1935). Although immature oocytes

can resume meiosis following isolation, cytoplasmic maturation; important machineries

which are crucial to support preimplantation embryo development, lags behind nuclear

maturation (Hubbard and Terranova 1982).

It is generally accepted that the second messenger cyclic adenosine mono-phosphate (cAMP)

plays an important role in oocyte maturation in mammalian oocytes. Levels of cAMP are

maintained by synthesis, via adenylate cyclase (AC), and degradation, via the cyclic

nucleotide phosphodiesterases (PDEs). The PDE gene family catalyze the hydrolysis of 3`,5`-

cyclic nucleotide to inactive 5` -nucleotide metabolites by cleaving the phosphodiester bond

of the cyclic phosphate ring. This large group of proteins consists of 11 gene families in

mammals (Conti 2000). In vitro, cAMP modulators, including membrane permeable

derivatives of cAMP, specific PDE subtype inhibitors and AC activators, have been shown to

inhibit or delay spontaneous maturation of murine (Nogueira et al. 2003b), bovine (Aktas et

al. 1995b; Thomas et al. 2002; Luciano et al. 2004), porcine (Funahashi et al. 1997;

Sasseville et al. 2006) and human oocytes (Nogueira et al. 2003a; Shu et al. 2008).


90

Oocyte quality remains a poorly defined determinant of subsequent embryonic development

(Hyttel et al. 1997; Sirard et al. 2006). In particular, current IVM techniques fail to enable the

majority of oocytes to develop and support embryo competence. In contrast, in vivo matured

oocytes lead to higher rates of development than in vitro matured oocytes (Leibfried-Rutledge

et al. 1987; Gilchrist and Thompson 2007).

A possible strategy to improve developmental competence of IVM oocytes is to align meiotic

and cytoplasmic maturation by retarding spontaneous meiotic resumption. It is hypothesized

that this provides time for cytoplasmic modifications (e.g. storage of mRNA and proteins,

morphological changes, ultra structural remodelling) and might enhance synchronization of

the starting population of immature oocytes (Luciano et al. 1999; Nogueira et al. 2004a;

Nogueira et al. 2003b; Luciano et al. 2004; Thomas et al. 2004b; Gilchrist and Thompson

2007).

The aim of this chapter was to establish an induced IVM model in bovine, by manipulating

oocyte cAMP immediately following collection and during the maturation process, and

examine the effect of induced IVM on oocyte/cumulus cells functions and hence oocyte

developmental competence.


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3.3 Materials and Methods

Unless otherwise specified, all chemicals and reagents were purchased from Sigma (St. Louis,

MO).

3.3.1 Oocyte collection and selection (pre-IVM phase)


saline (37ºC). Antral follicles (2 to 8 mm diameter) were aspirated using an 18-gauge needle

and a 10-ml syringe. For follicular fluid treatment, COCs were aspirated, collected and

incubated in pure freshly aspirated follicular fluid. For other pre-IVM treatments, shortly after

COC removal from the follicle (< 5 min), the pellet of sediment from the follicular aspirate

containing COCs was transferred to collection medium, Bovine VitroCollect (IVF Vet

solutions, Adelaide, Australia) (Appendix 2) in the presence or absence of cAMP modulators,

. Cyclic AMP modulators which were used during the pre-IVM phase were: the adenylate

cyclase activator, forskolin (100 µM), and a non-specific phosphodiesterase inhibitor, IBMX

(500 µM). Stock concentrations of the cAMP modulators were stored at -20�C dissolved in

anhydrous dimethyl-sulphoxide (DMSO). Solutions containing modulators were diluted fresh

for each experiment. Only COCs with intact cumulus vestments were selected under a

dissecting microscope for experiments and were maintained in pre-IVM media for up to 2

hours.

3.3.2 Oocyte in vitro maturation (IVM phase)

The basic medium for oocyte maturation was Bovine VitroMat (IVF Vet Solutions)

(Appendix 2). All IVM treatments were supplemented with 100 mIU/ml FSH (Puregon,


92

Organon, Oss, Netherlands). Where indicated, the type 3 PDE specific inhibitor cilostamide

(20 �M; Biomol Plymouth Meeting, PA) or the EGF receptor (EGFR) kinase inhibitor,

AG1478 (Calbiochem, La Jolla, CA) were added from a millimolar stock solution stored at -

20�C dissolved in DMSO. Thirty COCs were cultured in 4-well multidishes in pre-

equilibrated 300 µl drops of VitroMat overlaid with mineral oil and incubated at 39 ºC with

6% CO2 in humidified air.

3.3.3 In vitro fertilization and embryo culture

At 24 or 30 hrs after IVM COCs were removed from the IVM wells and washed twice using

Bovine VitroWash (IVF Vet Solutions), and transferred to insemination dishes containing

Bovine VitroFert (IVF Vet Solutions) supplemented with 0.2 mM penicillamine, 0.1 mM

hypotaurine, and 2 mg/ml heparin. Frozen semen from a single bull of proven fertility was

used in all experiments. Briefly, thawed semen was layered over a discontinuous 45%: 90%

Percoll gradient (Amersham Bioscience) and centrifuged at room temperature for 20-25 mins

at 700 g. The supernatant was removed and the sperm pellet was washed with 500 μl Bovine

VitroWash and centrifuged for a further 5 minutes at 200 g. Spermatozoa were resuspended

with VitroFert, then added to the fertilization media drops (Bovine VitroFert, supplemented

with 0.01 mM heparin, 0.2 mM penicillamine and 0.1 mM hypotaurine) at a final

concentration of 1 x 106 spermatozoa/ml. COCs were inseminated at a density of 10 μl of IVF

medium per COC for 24 h, at 39�C in 6% CO2 in humidified air.

Cumulus Cells were removed by gentle pipetting 23–24 h post insemination. Presumptive

zygotes were washed, transferred in groups of 5 into 20 µl drops of pre-equilibrated Bovine

VitroCleave (IVF Vet Solutions) and cultured under mineral oil at 38.5°C in 7% O2, 6%


93

CO2, balance N2, for five days (day 1 to day 5). On day 5, embryos were washed and

transferred in groups of 5-6 to 20 μl drops of pre-equilibrated Bovine VitroBlast (IVF Vet

Solutions) overlaid with mineral oil and cultured at 38.5°C in 7% O2, 6% CO2, balance N2

until Day 8. Embryos were assessed for quality at Day 8 according to the definitions

presented in the Manual of the International Embryo Transfer Society (Stingfellow 1998) and

were performed independently by an experienced bovine embryologist blinded to treatments.

3.3.4 Blastocyst differential staining

Blastocysts were placed into 0.5% pronase at 37°C to remove the zona, followed by a brief

wash in 4 mg/ml poly-vinyl alcohol (PVA) in phosphate-buffered saline (PBS/PVA). Zona-

free blastocysts were then incubated in 10 mM trinitrobenzene sulfonic acid in PBS/PVA at

4ºC for 10 min. Blastocysts were subsequently incubated with 0.1 mg/ml anti-dinitrophenol–

BSA antibody (Molecular Probes, Eugene, OR, USA) at 37°C for 10 min., then placed in

guinea pig serum with propodium iodide for 5 min at 37°C. Blastocysts were washed and

incubated in 10 μg/ml propidium iodide for 20 min at 37°C (to stain the trophectoderm),

followed by 4 μg/ml bisbenzimide (Hoechst 33342) in 100% ethanol at 4°C overnight (to

stain both the inner cell mass (ICM) and trophectoderm). Blastocysts were then whole

mounted in a drop of 80% glycerol in PBS on microscope slides and coverslips were sealed

with nail polish. Blastocysts were then examined under a fluorescence microscope (Olympus,

Tokyo, Japan) at 400x equipped with an ultraviolet filter and a digital camera attached, to

determine total and compartment cell counts, where inner cell mass (ICM) nuclei appeared

blue and trophectoderm (TE) nuclei stained pink.


94


Cyclic AMP content of COCs and denuded oocytes (those cultured as COCs but denuded of

their cumulus vestment prior to assay (DO)) was measured using a radioimmunoassay

described and validated previously (Reddoch et al. 1986). Following culture, 6-10 COC or

21-24 DO were washed in VitroCollect (IVF Vet solutions), transferred to 0.5ml of ethanol

(100%) and stored at –20�C. Before cAMP measurements, samples were vortex agitated for

30 seconds and then centrifuged at 3000g for 15 min at 4�C. Briefly, supernatants were

collected, evaporated, resuspended in assay buffer (50mM sodium acetate, pH 5.5) and

acetylated by the addition of triethylamine (AJAX Chemicals, Sydney, Australia) and acetic

anhydride (BDH Laboratory Supplies, Poole, England) 2:1 v/v. cAMP was measured in

duplicates after appropriate dilution. 125I-labelled cAMP (specific activity of 2175 Ci/mM)

and cAMP antibody as prepared by (Reddoch et al. 1986) were added to samples and left

overnight at 4�C. The following day, 1ml cold 100% ethanol was added and samples were

centrifuged at 3000g. The supernatant was removed and the pellet dried and radioactivity

counted using a gamma counter. Duplicates of known concentration samples were used to

produce a standard curve (0-1024 fmol cAMP).

3.3.6 Oocyte nuclear morphology assessment

After 2, 5 and 7 hours of culture, COCs were denuded and oocytes were fixed for 30 min in

4% paraformaldehyde in PBS (pH 7.4). Oocytes were then permeabilized in 0.1% Triton X-

100 in 0.1% sodium citrate for 1 hour, then transferred to 0.001% 4',6-diamidino-2-

phenylindole (DAPI), a fluorescence stain for nuclear material, for 15 min. Oocyte were

rinsed 3 times in PBS+0.03% BSA, mounted on slides and evaluated for nuclear status at


95

400x using an Olympus fluorescence microscope. Stages of GV and subsequent meiotic

development were assessed based on chromatin configurations described by (Chohan and

Hunter 2003). In brief, GV chromatin of bovine oocytes was classified into: GV I, condensed

filamentous chromatin around the nucleolus and nuclear membrane; GV II, filamentous

chromatin surrounding the nucleolus; GV III, filamentous chromatin are distributed in the

nucleus and the nucleolus has disappeared; GV IV, Chromatin is condensing into thick

clumps; diakenesis, chromatin condensed into single clump; metaphase I, tetrads are aligned

on the spindle; metaphase II, the metaphase chromatin is evident as well as a small

chromatin-containing polar body.

3.3.7 Oocyte-cumulus cells gap junctional communication assay

Oocyte-cumulus cell gap junctional communication (GJC) was measured by quantitative

fluorescence microscopy of calcein transfer to the oocyte as described previously (Thomas et

al. 2004b). GJC was measured either after 2 hours of pre-IVM phase or followed by 3 hours

of IVM with or without cilostamide (20 µM). After culture, COCs were transferred to a

solution of 1 �M calcein-AM (3’,6’-di(O-acetyl)-2’,7’-bis[N,N-bis(carboxymethyl) amino

methyl]-fluorescein, tetraacetoxy methyl ester; C-3100; Molecular Probes; Eugene, OR,

USA) freshly prepared in a BSA-free VitroCollect (IVF Vet solutions) supplemented with

polyvinyl alcohol (PVA; 0.3 mg/ml). COCs were cultured with the dye for 15 min, then

unincorporated dye was removed by three washes in calcein-AM-free VitroCollect (IVF Vet

solutions) and incubated for a further 25 min to allow dye transfer from the cumulus cells to

the oocyte. Prior to fluorescence microphotometry, COCs were completely denuded of their

surrounding cumulus cells using vigorous pipetting so that only dye confined within the

denuded oocyte after transport via gap junctions was measured. Within 30 min of denuding,


96

the intra-oocyte fluorescence emission of calcein in oocytes was measured using a

fluorophotometric-inverted microscope (Leica, Wetzlar, Germany).

3.3.8 Statistical analysis


Software, San Diego, CA, USA, www.graphpad.com). Statistical significance was assessed

by ANOVA followed by either Dunnett's or Bonferroni’s multiple-comparison post-hoc tests to

identify individual differences between means. All values are presented with their

corresponding standard error of the mean (SEM).


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3.4 Results

3.4.1 cAMP content of COC and DO during pre-IVM phase

After 5 min of the pre-IVM phase, cAMP levels in COCs collected in presence of cAMP

modulators were 9 fold higher than either the control group (absence of cAMP modulators) or

follicular fluid (figure 1A) (P < 0.01). This increase was similar after 30 min and at the end of

the pre-IVM phase (2 hours). Collection and processing of COCs in follicular fluid

maintained levels of cAMP in COCs for 30 min but dropped by the end of pre-IVM phase (2

hours) (20±3 and 10±3 fmol/COC, respectively) (P < 0.05). In contrast, COCs collected in the

absence of cAMP modulators, cAMP levels dropped sharply (from 15±4 to 2±1 fmol/COC)

(figure 1A)(P < 0.05) and continued to decrease during the 2 hour incubation period.

Denuded oocytes were used to evaluate the effect of the surrounding cumulus cells on intra-

oocyte cAMP (figure 1b). Intra-oocyte cAMP levels were comparable (0.9±0.1 fmol/DO) in

all treatments 5 min post-isolation from the follicle. In contrast, after 30 min of processing in

the pre-IVM phase, intra-oocyte cAMP of COCs treated with cAMP modulators was

significantly higher than in the control group and increased substantially with incubation time

(16±0.1 fmol/DO) at the end of the pre-IVM phase. Follicular fluid maintained intra-oocyte

cAMP levels during the whole period in pre-IVM phase (2 hours) (0.8±3 fmol/DO), whereas

COCs collected and processed in absence of cAMP modulators, intra-oocyte cAMP dropped

significantly within 30 min and remained depleted at the end of the pre-IVM phase (0.3±0.2

fmol/DO)(P < 0.05).


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3.4.2 Effect of cAMP modulators in pre-IVM phase and type-3 PDE inhibition in IVM

phase on spontaneous oocyte maturation (GV/GVBD)

COCs were incubated for 2 hours in the pre-IVM phase treatments, followed by 7 hours

culture in the presence of FSH and either the presence or absence of cilostamide (20 µM).

Combined treatment of FSH and cilostamide during IVM phase significantly lowered the

rates of GVBD amongst all pre-IVM treatments compared to the absence of cilostamide

during the IVM (figure 2). The majority of oocytes had commenced GVBD in absence of

cilostamide, except those processed in collection medium with cAMP modulators during pre-

IVM which were significantly delayed in the time of GVBD compared to other treatment

groups (P < 0.05) (figure 2), which lead us to investigate GV configuration changes at the end

of pre-IVM and during IVM phase.


phase on oocyte germinal vesicle configurations

As seen in (figure 3A, table A1 Appendix 1), at the end of pre-IVM phase (2 hours), the

majority of the oocytes processed in follicular fluid (61%±5) or in collection medium

supplemented with cAMP modulators (67%±5) (P < 0.05) were at the GV II stage. However,

oocytes processed in collection medium alone had a decreased GV II percentage (P < 0.05)

and had progressed to GV III (66%±5) (P < 0.01).

After 5 hours (2 hours of Pre-IVM and 3 hours of IVM+cilostamide) collected oocytes were

still arrested at the same previous GV stages prior to the IVM incubation (Figure 3C, table A1

Appendix 1). When compared to Figure 3B, this indicates that the presence of FSH and


99

cilostamide in the culture media during the first hours of IVM incubation prevented COCs

from progressing through their GV configurations compared to COCs cultured in the

presence of FSH alone (IVM-cilostamide). As seen in (figure 3B, table A1 Appendix 1),

oocytes processed in pure follicular fluid were progressing to GV III (58%±8), whereas most

of the oocytes which were processed in the absence of cAMP modulators during pre-IVM

phase were at GV III (41%±10) or at GV IV (52%±12) (P < 0.05). Surprisingly, even in the

absence of cilostamide during IVM, COCs were still arrested at GV II (55%±3) when

processed with cAMP modulators (figure 3B, table A1 Appendix 1).

As seen in figure 3E, after 9 hours of oocyte culture (2 hours of pre-IVM and 7 hours of

IVM+cilostamide), COCs processed in media comprising cAMP modulators were progressed

in maturation with the majority of COCs at GV II (44±3%) or at GV III (42±3%). This was

compared to oocytes which were selected in pure follicular fluid where the majority of

oocytes were at GV III (63±6), and oocytes which were processed in collection medium

lacking cAMP modulators where the oocytes had progressed to GV IV (51%±10)(P <

0.05)(figure 3E, table A1 Appendix 1). After 9 hours and in the presence of FSH alone (IVM-

cilostamide), most oocytes which were treated with cAMP modulators had progressed to the

GV III stage (58%±4) (figure 3D, table A1 Appendix 1), whereas the majority of oocytes

which were processed in pure follicular fluid progressed to diakenesis (40%±4) and M I

(38%±4)(figure 3D, table A1 Appendix 1). In contrast, of those oocytes processed with

collection medium lacking cAMP modulators, 23%±6 were at the diakenesis stage and

57%±5 had progressed to the M I stage (P < 0.05) (figure 3D, table A1 Appendix 1).


100

3.4.4 Effect of cAMP modulators during pre-IVM and IVM on oocyte-cumulus cell

gap junctional communication

The oocyte-cumulus GJC assay was performed at the end of the pre-IVM phase and after 5

hours of oocyte culture (2 hours of pre-IVM and 3 hours of IVM ± cilostamide). At the end of

pre-IVM phase (2 hours), levels of gap junctional communication between the oocyte and the

cumulus cells significantly higher for oocytes aspirated and processed in collection medium

supplemented with cAMP modulators compared with oocytes aspirated and processed in

follicular fluid or collection medium (P < 0.05) (figure 4).

Moreover, levels of GJC dramatically decreased after 3 hours of IVM post pre-IVM phase

when COCs were collected either in follicular fluid or collection medium. In contrast, COCs

collected in presence of cAMP modulators during pre-IVM phase had higher levels of GJC,

regardless of the presence or absence of cilostamide during IVM (P < 0.05) (figure 4).


phase on oocyte meiotic progression to MII stage

As seen in (figure 5A), in absence of FSH, cilostamide treatment delays oocyte meiotic

progression to M II stage (50%) at 24 hours of IVM either in the presence or absence of

cAMP modulators in the pre-IVM phase. However at 20 hours of IVM, 83%±7 of the oocytes

were at M I stage when the oocytes were processed in collection medium supplemented with

cAMP modulators in the pre-IVM phase, compared with 36%±4 of oocytes which were at M

I stage when the oocytes were processed in collection medium lacking cAMP modulators

(figure 5A).


101

As seen in (figure 5B), in the presence of FSH, the inhibitory effect of cilostamide in delaying

oocyte progression to the M II stage was overridden by FSH present in the IVM culture

media. After 20 hours of IVM+cilostamide treatment, 72%±5 of oocytes were at M I stage

when they were processed in collection medium supplemented with cAMP modulators during

the pre-IVM phase (figure 5B) compared to 21%±4 of oocytes when they were processed in

collection medium alone (figure 5B).

3.4.6 Intracellular concentration of cAMP in oocyte after pre-IVM and IVM phase

As seen in Figure 6, after 24 hrs of culture, the intracellular concentration of cAMP in DOs

remained significantly higher (up to 15 fold)(P < 0.05) when the oocytes were collected and

processed in collection medium supplemented with cAMP modulators during pre-IVM (as

opposed to those cultured in pure follicular fluid, or collection medium lacking cAMP

modulators), provided cilostamide was also present in the culture media.

3.4.7 Effect of (EGFR) kinase inhibitor on FSH-induced oocyte maturation in the

presence of the type-3 PDE inhibitor (Cilostamide)

As shown in (figure 5), the inhibitory effect of cilostamide on oocyte meiotic progression was

overridden by the addition of FSH in culture. It was therefore an interest to examine if the

FSH-induced maturation (meiotic induction) was suppressed by antisera to the Epidermal

Growth Factor Receptor (EGFR), for example by examining if the EGFR kinase inhibitor,

AG1478, could affect this pattern of maturation.


102

As seen in Figure 7, the addition of increasing doses of AG1478 in IVM significantly reduced

the maturation percentage (from >80% to approximately 5%)(P < 0.05), thereby completely

suppressing the induction response. This indicates that EGF signalling is required in FSH-

induced oocyte maturation.

3.4.8 Effect of cAMP modulators during pre-IVM and IVM phase on oocyte

developmental competence

Inclusion of cAMP modulators during pre-IVM phase improves cleavage rates compared to

their absence (89 ± 2.0% vs. 78±2%, respectively, P<0.05) and blastocyst development

(32±3% vs. 26±3%, P<0.05) when oocytes are matured in standard IVM medium in presence

of FSH for 24 hours (Table.1).

As previously shown in (figure 5), pre-treating oocytes with cAMP modulators during pre-

IVM phase and inclusion of cilostamide in IVM medium of isolated oocytes delays the onset

of M II by 4 hours in the combined presence of FSH. Consequently, oocytes were

inseminated at 24 or 30 hours after IVM with FSH and PDE inhibitor cilostamide. At 24

hours, the cleavage rate for oocytes pre-treated either in follicular fluid or medium

supplemented with cAMP modulators was approximately 80% and significantly higher than

when oocytes were collected in the absence of cAMP modulators during pre-IVM phase and

matured with cilostamide + FSH (67%) (P < 0.05). Moreover, the rate of development to the

blastocyst stage was also significantly higher (48%) in the presence of cAMP modulators in

pre-IVM compared to their absence (22%). However, oocytes fertilized at 30 hours yielded a

dramatic increase in blastocysts when pretreated with cAMP modulators and matured with


103

FSH + cilostamide (42% increase in yield to 69% blastocysts) compared to oocytes collected

and handled in absence of cAMP modulators (27% blastocysts) (P < 0.05).

3.4.9 Effect of cAMP modulators during pre-IVM and IVM on blastocyst cell

numbers

In the presence of cilostamide in IVM phase, oocytes processed in pure follicular fluid or in

presence of cAMP modulators in pre-IVM phase significantly increased blastocyst total , TE

and ICM cell numbers compared to oocytes processed in the absence of cAMP modulators in

the pre-IVM phase (P < 0.05) (figure 9).


104

3.5 Discussion

The current study was undertaken to test the hypothesis that developing a process for

inducing oocyte maturation in vitro by manipulating intracellular cAMP levels in the oocyte

immediately following collection and during the maturation process, presumably

synchronizes nuclear and cytoplasmic compartments, thereby leading to improved

developmental competence of maturing oocytes.

The participation of cAMP in controlling oocyte maturation in mammals has been shown in

the past few years. Nevertheless, the molecular mechanisms of the series of coordinate events

guided by cAMP have not been clearly defined. The experimental data showed that the

efficacy of PDE inhibitors may be enhanced by modulating cAMP levels during the period

preceding IVM. In the first part of this study (figure 1), results obtained from measuring

cAMP levels in whole COCs or DOs were consistent with previously measured data in

bovine at the time of follicle removal (Bilodeau et al. 1993; Aktas et al. 1995a; Aktas et al.

1995b; Luciano et al. 1999). In particular, results from the present experiments showed that

the intracellular cAMP concentration during the interval between oocyte isolation from the

follicle and the beginning of in vitro maturation (IVM) appears critical for oocyte kinetics and

for the achievement of higher developmental competence. Indeed, modulating oocyte cAMP

levels in the pre-IVM phase appeared to have a long effect in maintaining high oocyte cAMP

levels after 24 hours of IVM in the presence of cilostamide in culture (figure 6).

The different collection conditions used in this study have been found to exert a profound

effect on oocyte intracellular cAMP levels, oocyte meiotic progression, oocyte-cumulus cells

gap junctional communication and finally oocyte developmental competence. The present


105

results demonstrated that modulating cAMP for a short period of time from the time of COC

isolation can lead to sustained gap junctional communication between cumulus cells and

oocyte (figure 4). Although the exact amount of time required for adequate cytoplasmic

maturation remains to be determined, beneficial effects of maintenance of gap junctional

communication during IVM process on the promotion of cytoplasmic maturation and

subsequent embryonic development have been observed in different species (Hashimoto et al.

1998; Thomas et al. 2004b; Atef et al. 2005).

The increase in gap junctional communication observed in this study, when cAMP levels

were modulated during the pre-IVM phase, was due to prolonged gap junction conductance

and/or the prevention of gap junction removal from the cumulus cell-derived meiosis-

modulating factors to the oocyte, thereby delaying GV configurations or GVBD after 7 hours

of oocyte culture (figures 2 and 3).

An intriguing question was how the exogenous modulation of cAMP can evoke a stimulatory

or inhibitory response in germinal and somatic compartments. In described experiments, the

intra-oocyte cAMP level was 15 fold higher when cAMP modulators were present in culture

media during the pre-IVM and IVM phases compared to other treatments (figure 6). This had

further been supported from the experiments testing the EGFR kinase inhibitor, AG1478,

during IVM (figure 7). Previous studies have shown that the epidermal growth factor (EGF)

can be produced by ovarian cells (Skinner et al. 1987), and is the most potent stimulator of

both cumulus expansion and meiotic resumption amongst a number of other growth-

promoting factors (Downs 1989). It has also been shown that EGF-like peptides can mediate

the actions of gonadotrophins in pre-ovulatory follicles. EGF-like peptides therefore can play


106

an intimate role in the paracrine signalling between metabolic pathways of different cell types

leading to oocyte maturation and follicular rupture (Park et al. 2004; Ashkenazi et al. 2005).

Therefore, the aim of one of the experiments, the results of which are shown in figure 7, was

to examine the role of EGF-like peptides in follicle-stimulating hormone-induced maturation

of cilostamide-cultured COCs. As mentioned previously, an increase in intra-oocyte cAMP

levels up to 15 fold delays meiotic progression to M II up to 28 hours (figure 5). Interestingly,

the inhibitory effect of cilostamide on oocytes in this study was overcome by addition of FSH

in culture (figure 5B). In the absence of FSH, 40-50% of cilostamide-treated oocytes remain

arrested at the M I stage after 24 hrs of culture (figure 5A). However, the addition of FSH,

whilst initially delaying GVBD (figure 2), eventually induced the majority of cilostamide-

treated oocytes to progress to M II after 24 hrs of culture. The exception were oocytes

collected in media supplemented with cAMP modulators during the pre-IVM phase, where

even in the presence of FSH there was a delay in GVBD. However, oocytes treated in this

way eventually progressed to the M II stage after 28 hrs of culture (figure 5B), indicating that

the efficacy of including cAMP modulators in IVM media can be enhanced by modulating

COC cAMP levels following collection, thereby leading to further meiotic inhibition which

may be induced by the presence of FSH in culture.

This observation may be the first model described in a ruminant species for induced oocyte

maturation, as opposed to spontaneous maturation where mechanically removing oocytes

from their follicle results in spontaneous meiotic resumption (Pincus and Enzmann 1935).

However, although it is well established that in vivo, meiotic resumption occurs due to the

pre-ovulatory surge of gonadotrophins, the mechanism(s) by which this occurs is not fully

understood.


107

When oocytes were cultured in the presence of cilostamide, FSH treatment resulted initially

in an inhibitory effect that later becomes stimulatory (figure 5). It has been proposed that

cAMP generated by FSH has a paradoxical effect in that it initially blocks maturation before

downstream participants in the signal cascade eventually exert a positive influence. To test if

EGF-like peptides could be responsible for this observation, the experiment of induced IVM

was repeated, with FSH treatment, and also with exposure to increasing doses of the EGFR

tyrosine kinase inhibitor, AG1478. Increasing doses of AG1478 completely prevented the

meiotic recovery of FSH and cilostamide treated COCs, indicating that the synthesis and

release of EGF-like peptides and EGF signalling is required for FSH-induced oocyte

maturation.

An improvement in cleavage and blastocyst rates has been noted when intra-oocyte cAMP is

maintained (oocytes were collected and selected in pure follicular fluid) during pre-IVM

phase and then mature in presence of cilostamide during IVM phase. This is consistent with

our previously approach (Thomas et al. 2004b), where bovine oocyte developmental

competence is also significantly improved when collected in pure follicular fluid and matured

in the presence of another specific PDE3 inhibitor milrinone. A substantial increase (up to 3

fold) in blastocyst percentage (69%) was generated when COCs were collected in pre-IVM

with cAMP modulators (forskolin+IBMX) and mature in presence of cilostamide. This is also

consistent with previous reports in bovine, where the addition if invasive adenylate cyclase in

the washing medium results in an improved blastocyst rate (Luciano et al. 1999; Guixue et al.

2001; Luciano et al. 2004). The substantial increase in cleavage and blastocyst percentages in

this chapter, works on the same principle as these previous demonstrations and is due to an

increase in intra-oocyte cAMP during the pre-IVM phase. However, this was enhanced by the


108

presence of cilostamide during IVM, in addition IBMX+forskolin in the pre-IVM phase.

Moreover, as shown in Table 2, modulating cAMP during pre-IVM alone, or during IVM

alone, generated a blastocyst percentage around 30%. It is interesting to note that in the

absence of cAMP modulators in the pre-IVM, and culturing the oocytes in the presence of

cilostamide decreased blastocyst percentage (29%) compared to their presence in the pre-

IVM media (69%). The substantial improvement in blastocyst percentage is in parallel with

previous report on coasting in vivo, in which 80% blastocysts have been developed, when

animals received leutinising hormone 6 hours before oocyte aspiration (Blondin et al. 2002).

Collectively, the evidence presented in this study demonstrated that intracellular cAMP levels

during the pre-IVM and the IVM phase has a profound effect on oocyte developmental

competence and blastocyst quality, starting from COC removal from the follicle to the end of

maturation. A stable modulation of intracellular cAMP levels within a physiological range in

the oocyte and cumulus cells during in vitro maturation can create an induced maturation in

vitro that in turn can lead to acquisition of high developmental potential.


109

3.6 Figures

Figure 1. The effect of various pre-in vitro maturation (pre-IVM) phase treatments over time on the cAMP content of two different types of oocytes: (A) oocytes collected and assayed with their cumulus vestments intact (COC); and (B) oocytes collected as COCs but denuded of their cumulus vestment prior to assay (DO). Oocytes of the two types were collected and selected in pure follicular fluid, collection medium, or collection medium supplemented with cAMP modulators (100 µM forskolin (FSK) and 500 µM IBMX). Values are expressed as the mean concentration of cAMP per oocyte or complex ± SEM of three replicates using 6 -10 COCs or 21-24 DOs per treatment replicate. Means within the same graph/oocyte type with different letters indicate significantly different amounts of cAMP between individual treatments or end time points (two-way ANOVA, P <0.05).

5 30 120 0.0

0.3

0.6

0.9

369

12151821

fmol

cAM

P pe

r DO

aa

aa

b

c

a

b

d

pre-IVM incubation time (min)

5 30 120 0

10

20

60

120

180

240

follicular fluidcollection mediumcollection medium +cAMP modulators

a a

b

a

c

b

a

c

b

pre-IVM phase

fmol

cAM

P pe

r CO

C

COCA.

B . DO


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Figure 2. The effect on spontaneous oocyte maturation (GV/GVBD) following incubation of cumulus-oocyte complexes in various pre-in vitro maturation (pre-IVM) and IVM phase media. Bovine COCs were collected and selected in pure follicular fluid, collection medium, or collection medium supplemented with cAMP modulators (100 µM forskolin (FSK) and 500 µM IBMX), followed by 7 hours culture in the presence of FSH, and in the presence or absence of cilostamide (20 µM). Oocytes were then fixed and assessed for meiotic progression and classified as GV (germinal vesicle) or GVBD (germinal vesicle break down). A mean number of 45 oocytes were used in each treatment group and time-point from four replicate experiments. The presence of letters above each column indicate a statistical difference between the means as determined by two-way ANOVA analysis followed by Bonferronni’s post hoc test, P < 0.05.

0

20

40

60

80

100+ cilostamide

- cilostamide

a

b

a

b

a a

follicular fluid collection medium collection medium + cAMP modulators

Pre-IVM treatments

IVM phase

% G

V at

7 h

ours


111

End of pre-IVM (2 hrs)

0

20

40

60

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DiakenesisM I

GV IIGV I

GV IVGV III


Mei

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inde

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5 hrs (pre-IVM + 3 hrs IVM)- cilostamide

0

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40

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Mei

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5 hrs (pre-IVM + 3 hrs IVM)+ cilostamide

0

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Mei

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x

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9 hrs (pre-IVM + 7 hrs IVM)- cilostamide

Mei

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inde

x

0

20

40

60

80

100


9 hrs (pre-IVM + 7 hrs IVM)+ cilostamide

Mei

otic

inde

x

A.

B. C.

D. E.

Figure 3. Germinal vesicle (GV) configurations of cumulus-oocyte complexes cultured in various pre-IVM and IVM phase. Oocytes were exposed to pre-IVM phase comprising pure follicular fluid, collection medium, or collection medium supplemented with cAMP modulators (100 µM forskolin (FSK) and 500 µM IBMX) (A), followed by extended culture in the presence (C; E) or absence (B; D) of the type-3 PDE inhibitor, cilostamide (20 µM), plus follicle stimulating hormone (FSH, 100 mIU/ml). Oocytes were then fixed and assessed for GV configuration at 2, 5 & 9 hrs. A mean number of 40 oocytes were used in each treatment group and time-point from four replicate experiments.


112

Figure 4. Effect of cAMP modulators in pre-IVM phase and type-3 PDE inhibitor (Cilostamide) in IVM on oocyte-cumulus cell gap junctional communication during 2 hrs of pre-IVM phase and during 5 hrs of culture (2 hrs of pre IVM+3 hrs of IVM); in the presence or absence of type 3 PDE inhibitor (cilostamide; 20 µM). A mean number of 10-12 oocytes were used in each treatment group in each of four replicate experiments. Different superscripts represent a statistical difference between the means as determined by (two-way ANOVA, P < 0.05) analysis followed by Bonferronni’s post hoc test.

0

500

1000

follicular fluid

collection medium

collection medium + cAMP modulators

c

b

c

b

a

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a

Phase pre-IVM IVM+cilostamide IVM-cilostamide

IVM time (hrs) - 5 5

c

pre-IVM treatmentsRe

lativ

e In

terc

ellu

lar G

JC (f

luor

esce

nce

inte

nsity

)


113

Figure 5. The effect of follicle stimulating hormone (FSH) on inducing meiotic maturation of cumulus-oocyte complexes (COCs) exposed to various pre-IVM and IVM phase. Oocytes were aspirated and selected in either follicular fluid, collection medium or collection medium supplemented with 100 µM forskolin (FSK) and 500 µM IBMX for 2 hrs. COCs were then cultured in 2 media (+/- cilostamide) in the absence of FSH (A) or presence of FSH (B). Oocytes were then fixed and assessed for meiotic progression at 20, 24 & 28 hrs. A mean number of 45 oocytes were used in each treatment group and time-point from four replicate experiments.

24 24 20 24 28 20 24 28 20 24 280

20

40

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M II

M I

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% M

eiot

ic S

tage

24 24 20 24 28 20 24 28 20 24 280

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Pre-IVM phase follicular fluid collection medium

cAMP modulators - - - + +

IVM phase - - + - +(+/- cilostamide)

% M

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tage

A - FSH

B +FSH


114

0.0

0.2

0.4

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1.2

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a

a

b

cc

a

follicular fluidcollection mediumcollection medium+cAMP modulators

pre-IVM phase

IVM phase + cilostamide - cilostamide

fmol

cAM

P pe

r DO

Figure 6. The intra-oocyte cAMP concentration of oocytes exposed to various pre-IVM and IVM phase. Oocytes were first denuded of their cumulus vestment prior to the assay (DOs), and were then collected and handled in 3 different pre-IVM phase (follicular fluid, collection medium, and collection medium supplemented with 100 µM forskolin (FSK) and 500 µM IBMX), followed by extended culture in the presence or absence of the type-3 PDE inhibitor cilostamide (20 µM) and FSH (100 mIU/ml) for 24 hrs. The data represent the mean cAMP level per DO ± SEM of 4 replicates. Each measurement was conducted on 24 DO. The presence of letters (a, b or c) above each column indicate a statistical difference between the means as determined by (one-way ANOVA, P < 0.05) analysis followed by Dunnett’s post hoc test.


115

Figure 7. The effect of an epidermal growth factor receptor (EGFR) inhibitor on follicle stimulating hormone (FSH)-induced oocyte maturation in the presence of the FSH (100 mIU/ml), the PDE inhibitor cilostamide (20 µM), and increasing doses of the EGFR inhibitor AG1478. Oocytes were then fixed and assessed for meiotic progression at 24 hrs. A mean number of 40 oocytes were used in each treatment group and time-point from three replicate experiments. The presence of letters (a, b or c) above each column or data point indicate a statistical difference between the means as determined by ANOVA analysis followed by Dunnett’s post hoc test.

0

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FSH - + + + + + +cilostamide + + + + + + +AG1478 (µM) 0 0 0.1 0.25 1 2.5 10

a

b b b

a

c c

% M

II


116

Figure 8. The effect of cAMP modulators during pre-IVM & IVM phase on oocyte developmental capacity. COCs were aspirated and selected in either follicular fluid, collection medium and collection medium supplemented with forskolin (FSK); 100µM & IBMX; 500µM) for 2 hrs. Following, COCs were matured with FSH for 24 or 30 hours with cilostamide (20 μM). Subsequently, oocyte developmental capacity was assessed after in vitro fertilization and embryo development by the cleavage rate (A) and the blastocyst rate on day 8 (B). The data represent a mean number of 45 oocytes were used in each treatment group and from four replicate experiments. Statistical analysis was performed by (two-way ANOVA, P < 0.05) followed by Bonferronni’s post hoc test.

0

20

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follicular fluidcollection mediumcollection medium+ cAMP modulators

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% c

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age

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117

0

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collection medium

collection medium+cAMP modulators

Total cells Trophoectoderm Inner Cell Mass

pre-IVM phase

aa

b h h

i

x

yxy

Cel

l No.

Figure 9. The effect cAMP modulators in pre-IVM and IVM phase on blastocyst cell numbers. Cumulus-oocyte complexes (COCs) were aspirated and selected in either follicular fluid, collection medium or collection medium supplemented with 100 µM forskolin (FSK) and 500 µM IBMX for 2 hrs. The COCs were then matured by the addition of FSH for 30 hours in the presence of cilostamide (20 µM). The number of total, inner cell mass and trophectoderm cells (mean ± SEM) of Day 8 expanded and hatched blastocysts was determined. A mean number of 20-30 expanded or hatched blastocyst were used in each treatment group. The presence of letters (a, b, h, I, x or y) above each column indicate a statistical difference between the means as determined by ANOVA analysis followed by Bonferronni’s post hoc test.


118

3.7 Tables

Table 1. Effects of pre-IVM phase before oocyte culture on oocyte developmental

competence.

Pre-IVM Treatments

Number of

oocytes

% Cleavage Rate

% Blastocysts from

cleaved follicular fluid 186 89.5 ± 1.4a 38.5 ± 5.7a collection medium 205 77.8 ± 3.6b 26.5 ± 4.0b

collection medium +cAMP modulators

200 88.5 ± 1.9a 32.0 ± 2.1ab

a-b Values with different superscripts within the same column represent a statistically significant difference (P < 0.05). Values are expressed as (mean ± SEM).


119

Table 2. Summary of the effects of cAMP modulators during pre-IVM phase and IVM on

blastocyst yield.

Pre-IVM phase IVM phase maturation time(h)

Blastocyst (%)

collection medium control 24 27% collection medium +cilostamide 24 22% collection medium +cilostamide 30 29%

follicular fluid control 24 39% follicular fluid +cilostamide 24 38% follicular fluid +cilostamide 30 61%

collection medium+cAMP modulators Control 24 32%

collection medium+cAMP modulators +cilostamide 24 48%

collection medium+cAMP modulators +cilostamide 30 69%


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3.8 References Aktas H, Wheeler MB, First NL, Leibfried-Rutledge ML (1995a) Maintenance of meiotic arrest by increasing [cAMP]i may have physiological relevance in bovine oocytes. J Reprod Fertil 105, 237-45. Aktas H, Wheeler MB, Rosenkrans CF, Jr., First NL, Leibfried-Rutledge ML (1995b) Maintenance of bovine oocytes in prophase of meiosis I by high [cAMP]i. J Reprod Fertil 105, 227-35. Ashkenazi H, Cao X, Motola S, Popliker M, Conti M, Tsafriri A (2005) Epidermal growth factor family members: endogenous mediators of the ovulatory response. Endocrinology 146, 77-84. Bilodeau S, Fortier MA, Sirard MA (1993) Effect of adenylate cyclase stimulation on meiotic resumption and cyclic AMP content of zona-free and cumulus-enclosed bovine oocytes in vitro. J Reprod Fertil 97, 5-11. Blondin P, Bousquet D, Twagiramungu H, Barnes F, Sirard MA (2002) Manipulation of follicular development to produce developmentally competent bovine oocytes. Biol Reprod 66, 38-43. Chohan KR, Hunter AG (2003) Meiotic competence of bovine fetal oocytes following in vitro maturation. Anim Reprod Sci 76, 43-51. Conti M (2000) Phosphodiesterases and cyclic nucleotide signaling in endocrine cells. Mol Endocrinol 14, 1317-27. Downs SM, Daniel SA, Bornslaeger EA, Hoppe PC, Eppig JJ (1989) Maintenance of meiotic arrest in mouse oocytes by purines: modulation of cAMP levels and cAMP phosphodiesterase activity. Gamete Res 23, 323-34. Funahashi H, Cantley TC, Day BN (1997) Synchronization of Meiosis in Porcine Oocytes By Exposure to Dibutyryl Cyclic Adenosine Monophosphate Improves Developmental Competence Following in vitro Fertilization. Biology of Reproduction 57, 49-53. Gilchrist RB, Thompson JG (2007) Oocyte maturation: emerging concepts and technologies to improve developmental potential in vitro. Theriogenology 67, 6-15. Hashimoto S, Saeki K, Nagao Y, Minami N, Yamada M, Utsumi K (1998) Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes. Theriogenology 49, 1451-63. Hubbard CJ, Terranova PF (1982) Inhibitory action of cyclic guanosine 5'-phosphoric acid (GMP) on oocyte maturation: dependence on an intact cumulus. Biol Reprod 26, 628-32. Hyttel P, Fair T, Callesen H, Greve T (1997) Oocyte growth, capacitation, and final maturation in cattle. Theriogenology 47, 23-32.


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Leibfried-Rutledge ML, Critser ES, Eyestone WH, Northey DL, First NL (1987) Development potential of bovine oocytes matured in vitro or in vivo. Biol Reprod 36, 376-83. Luciano AM, Modina S, Vassena R, Milanesi E, Lauria A, Gandolfi F (2004) Role of intracellular cyclic adenosine 3',5'-monophosphate concentration and oocyte-cumulus cells communications on the acquisition of the developmental competence during in vitro maturation of bovine oocyte. Biol Reprod 70, 465-72. Luciano AM, Pocar P, Milanesi E, Modina S, Rieger D, Lauria A, Gandolfi F (1999) Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization. Mol Reprod Dev 54, 86-91. Nogueira D, Albano C, Adriaenssens T, Cortvrindt R, Bourgain C, Devroey P, Smitz J (2003a) Human oocytes reversibly arrested in prophase I by phosphodiesterase type 3 inhibitor in vitro. Biol Reprod 69, 1042-52. Nogueira D, Cortvrindt R, De Matos DG, Vanhoutte L, Smitz J (2003b) Effect of Phosphodiesterase Type 3 Inhibitor on Developmental Competence of Immature Mouse Oocytes In vitro. Biol Reprod 20, 20. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M (2004) EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 303, 682-4. Pincus G, Enzmann E (1935) The comparative behaviour of mammalian eggs in vivo and in vitro. J. Exp. Med. 62, 665-675. Reddoch RB, Pelletier RM, Barbe GJ, Armstrong DT (1986) Lack of ovarian responsiveness to gonadotropic hormones in infantile rats sterilized with busulfan. Endocrinology 119, 879-86. Richard FJ (2007) Regulation of meiotic maturation. J Anim Sci 85, E4-6. Sasseville M, Cote N, Guillemette C, Richard FJ (2006) New insight into the role of phosphodiesterase 3A in porcine oocyte maturation. BMC Dev Biol 6, 47. Shu YM, Zeng HT, Ren Z, Zhuang GL, Liang XY, Shen HW, Yao SZ, Ke PQ, Wang NN (2008) Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes. Hum Reprod 23, 504-13. Sirard MA, Richard F, Blondin P, Robert C (2006) Contribution of the oocyte to embryo quality. Theriogenology 65, 126-36. Skinner MK, Lobb D, Dorrington JH (1987) Ovarian thecal/interstitial cells produce an epidermal growth factor-like substance. Endocrinology 121, 1892-9. Stingfellow DA, and Seidel, S. M (1998) Manual of the International Embryo Transfer Society. In. (IETS: Savoy, IL, USA)


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Thomas RE, Armstrong DT, Gilchrist RB (2002) Differential effects of specific phosphodiesterase isoenzyme inhibitors on bovine oocyte meiotic maturation. Dev Biol 244, 215-25. Thomas RE, Thompson JG, Armstrong DT, Gilchrist RB (2004) Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity. Biol Reprod 71, 1142-9.

CHAPTER 4

SPECIFIC PRE-IVM CONDITIONS THAT IMPROVE BOVINE OOCYTE

DEVELOPMENTAL COMPETENCE

Chapter 4. Specific pre-IVM conditions that improve bovine oocyte developmental competence

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4.1 Abstract

It has previously been shown that supplementation of cyclic adenosine mono-phosphate

(cAMP) modulators during IVM resulted in delayed meiosis, allowing improved oocyte

cytoplasmic maturation and increased developmental capacity. This study was designed to

evaluate the effects of increasing intra-oocyte cAMP level in immature bovine oocytes during

pre-in vitro oocyte maturation (pre-IVM) using modulators of cAMP on the acquisition of

embryonic developmental competence. During the pre-IVM phrase, the non-specific

phosophodiesterase (PDE) inhibitor, 3-isobutyl-methylxanthine (IBMX, 500 µM), and

increasing doses of the adenylate cyclase activator, forskolin (FSK, 0.4-100 µM), were used.

Combinations of these cAMP modulators and duration of exposure during the pre-IVM phase

were examined on cumulus oocyte complex (COC) and denuded oocyte (DO) cAMP levels.

After 2 hours in pre-IVM, cAMP in control COCs was significantly lower (1 fmol/COC)

compared to the follicular fluid control (9 fmol/COC) (P < 0.01), whereas FSK significantly

elevated (P < 0.05) cAMP levels within COCs in a dose dependent manner (10-100 µM). In

combination with IBMX, increasing concentrations of FSK (10, 20, 50 and 100 µM)

substantially increased cAMP levels up to 20 fold higher than levels observed in absence of

IBMX or the follicular fluid control (P < 0.05). Next we examined the effect of pre-IVM

duration when COCs are incubated with IBMX and increasing doses of forskolin (10-100

µM). Higher concentrations of forskolin (50, 100 µM) and IBMX (500 µM) led to a

substantial increase in cAMP levels in COCs and denuded oocytes DO. However, this

increase in cAMP levels was time-dependent in the oocyte but not in the COC. Moreover,

treatments that elevated intra-oocyte cAMP levels during pre-IVM led to a substantial

improvement in oocyte developmental competence as observed through increased cleavage

and blastocyst development rates. These results indicate that increases in intra-oocyte cAMP


125

concentrations in the collection period prior to the commencement of IVM are critical for

obtaining substantial improvements in developmental competence.


126

4.2 Introduction

During mammalian ovarian follicle growth, oocyte maturation is characterized by prolonged

meiotic arrest at the germinal vesicle (GV) stage and undergoes gonadotrophin induced

resumption of meiosis when exposed to the pre-ovulatory surge of luteinising hormone (LH)

during each ovarian cycle. In response to the LH surge, the oocyte within the dominant

follicle undergoes germinal vesicle breakdown (GVBD) and proceeds through metaphase I

(MI), only to subsequently arrest again at the metaphase II (MII) stage of meiosis.

Alternately, artificial oocyte isolation from an antral follicle in vitro leads to spontaneous

resumption of meiosis independent of gonadotrophins. Previous studies implicate the crucial

role of the follicular environment in maintaining oocyte arrest via inhibitory molecules and

signals produced by the follicle, the suppression of stimulatory factors, or by a combination

of both (Downs 1996).

The second messenger cyclic adenosine mono-phosphate (cAMP) plays an important role in

keeping mammalian oocytes in a meiotically arrested state (Tsafriri et al. 1972; Downs et al.

1989; Eppig 1989). There are two hypothesized mechanisms by which cAMP maintains

meiotic arrest. Firstly, cAMP diffuses from the somatic cells (namely granulosa and cumulus

cells) to the oocyte and increased intra-oocyte cAMP levels prevent oocyte maturation

(Anderson and Albertini 1976). Secondly, phosphodiesterases (PDEs) degrade cAMP and the

specific type 3 phosphodiesterase (PDE3A) in the oocyte (Tsafriri et al. 1996) is inhibited by

cyclic guanine monophosphate (cGMP) (Hambleton et al. 2005; Norris et al. 2009). Somatic

cell derived cGMP enters the oocyte through the gap junctions, thus inhibiting PDE3A and

maintaining meiotic arrest (Tornell et al. 1991; Norris et al. 2009). High cAMP levels within

the oocyte maintain protein kinase A (PKA) in an active state and lead to phosphorylation of


127

two Cdc2 kinase regulators (Cdc25 phosphatase and Wee1 kinase), keeping the oocyte in an

arrested state (Han and Conti 2006). The LH surge causes a rapid and complete closure of the

gap junctions between the granulosa cells in the follicle (Norris et al. 2008), cumulus cell

derived-cAMP within the oocyte is degraded by PDE3A (Sela-Abramovich et al. 2006),

decreasing PKA activity and inactivation of Wee1 and hence activation of Cdc25. Active

Cdc25 dephosphorylates the Cdc2/cyclin B complex (M phase promoting factor (MPF)

complex) that is maintained by Wee 1 B phosphorylation (Han and Conti 2006). As a result,

kinase activity of the Cdc2/cyclin B complex increases and oocytes can undergo GVBD.

Other kinases such as Akt/PKB and polo-like kinase (PLk) may be involved in the regulation

of Cdc25 phosphatase and Wee 1 B kinase (Schultz et al. 1983; Vivarelli et al. 1983; Han and

Conti 2006).

Basal physiological levels of cAMP are maintained by adenylate cyclase gene family

members and degraded by the cyclic nucleotide PDE to its inactive form 5`- adenosine

monophosphate (5`AMP). The mammalian PDE family currently constitutes of 11 distinct

sub-family isoenzymes (PDE1-11) transcribed by at least 23 gene types (Conti and Beavo

2007). Compartmentalisation of two PDE types has been demonstrated in rat follicles, where

PDE3A has been localized and restricted to the oocyte, while PDE4D is detected in the

granulosa/cumulus cell compartment (Tsafriri et al. 1996) .

The spontaneous nature of IVM is thought to result in compromised oocyte developmental

potential compared to in vivo matured oocytes, as demonstrated by reduced blastocyst

development post-fertilization (Greve et al. 1987; Leibfried-Rutledge et al. 1987; Lonergan et

al. 2003). This strongly suggests major differences between oocytes matured in vivo and in

vitro in the degree of cytoplasmic maturation, which is dependent on the accumulation of


128

important factors in the form of proteins or stable mRNA involved in the acquisition of

oocyte developmental potential (Hyttel et al. 1986a; Hyttel et al. 1986b; Hyttel et al. 1989).

Delaying or temporarily blocking the process of spontaneous in vitro meiotic maturation may

permit a more complete oocyte cytoplasmic maturation to occur, granting the oocyte a greater

capacity for fertilization and subsequent development (Fouladi Nashta et al. 1998). The

significance of this approach is to allow sufficient mRNA and protein accumulation within

the ooplasm. Spontaneous oocyte maturation has been prevented using cAMP modulators

during culture, such as cAMP analogues (e.g. dibutyryl cAMP), adenylate cyclase activators

(e.g. FSH and forskolin) and PDE inhibitors, such as the non-specific inhibitor IBMX, the

PDE type4 inhibitor rolipram or the PDE3 inhibitors e.g cilostamide, milrinone or Org9935.

The presence of cAMP modulators during IVM blocked meiotic resumption of mouse (Eppig

1989; Nogueira et al. 2003b; Vanhoutte et al. 2008) and human oocytes (Nogueira et al.

2003a; Shu et al. 2008), but temporarily attenuate meiotic resumption in bovine oocytes

(Sirard and First 1988; Aktas et al. 1995b; Thomas et al. 2004a; Thomas et al. 2004b).

Exposure of mouse immature oocytes to Org9935 (Nogueira et al. 2003b) or cilostamide

(Vanhoutte et al. 2008) improves their quality and developmental potential. In bovine, the

addition of milrinone delays also the rupture of gap-junctional communication between the

oocyte and the surrounding cumulus cells, resulting in increased blastocyst yield and quality

(Thomas et al. 2004a; Thomas et al. 2004b). In addition, enhancement of nuclear maturation

rates occur when human oocytes are matured in the presence of Org9935 (Nogueira et al.

2006). Furthermore, combined exposure of human oocytes to cilostamide and forskolin has a

positive effect on fertilization rate following IVM (Shu et al. 2008). Although, there is a

slight improvement in oocyte quality and competence, the results are still suboptimal

compared with the in vivo situation.


129

In the previous chapter (chapter 3), it was demonstrated that the efficacy of cAMP modulators

during IVM is substantially improved by management of COC cAMP levels during the pre-

IVM phase. Moreover, careful modulation of intracellular cAMP levels within a

physiological range in the oocyte and cumulus cells during collection and maturation, along

with specific FSH concentrations during IVM, generate a form of induced maturation in vitro

that leads to acquisition of high oocyte developmental competence.

The aim of this study was to evaluate the effects of treating immature bovine oocytes with

various forms of cAMP modulators before and during bovine IVM. A similar approach has

been tested in the previous chapter (chapter 3), but in this chapter we aimed to optimize

specifically the pre-IVM phase: the duration of exposure and effect of maintaining or

increasing intra-oocyte cAMP before IVM on the acquisition of embryonic developmental

competence.


130

4.3 Materials & methods

Chemicals

Unless otherwise noted, all chemicals were bought from Sigma Chemical Co. (St. Louis, MO,

USA). All pharmacological inhibitors were stored in aliquots at -20°C until use.

4.3.1 Oocyte recovery, selection and incubation (pre-IVM phase)


saline (37ºC). Antral follicles (2 to 8 mm diameter) were aspirated using an 18-gauge needle

and a 10-ml syringe. Shortly after COC removal from the follicle (< 5 min), the pellet of

sediment from the follicular aspirate containing COCs was transferred to collection medium,

Bovine VitroCollect (IVF Vet solutions, Adelaide, Australia) in the presence or absence of

cAMP modulators (pre-IVM phase). Modulators which were used during the pre-IVM phase

were: the adenylate cyclase activator, forskolin (0.4-100 µM), and a non-specific

phosphodiesterase inhibitor, IBMX (500 µM). Stock concentrations of the cAMP modulators

were stored at -20�C dissolved in 95% ethanol. Solutions containing modulators were diluted

fresh for each experiment. Only COCs with intact cumulus vestments were selected under a

dissecting microscope for experiments and were maintained in pre-IVM media for up to 2

hours.

4.3.2 In vitro oocyte maturation, fertilization and embryo culture

The basic medium for oocyte maturation was Bovine VitroMat (IVF Vet Solutions)

supplemented with 100 mIU/ml FSH (Puregon, Organon, Oss, Netherlands) and 20 µM


131

cilostamide (Biomol Plymouth Meeting, PA), a PDE3 inhibitor. Stock concentration of

cilostamide was stored at -20�C dissolved in 95% ethanol. Solutions containing cilostamide

were diluted fresh for each experiment. Thirty COCs were cultured in 4-well multidishes in

pre-equilibrated 300 µl drops of VitroMat overlaid with mineral oil and incubated at 38.5ºC

with 6 CO2 in humidified air for 30 hours (delayed IVM, see chapter 3).

At the end of IVM, COCs were removed from the IVM wells and washed twice using Bovine

VitroWash (IVF Vet Solutions), and transferred to insemination dishes containing Bovine

VitroFert (IVF Vet Solutions) supplemented with 0.2 mM penicillamine, 0.1 mM

hypotaurine, and 2 mg/ml heparin. Frozen semen from a single bull of proven fertility was

used in all experiments. Briefly, thawed semen was layered over a discontinuous 45%: 90%

Percoll gradient (Amersham Bioscience) and centrifuged at room temperature for 20-25 mins

at 700 g. The supernatant was removed and the sperm pellet was washed with 500 μl Bovine

VitroWash and centrifuged for a further 5 minutes at 200 g. Spermatozoa were resuspended

with VitroFert, then added to the fertilization media drops (Bovine VitroFert, supplemented

with 0.01 mM heparin, 0.2 mM penicillamine and 0.1 mM hypotaurine) at a final

concentration of 1 x 106 spermatozoa/ml. COCs were inseminated at a density of 10 μl of IVF

medium per COC for 24 h, at 39�C in 6% CO2 in humidified air.

Cumulus Cells were removed by gentle pipetting 23–24 h post insemination. Presumptive

zygotes were washed, transferred in groups of 5 into 20 µl drops of pre-equilibrated Bovine

VitroCleave (IVF Vet Solutions) and cultured under mineral oil at 38.5°C in 7% O2, 6%

CO2, balance N2, for five days (day 1 to day 5). On day 5, embryos were washed and

transferred in groups of 5-6 to 20 μl drops of pre-equilibrated Bovine VitroBlast (IVF Vet

Solutions) overlaid with mineral oil and cultured at 38.5°C in 7% O2, 6% CO2, balance N2


132

until Day 8. Embryos were assessed for quality at Day 8 according to the definitions

presented in the Manual of the International Embryo Transfer Society (Stingfellow 1998) and

were performed independently and blinded by an experienced bovine embryologist.


Cyclic AMP quantification was performed by using a radioimmunoassay method described

and validated previously (Reddoch et al. 1986) At specific time intervals, 10 COCs were

washed twice with VitroCollect, transferred to 0.5ml 100% ethanol and stored at –20�C.

Before cAMP measurements, samples were vortexed for 30 seconds and then centrifuged at

3000g for 15 min at 4�C. The supernatants were evaporated and the pellets resuspended in

assay buffer (50mM sodium acetate, pH 5.5), acetylated using triethylamine (AJAX

Chemicals, Sydney, Australia) and acetic anhydride (BDH Laboratory Supplies, Poole,

England) (2:1) and assayed. 125I-labelled cAMP (specific activity of 2175 Ci/mM) and cAMP

antibody (Reddoch et al. 1986) were added to samples and left overnight at 4�C. The

following day, 1ml of cold 100% ethanol was added to samples which were centrifuged at

3000g. The supernatant was removed and the pellet dried and counted using a gamma

counter. Samples were assayed in duplicate and compared to a standard curve of known

cAMP concentration (0-1024 fmol cAMP).

4.3.4 Experimental design

Experiment 1. The effects of increasing doses of forskolin with and without IBMX during

the pre-IVM phase on cAMP levels in COCs were investigated. COCs were aspirated and

selected either in follicular fluid (control) or collection medium (VitroCollect) supplemented


133

with 0, 0.4, 2, 10, 20, 50 or 100 µM forskolin, in the presence or absence of 500 µM IBMX.

After 2 hours of COCs incubation in treatments, 6-10 COCs were collected and snap frozen

for cAMP assay.

Experiment 2. The aim of this experiment was to determine the effect of increasing

incubation (pre-IVM) time on COC cAMP levels when pre-treated by increasing

concentrations of forskolin (0, 10, 20, 50 and100 µM) and 500 µM IBMX. Only the

concentrations of forskolin that had been shown to increase COC cAMP levels in Experiment

1 were used. cAMP measurements were carried out after 5, 30, 60 and 120 minutes of pre-

IVM phase. 6-10 COCs were collected from each time point/treatment and snap frozen for

cAMP assay.

Experiment 3. The aim of this experiment was to determine the effect of increasing

incubation (pre-IVM) time on intra-oocyte (DO) cAMP levels. COCs were collected and

incubated in collection medium for 30 or 120 minutes with increasing concentrations of

forskolin (0, 10, 20, 50 and100 µM) and 500 µM IBMX. After 30 or 120 minutes, COCs

were gently denuded of cumulus cells and 21-24 oocytes were collected from each treatment

and snap frozen for cAMP assay.

Experiment 4. The aim of this experiment was to examine the presence of IBMX and

increasing doses of forskolin (0, 10, 50 and 100 µM) in pre-IVM phase before oocyte culture

(1 hour of incubation), and the presence of cilostamide (20 µM) and 100 mIU/ml FSH in the

IVM phase (30 hours of IVM, see chapter 3) on oocyte developmental capacity.


134


Statistical analyses were conducted using Prism 5.00 GraphPad for Windows version 5

(GraphPad Software, San Diego, CA, USA, www.graphpad.com). Statistical significance was

assessed by ANOVA followed by either Dunnett's (figures 1 and 4) or Bonferroni’s (figure 2-

3) multiple-comparison post-hoc tests to identify individual differences between means.

Probabilities of P < 0.05 were considered statistically significant. All values are presented as

means with their corresponding standard error of the mean (SEM).


135

4.4 Results

4.4.1 Experiment 1. The effect of increasing concentrations of forskolin and the

presence/absence of IBMX in collection medium during the pre-IVM phase on COC

cAMP levels

As shown in figure 1A, after 2 hours of pre-IVM, forskolin significantly elevated (P < 0.05)

cAMP levels within COCs in a dose dependent manner. Compared to the follicular fluid

control, only the highest concentration of forskolin (100 µM) gave a similar value. However,

neither 0.4 µM, nor 2 µM forskolin showed any increase in cAMP levels, which was not

significantly different from the control treatment (collection medium without cAMP

modulators). In combination with IBMX, increasing concentrations of forskolin induced a 20-

fold increase in cAMP in a dose dependent manner over the levels observed in absence of

IBMX and presence of forskolin alone (figure 1B). Hence, forskolin or IBMX alone

maintains but does not elevate cAMP levels substantially higher than the controls.

4.4.2 Experiment 2. The effect of pre-IVM duration on COC cAMP when incubated

with increasing concentrations of forskolin and IBMX

The aim of this experiment was to examine the effect of pre-IVM duration when COCs are

incubated with IBMX and increasing doses of forskolin (10-100 µM) on COC cAMP levels.

As shown in figure 2, COC cAMP levels were maintained during the pre-IVM period among

all treatment groups except the control group (collection medium without cAMP modulators).

When COCs were incubated in collection medium alone, cAMP levels dropped significantly

(P < 0.05) after 30 minutes from 14 to 4 fmol/COC, and continued to drop significantly with


136

increasing time (0.4 fmol/COC) up to 2 hours of incubation. Treating COCs with IBMX or

forskolin alone maintained cAMP levels for 2 hours. However, a dramatic induction of up to

20 fold in cAMP levels was noted when COCs were incubated in the presence of IBMX and

increasing doses of forskolin, and this increase in cAMP levels was notable regardless of

incubation period. The highest cAMP levels were noted when COCs were incubated in the

presence of IBMX and 50 or 100 µM forskolin (approximately 165 fmol/COC). There was no

effect of time in increasing cAMP in COC except in the control group (no cAMP

modulators).

4.4.3 Experiment 3. The effect of pre-IVM duration on intra-oocyte cAMP when

incubated with increasing concentrations of forskolin and IBMX

The aim of this experiment was to examine cAMP levels in the oocyte (COCs denuded after

pre-IVM) and how these levels change in the presence of cAMP modulators with extending

the incubation time in the pre-IVM phase. As shown in figure 3, after 30 minutes of

incubation, cAMP levels were significantly higher (12 fmol/oocyte) when COCs were

incubated in the presence of IBMX and 10, 50 or 100 µM forskolin, compared to control (0.5

fmol/oocyte, P < 0.05). After 2 hours, levels of cAMP are further significantly increased up to

34 fmol/oocyte, compared to control (0.1 fmol/oocyte, P <0.05). It appears that increasing the

incubation time in the pre-IVM phase, leads gradually to a substantial increase in intra-oocyte

cAMP in the presence of IBMX and high concentrations of forskolin.


137

4.4.4 Expeirment 4. The effect of increasing cAMP levels prior to IVM on oocyte

developmental competence

As shown in figure 4, the cleavage rates of oocytes incubated in the presence of IBMX and 50

or 100 µM forskolin for 30-60 minutes prior to IVM were significantly greater than that of

oocytes incubated in the presence of IBMX alone or control treatment (P < 0.05).

Development to the blastocyst stage was similarly improved for oocytes incubated in the

presence of IBMX and 50 or 100 µM forskolin for 30-60 minutes prior to IVM compared to

control treatment (P < 0.05). Moreover, the rate of development to the blastocyst stage was

not significantly different between IBMX alone or IBMX and 10 µM forskolin group,

however were significantly higher than control (no cAMP modulators) (P < 0.05). Hence,

increasing cAMP levels within the oocyte prior to IVM leads to a substantial improvement in

developmental outcomes when COCs are matured in the presence of cilostamide.


138

4.5 Discussion

The results of this study clearly demonstrate that the level of intracellular oocyte cAMP after

follicle isolation and prior to IVM notably affects oocyte developmental competence. We

have shown following collection from the follicle, increases in cAMP levels are time

dependent in the oocyte, but not in COCs and that this increase in cAMP during the first

hours of IVM has profound long-term consequences for oocyte potential. This increase in

intra-oocyte cAMP could be required prior to oocyte culture in order to synchronize nuclear

and cytoplasmic maturation which lead to improved developmental outcomes.

This chapter highlights the importance of the time period preceding IVM: the oocyte

collection and the selection phase (pre-IVM phase). The importance of this period is crucial

in attempting to improve developmental outcomes from oocytes undergoing IVM. In clinical

human IVM applications, the collection period is a long process in which immature oocyte

retrieval usually occurs in buffered tissue culture medium. In cattle, oocytes are retrieved

from the ovary into simple phosphate buffered saline (PBS) using Trans-Vaginal Oocyte

Recovery (TVOR). In either case, the follicular fluid in which the oocyte was bathed in vivo

is rapidly diluted and eliminated, and the oocyte is then exposed to a far from physiological

environment. These oocyte collection procedures, together with IVM media most commonly

used, actually promotes a form of precocious depletion in cAMP (figure 2), leading to

precocious meiotic resumption. In contrast, in vivo it has been shown that there is a sharp

increase in cAMP followed by a gradual decrease, following the LH surge (Mattioli et al.

1994).

In the laboratory environment, bovine COC aspiration for IVM usually occurs in pure

follicular fluid, which is known to have a higher meiosis-inhibiting effect (Bilodeau et al.


139

1993; Hinrichs et al. 1995; Dostal and Pavlok 1996). A previous study by Thomas and

collaborators showed when COCs are retrieved from follicles in pure follicular fluid and

matured in the presence of a specific PDE3 inhibitor, milrinone, or specific PDE4 inhibitor,

rolipram, oocyte developmental competence significantly improved when COCs were

fertilized after 28 hours of IVM (Thomas et al. 2004b). In the mouse model, the application

of a specific PDE3 inhibitor during collection and biphasic IVM has been studied using Org

9935 or cilostamide (Nogueira et al. 2003b; Vanhoutte et al. 2008). The application of these

specific PDE 3 inhibitors during IVM resulted in an improvement in developmental outcomes

compared to their absence (control) but significantly poorer outcomes compared to in vivo

matured oocytes (Nogueira et al. 2003b; Vanhoutte et al. 2008). The aim of the inclusion of

PDE inhibitors in these experiments was to prevent the precocious drop in intra-oocyte

cAMP, which would negatively affect the oocyte’s developmental competence. In this study,

the inclusion of PDE inhibitor (IBMX) alone, although it maintains cAMP levels during pre-

IVM phase, cleavage and blastocyst rates were not improved and were similar to control

(figure 4).

It has been reported that the addition of an adenylate cyclase activator (invasive adenylate

cyclase) in the washing medium before culture results in an improved blastocyst rate

(Luciano et al. 1999; Guixue et al. 2001; Luciano et al. 2004). The substantial increase in

cleavage and blastocyst percentages in our results, works on the same principle as these

previous demonstrations and is due to an increase in cAMP levels during the pre-IVM phase.

However, this was achieved through the presence of the specific PDE3 inhibitor, cilostamide

in the IVM medium, in addition to cAMP modulators IBMX and forskolin in the pre-IVM

phase.


140

In chapter 3, addition of cAMP modulators during pre-IVM and IVM led to significant effects

on intra-oocyte cAMP levels, oocyte meiotic progression and oocyte-cumulus cell gap

junctional communication. These differences resulted in substantial improvement in oocyte

developmental competence. A significant improvement in embryonic development was

achieved when COCs were incubated in the presence of IBMX and forskolin during pre-IVM

and then matured in the presence of cilostamide (delayed IVM). We concluded that cAMP

modulation during IVM is particularly enhanced with the presence of cAMP modulators

during the pre-IVM phase.

A substantial improvement specifically in cleavage and blastocyst rates has been noted when

intra-oocyte cAMP is elevated by forskolin during pre-IVM. We have shown that this

elevation in the oocyte does not happen as rapidly as in the cumulus cells (figure 2). This

indicates that cAMP transfer from the cumulus cells to the oocyte is the limiting step to this

effect rather than cAMP synthesis. The overall effect of this elevation of cAMP was a

significant increase in the cleavage rate and blastocyst development rate of subsequent

embryos. This is a clear effect of the inclusion of high concentrations of forskolin in the pre-

IVM phase. Forskolin is an activator of adenylate cyclase and has a well known role in

delaying oocyte meiotic progression in rodents (Dekel et al. 1984; Racowsky 1984), porcine

(Racowsky 1985; Xia et al. 2000) and bovine (Thomas et al. 2004b). It has also been

previously observed that that there is an increase in intra-oocyte cAMP after forskolin

treatment of COCs but not of cumulus-free oocytes (denuded oocytes) (Dekel et al. 1984),

and that, the inclusion of an adenylate cyclase activator such as invasive adenylate cyclase

during collection, selection and the maturation process leads to an improvement in oocyte

developmental competence (Luciano et al. 1999; Guixue et al. 2001; Luciano et al. 2004).


141

As shown in figures 2 and 3, the incubation period during the pre-IVM phase plays an

important role in increasing cAMP in the oocyte but not in the COCs. It is interesting how

cAMP is acutely elevated by forskolin and IBMX in the COC, and this increase is maintained

without any drop. Whereas it takes around 30 minutes until intra-oocyte cAMP is increased,

supporting the hypothesis that cAMP is synthesized in the cumulus and then transferred to the

oocyte.

Overall, we have shown that the pre-IVM phase is a very important period. An increase in

cAMP may allow the oocyte to synchronize nuclear and cytoplasmic maturation and hence

supporting further oocyte development. Such an approach could profoundly change the

routine practice of usual oocyte collection and change overall the way to initiate maturation in

vitro which might mimic some aspects of what is happening in vivo.


142

4.6 Figures

Figure 1. Effect of increasing doses of forskolin without (A) or with (B) (IBMX; 500 µM) during pre-IVM phase on the cAMP content of the whole COCs. COCs were collected and selected for 2 hours either in follicular fluid or collection medium supplemented ± (0.4-100 µM forskolin, 500 µM IBMX). Values are expressed as the mean concentration of cAMP/COC ± SEM of three replicates using 6-10 COCs per treatment replicate. Different superscripts indicate significantly different amounts of cAMP between treatments (one-way ANOVA, P < 0.05).

0

4

8

12

follicular fluidmedium

forskolin (µM) - 0 0.4 2 10 20 50 100

a

b b b

c cc

ac

fmol

cAM

P pe

r CO

C

05

10152025306090

120150180

a

b

aa

c c

d d

forskolin (µM) - 0 0.4 2 10 20 50 100IBMX - - + + + + + +

fmol

cAM

P pe

r CO

C

A.

B.


143

Figure 2. The effect of pre-IVM duration on COC cAMP when incubated with increasing concentrations of forskolin and IBMX. COCs were collected and selected for up to 2 hours incubation time either in follicular fluid or collection medium supplemented with increasing doses of forskolin (0-100 µM) and IBMX (500 µM). Values are expressed as the mean concentration of cAMP/COC ± SEM of three replicates using 6-10 COCs per treatment replicate. Different superscripts indicate significantly different amounts of cAMP between treatments (two-way ANOVA, P < 0.05).

0

5

10

15

20

25

306080

100120140160180200220

5 min

30 min

1 hr

2 hrs

follicular fluid medium

IBMX - - - + + + + + forskolin (µM) - 0 100 0 10 20 50 100

Incubation time

a

a

a

a

a

bbc

c

a

a

a

a

ab a aa

d d d dd

dd d

e e ee

e e

e e

cAM

P(fm

ol/C

OC

)


144

Figure 3. The effect of pre-IVM duration on intra-oocyte cAMP content: those collected and incubated with their cumulus vestments intact (COCs) and denuded prior to assay (DO). COCs were collected and selected for 30 minutes or 2 hours incubation time either in collection medium supplemented with (increasing doses of forskolin, 10-100 and IBMX, 500 µM). Values are expressed as the mean concentration of cAMP/DO ± SEM of three replicates using 21-24 DO per treatment replicate. Different superscripts indicate significantly different amounts of cAMP between treatments (two-way ANOVA, P < 0.05).

0

10

20

30

40

IBMX + + + + + + + +forskolin (µM) 0 10 50 100 0 10 50 100

Incubation time (min) 30 min 120 min

ab

c c

a

c

dd

fmol

cAM

P pe

r DO


145

Figure 4. The effect of IBMX and increasing doses of forskolin in the pre-IVM phase on oocyte developmental capacity. COCs were aspirated and selected either in collection medium alone or collection medium supplemented with increasing doses of forskolin; 10-100 µM ± 500 µM IBMX; for 1 hour. COCs were then matured with in the presence of 100 mIU/ml FSH and 20 µM cilostamide for 30 hours. Subsequently, oocyte developmental capacity was assessed after in vitro fertilization and embryo development by the cleavage rate (A) and the blastocyst rate on day 8 (B). Different superscripts indicate significantly different development percentages between treatments (one-way ANOVA, P < 0.05).

20

40

60

80

100a a

ab b b

% c

leav

age

20

40

60

80

aab b

cc

IBMX - + + + + forskolin (�M) 0 0 10 50 100

% b

last

ocys

t fro

m c

leav

ed

A.

B.


146

4.7 References

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Norris RP, Freudzon M, Mehlmann LM, Cowan AE, Simon AM, Paul DL, Lampe PD, Jaffe LA (2008) Luteinizing hormone causes MAP kinase-dependent phosphorylation and closure of connexin 43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption. Development 135, 3229-38. Norris RP, Ratzan WJ, et al. (2009) Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte. Development 136, 1869-78. Racowsky C (1984) Effect of forskolin on the spontaneous maturation and cyclic AMP content of rat oocyte-cumulus complexes. J Reprod Fertil 72, 107-16. Racowsky C (1985) Effect of forskolin on maintenance of meiotic arrest and stimulation of cumulus expansion, progesterone and cyclic AMP production by pig oocyte-cumulus complexes. J Reprod Fertil 74, 9-21. Reddoch RB, Pelletier RM, Barbe GJ, Armstrong DT (1986) Lack of ovarian responsiveness to gonadotropic hormones in infantile rats sterilized with busulfan. Endocrinology 119, 879-86. Schultz RM, Montgomery RR, Belanoff JR (1983) Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis. Dev Biol 97, 264-73. Sela-Abramovich S, Edry I, Galiani D, Nevo N, Dekel N (2006) Disruption of gap junctional communication within the ovarian follicle induces oocyte maturation. Endocrinology 147, 2280-6. Shu YM, Zeng HT, Ren Z, Zhuang GL, Liang XY, Shen HW, Yao SZ, Ke PQ, Wang NN (2008) Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes. Hum Reprod 23, 504-13. Sirard MA, First NL (1988) In vitro inhibition of oocyte nuclear maturation in the bovine. Biol Reprod 39, 229-34. Stingfellow DA, and Seidel, S. M (1998) Manual of the International Embryo Transfer Society. In. (IETS: Savoy, IL, USA) Thomas RE, Thompson JG, Armstrong DT, Gilchrist RB (2004) Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity. Biol Reprod 71, 1142-9. Tornell J, Billig H, Hillensjo T (1991) Regulation of oocyte maturation by changes in ovarian levels of cyclic nucleotides. Hum Reprod 6, 411-22. Tsafriri A, Chun SY, Zhang R, Hsueh AJ, Conti M (1996) Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors. Dev Biol 178, 393-402. Tsafriri A, Lindner H, Zor U, Lamprecht S (1972) In vitro induction of meiotic division in follicle-enclosed oocytes by LH, cyclicAMP and prostaglandin E2. J. Reprod. Fert. 31, 39-50.


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CHAPTER 5

INDUCED OOCYTE IVM SUBSTANTIALLY IMPROVES MOUSE

EMBRYO YIELD AND PREGNANCY OUTCOMES

Chapter 5. Induced oocyte IVM substantially improves mouse embryo yield and pregnancy outcomes

151

5.1 Abstract In vitro oocyte maturation (IVM) is an alternative technique to ovarian hyperstimulation in

the treatment of infertility and has the potential to alter reproductive biotechnologies and

human infertility treatment if success rates were notably higher. Oocyte maturation in vivo is

a highly orchestrated, induced process, whereby 3'-5'-cyclic adenosine monophosphate

(cAMP)-mediated meiotic arrest is overridden by the gonadotrophin surge prior to ovulation.

However, during IVM oocytes undergo spontaneous meiotic resumption hence compromising

developmental competence as a consequence of asynchronous oocyte cytoplasmic

maturation. Hence we hypothesized that establishing an induced IVM system will improve

mouse oocyte quality leading to improved developmental outcomes.

Mouse immature oocytes were treated for the first hour in vitro (pre-IVM phase) with the

adenylate cyclase activator, forskolin (FSK, 50 µM) and a non-specific phosphodiesterase

(PDE) inhibitor, 3-isobutyl-methylxanthine, (IBMX, 50 µM), which substantially increased

cumulus-oocyte complex (COC) cAMP to levels comparable to in vivo physiological levels.

To maintain oocyte cAMP levels and prevent precocious oocyte maturation, oocytes were

then cultured during IVM with an oocyte-specific (type 3) PDE inhibitor, cilostamide (0.1

µM) and induced to mature with follicle stimulating hormone (FSH, 100 mIU/ml). The

induced IVM system slowed meiotic progression through prophase I to metaphase II,

extending the normal IVM interval (22 vs. 18 h; treated vs. control). Induced IVM increased

blastocyst rate (86% vs. 55%; treated vs. control, p<0.05), implantation rate (53 vs. 28%),

fetal yield (26% vs. 8%) and fetal weight (0.9g vs. 0.5g, p<0.05). All these developmental

outcomes were restored, by using induced IVM, to levels obtained from in vivo matured

control oocytes (conventional IVF).


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In conclusion, Induced IVM mimics some of the characteristics of oocyte maturation in vivo

and substantially improves oocyte developmental outcomes. This should lead to an increase

in the use of this technique in reproductive biotechnologies.


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5.2 Introduction In vitro oocyte maturation (IVM) is a promising technology for infertility treatment or

fertility preservation, as it does not require expensive and potentially harmful hormonal

ovarian hyperstimulation. (Chian et al. 2004c; Jurema and Nogueira 2006). However, the

application of this technology in clinical settings is limited by the lower oocyte

developmental capacity due to nuclear and cytoplasmic maturation asynchrony (Eppig et al.

1994a). Temporarily arresting or delaying spontaneous meiotic resumption might improve

the synchronization of nuclear and cytoplasmic maturation during culture of immature

oocytes and ultimately improving oocyte developmental competence (Anderiesz et al. 2000).

Several pharmacologic approaches have been attempted to attenuate meiotic resumption and

allow cytoplasmic maturation during IVM (Faerge et al. 2001; Guixue et al. 2001; Sirard

2001; Wu et al. 2002; Le Beux et al. 2003; Shimada et al. 2003).

Phosphodiesterase (PDE) 3A enzyme is highly expressed in the oocyte (Tsafriri et al. 1996)

and is responsible for the degradation of 3'-5'-cyclic adenosine monophosphate (cAMP),

which is a key molecule regulating oocyte maturation (Conti et al. 1998). Inhibition of

oocyte-specific PDE3 maintains intra-oocyte cAMP levels at high levels, thus keeping

oocytes arrested at the germinal vesicle (GV) stage, avoiding interference with the cAMP

pathway in the surrounding somatic cells (Richard 2007). Previous studies demonstrated that

spontaneous meiotic resumption can be arrested or attenuated by administration of a PDE3

inhibitor in vivo (Wiersma et al. 1998) or in vitro in mouse (Tsafriri et al. 1996; Coticchio et

al. 2004), bovine (Mayes and Sirard 2002; Thomas et al. 2002), macaque (Jensen et al. 2002)

and humans oocytes (Nogueira et al. 2003a).


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Recent reports demonstrate that culturing immature oocytes with a specific PDE3 inhibitor

during IVM had a positive effect in improving developmental outcomes in mouse (Nogueira

et al. 2003b; Vanhoutte et al. 2008) and bovine oocytes (Thomas et al. 2004b). Based on

theses promising animal outcomes, five studies were conducted with human

oocytes(Nogueira et al. 2006; Vanhoutte et al. 2007; Shu et al. 2008; Vanhoutte et al. 2009a;

Vanhoutte et al. 2009b). Although, oocytes temporarily arrested by PDE3 inhibitors had

similar maturation rates compared to non-arrested control, there appeared to be no substantial

beneficial effects of pre-maturation culture or the temporal blockage of spontaneous meiotic

resumption on the developmental competence of human immature oocytes.

To date, specific PDE3 inhibitors supplementation during IVM have been applied for 24-hour

periods. However, the optimal duration of pre-maturation culture or time for appropriate

cytoplasmic maturation is unknown. The human trials demonstrated that there were no

differences in maturation rates of oocytes cultured in the presence of PDE inhibitors for 24-

hour and 48-hour, but 24 hours generated better embryonic integrity (Nogueira et al. 2006;

Vanhoutte et al. 2009a).

Delaying spontaneous IVM may improve cytoplasmic maturation by prolonging cumulus

cell-oocyte gap jucntional communication during meiotic resumption. For example, in bovine

PDE3 inhibitor supplementation slightly extended cumulus-oocyte gap jucntional

communication over the first 9 hours (Thomas et al. 2004b; Shu et al. 2008; Vanhoutte et al.

2009b). Therefore, it can be postulated that a shorter period of exposure would be beneficial

for immature oocytes to acquire developmental competence.


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In chapter 3, it was demonstrated that the efficacy of cAMP modulators during IVM is

substantially improved by management of COC cAMP levels during the pre-IVM phase.

Moreover, careful modulation of intracellular cAMP levels within a physiological range in

the oocyte and cumulus cells during collection and maturation, along with specific FSH

concentrations during IVM, generate a form of induced IVM that leads to acquisition of high

oocyte developmental competence.

In chapter 4, the results clearly demonstrated that the level of intracellular oocyte cAMP after

follicle isolation and prior to IVM and the period of exposure to a pre-IVM phase notably

affected oocyte developmental competence. The increased intra-oocyte cAMP during pre-

IVM could be required prior to oocyte culture in order to synchronize nuclear and

cytoplasmic maturation, leading to improved developmental outcomes.

The aim of this chapter is to devise a murine induced IVM system and to examine the effects

of induced IVM on oocyte maturation, developmental competence, pregnancy outcomes and

fetal parameters.


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5.3 Materials and Methods All experiments were approved by local animal ethics committees and conducted in

accordance with the Australian Code of Practice for the Care and Use of Animals for

scientific Purposes.

5.3.1 Source of oocytes

Juvenile (21-26 day old) 129/SV mice were injected with 5 I.U. of equine chorionic

gonadotrophin (eCG, Folligon, Intervet, The Netherland) and immature COCs were collected

46 hours later. In vivo matured COCs (control), were collected from mice that were primed

with 5 I.U. eCG then 46 hours later with 5 I.U. human CG (Pregnyl, Organon, Oss, The

Netherlands), and ovulated COCs were collected 14 hours later from oviducts in Research

Vitro Wash (COOK Australia, Eight Mile Plains, Qld, Australia).

5.3.2 Oocyte collection (pre-IVM phase)

A pre-IVM phase was developed (see chapter 2 and 3). The aim of this phase is 1) to prevent

rapid COC cAMP degradation that occurs during standard IVM, and 2) to substantially

elevate COC cAMP levels, as occurs during oocyte maturation in vivo (figure 1). Ovarian

follicles were punctured and COCs were collected in HEPES-buffered alpha minimal

essential medium (HEPES αMEM; Invitrogen, Carlsbad, USA) supplemented with 3 mg/ml

fatty acid-free bovine serum albumin (BSA; ICPbio Ltd, Auckland, NZ) and 1 mg/ml fetuin

(Sigma). Depending on individual experimental design, COCs were exposed during pre-IVM

to the adenylate cyclase activator, forskolin (FSK, Sigma: 50 µM), which potently increases

whole COC and intra-oocyte cAMP levels. COCs were also treated during pre-IVM with or

without the PDE inhibitor, 3-isobutyl 1-methylxanthine (IBMX, Sigma: 50 µM), which is a

non-specific PDE inhibitor acting on oocyte and cumulus cell PDEs. Millimolar stock

concentrations of the cAMP modulators were stored at -20 °C dissolved in anhydrous


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dimethyl-sulphoxide (DMSO, Sigma) solutions and fresh aliquots were used for each

experiment. COCs were maintained in pre-IVM treatments under atmospheric conditions at

37 °C for 1 hour. At the end of the pre-IVM phase, COCs were washed twice in their

respective IVM treatments, before transfer to IVM drops.

5.3.3 Oocytes in vitro maturation (IVM phase)

COCs were matured in IVM medium (bicarbonate buffered αMEM medium supplemented

with 50 µg/ml Streptomycin, 75 µg/ml Penicillin G, 3 mg/ml BSA, 1 mg/ml fetuin and FSH

(Puregon; Organon, Oss, The Netherlands); (50 mIU/ml, standard IVM or 100 mIU/ml,

induced IVM) + 0.1 µM cilostamide). Groups of 30-40 COCs were matured in pre-

equilibrated 500 µl drops of IVM medium overlaid with mineral oil and incubated with 5 %

CO2 in humidified air for 18 h (standard IVM) or 22 h (induced IVM).

5.3.4 In vitro fertilization

At the end of culture, COCs were washed twice in Research Vitro Wash (COOK Australia),

twice in Research Vitro Fert (IVF medium, COOK Australia) and fertilized in 40 �l drops of

IVF medium using fresh sperm (final dilution of 2x106/ml) obtained from a CBAB6F1 male

donor. After incubation with sperm for 4 hour at 37 οC in 6% CO2, 5% O2, 89% N2,

presumptive zygotes were washed once in Research Vitro Wash, once in Research Vitro

Cleave (COOK Australia) and cultured in groups of 10-12 in 20 µl Research Vitro Cleave

drops over-layered with mineral oil at 37 οC in 6% CO2, 5% O2 and 89% N2. Fertilization

rates were scored 20 hours after IVF and 2-cell embryos were transferred into fresh Research

Vitro Cleave drops for another 25-27 hours. Embryos were assessed for their rate of

development to distinguish percentage of faster developing embryos and transferred into fresh

Research Vitro Cleave drops for 47-49 hours. Embryonic morphology was assessed at the end

of the culture period at 96-100 hours post-fertilization to determine blastocyst development.


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5.3.5 Differential nuclear staining

The number and proportion of blastocyst inner cell mass (ICM) and trophectoderm (TE) cells

were determined using a differential nuclei staining protocol described by Gardner et al.

(2000). Briefly, blastocysts were placed in 0.5% pronase solution at 37°C for up to 5 minutes

to dissolve the zona pellucida and then incubated in 10 mM 2,4,6-trinitrobenzenesulfonic acid

at 4°C for 10 minutes. Blastocysts were then transferred to 0.1 mg/ml anti-dinitrophenyl-BSA

for 10 minutes at 37°C and placed in guinea pig serum with propidium iodide for 5 minutes at

37°C. Blastocysts were then stained with bisbenzimide in ethanol at 4°C overnight. Embryos

were washed in 100% alcohol in the dark at 4°C until visualized. Stained embryos were

mounted in glycerol on a microscope slide and overlayed with a coverslip. Analyses were

performed using a fluorescent microscope at x400 magnification and a UV filter, where ICM

and TE cells appeared blue and pink, respectively.

5.3.6 Embryo transfer

Naturally ovulating female Swiss mice aged between 8 and 12 weeks were mated with

vasectomised CBAB6F1 males to induce pseudopregnancy. On day 3.5 of pseudopregnancy,

the female recipients were anaesthetized with 2% avertin (2,2,2-tribromoethanol in 2-methyl-

2-butanol, diluted to 2% in sterile saline; 0.015 ml/g body weight). Six blastocysts from a

treatment were randomly assigned to each uterine horn. A total of 192 blastocysts across the

three treatments were transferred to 16 recipients. Pregnancy outcomes were assessed

following euthanasia 3 days prior to term, on day 18.5 of pregnancy.


Cyclic AMP quantification was performed by using a radioimmunoassay method described

and validated previously (Reddoch et al. 1986). At specific time intervals, 30 COCs were

washed twice with Research Vitro Wash, transferred to 0.5 ml 100% ethanol and stored at –


159

20�C. Samples were vortexed for 30 seconds and centrifuged at 3000g for 15 minutes at 4�C.

The supernatants were evaporated and the pellets were resuspended in assay buffer (50 mM

sodium acetate, pH 5.5), acetylated using triethylamine (AJAX Chemicals, Sydney, Australia)

and acetic anhydride (BDH Laboratory Supplies, Poole, England) (2:1) and assayed. 125I-

labelled cAMP (specific activity of 2175 Ci/mM) and cAMP antibody (Reddoch et al. 1986)

were added to samples and left overnight at 4�C. The following day, 1 ml of cold 100%

ethanol was added to samples which were centrifuged at 3000g. The supernatant was

removed and the pellet dried and counted using a gamma counter. Samples were assayed in

duplicate and compared to a standard curve of known cAMP concentration (0-1024 fmol

cAMP).

5.4 Experimental Design

5.4.1 COC cAMP levels and initiation of maturation

The aim of this experiment is to compare levels of cAMP over the first 2 hours of initiation of

oocyte maturation (in vivo vs. in vitro). For IVM derived oocytes, COCs were collected and

kept as a pool in a control pre-IVM phase (collection medium without cAMP modulators) and

at 30 minutes intervals, 12 COCs were collected and snap frozen for cAMP quantification.

For in vivo cAMP measurement, mice were killed at 30 minutes intervals (from 0 minutes-2

hours post hCG) and 12 COCs were collected and snap frozen for cAMP measurement.

5.4.2 The effect of increasing doses of FSH to induce meiotic maturation of COCs

matured in the presence of the type 3 PDE inhibitor (cilostamide)

This experiment was designed to determine the effective dose of FSH that efficiently

overrides the inhibitory effect of two arresting doses of cilostamide (0.1, 1 µM). IVM media

were supplemented with 0.01, 0.1, 1, 10, 50, 100 and 200 mIU/ml FSH. After 24 hours of


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culture, oocytes were denuded of cumulus cells by gentle pipetting to facilitate visualization

of the nuclear stage. Maturation stage in these oocytes was classified and recorded by light

microscopy as the percentage of oocytes that had reached M II (polar body extrusion).

5.4.3 Effect of pre-IVM phase on oocyte meiotic resumption that matured by

induced IVM (22 hours IVM)

This experiment was designed to determine if a pre-IVM phase would further delay oocyte

meiotic progression when combined with IVM + cilostamide. COCs were aspirated and

selected in collection medium supplemented with 0.1 µM cilostamide or 50 µM forskolin +

50µM IBMX for 1 hour. COCs were then matured in IVM medium + 100 mIU/ml FSH + 0.1

µM cilostamide for 18-26 hours. After IVM oocytes were denuded of cumulus cells by gentle

pipetting to facilitate visualization of the nuclear stage. Maturation stages in these oocytes

were assessed using a light microscopy and classified as the percentage of M II oocytes.

5.4.4 The effect of standard or induced IVM on oocyte meiotic maturation

To determine the effect of the induced IVM system on the rate of meiotic resumption and

completion, COCs were matured in vivo: mice were primed at 9 pm with 5 I.U. eCG then 46

hours later with 5 I.U. hCG; or in vitro either by standard IVM (control pre-IVM, without

cAMP modulators for 1 hour and control IVM with 50 mIU/ml FSH), or by induced IVM

(pre-IVM, 50 µM FSK+50µM IBMX for 1 hour, followed by IVM with 0.1 µM cilostamide

+100 mIU/ml FSH). From 0 to 14 hours and after 18 or 22 hours, COCs were collected and

denuded of cumulus cells by gentle pipetting to facilitate visualization of the nuclear stage.

Maturation stages in these oocytes were classified by light microscopy as immature oocytes

that progressed into germinal vesicle breakdown (% GVBD, 0-14 hours) and the percentage

of oocytes that had reached M II (18 hours and 22 hours).


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5.4.5 The effect of time of maturation (18 or 22 hours) on oocytes matured by

standard or induced IVM on oocyte developmental capacity

Oocytes were matured either by standard or induced IVM up to 18 or 22 hours of culture.

Oocytes then were fertilized and developmental capacity was assessed by embryo

development cleavage rate, blastocyst rate on day 5 and blastocyst quality.

5.4.6 The effect of different cAMP modulators during the pre-IVM & IVM phases on

oocyte developmental capacity

As shown from chapters 3 and 4, increasing cAMP levels during the pre-IVM phase

positively affects oocyte developmental competence. Hence, the effect of using different

cAMP modulators during pre-IVM and IVM on oocyte developmental competence was

examined. COCs were subjected to pre-IVM for 1 hour (control medium or medium

supplemented with 50 µM IBMX +/- 50 µM FSK). COCs were then matured in IVM medium

+ 50 mIU/ml FSH for 18 hours (control) or with 0.1 µM cilostamide + 100 mIU/ml FSH for

22 hours. Oocyte developmental capacity was assessed after IVF and embryo development by

cleavage rate, blastocyst rate and hatching blastocyst rate on day 6.

5.4.7 Embryo developmental capacity, pregnancy outcomes & fetal parameters of

oocytes derived from induced and standard IVM and from in vivo matured oocytes

The aim of this experiment is to compare the developmental competence of oocytes derived

from induced IVM to oocytes matured in vivo, as assessed by embryo development,

pregnancy outcomes and fetal parameters. In vivo matured COCs (collected from oviducts 14

hours after hCG administration), COCs matured in vitro (standard or induced IVM) were

collected, fertilized (IVF) and cleavage and blastocyst rates were assessed on day 3.5.

Blastocysts were transferred to pseudo pregnant recipients and outcomes were analysed on

day 18.5 of pregnancy.


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Software, San Diego, CA, USA). Treatment effects were assessed by 1-way or 2-way

ANOVA followed by either Dunnett's or Bonferroni’s multiple-comparison post-hoc tests to

identify individual differences between means. All values are presented as means with their

corresponding standard error of the mean (SEM). Implantation rate and fetal survival were

considered as binomial data and analysed using Chi-square.


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5.5 Results

5.5.1 COC cAMP measurements in vitro versus in vivo

The levels of cAMP of COCs during in vivo and in vitro maturation were determined over a 2

hour period. Cyclic AMP levels within in vivo maturing COCs started to increase and were

200 fold higher two hours after hCG administration compared to time zero (figure 1A,

P<0.05). In comparison, cAMP levels in COCs incubated in vitro (control pre-IVM phase)

dropped significantly (P<0.05) 30 minutes after isolating from the follicle and continued to

decrease during the 2 hour incubation period (figure 1B).

5.5.2 Increasing doses of FSH to induce meiotic maturation of COCs matured in the

presence of the type 3 PDE inhibitor (cilostamide)

As shown in figure 2B, when COCs were matured in the presence of 0.1 µM cilostamide,

increasing concentrations of FSH during IVM significantly increased the percentage of

oocytes reaching MII (P<0.05). The inhibitory effect of cilostamide was completely

overridden when 100 mIU/ml FSH or higher were added to IVM media. However, high doses

of FSH did not override the inhibitory effect of 1 µM cilostamide (figure 2A). These results

indicate that FSH can induce nuclear maturation of oocytes arrested by cilostamide, provided

the appropriate combination of cilostamide and FSH are used.

5.5.3 Effect of pre-IVM phase on oocyte meiotic resumption that matured by

induced IVM

In chapter 3, the combination of forskolin and IBMX during pre-IVM phase and cilostamide

during IVM phase further delayed bovine oocyte nuclear maturation. As shown in figure 3,

there were no further delays in oocyte meiotic progression to MII when COCs were treated

with forskolin and IBMX during the pre-IVM phase compared to cilostamide alone. Most


164

oocytes reached MII (80%) after 22 hours of IVM, regardless of the pre-IVM phase

conditions.

5.5.4 The effect of Induced IVM on GV/GVBD (0-14 hours of IVM)

As shown in figure 4, within 60 mins, 40% of oocytes in the control pre-IVM group (standard

IVM) commenced GVBD whereas all the oocytes matured in vivo (collected 60 minutes post

hCG) and all oocytes in the induced IVM group (60 mins pre-IVM culture in the presence of

cAMP modulators) were still arrested at GV (standard IVM = 60% GV vs. induced IVM and

in vivo matured = 100% GV, P<0.05). Two hours post-hCG, 50% of in vivo matured oocytes

had resumed meiosis, compared to 85% of oocytes after 2 hours of culture in the standard

IVM system (figure 4, P<0.05). The induced IVM system significantly delayed the

resumption of meiosis, with 25% of oocytes at GVBD after 8 hours and 90% GVBD after 12

hours of culture (figure 4, P < 0.05).

5.5.5 The effect of Induced IVM on oocyte maturation (18 and 22 hours of IVM)

Oocytes were matured by standard or induced IVM and nuclear maturation was assessed at

18 and 22 hours. As shown from figure 5, after 18 hours of culture, 82% of the oocytes were

at MII stage when oocytes were matured by standard IVM, whereas, only 45% of the oocytes

reached MII when matured by induced IVM (P<0.05). By 22 hours of IVM, majority of

oocytes were reached MII stage, regardless IVM systems (induced or spontaneous IVM).

5.5.6 The effect of induced IVM vs. standard IVM on oocyte developmental capacity

& blastocyst quality

After 18 hours of IVM, there was no significant difference in cleavage rate between induced

or standard IVM groups (figure 6A). However, after 22 hours, cleavage rate was significantly

higher (85%) in induced IVM group compared to standard IVM (40%) (figure 6A)(P<0.05).

Blastocyst rates assessed on day 5 were higher in induced IVM group when oocytes are


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matured for 22 hours compared to 18 hours and significantly higher standard IVM regardless

culture peroid (22 hours induced IVM, blastocyst = 81% vs. 18 hours induced IVM= 71% ,

18 hours standard IVM = 62% and 22 hours standard IVM = 43%; P<0.05).

Improved oocyte developmental competence was also reflected in blastocyst quality. After

culture for 22 hours in the induced IVM system, the number of total cells, inner cell mass and

the proportion of inner cell mass to total cells in blastocysts were greater than that for

standard IVM (figure 7, P<0.05).

Figure 8 demonstrates the effect of using different cAMP modulators during the pre-IVM and

IVM phases on oocyte developmental capacity. Collecting COCs in presence of IBMX or

IBMX+forskolin during pre-IVM followed by IVM in presence of cilostamide resulted in a

significant increase in cleavage (83%) and blastocyst rate (84%) compared to oocytes

pretreated during pre-IVM or matured during IVM in control treatment (without cAMP

modulators) (58%) (P<0.05). Interestingly, a substantial improvement were seen on day 6 in

the percentage of hatching blastocysts when COCs were pretreated in the presence of

forskolin and IBMX (65%) compared to IBMX alone (33%) during the pre-IVM phase and

maturation in induced IVM (figure 8, P <0.05).

5.5.7 The effect of maturing oocytes by induced or standard IVM compared to in

vivo matured oocytes on developmental capacity, pregnancy outcomes & fetal

parameters

Figure 9A shows developmental competence of oocytes matured either in vivo, by induced

IVM or by standard IVM. Cleavage and blastocyst rates of oocytes cultured in the induced

IVM system were similar to oocytes matured in vivo (obtained from ovulated follicles) and

were significantly higher than the standard IVM group (figure 9A, P<0.05). Furthermore, this

improved oocyte developmental competence was also reflected in blastocyst quality, as total

cell number, inner cell mass number and the proportion of inner cell mass to total cells, were


166

all similar after in vivo maturation or induced IVM and were significantly greater than that for

the standard IVM (figure 9B, P<0.05).

Pregnancy outcomes such as implantation rate, fetal survival and fetal weight from oocytes

which were matured by induced IVM were significantly higher than oocytes matured by

standard IVM (figure 10, P<0.05). There was no significant difference in these parameters

between induced IVM and in vivo matured oocytes. Moreover, fetal parameters such as fetal

crown-rump length and fetal: placental weight ratio were similar to in vivo matured controls,

when oocytes were matured by induced IVM and both treatments were significantly higher

than standard IVM rates (figure 11, P<0.05).


167

5.6 Discussion The fact that oocyte quality is compromised after standard IVM remains an intriguing

problem. Recent results offer some prospects for the application of cAMP modulators such as

specific PDE inhibitors to extend the developmental period of the immature oocyte before

IVM (Nogueira et al. 2003b; Thomas et al. 2004b; Nogueira et al. 2006; Shu et al. 2008).

Nevertheless, given the obvious differences in developmental potential to in vivo matured

oocytes, further studies are mandatory to clarify precisely the effect of cAMP modulators on

the maturation itself, in order to improve IVM culture systems.

The results in this chapter demonstrate that cAMP levels within the COC during IVM have

major effects on oocyte developmental competence, pregnancy outcomes and fetal survival.

In this chapter we have adapted the bovine induced IVM system and developed an induced

IVM system in mouse. Indeed, immature mouse oocytes cultured in the induced IVM system

have significantly higher fertilization, cleavage and blastocyst rates compared to oocytes

cultured in the standard IVM system. As previously shown, total blastocyst cell numbers

reflect embryo viability, as demonstrated by increased capacity to develop into a viable fetus

after embryo transfer (Lane and Gardner 1997). In the current study, induced IVM resulted in

significant increases in blastocyst total cell numbers, inner cell mass and the proportion of

inner cell mass to total cells, compared to standard IVM, which were all similar to in vivo

matured oocytes. Following embryo transfer, implantation rate was almost doubled, and fetal

number was 6-fold higher compared to the standard IVM group and were similar to the in

vivo matured oocyte group. Thus, induced IVM has a substantial effect on developmental

programming of oocyte and this persists through late preimplantation development and into

fetal development. This is the first study in mouse reporting developmental outcomes from

oocytes matured in vitro similar to in vivo matured oocytes.


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A key result in this chapter is the level of cAMP that accompanies the initiation of maturation

in vivo vs. in vitro (figure 1). During the initiation of in vivo maturation, there is a spike or a

dramatic surge in cAMP levels 2 hours after the hCG administration. This supports previous

observation in pig, where in vivo, it has been shown that there is a sharp increase in cAMP

followed by a gradual decrease, following the LH surge (Mattioli et al. 1994). In contrast, in

the standard IVM system, cAMP levels within the COC dropped and were significantly

depleted after 2 hours of maturation. The results in this chapter and also in the previous

chapters 3+4 support the hypothesis that a surge in cAMP levels is correlated with acquiring

oocyte developmental competence and the decrease in COC cAMP levels seen in the standard

IVM appear to be associated with reduced developmental competence.

This is the first report to demonstrate the use of higher concentrations of FSH to override the

inhibitory effect of cilostamide on resumption of meiosis (figure 2). Previous studies in

mouse used specific PDE-3 inhibitors in a bi-phasic IVM system, where nuclear maturation is

inhibited in the first phase and then oocytes are cultured without PDE3 inhibitors to allow

oocytes to undergo nuclear maturation (Downs et al. 1986a; Nogueira et al. 2003b; Vanhoutte

et al. 2008). These studies have led to small improvements in developmental competence, but

not to levels similar to in vivo matured oocytes. In contrast, this study has used continuous

exposure to PDE3 inhibitor and high dose of FSH to override the inhibitory effect of the PDE

inhibitor cilostamide (figure 2).

As shown in figure 2, in the absence of FSH > 80% of oocytes had not reached M II by 18

hours in the induced IVM system. However, by increasing doses of FSH, the percentage of

oocytes that reached M II increased and the majority of cilostamide-treated oocytes

progressed to M II after 22 hours of culture, despite delayed GVBD (Figure 4). This


169

observation was also seen in chapter 3, where in the absence of FSH, 40-50% of cilostamide-

treated bovine oocytes arrested at the M I stage after 24 hours of culture. However, the

addition of FSH, delayed GVBD, and the majority of cilostamide-treated oocytes to progress

to M II after 24 hours of culture. The exception were oocytes collected in medium

supplemented with cAMP modulators during the pre-IVM phase, where there was a delay in

GVBD regardless of the presence or absence of FSH. However, oocytes treated in this way

progressed to the M II stage after 28 hours of culture (chapter 3). This indicates that the

efficacy of including cAMP modulators in IVM media can be enhanced by modulating COC

cAMP levels following collection, thereby leading to further meiotic inhibition, which can be

overridden by the presence of FSH in culture. Together these leads to a form of induced

oocyte maturation in vitro.

In vivo the ovulatory gonadotrophin surge induces a secondary cascade of epidermal growth

factor (EGF)-like proteins that override cAMP mediated meiotic arrest to induce oocyte

maturation (Park et al. 2004). Thus the main aim of this study was to establish an induced

IVM system which would recapitulate some of the intercellular signals that occur in the

COCs in vivo and thereby improve oocyte quality in vitro. As previously discussed in bovine

(chapter 3), progression to MII with cAMP modulator supplementation during pre-IVM +

IVM required FSH, suggesting a form of induced maturation. Confirming this, FSH-induced

maturation of cAMP modulator-treated COCs was prevented by the EGF-receptor kinase

inhibitor AG1478, demonstrating autocrine ligand-induced oocyte maturation. Moreover,

previous studies have shown that the EGF can be produced by cumulus cells and is the most

potent stimulator of cumulus expansion and meiotic resumption. It has also been shown that

EGF-like peptides can mediate the actions of gonadotrophins in pre-ovulatory follicles. EGF-

like peptides therefore can play an intimate role in the paracrine signalling between metabolic


170

pathways of different cell types leading to oocyte maturation and follicular rupture (Park et

al. 2004; Ashkenazi et al. 2005).

The current study also shows that specifically the pre-IVM phase substantially improved

oocyte developmental competence (figure 8), as previously shown in chapter 3+4. The

presence of forskolin during pre-IVM has resulted in a greater percentage in hatching

blastocyst on day 6 compared to other treatments (figure 8).

The significance of the pre-IVM phase is further supported by in vivo levels of cAMP at the

initiation of maturation in mouse (figure 1) and pig (Mattioli et al. 1994). The pre-IVM phase

may mimic some aspects of in vivo maturation. This is the first study to report the concept of

induced IVM in mouse, by the inclusion of different cAMP modulators (adenylate cyclase

activator and non specific PDE inhibitor) at the collection phase (pre-IVM phase) and the

inclusion of lower doses of specific PDE 3 inhibitor (cilostamide) and high doses of FSH

during maturation (delayed IVM).

Recent reports clearly demonstrated that mouse standard IVM protocols are suboptimal.

(Eppig et al. 2009) demonstrated that current efficiency of mouse IVM, as measured by live

birth rate, is considerably lower than that of conventional IVF (21% vs 52%). Moreover,

(Vanhoutte et al. 2009b) demonstrated that fertilization (2 cell stage) and blastocyst rate are

significantly lower in in vitro matured oocytes compared to in vivo matured oocytes control.

In the mouse model, the application of a specific PDE3 inhibitor during collection (pre-IVM)

and biphasic IVM has been studied using Org 9935 or cilostamide (Nogueira et al. 2003b;

Vanhoutte et al. 2008; Vanhoutte et al. 2009b). The application of these specific PDE 3

inhibitors during IVM resulted in an improvement in developmental outcomes compared to


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standard IVM but significantly poorer outcomes compared to in vivo matured oocytes

(Nogueira et al. 2003b; Vanhoutte et al. 2008). The aim of the inclusion of PDE inhibitors in

during collection and selection prior IVM was to prevent the precocious drop in intra-oocyte

cAMP, which would negatively affect the oocyte’s developmental competence. In this study,

the inclusion of IBMX alone during pre-IVM phase, resulted in significantly lower hatching

blastocyst rate assessed on day 6 compared to the inclusion of forskolin and IBMX during

pre-IVM (figure 8). Moreover, in chapter 4, in bovine the inclusion of IBMX during pre-IVM

phase resulted in cleavage and blastocyst rates which were not improved and were similar to

control.

Although live human births have resulted from oocytes in vitro matured, (Barnes et al. 1995;

Cha and Chian 1998; Chian et al. 2000; Mikkelsen et al. 2000), current success rates are

significantly lower compared with in vivo matured oocytes collected after ovarian

hyperstimulation (Mikkelsen et al. 2000; Trounson et al. 2001). Current IVM systems also

result in reduced embryo quality with embryos displaying frequent cleavage retardation and

blockage to development (Trounson et al. 1998; Nogueira et al. 2000). Given the current

limitations of IVM, it is thus essential that systems be devised to improve the quality of COCs

in order to advance IVM proficiency in the human. Therefore, the results from this study may

provide insight into a novel induced IVM system for human immature oocytes.

Hence, human studies are necessary to examine the effect of induced IVM in human oocytes

and the effect on oocyte developmental competence. This potentially has great implications

for our understanding of human oocyte biology as well as for the development of IVM

technology and the improvement of clinical IVM success rates.


172

5.7 Figures

Figure 1. A comparison between in vivo cAMP concentrations in mouse cumulus-oocyte complexes (COCs) following follicular growth induced by equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG)-induced oocyte maturation (A), compared with cAMP concentrations in in vitro cultured COCs during a control pre-IVM phase (2 hrs which includes COC collection and selection) (B). Values are expressed as the mean concentration of cAMP per COCs � SEM of three replicates using 30 COCs per time point/replicate. Different superscripts are significantly different (One-Way ANOVA, P<0.05).

0 30 60 90 120 150 18002468

10

50

70

90

110

Post hCG time (min)

a a a a a

b

A.

fmol

cAM

P pe

r C

OC

0 30 60 90 120 150 1800.0

0.2

0.4

0.6

0.8

1.0

incubation time (min)

a

b

bb b

b

B.

fmol

cAM

P pe

r C

OC


173

Figure 2. Effect of increasing doses of FSH to induce meiotic maturation of COCs matured in the presence of the type 3 PDE inhibitor (cilostamide; 1µM (A) or 0.1 µM (B)). COCs were cultured in media + (1µM (A) or 0.1 µM (B) cilostamide) and increasing doses of FSH (0.01-200 mIU/ml) for 24 hours of IVM. Oocytes were the fixed and assessed for meiotic progression at 18 and 24 hrs. A mean number of 40 oocytes were used in each treatment group and time-point from four replicate experiments. The bars in figure A and B represents further control groups: COCs matured with 50 mIU/ml FSH alone (18 hrs of IVM), COCs matured with 1 µM (A) or 0.1 (B) µM. Different superscript are significantly different (One-Way ANOVA, P<0.05).

0

20

40

60

80

100

a

b b b b b b

c c

A. 1 �M cilostamide

% M

II

0

20

40

60

80

100

FSH (mIU/ml) 50 - 0.01 0.1 1 10 50 100 200cilostamide - + + + + + + + +IVM time (hours) 18 18 24 24 24 24 24 24 24

a

b b b b

d

c

e

aeB. 0.1 �M cilostamide

% M

II


174

Figure 3. Effect of cAMP modulators during pre-IVM & IVM on oocyte meiotic resumption. COCs exposed to different pre IVM phases following extended culture in the presence of the type 3 (cilostamide; 0.1 µM) PDE inhibitor. COCs were aspirated and selected in either collection medium supplemented with 0.1 µM cilostamide or collection medium supplemented with 50µM forskolin (FSK); & 50µM IBMX for 1 hr. COCs were then matured in presence of 100 mIU/ml FSH & 0.1 µM cilostamide for 18-26 hours. Oocytes were then fixed and assessed for meiotic progression at each time point. A mean number of 45 oocytes were used in each treatment group and time-point from four replicate experiments. Different superscript are significantly different (One-Way ANOVA, P<0.05).

0

20

40

60

80

100

0.1 µM CM 50 µM IBMX+ 50 µM FSK

FSH (mIU/ml) 50 100 100 100 100 100 100cilostamide (0.1µM) - + + + + + +

IVM time (hours) 18 24 18 20 22 24 26

Pre-

IVM

IVM

a a

b

c

a aa

% M

II


175

0 0.5 1 2 4 6 8 10 12 140

20

40

60

80

100

in vivo matured control

induced IVMstandard IVM

Time (hrs)

a a a a a a

bb

b

c cc c c c c

cd

% G

VB

D

Figure 4. Meiotic maturation of COCs matured either in vivo, by induced IVM or by standard IVM for 14 hours. Oocytes were then assessed for meiotic progression and classified as GVBD stage. Standard IVM = control pre-IVM and control IVM with 50 mIU/ml FSH. Induced IVM = pre-IVM with FSK+IBMX and IVM with cilostamide + 100 mIU/ml FSH. In vivo matured control = COCs collected from ovary 0-14h after hCG administration. Oocytes were then assessed for meiotic progression at each time point. A mean number of 30 oocytes were used in each treatment group and time-point from three replicate experiments. Different superscript are significantly different (Two-Way ANOVA, P<0.05).


176

Figure 5. Meiotic maturation of COCs matured either in induced IVM or standard IVM following 18 and 22 of culture. Oocytes were then assessed for meiotic progression and classified as MII stage. Standard IVM = control pre-IVM and control IVM with 50 mIU/ml FSH. Induced IVM = pre-IVM with FSK+IBMX and IVM with cilostamide + 100 mIU/ml FSH. A mean number of 30 oocytes were used in each treatment group and time-point from 3 replicate experiments. Means at the same time point or between treatments and marked with an asterisk are significantly different (Two-Way ANOVA, P<0.05).

0

20

40

60

80

100standard IVMinduced IVM

18 hrs IVM 22 hrs IVM

*

% M

II


177

Figure 6. The effect of maturing oocytes in either standard or induced IVM either for 18 or 22 hours of IVM on oocyte developmental capacity. Oocyte developmental capacity was assessed after in vitro fertilization and embryo development by cleavage rate (day 2) and blastocyst rate (day 5). Standard IVM = control pre-IVM; control IVM with 50 mIU/ml FSH. Induced IVM = pre-IVM with FSK+IBMX, IVM with cilostamide + 100 mIU/ml FSH. A mean number of 45 oocytes were used in each treatment group and time-point from three replicate experiments. Different superscript are significantly different between treatments (One-Way ANOVA, P<0.05).

0

20

40

60

80

100 C

leav

age

(%)

a a

b

c

standard IVMinduced IVM

0

20

40

60

80

100

a a

b

c

18 hrs IVM 22 hrs IVM

Bla

stoc

yst /

2 C

ell (

%)

A.

B.


178

Figure 7. The effect of maturing oocytes in either standard or induced IVM either for 18 or 22 hours of IVM on blastocyst quality. COCs were matured using either induced and standard IVM for 18 or 22 hours, then fertilized and embryos cultured for 5 days and blastocyst quality was quantified by total cell counts and cell allocation to trophoectoderm (TE) or inner cell mass (ICM). Standard IVM = control pre-IVM, control IVM with 0.05 IU/ml FSH. Induced IVM = pre-IVM with FSK+IBMX, IVM with cilostamide + 0.1 IU/ml FSH. A mean number of 20 blastocysts were used in each treatment group and time-point from three replicate experiments. Means at the same time point or between treatments and marked with an asterisk are significantly different (Two-Way ANOVA, P<0.05).

0

20

40

60

80Induced IVMStandard IVM

Total cells TE ICM

Cel

l No. 0

10

20

30

% IC

M /

tota

l

18 hrs IVM

*

*

*

0

20

40

60

80

Total cells TE ICM

Cel

l No. 0

10

20

30

40 *

% IC

M /

tota

l

22 hrs IVM

*

*


179

Figure 8.

The effect of using different cAMP modulators during the pre-IVM & IVM phases onmouse oocyte developmental capacity. COCs were subjected to Pre-IVM for 1 hour ineither control medium or medium supplemented with 50 µM IBMX +/- 50 µMforskolin (FSK). COCs were then matured either without cilostamide for 18 (0.05IU/ml FSH) or for 22 hours in the presence of cilostamide (0.1 μM) and FSH (0.1IU/ml). Subsequently, oocyte developmental capacity was assessed after in vitrofertilization and embryo development by cleavage rate, blastocyst rate and hatchingblastocyst rate on day 6.

0

20

40

60

80

100

cleavage blastocyst (day 6)

hatching blastocyst (day 6)

Pre-IVM IVM IVM Time- - 18

- +CM 22+IBMX +CM 22

+IBMX+FSK +CM 22

% o

ocyt

e de

velo

pmen

tal c

ompe

tenc

e

* * * *

*

**


180

Figure 9. Developmental competence of oocytes matured either in vivo, by induced IVM or by standard IVM. Following IVM, COCs were fertilized and embryos cultured until day 5 (A) and then blastocyst quality was quantified by total cell counts and cell allocation to trophoectoderm (TE) or inner cell mass (ICM) (B). Standard IVM = control pre-IVM, control IVM with 50 mIU/ml FSH and fertilization at 18 h. Induced IVM = pre-IVM with FSK+IBMX, IVM with cilostamide + 100 mIU/ml FSH and fertilized at 22 h. In vivo matured control = COCs collected from oviducts 14h after hCG administration. Mature COC (42-210/treatment/replicate, 4 replicates) were fertilised and embryos cultured until day 5 (A). A mean number of 15-20 blastocysts were used in each treatment group and time-point from four replicate experiments (B). Means between treatments and marked with an asterisk are significantly different (One2-Way ANOVA, P<0.05).

0

20

40

60

80

100 induced IVMstandard IVMin vivo matured control

cleavage blastocyst(day 5)

hatching blastocyst (day 5)

* *

*

A.

% o

ocyt

e de

velo

pmen

tal c

ompe

tenc

e

0

20

40

60

80

Total cells TE ICM

Cel

l No. 0

10

20

30

40

% IC

M /

tota

l

*

*

*

B.


181

Figure 10. The effect of induced vs. standard IVM on pregnancy outcomes. Day 3.5 blastocysts developed from COCs matured in vivo or by induced or standard IVM were transferred to pseudo-pregnant recipients and outcomes analysed on day 18.5 of pregnancy. Implantation rate = implantation sites / embryos transferred. Fetal yield = day 18.5 fetuses / embryo transferred. Standard IVM = control pre-IVM, control IVM with 50 mIU/ml FSH and fertilization at 18 h. Induced IVM = pre-IVM with FSK+IBMX, IVM with cilostamide + 100 mIU/ml FSH and fertilized at 22 h. In vivo matured control = COCs collected from oviducts 14h after hCG administration. Six blastocysts from one treatment were randomly assigned to each uterine horn. A total of 192 embryos across the three treatments were transferred to 16 recipients. Pregnancy outcomes were assessed following euthanasia 3 days prior to term, on day 18.5 of pregnancy. Means with no common superscripts are significantly different (Chi squared analysis, P<0.05).

0

20

40

60induced IVMstandard IVM

aa

in vivo matured control

bIm

plan

tatio

n R

ate

(%)

0

10

20

30

a,ba

b

Feta

l Yie

ld (%

)

0.0

0.2

0.4

0.6

0.8

1.0 a a

b

Feta

l Wei

ght (

g)


182

Figure 11. The effect of induced vs. standard IVM on fetal parameters. Day 3.5 blastocysts developed from COCs matured in vivo or by induced or standard IVM were transferred to pseudo-pregnant recipients and outcomes analysed on day 18.5 of pregnancy. Standard IVM = control pre-IVM, control IVM with 50 mIU/ml FSH and fertilization at 18 h. Induced IVM = pre-IVM with forskolin+IBMX, IVM with cilostamide + 100 mIU/ml FSH and fertilized at 22 h. In vivo matured control = COCs collected from oviducts 14h after hCG administration. Six blastocysts from one treatment were randomly assigned to each uterine horn. A total of 192 embryos across the three treatments were transferred to 16 recipients. Pregnancy outcomes were assessed following euthanasia 3 days prior to term, on day 18.5 of pregnancy.. Means between treatments and marked with an asterisk are significantly different (One-Way ANOVA, P<0.05).

0

5

10

15

20

25 induced IVMstandard IVMin vivo matured control

*

Cro

wn to

Rum

p Le

ngth

(mm

)

0

2

4

6

8

*

Feta

l to

plac

etal

wei

ght r

atio


183

5.8 References Anderiesz C, Fong CY, Bongso A, Trounson AO (2000) Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine. Hum Reprod 15, 379-88. Ashkenazi H, Cao X, Motola S, Popliker M, Conti M, Tsafriri A (2005) Epidermal growth factor family members: endogenous mediators of the ovulatory response. Endocrinology 146, 77-84. Barnes FL, Crombie A, Gardner DK, Kausche A, Lacham-Kaplan O, Suikkari AM, Tiglias J, Wood C, Trounson AO (1995) Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum Reprod 10, 3243-7. Cha KY, Chian RC (1998) Maturation in vitro of immature human oocytes for clinical use. Hum Reprod Update. 4, 103-20. Chian RC, Buckett WM, Tulandi T, Tan SL (2000) Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 15, 165-70. Chian RC, Lim JH, Tan SL (2004) State of the art in in-vitro oocyte maturation. Curr Opin Obstet Gynecol 16, 211-9. Conti M, Andersen CB, Richard FJ, Shitsukawa K, Tsafriri A (1998) Role of cyclic nucleotide phosphodiesterases in resumption of meiosis. Mol Cell Endocrinol 145, 9-14. Coticchio G, Rossi G, Borini A, Grondahl C, Macchiarelli G, Flamigni C, Fleming S, Cecconi S (2004) Mouse oocyte meiotic resumption and polar body extrusion in vitro are differentially influenced by FSH, epidermal growth factor and meiosis-activating sterol. Hum Reprod 19, 2913-8. Downs S, Schroeder A, Eppig J (1986) Developmental capacity of mouse oocytes following maintenance of meiotic arrest in vitro. Gamete Research 15, 305-316. Eppig JJ, O'Brien MJ, Wigglesworth K, Nicholson A, Zhang W, King BA (2009) Effect of in vitro maturation of mouse oocytes on the health and lifespan of adult offspring. Hum Reprod 24, 922-8. Eppig JJ, Schultz RM, O'Brien M, Chesnel F (1994) Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes. Dev Biol 164, 1-9. Faerge I, Mayes M, Hyttel P, Sirard MA (2001) Nuclear ultrastructure in bovine oocytes after inhibition of meiosis by chemical and biological inhibitors. Mol Reprod Dev 59, 459-67. Gardner DK, Lane MW, Lane M (2000) EDTA stimulates cleavage stage bovine embryo development in culture but inhibits blastocyst development and differentiation. Mol Reprod Dev 57, 256-61.


184

Guixue Z, Luciano AM, Coenen K, Gandolfi F, Sirard MA (2001) The influence of camp before or during bovine oocyte maturation on embryonic developmental competence. Theriogenology 55, 1733-43. Jensen JT, Schwinof KM, Zelinski-Wooten MB, Conti M, DePaolo LV, Stouffer RL (2002) Phosphodiesterase 3 inhibitors selectively block the spontaneous resumption of meiosis by macaque oocytes in vitro. Hum Reprod 17, 2079-84. Jurema MW, Nogueira D (2006) In vitro maturation of human oocytes for assisted reproduction. Fertil Steril 86, 1277-91. Lane M, Gardner DK (1997) Differential regulation of mouse embryo development and viability by amino acids. J Reprod Fertil 109, 153-64. Le Beux G, Richard FJ, Sirard MA (2003) Effect of cycloheximide, 6-DMAP, roscovitine and butyrolactone I on resumption of meiosis in porcine oocytes. Theriogenology 60, 1049-58. Mattioli M, Galeati G, Barboni B, Seren E (1994) Concentration of Cyclic Amp During the Maturation of Pig Oocytes in vivo and in vitro. Journal of Reproduction & Fertility 100, 403-409. Mayes MA, Sirard MA (2002) Effect of type 3 and type 4 phosphodiesterase inhibitors on the maintenance of bovine oocytes in meiotic arrest. Biol Reprod 66, 180-4. Mikkelsen AL, Smith S, Lindenberg S (2000) Possible factors affecting the development of oocytes in in-vitro maturation. Hum Reprod 15 Suppl 5, 11-7. Nogueira D, Albano C, Adriaenssens T, Cortvrindt R, Bourgain C, Devroey P, Smitz J (2003a) Human oocytes reversibly arrested in prophase I by phosphodiesterase type 3 inhibitor in vitro. Biol Reprod 69, 1042-52. Nogueira D, Cortvrindt R, De Matos DG, Vanhoutte L, Smitz J (2003b) Effect of Phosphodiesterase Type 3 Inhibitor on Developmental Competence of Immature Mouse Oocytes In vitro. Biol Reprod 20, 20. Nogueira D, Ron-El R, Friedler S, Schachter M, Raziel A, Cortvrindt R, Smitz J (2006) Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development. Biol Reprod 74, 177-84. Nogueira D, Staessen C, Van de Velde H, Van Steirteghem A (2000) Nuclear status and cytogenetics of embryos derived from in vitro-matured oocytes. Fertil Steril 74, 295-8. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M (2004) EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 303, 682-4. Reddoch RB, Pelletier RM, Barbe GJ, Armstrong DT (1986) Lack of ovarian responsiveness to gonadotropic hormones in infantile rats sterilized with busulfan. Endocrinology 119, 879-86.


185

Richard FJ (2007) Regulation of meiotic maturation. J Anim Sci 85, E4-6. Shimada M, Nishibori M, Isobe N, Kawano N, Terada T (2003) Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes. Biol Reprod 68, 1142-9. Shu YM, Zeng HT, Ren Z, Zhuang GL, Liang XY, Shen HW, Yao SZ, Ke PQ, Wang NN (2008) Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes. Hum Reprod 23, 504-13. Sirard MA (2001) Resumption of meiosis: mechanism involved in meiotic progression and its relation with developmental competence. Theriogenology 55, 1241-54. Thomas RE, Armstrong DT, Gilchrist RB (2002) Differential effects of specific phosphodiesterase isoenzyme inhibitors on bovine oocyte meiotic maturation. Dev Biol 244, 215-25. Thomas RE, Thompson JG, Armstrong DT, Gilchrist RB (2004) Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity. Biol Reprod 71, 1142-9. Trounson A, Anderiesz C, Jones G (2001) Maturation of human oocytes in vitro and their developmental competence. Reproduction 121, 51-75. Trounson A, Anderiesz C, Jones GM, Kausche A, Lolatgis N, Wood C (1998) Oocyte maturation. Hum Reprod 13 Suppl 3, 52-62; discussion 71-5. Tsafriri A, Chun SY, Zhang R, Hsueh AJ, Conti M (1996) Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors. Dev Biol 178, 393-402. Vanhoutte L, De Sutter P, Nogueira D, Gerris J, Dhont M, Van der Elst J (2007) Nuclear and cytoplasmic maturation of in vitro matured human oocytes after temporary nuclear arrest by phosphodiesterase 3-inhibitor. Hum Reprod 22, 1239-46. Vanhoutte L, Nogueira D, De Sutter P (2009a) Prematuration of human denuded oocytes in a three-dimensional co-culture system: effects on meiosis progression and developmental competence. Hum Reprod 24, 658-69. Vanhoutte L, Nogueira D, Dumortier F, De Sutter P (2009b) Assessment of a new in vitro maturation system for mouse and human cumulus-enclosed oocytes: three-dimensional prematuration culture in the presence of a phosphodiesterase 3-inhibitor. Hum Reprod 24, 1946-59. Vanhoutte L, Nogueira D, Gerris J, Dhont M, De Sutter P (2008) Effect of temporary nuclear arrest by phosphodiesterase 3-inhibitor on morphological and functional aspects of in vitro matured mouse oocytes. Mol Reprod Dev 75, 1021-30. Wiersma A, Hirsch B, Tsafriri A, Hanssen RG, Van de Kant M, Kloosterboer HJ, Conti M, Hsueh AJ (1998) Phosphodiesterase 3 inhibitors suppress oocyte maturation and consequent pregnancy without affecting ovulation and cyclicity in rodents. J Clin Invest 102, 532-7.


186

Wu GM, Sun QY, Mao J, Lai L, McCauley TC, Park KW, Prather RS, Didion BA, Day BN (2002) High developmental competence of pig oocytes after meiotic inhibition with a specific m-phase promoting factor kinase inhibitor, butyrolactone I. Biol Reprod 67, 170-7.

CHAPTER 6

FINAL DISCUSSION

Chapter 6. Final discussion

188

Oocyte in vitro maturation (IVM) is a methodology that has been applied for a range of

applications including human infertility treatment, embryo production for livestock artificial

breeding programs, nuclear transfer (cloning), stem cell and transgenic animal production

technologies. It is also an important research tool in reproductive and developmental biology.

For clinical applications, IVM has been applied in assisted reproductive technologies (ART)

as an alternative to conventional IVF, as it does not require expensive and potentially harmful

hormonal ovarian hyperstimulation. However, the efficiency of IVM, as measured by embryo

yield or live births, is considerably lower than that of conventional IVF (Child et al. 2002;

Rizos et al. 2002; Eppig et al. 2009). This poor efficiency has restricted widespread use of

IVM, particularly in the treatment of human infertility. In particular, all IVM studies

published so far using many different species indicate that developmental outcomes for in

vitro matured oocytes remain suboptimal when compared to oocytes matured in vivo (Table

1). In human clinics, implantation rates from IVM cycles are reduced by half compared to

IVF cycles (Child et al. 2002), and IVM cycles present an increased incidence of early

pregnancy loss (Buckett et al. 2008).

Table 1: A comparison between in vivo vs in vitro matured oocytes and outcomes among different species Mode of oocyte maturation Species Outcomes In vitro (IVM) In vivo References Human Implantations 9.5% 17% (Child et al. 2002) Ovine Blastocysts 35% 74% (Thompson et al. 1995) Bovine Blastocysts 39% 58% (Rizos et al. 2002) Murine Live births 21% 52% (Eppig et al. 2009)


189

For human IVM to become an accepted routine technology, the efficiency rates in embryo

yield and viability require improvement by translating basic research generated in animal

models to human IVM. Animal studies have shown that oocyte developmental competence is

the rate-limiting factor in the production of embryos using in vitro techniques (Thompson et

al. 1995; Rizos et al. 2002; Eppig et al. 2009). Studies designed to optimize the culture

conditions for oocyte maturation during IVM can significantly improve developmental

outcomes (Sirard et al. 2006; Gilchrist and Thompson 2007; Banwell and Thompson 2008).

Some recent advances that improve the efficiency of IVM include the inclusion of FF-MAS

(Marin Bivens et al. 2004b; Smitz et al. 2007), oocyte-secreted factors such as GDF-9 and

BMP-15 (Hussein et al. 2006; Yeo et al. 2008) and cAMP modulators (Nogueira et al. 2003b;

Luciano et al. 2004; Thomas et al. 2004b; Shu et al. 2008). Although these approaches have

led to incremental improvements, there remains a disparity in competence with in vivo

matured oocytes. Therefore, further studies are mandatory to bring IVM success rates to

levels achieved following maturation in vivo.

The crucial role of the second messenger cAMP in the maturation of mammalian oocytes has

long been recognized, however its exact role in the regulation of oocyte maturation remains

unclear. As one of the primary means for determining intracellular cAMP levels, original

experiments, beginning in the late 1980s, utilized non-specific phosphodiesterase inhibitors

(such as IBMX and theophylline) to modulate intracellular cAMP levels. These initial studies

suggested PDE involvement in the regulation of oocyte maturation; however it was the lack

of specificity toward specific PDE subtypes, in addition to the toxic side effects of these

inhibitors, which restricted their usefulness in IVM.


190

In 1995, a landmark study demonstrated the differential localization of phosphodiesterase

subtypes within the two compartments of the rodent follicle - the germ cell (oocyte; PDE3)

and the somatic cell (cumulus and mural granulosa cells; PDE4) (Tsafriri et al. 1996).

Subsequently, subtype-specific PDE inhibitors were used against the type 3 and 4 PDE

subtypes of the rodent ovarian follicle – the results of which were compiled in two key

publications (Tsafriri et al. 1996; Wiersma et al. 1998). However, within the bovine ovarian

follicle, there is a recently discovered alternate distribution of PDE families (Sasseville et al.

2009), in which the PDE’s expressed and functional within the oocyte is the PDE3 and

PDE8A and PDE8B are highly expressed in the cumulus cells with low levels of PDE4D.

Results demonstrated that bovine cumulus cell cAMP-PDE activity is predominantly

insensitive to inhibition by IBMX, indicating PDE8 activity in cumulus cells and a minor role

for PDE4. Furthermore, PDE8A, PDE8B and PDE4D proteins were found in bovine mural

granulosa cells and COCs (Sasseville et al. 2009).

Given this new perspective on the distribution of PDE subtypes, the first aim of this thesis

was to determine how inhibition of PDE8 degradation during IVM effects bovine cAMP

levels, oocyte meiotic resumption and hence oocyte developmental competence (chapter 2).

The inhibition of PDE8 degradation using dipyridamole resulted in a dose-dependent increase

in cAMP levels within the COC and delayed spontaneous meiotic resumption. However,

dipyridamole supplementation during IVM failed to enhance oocyte developmental potential.

These results are the first to demonstrate the functional presence of PDE8 in the bovine

ovarian follicle and explain why IBMX is relatively inefficient in maintaining bovine oocyte

meiotic arrest.


191

Current routine application of IVM uses a system of ‘spontaneous IVM’. Using this approach,

COCs are aspirated and rapidly removed from the follicular environment and hence oocyte

mature spontaneously in vitro and undergoing precociously meiotic resumption in the absence

of the important endocrine and paracrine signaling that synchronize oocyte maturation in vivo

(Park et al. 2004; Ashkenazi et al. 2005; Shimada et al. 2006; Norris et al. 2009; Vaccari et

al. 2009). This mode of maturation lead to asynchrony between oocyte nuclear and

cytoplasmic maturation and hence will compromised oocyte developmental competence (Fair

et al. 1995). Moreover maturing COCs in current IVM system, causes an early breakdown of

oocyte CC gap junctions (Thomas et al. 2004a), leading to loss of essential cumulus cells

metabolites, such as nucleotides and nutrients (Gilchrist and Thompson 2007).

Understanding the biological aspects of oocyte maturation and subsequently attempting to

mimic the in vivo environment and adapt it in vitro, is an approach successfully used in the

development of improved embryo culture conditions which could be applied to IVM. Hence,

the major aim of this thesis was to understand cAMP levels during oocyte maturation in vivo,

and trying to mimic it in vitro by establishing a novel IVM system using bovine and murine

immature oocytes as the two most widely studied models of mammalian oocyte biology. The

experiments in this thesis described a new approach to IVM technique that substantially

improve oocyte developmental competence (chapter 3-5) including pregnancy outcomes

(chapter 5). Induced IVM is characterized by firstly, a pre-IVM phase that substantially

increased COC cAMP levels (chapter 3-5). This substantial spike in cAMP resembles that

which occurs during in vivo oocyte maturation (chapter 5), and notably contrasts the

immediate drop that occurs during standard IVM (chapter 3-5). Secondly, the induced IVM is

characterized by an extended delayed IVM phase containing sufficient FSH override


192

cilostamide and induce meiotic maturation in the presence of cilostamide (chapter 3-5)

(Figure 1).

Pre-IVM Extended-IVM(forskolin+IBMX) (cilostamide+FSH)

-Substantial increase in COC cAMP(mimic in vivo cAMP spike)

-Increase oocyte-cumulus cell gap junctionalcommunication

- cilostamide retards the rapid decline in intra-oocyte cAMP

-FSH induces oocyte maturation

-Extend oocyte-cumulus cell gap junctional communication

-Delay spontaneous meiotic resumption (extend maturation interval)

Delay maturation

Preserve oocyte cAMP

Induce maturation

IVF

Induced-IVM

The results in chapter 3+4 demonstrated that induced IVM improves bovine blastocyst rate to

>60% in a completely serum-free system which reflects a major increase in efficiency on

current methodologies (Figure 2). In chapter 3, we choose two important controls: standard

clinical IVM control (COC collection and selection in control medium) and a standard

research IVM control (collection and selection in follicular fluid). In the cattle and human

collection conditions are comparable, as in both cases COCs are always removed from

follicular compartments and placed in simple media. This causes a rapid depletion in COC

Figure 1: Schematic demonstrates the induced IVM system for bovine and murine oocytes.


193

cAMP resulting in precocious meiotic resumption. In contrast, in cattle IVM research, COCs

are collected and processed in pure follicular fluid, which maintains COCs cAMP levels but

does not enhance it, which resulted in blastocyst rates of 40 % (Hussein et al. 2006). The

results in this thesis have the potential to increase the outcomes in cattle artificial breeding

programs where IVM is commonly applied and used. As previously reported, in 2005

approximately 133,000 viable cattle offspring were produced by using IVM (Thibier 2006).

IVM

Conventional IVF

IVF

Pre-IVM Extended-IVM(Forskolin+IBMX) (cilostamide+FSH)

IVF

IVF

IVFOvarian hyperstimulation

Standard IVM

Induced IVM

References

58%

27%

69%

Blastocyst Rate

Rizos et al. 2002

Current (chapter 3)

Current (chapter 3)

Figure 2: Schematic demonstrates a comparison in blastocyst outcome between bovine oocytes matured either

in vivo, by standard IVM or induced IVM.

In contrast to cattle, blastocyst rates is less significant as an indicator of developmental

competence in the mouse model, where rates are varied depending on the mouse strain and

the oocyte/embryo culture condition such as serum. As oocytes from hybrids of inbred strains

(F1 mice) are more robust, in the current study we used relatively sensitive mouse


194

oocyte/embryo conditions by choosing an inbred mouse strain (129/Sv) and serum-free

culture conditions throughout. Consistent with other serum-free standard IVM systems (Preis

et al. 2007), this yielded approximately 50% control blastocyst and poor-transfer outcomes.

Despite these adverse conditions, Induced IVM improved fertilization and blastocyst rates

compared to standard IVM, and substantially increased implantation rate and fetal yield.

Developmental outcomes were improved to such an extent using induced IVM that they

matched outcomes from in vivo matured oocytes (figure 3).

22%IVF/ET

Conventional IVF

8%IVF/ET

26%IVF/ETExtended-IVMPre-IVM

Induced IVM

IVMStandard IVM

Ovarian HyperstimulationOvarian Hyperstimulation

Figure 3: Schematic demonstrates fetal yield in mouse oocytes matured either in vivo, by standard IVM

or induced IVM (chapter 5).


195

The first key factor in the induced IVM system is the 1-2 hours pre-IVM phase containing

cAMP modulators (forskolin and IBMX) that generates a substantial (>100 fold) increase in

COCs cAMP levels within minutes of COCs retrieval (chapter 3+4). In bovine, collecting

COCs in pure follicular fluid protects the rapid loss of cAMP (chapter 3+4) but does increase

COC cAMP levels. Collecting bovine COCs in the presence of adenylate cyclase activator

has previously reported (Aktas et al. 1995a; Luciano et al. 1999; Guixue et al. 2001), in

parallel with the current study the adenylate cyclase activator presence during pre-IVM phase

which targets the cumulus cell compartment and mandatory to generate the rapid increase in

cAMP (chapter 3+4) that resembles the in vivo increase in COC cAMP levels (chapter5) and

to achieve high embryo outcomes (chapter 3-5). The presence of forskolin and IBMX during

pre-IVM phase had significant effects on COC functions and developmental programming of

the oocyte. Consistent with previously studies, these modulators increased oocyte-CC gap

junctional communication, elevating intraoocyte cAMP levels at the end of IVM and hence

delaying the completion of meiotic maturation (Thomas et al. 2004a; Thomas et al. 2004b).

The exploitation of subtype-specific PDE inhibitors is a powerful principle to study the

oocyte and surrounding cumulus cells in separation, and to investigate the functions of cAMP

in the two follicular compartments (Gilchrist and Thompson 2007). In the induced IVM

system, the non-specific PDE inhibitor was used during the pre-IVM phase to inhibit both CC

and oocyte PDE, whereas in the IVM phase the use of the PDE3-specific inhibitor

cilostamide is used to target and regulate the oocyte PDE only. The application of using

PDE3 inhibitor in IVM have been studied previously, by temporarily blocking spontaneous

meiotic resumption using biphasic IVM (Nogueira et al. 2003a; Nogueira et al. 2003c;

Nogueira et al. 2006; Shu et al. 2008; Vanhoutte et al. 2009a; Vanhoutte et al. 2009b). In this

study, oocytes were continuously exposed throughout IVM to moderate concentrations of


196

PDE3 inhibitor cilostamide. In addition to cilostamide in the culture, a relatively high dose of

FSH (100 mIU/ml) was used to override the meiotic inhibiting effects of cilostamide. As

shown from the experiments in this thesis, the IVM phase in the induced IVM required a

balance between the low doses of cilostamide and high doses of FSH. This feature shares

some similarities with the well established induced rodent model to study oocyte meiosis. In

rodents, oocytes are stimulated to mature by FSH or EGF to override a meiotic arresting

agent such as hypoxanthine (Downs et al. 1988). However these experiments were focusing

in studying meiosis but rarely been used to generate embryos.

In vivo, the ovulatory gonadotrophin surge overrides the meiotic-inhibiting effects of the

preovulatory follicle to induce oocyte maturation. Recent studies have demonstrated that

gonadotrophins cause a secondary cascade of follicular EGF-like peptides which act on

cumulus cells EGF receptor to induce maturation requiring ERK1/2-dependent mechanism

(Park et al. 2004; Ashkenazi et al. 2005; Shimada et al. 2006; Downs and Chen 2008; Fan et

al. 2009). Consequently inhibition of signalling through the EGF receptor using AG1478

blocks in vivo oocyte maturation (Park et al. 2004; Ashkenazi et al. 2005). In the current

study, FSH induced maturation of treated COCs was blocked by the EGF receptor kinase

inhibitor AG1478. Hence, in the current system, FSH is inducing meiotic resumption by a

mechanism that requires the EGF receptor, which is likely to involve FSH/cAMP induced

expression of EGF-like peptides by CC (Downs and Chen 2008).

Induced IVM captures some of the characteristics of oocyte maturation in vivo and that

substantially improves embryo yield, fetal survival and pregnancy outcomes, compared to

standard IVM. The novel system will provide new options for a wide range of reproductive


197

biotechnologies including livestock breeding. Over the past 25 years, artificial reproductive

technologies (ART) have come to the forefront of reproductive research in livestock industry.

Although many techniques are now available that promise to enhance reproductive

performance, additional refinement and optimization allowing for subsequent embryo

development and well being of the offspring are needed for these technologies to influence

the dairy industry.

Induced IVM has also potential for conservation applications of rare and endangered species.

Oocyte collection and maturation are potentially important means of preserving genetic

resources for these species. Applying induced IVM in conservation biotechnology field may

add significantly to the ability to rescue genetic material from valuable or endangered species.

The outcomes of the research presented in this thesis have made a substantial contribution to

our understanding of the mechanisms regulating oocyte maturation and the acquisition of

developmental competence. Results from the present study demonstrate a means of

improving oocyte maturation during IVM where may prove to be a beneficial component of

oocyte IVM culture systems, increasing the quality and quantity of in vitro embryo

production.


198

Future directions

So far the data presented in this thesis demonstrated the outcomes of induced IVM in research

setting. To apply induced IVM in cattle industry, the pre-IVM and extended IVM phase will

be applied on field. In live animals, the most commonly employed technique for harvesting

oocytes is TVOR, which is dependent mostly in PBS as an aspiration medium. Instead, the

application of induced IVM involves the pre-IVM phase including the cAMP modulators

which will be employed as a flushing medium during aspiration procedure prior to oocyte

culture. More studies are required to examine the effect also of other factors such as hormonal

stimulation prior oocyte retrieval, time interval between successive aspiration and the

combination of needle gauge and vacuum pressure applied. Additionally, in the laboratory

setting, further demonstration should be required and longer hours is required to extend the

culture (IVM) time compared to the routine applied technique. This will require further hours

of work and experienced personnel who understand the need of extending the culture time

frame.

In cattle, the experimental end points in this thesis have been embryo blastocyst production

and quality. It will be of interest to examine the effect of induced IVM on fetal outcomes after

embryo transfer, and verify if this modified system improves live birth rates in cattle. These

future experiments will provide us with high quality safety and efficacy data by using this

system.

Moreover, a major hurdle to the application of cattle IVP embryos with standard IVM is the

poor freeze-thaw survival of embryos, compared to embryos generated in vivo. An essential

measure of using an induced IVM will be the freeze-thaw embryo survival. This is considered


199

another parameter in measuring oocyte developmental competence and examines the benefit

of the induced IVM system.

In mouse, the experimental endpoints in this thesis have been fetal survival and fetal/placental

morphometrics which were assessed on day 18 of pregnancy. Future directions should allow

development these foetuses to term and then examine post-natal growth trajectories (weight

gain) of offspring. Moreover, reproductive potential of induced IVM offspring should be

assessed by allowing natural mating and assessing 2nd generation litter sizes and growth

trajectories. This would establish if the Induced IVM system has any long-term

consequences, perhaps mediated by epigenetic effects. There is a lack of information

regarding the epigenetic state of oocytes following IVM. As a part of future work, using day

18 fetal and placental tissues from chapter 5 will give a better idea and safety data for the

application of the induced IVM by examining methylation status of genes encoding, IGF-1,

IGF-II, IGFR.

In this thesis the primary endpoints were embryo production, quality and pre-natal fetal

survival. Other studies should focus on gene arrays of cumulus cells, metabolic profiles and

energy substrate usage of cumulus oocyte complexes matured by induced IVM. Moreover,

investigate the molecular mechanisms in cumulus cells and oocytes mediating induced IVM

such as the EGF-like peptides, MAPK kinase signalling and MPF pathways will be desirable

to understand the molecular mechanisms for oocytes mature by induced IVM.

Finally, the anticipated outcome of this work is to validate the new concept of induced IVM

using human oocytes. Data based on human studies are mostly insufficient for final

conclusion to be drawn neither can they provide that current IVM technology can be applied


200

as a routine. Oocytes derived from small follicles without mild ovarian stimulation would

need a proper culture system which mimics the in vivo situation. Therefore, applying cultures

that improve cytoplasmic maturation processes will benefit IVM technology in human ART.

Currently, in clinical practice, patients undergoing IVM mostly receive minimal human

chorionic gonadotrophin (hCG) stimulation 36 hours prior to ovum pick-up or in combination

of FSH and hCG which leads to maturation initiation cascade before oocyte culture. Thus, to

devise induced IVM in clinical setting. One hurdle that must be overcome before oocyte

culture (IVM) is the technical aspects of aspirating and handling immature oocytes during the

pre-IVM phase. Specifically if the patients receive no hormonal stimulation, aspiration of

these immature oocytes from unstimulated ovaries will be more technically demanding,

requiring adjustments in the aspiration needles, the pressure used, skills and longer time

required to navigate an ovary with small follicles.

Additionally, cAMP modulators concentrations in pre-IVM and IVM need to be tested.

Moreover, meiotic maturation after culture (IVM) will be assessed to examine the time in

which these matured oocytes are ready for insemination. This delay in the maturation interval

compared to the control (standard IVM) requires additional hours of work and experienced

personnel.

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CHAPTER 7

APPENDICES

221

Appendix 1: Additional Experiments

1.1 The effect of cAMP modulator inclusion during pre-IVM phase on bovine COC

cAMP.

The aim of this experiment was to determine the effect of different cAMP modulators

inclusion during the pre-IVM phase on cAMP levels in COCs. COCs were aspirated and

selected in collection medium supplemented with/without forskolin (100 µM), with/without

IBMX (500 µM) and with/without dipyridamole (50 µM) for 2 hrs. After pre-IVM, 6-10

COCs from each treatment were collected and snap frozen for cAMP assay.

As shown in Fig A1, cAMP levels were significantly higher when COCs were collected in

presence of forskolin, IBMX compared to their absence in the pre-IVM phase and compared

to the presence of dipyridamole alone (P<0.05). However, in combination of forskolin and

IBMX in pre-IVM phase, cAMP levels were significantly higher (20 fold increase) over the

levels observed in absence of IBMX and presence of forskolin alone (P<0.05). Moreover, the

presence of dipyridamole, didn`t show an additional increase in cAMP levels in combination

of forskolin and IBMX.

1.2 The effect of cAMP modulator inclusion during pre-IVM phase on % germinal

vesicle breakdown of bovine oocyte matured for 9 hours in IVM.

Preliminary experiments were performed to examine the effect of different cAMP modulators

during pre-IVM (collection and selection) phase on % GVBD of bovine oocytes matured for

9 hours in IVM. During the pre-IVM phase, COCs were collected and selected either in

follicular fluid, collection medium, collection medium supplemented with IBMX (500 µM),

collection medium supplemented with forskolin (100 µM) or collection medium

supplemented with (forskolin; 100µM & IBMX; 500µM) for 2 hrs. After pre-IVM COCs

were matured with FSH (100 mIU/ml). After 9 hours oocytes were then fixed and assessed

for meiotic progression and classified as GVBD (resumption of meiosis).

222

As shown in Fig A2, there were no obvious significant differences in % GVBD among the

different pre-IVM treatments.

1.3 The effect of cAMP modulator inclusion in pre-IVM phase on germinal vesicle (%) of

bovine oocytes following 7 hours of IVM

The aim of this experiment was to examine the effect of cAMP modulators during pre-IVM

(collection and selection) phase on % GV of bovine oocytes matured for 7 hours in IVM.

COCs were aspirated and selected (pre-IVM) in either follicular fluid, collection medium,

collection medium with DMSO (carrier control), collection medium supplemented with

IBMX (500 µM), collection medium supplemented with forskolin (100 µM), or collection

medium supplemented with (forskolin; 100µM & IBMX; 500µM) for 2 hrs. Following,

COCs were matured with FSH (100 mIU/ml) for 7 hours. Oocytes were then fixed and

assessed for meiotic progression and classified as GV (germinal vesicle).

As shown in Fig A3, COCs which were selected during pre-IVM phase in collection medium

containing forskolin or forskolin+IBMX, showed significantly higher rates of oocytes which

were at the GV stage compared to other treatments (P<0.05).

1.4 The effect of inclusion of IBMX during IVM on spontaneous meiotic resumption of

isolated bovine oocytes following 9 hours of culture.

The aim of this experiment was to examine the effect of the non selective PDE inhibitor

IBMX inclusion during IVM on % GV of bovine oocytes matured for 9 hours of culture.

During pre-IVM phase COCs were collected and selected either in follicular fluid, collection

medium, collection medium supplemented with IBMX (500 µM), collection medium

supplemented with forskolin (100 µM) or collection medium supplemented with forskolin;

100µM & IBMX; 500µM) for 2 hrs. After pre-IVM COCs were matured with IBMX (500

223

µM) and FSH (100 mIU/ml). After 9 hours oocytes were fixed and assessed for meiotic

progression and classified as % GV.

As shown in Fig A4, the majority of oocytes which were selected in different pre-IVM phase

treatments and matured in presence of IBMX were blocked at the GV stage compared to the

control (oocytes collected in follicular fluid and matured in standard IVM), indicating that

presence of IBMX in IVM, blocked oocytes to resume spontaneous meiotic resumption after

9 hours of culture.

1.5 The effect on spontaneous oocyte maturation (GV/GVBD) following incubation of

bovine cumulus-oocyte complexes in various (pre-IVM) and IVM phase treatments

The aim of this experiment was to examine the effect on spontaneous oocyte maturation

(GV/GVBD) following incubation of cumulus-oocyte complexes in various (pre-IVM) and

IVM phase media. Bovine COCs were incubated for 2 hours in the pre-IVM phase media,

followed by 7 hour cultures in the presence of FSH, in the presence or absence of milrinone

(100 µM) during IVM. Oocytes were then fixed and assessed for meiotic progression and

classified as GV (germinal vesicle).

As shown in Fig 5A, after culture, significantly more oocytes were arrested at the GV stage

when oocytes were matured in presence of milrinone compared to absence during IVM

(P<0.05), regardless of pre-IVM phase treatment. Moreover, when IVM was performed with

milrinone, spontaneous meiotic resumption was further delayed by a pre-IVM phase with

forskolin+IBMX, compared to control (pre-IVM phase without forskolin+IBMX).

1.6 The effect of standard or induced IVM on mouse oocyte meiotic resumption To determine the effect of the induced IVM system on the rate of meiotic resumption and

completion, COCs were matured in vivo: primed at 2 pm with 5 I.U. eCG then 46 hours later

with 5 I.U. hCG; or in vitro either by standard IVM (control pre-IVM, without cAMP

224

modulators for 1 hour and control IVM with 50 mIU/ml FSH), or by induced IVM (pre-IVM,

50 µM FSK+50µM IBMX for 1 hour, followed by IVM with 0.1 µM cilostamide +100

mIU/ml FSH). From 0 to 14 hours, COCs were collected and denuded of cumulus cells by

gentle pipetting to facilitate visualization of the nuclear stage. Maturation stages in these

oocytes were classified by light microscopy as immature oocytes that progressed into

germinal vesicle breakdown (% GVBD).

As shown in figure A6, within 60 mins, 20% of oocytes in the control pre-IVM group

(standard IVM) commenced GVBD whereas all the oocytes matured in vivo (collected 60

minutes post hCG) and all oocytes in the induced IVM group (60 mins pre-IVM culture in the

presence of cAMP modulators) were still arrested at GV. 8 hours post-hCG, 60% of in vivo

matured oocytes had resumed meiosis, compared to 98% of oocytes after 10 hours of culture

in the standard IVM system. The induced IVM system significantly delayed the resumption

of meiosis with 85% of oocyte at GVBD after 10 hours and 97% GVBD after 12 hours of

culture.

1.7 The influence of BMP15 or glycine inclusion in induced IVM on oocyte developmental

competence

The purpose of this experiment is to examine the effect bovine COCs treatment with BMP-15

or glycine during induced IVM on subsequent oocyte developmental competence. COCs were

aspirated and selected in collection medium supplemented with (forskolin; 100µM & IBMX;

500µM) for 2 hrs. Following, COCs were matured with FSH and cilostamide (20 μM) for 30

hrs either with BMP15 (10% v/v) or glycine (4% v/v). Subsequently, oocyte developmental

capacity was assessed after vitro fertilization and embryo development by the cleavage rate

and the blastocyst rate on day 8.

225

As shown in Fig 7A, there was no obvious difference in percentages of cleavage or blastocyst

formation among treatment groups. Hence, the addition of BMP 15 or glycine did not show

an additional improvement above control.

226

Appendix 1: Figures

Figure A1 . The effect of different cAMP modulators inclusion during pre-IVM phase on the cAMP content of bovine oocytes cultured and assayed with their vestments intact (COCs) after 2 hours of pre-IVM. Values are expressed as the mean concentration of cAMP/COC ± SEM of three replicates using 6-10 COCs per treatment replicate. Means with different letters indicate significantly different amounts of cAMP between treatments (one-way ANOVA, P<0.05).

0

5

1020

40

60

80

forskolin (100 �M) - + - + - + - +IBMX (500 �M) - - + + - - + +dipyridamole (20�M) - - - - + + + +

a

b

b

c

a

d

b

cfm

ol c

AMP

per C

OC

227

Figure A2 . The effect of cAMP modulator inclusion during pre-IVM phase on % germinal vesicle breakdown of bovine oocyte matured for 9 hours in IVM. COCs were aspirated and selected (pre-IVM) in either follicular fluid, collection medium, collection medium supplemented with IBMX; 500 µM, collection medium supplemented with forskolin (FSK; 100 µM) and collection medium supplemented with forskolin (FSK); 100µM & IBMX; 500µM) for 2 hrs. Following, COCs were matured with FSH (100 mIU/ml) for 9 hours. Oocytes were then fixed and assessed for meiotic progression and classified as GVBD (germinal vesicle breakdown – resumption of meiosis). A mean number of 25 oocytes were used in each treatment. Each column represents a mean of only one replicate.

0

20

40

60

80

100

pre-IVM phase FF - IBMX FSK IBMX+FSK

% G

VBD

228

Figure A3 . The effect of cAMP modulator inclusion in pre-IVM phase on germinal vesicle (%) of bovine oocytes following 7 hours of IVM. COCs were aspirated and selected (pre-IVM) in either follicular fluid, collection medium, collection medium with DMSO (carrier control), collection medium supplemented with IBMX; 500 µM, collection medium supplemented with forskolin (FSK; 100 µM) or collection medium supplemented with forskolin (FSK); 100µM & IBMX; 500µM) for 2 hrs. Following, COCs were matured with FSH (100 mIU/ml) for 7 hours. Oocytes were then fixed and assessed for meiotic progression and classified as GV (germinal vesicle). A mean number of 25 oocytes were used in each treatment. Each column represents a mean of 3 replicates following culture.

0

20

40

60

% G

V

pre-IVM phase FF - DMSO IBMX FSK FSK+IBMX

aa

a

abb

b

229

Figure A4. The effect of IBMX on oocyte meiotic resumption of isolated bovine oocytes following 9 hours of IVM. COCs were aspirated and selected (pre-IVM) in either follicular fluid, collection medium, collection medium supplemented with IBMX; 500 µM, collection medium supplemented with forskolin (FSK; 100 µM) or collection medium supplemented with (forskolin; 100µM & IBMX; 500µM) for 2 hrs. Following, COCs were matured with FSH (100 mIU/ml) and IBMX (500 µM) for 9 hours. Oocytes were then fixed and assessed for meiotic progression and classified as GV (germinal vesicle). Each column represents a mean of only one replicate.

0

20

40

60

80

100%

GV

pre-IVM phase FF FF - IBMX FSK IBMX+FSK

IVM phase(+IBMX) - + + + ++

230

Figure A5. The effect on spontaneous oocyte maturation (GV/GVBD) following incubation of cumulus-oocyte complexes in various (pre-IVM) and IVM phase media. Bovine COCs were aspirated and selected (pre-IVM) in either follicular fluid, collection medium, collection medium with DMSO (carrier control) or collection medium supplemented with (forskolin (FSK); 100µM & IBMX; 500µM) for 2 hrs., followed by 7 hour culture in the presence of FSH, and in the presence or absence of milrinone (100 µM) during IVM. Oocytes were then fixed and assessed for meiotic progression and classified as GV (germinal vesicle). A mean number of 35 oocytes were used in each treatment group and time-point from three replicate experiments.

0

10

20

30

% G

V

pre-IVM phase - FSK+IBMX

IVM phase(+ milrinone) + + + -

- FSK+IBMX DMSO

follicular fluid

collection medium

- -

- - -

pre-IVM phase

ab

a

b

c c c c

231

Figure A6. Meiotic maturation of mouse COCs matured either in vivo, by induced IVM or by standard IVM for 14 hours. Oocytes were then assessed for meiotic progression and classified as GVBD stage. Standard IVM = control pre-IVM and control IVM with 50 mIU/ml FSH. Induced IVM = pre-IVM with FSK+IBMX and IVM with cilostamide + 100 mIU/ml FSH. In vivo matured control = COCs collected from ovary 0-14h after hCG administration. Oocytes were then meiotic progression was assesed at each time point. A mean number of 25 oocytes were used in each treatment group and time-point from three replicate experiments. Different superscript are significantly different.

0 0.5 1 2 4 6 8 10 12 140

20

40

60

80

100

in vivo matured controlstandard IVM

Time (hrs)

induced IVM% G

VBD

232

Figure A7. The effect of treatment of bovine COCs with BMP-15 or glycine during induced IVM on subsequent oocyte developmental competence. COCs were aspirated and selected collection medium supplemented with forskolin; 100µM & IBMX; 500µM) for 2 hrs. Following, COCs were matured with FSH and cilostamide (20 μM) and either BMP15 (10% v/v) or glycine (4% v/v) for 30 hrs. Subsequently, oocyte developmental capacity was assessed after vitro fertilization and embryo development by the cleavage rate and the blastocyst rate on day 8. The data represent a mean number of 50 oocytes were used in each treatment group and from four replicate experiments

0

20

40

60

80

100

% cleavage

% blastocyst

IVM phase - BMP15 Glycine

% o

ocyt

e de

velo

pmen

tal c

ompe

tenc

e

233

Table A1. Germinal vesicle (GV) configurations of COCs cultured in various pre-IVM and IVM phase. Oocytes were collected in pure follicular fluid, collection medium, or collection medium supplemented with cAMP modulators (100 µM forskolin (FSK) and 500 µM IBMX) (A), followed by extended culture in the presence (B;D) or absence (C;E) of cilostamide (20 µM), plus FSH, 100 mIU/ml. Oocytes were then fixed and assessed for GV configuration at 2, 5 & 9 hrs. A mean number of 40 oocytes were used in each treatment group and time-point from four replicate experiments.

(A) End of Pre-IVM (2hrs)

Pre-IVM Phase Meiotic Index (%) GV I GV II GV III GV IV Diakenesis MI Follicular Fluid 8±4 61±5 25±7 7±3 0 0 Collection Medium 7±4 12±3 66±5 15±2 0 0 Collection Medium+cAMP Modulators 7±5 67±5 20±9 4±2 0 0

(B) 5 hrs (pre-IVM+3hrs IVM with cilostamide)

Pre-IVM Phase Meiotic Index (%) GV I GV II GV III GV IV Diakenesis MI Follicular Fluid 6±2 53±3 32±3 7±3 0 0 Collection Medium 2±2 14±4 67±4 16±2 0 0 Collection Medium+cAMP Modulators 8±3 62±3 26±5 2±2 0 0

(C) 5 hrs (pre-IVM+3hrs IVM without cilostamide)

Pre-IVM Phase Meiotic Index (%) GV I GV II GV III GV IV Diakenesis MI Follicular Fluid 0 26±3 58±8 16±5 0 0 Collection Medium 0 7±3 42±9 52±12 0 0 Collection Medium+cAMP Modulators 6±2 55±4 38±4 2±2 0 0

(D) 9 hrs (pre-IVM+7hrs IVM with cilostamide)

Pre-IVM Phase Meiotic Index (%) GV I GV II GV III GV IV Diakenesis MI Follicular Fluid 0 11±3 63±6 12±4 3±2 0 Collection Medium 0 4±2 21±6 51±10 9±5 0 Collection Medium+cAMP Modulators 0 44±3 42±3 9±3 0 0

(E) 9 hrs (pre-IVM+7hrs IVM without cilostamide)

Pre-IVM Phase Meiotic Index (%) GV I GV II GV III GV IV Diakenesis MI Follicular Fluid 0 0 11±2 9±3 40±14 38±4 Collection Medium 0 0 3±2 17±6 23±6 52±5 Collection Medium+cAMP Modulators 0 7±3 58±4 14±3 15±3 5±2

234

Appendix 2: Culture Media

Stock Solutions for Bovine IVM/IVF/IVC

All chemicals and reagents were purchased for Sigma (St Louis, MO, USA) unless otherwise

stated.

Stock A (x4) Stock Heparin

2337.6 mg NaCl 20 mg heparin

59.64 mg KCl Dissolve in 2 ml 0.9% sterile saline

78.87 mg MgSO4�7H2O

108.87 mg KH2PO4 Stock Hypotaurine (10 mM)

Dissolve in 100 ml of Milli Q 10.9 mg hypotaurine Dissolve in 10 ml 0.9% sterile saline

Stock B

1.05 g NaHCO3 Stock Penicillamine (20 mM)

5 mg Phenol Red 29.84 mg penicillamine

Dissolve in 50 ml Milli Q Dissolve in 10 ml 0.9 % sterile saline

Stock C (x125) Stock S (x10)

51 mg Na pyruvate 5.5 g NaCl

Dissolve in 10 ml Milli Q 300 mg KCl 36 mg NaH2PO4

Stock GL (x10) 123 mg MgSO4.7H2O

405 mg D-glucose 100 mg Kanamycin

Dissolve in 100 ml Milli Q Dissolve in 100 ml Milli Q

Stock SH SPAD (x100)

600 mg HEPES (free acid) 292 mg NaCl

650 mg HEPES (sodium salt) 300 mg CaCl2.2H2O

Dissolve in 25 ml Stock S Dissolve in 10 ml Milli Q

235

Stock CA (x10) Stock H

85.08 mg Ca [lactate] 6 g HEPES (free acid)

Dissolve in 12 ml Milli Q 6.5 g HEPES (sodium salt)

20 mg phenol red

N-Acetyl-L-cysteine (x100)

Dissolve in 200 ml Milli Q

16.32 mg N-Acetyl –L-cysteine Cysteamine (x100)

Dissolve in 10 ml Milli Q 7.77 mg Mercaptoethylamine

Dissolve in 10 ml Milli Q

Bovine VitroCollect (Aspiration/Handling) Medium 26.5 mg CaCl2.2H2O

648.7 mg NaCl

50.7 mg KCl

19.7 mg MgSO4.7H2O

42.0 mg NaHCO3

1 ml Glutamax

12.0 mg NaH2PO4

221.9 mg MOPS acid

332.9 mg MOPS Na salt

2 ml MEM essential amino acid

41.4 mg Glucose

10 mg Gentamycin/ kanamycin

10 mg Heparin

Add MilliQ to make up to 100 ml and add 0.2 mg/ml BSA.

Osmolarity = 280 mOsm

236

Bovine In vitro Maturation Medium

25 ml Stock A

16 ml Stock B

800 �l Stock C

10 ml Stock CA

24.34 ml Stock GL

1 ml Glutamax I (Gibco Invitrogen Corporation, Carlsbad, CA, USA),

1 ml N-acetyl-cysteine

1 ml cysteamine

1 ml non-essential amino acids (100X, Gibco Invitrogen Corporation),

2 ml essential amino acids (50X, Gibco Invitrogen Corporation)

Add Milli Q to make up to 100 ml and add 4 mg/ml BSA.

Osmolarity = 294 Osm

Bovine IVF media

F-SOF

Bovine In vitro Fert, IVF Vet solutions.

5 ml Stock S

5 ml Stock B

0.5 ml Stock C

0.5 ml Stock D

0.5 ml Stock K

0.5 ml Stock L

0.5 ml Stock N


For 10 ml F-SOF, add:

100 µl Stock Penicillamine

100 µl Stock Hypotaurine

10 µl Stock Heparin

Osmolarity = 270-280 mOsm

237

90% Percoll

4.5 ml Percoll

400 µl Stock SH

50 µl Stock B

50 µl 100X SPAD

45% Percoll

Dilute 2 ml or 90% Percoll with 2 ml HSOF

All solutions used to make Percoll gradients were warmed to room temperature before use.

Sperm thawed in 37oC water.

H-SOF Bovine In vitro Wash; IVF Vet solutions. 5 ml Stock S

1 ml Stock B

4 ml Stock H

0.5 ml Stock C

0.5 ml Stock D

50 µl Stock G

0.5 ml Stock K

0.5 ml Stock L

0.5 ml Stock N

0.5 ml NEAA

1 ml Glutamax



E-SOF Bovine In vitro Cleave; IVF Vet solutions. 5 ml Stock S

5 ml Stock B

0.5 ml Stock C

238

0.5 ml Stock D

50 µl Stock G

0.5 ml Stock K

0.5 ml Stock L

0.5 ml Stock N

0.5 ml NEAA

0.25 ml EAA

0.5 ml Glutamax



L-SOF Bovine Blast Media, IVF Vet solutions. 5 ml Stock S

5 ml Stock B

0.2 ml Stock C

0.5 ml Stock D

0.5 ml Stock G

0.1 ml Stock K

0.5 ml Stock L

0.5 ml Stock M

0.5 ml Stock N

0.5 ml Stock PO

0.5 ml NEAA

1 ml EAA

0.5 ml Glutamax



239

Appendix 3: Reagents cAMP assay acetylation mix cAMP assay acetylation mix prepared fresh before each assay using triethylamine (Ajax Chemicals, Auburn, NSW, Australia): acetic anhydride (BDH Laboratory Supplies, Poole, Dorset, England) (2:1).

Assay Buffer cAMP assay buffer prepared using 0.05 M sodium acetate (4.1 g/L sodium acetate; BDH Laboratory Supplies), pH adjusted to 5.5 with 1M Acetic acid.

Calcein-AM Individual vials (50 �g) of Calcein-AM (Molecular Probes; C-3100) were stored desiccated at -20�C and reconstituted to a 5 mM solution with anhydrous dimethyl-sulphoxide (DMSO Hybrimax�; Sigma, D-2650). Calcein-AM was made up fresh for each experiment (Calcein-AM in solution is gradually hydrolysed over time to generate fluorescent Calcein).

cAMP 100 �l aliquots of a 10-8 mol/ml stock solution dissolved in 100% ethanol were lyophilised in a 1.6 ml Eppendorf tube (1x108mol per tube) for use in cAMP RIA.

��- CAMP Stock solution prepared by dissolving 100 mg of lyophilised antibody in 2.5 ml cAMP assay buffer. Stored frozen at -20�C in 0.1 ml aliquots (4 mg/100 �l). For use in cAMP RIA, one 0.1 ml aliquot diluted in 40 ml cAMP assay buffer containing 10 mg/ml BSA. 200 �l added per tube containing 100 �l standard or sample.

Cilostamide 20 mM stock solution of Cilostamide (BIOMOL, Plymouth Meeting, PA, USA) prepared by dissolving pure compound in DMSO or 95% ethanol. Stored at -20�C in desiccator in 3 and 10 �l aliquots.

Forskolin 125 mM stock solution (Sigma, F6886) prepared by dissolving pure compound in DMSO or 95% ethanol. Stored at -20�C in 30 �l aliquots.

Heparin (10 mg/ml stock) Dissolve 20 mg Heparin (Sodium salt, Grade I-A; Sigma, H-3393) in 2 mL 0.9 % sterile saline.

Hypotaurine (10 mM stock) Dissolve 10.9 mg hypotaurine (2-aminoethanesulfinic acid; Sigma, H-1384) in 10mL 0.9 % sterile saline and store filtered at 4°C. Lasts 1 week.

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125I-cAMP cAMP labelled by David Casley, Department of Medicine, The University of Melbourne, Austin and Repatriation Hospital. The cAMP-tyrosine methyl-ester was iodinated using the chloramine T method with resultant specific activity of 0.537 MBq/pmol. Stored in 70 % ethanol at -20�C.

IBMX 3-isobutyl-1-methyl-xanthine (Sigma, I-7018). 100 mM stock solution prepared by dissolving pure compound in DMSO. Stored at -20�C in desiccator in 3 and 100 �l aliquots.

Human Chorionic Gonadotrophin (hCG) hCG (Profasi; Serono, French’s Forest, NSW, Australia) dissolved in sterile saline containing 0.1 % BSA. Stored at -20�C.

Milrinone 250 mM stock solution of milrinone (Sigma, M-4659) prepared by dissolving pure compound in DMSO. Stored at 4�C in desiccator in 2 �l aliquots.

Mineral Oil Wash mineral oil (embryo-tested; Sigma, M-8410) with sterile Milli Q water and store at room temperature. Maintain sterility.

Penicillamine 20 mM stock solution prepared by dissolving DL-penicillamine (DL-2-amino-3-mercapto-3-methylbutanoic acid; Sigma, P-5125) in 0.9 % sterile saline and store filtered at 4°C. Lasts 1 week.

Penicillin-Streptomycin Aliquot out Penicillin-Streptomycin solution (JRH Biosciences, Parkvelle, Victoria, Australia; 050-81901) and store at -20�C until use.

rhFSH Recombinant human follicle stimulating hormone (rhFSH, Gonal-F, Serono, French’s Forest, NSW, Australia; L1932701A). 10 IU/ml stock solution prepared by dissolving 75 IU in 7.5 ml sterile saline containing 0.1 % BSA. Stored at -20�C in 0.1 and 0.5 ml aliquots (1 and 5 IU respectively).

Rolipram 10 mM stock solution prepared by dissolving pure rolipram compound (ICN Biomedicals Inc., Aurora, Ohio, USA; Catalogue Number 159810) in DMSO. Stored at -20�C in 13, 28, and 220 �L aliquots.

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Appendix 4: Bovine Blastocyst Scoring System Blastocyst scoring system used to select in vitro produced embryos for analysis (adapted from

the Manual of the International Embryo Transfer Society).

The assessment can be performed on a dissecting microscope.

Initially blastocysts are categorized according to their developmental stage:

1c one cell (oocyte/embryo); no cleavage has occurred and the embryo appears like a

single cell

CL cleaved embryo; one or more cleavage divisions has occurred

CM compact morula: embryo consisting of at least 16 cells that has compacted away

from the zona and cell definition is lost

eB early blastocyst; the blastocoel cavity being less than half the volume of the embryo

B blastyocyst; the blastocoel being greater than or equal to half the volume of the

embryo

XB expanded blastocyst; the blastocyst has initiated expansion, increasing the volume of

the bastocoel, thereby increasing in diameter, accompanied by zona pellucida thinning

HB hatched blastocyst; the blastocyst has completely escaped from the zona.

For compact morula and blastocyst stages, embryos are then graded from 1-3, or as

degenerating, depending on the development and appearance of the inner cell mass and

trophectoderm.

1 Excellent/Good. Symmetrical and spherical embryo mass with individual blastomeres

(cells) that are uniform in size, color and density. The embryo is consistent with its

expected stage of development. The embryo possesses a single, tightly packed inner

cell mass with many cells, translucent trophectoderm consisting of many cells, and

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little cellular debris. Any irregularities should be relatively minor, consisting of less

than 15% of the total embryonic mass. This judgment should be based on the

percentage of embryonic cells represented by extruded material in the perivitelline

space. The zona pellucida should be smooth and have no concave or flat surfaces that

may cause the embryo to adhere to a Petri dish.

2 Fair. Moderate irregularities in overall shape of the embryonic mass or in size, color

and density of individual cells. Loosely grouped inner cell mass with several cells or

spatially not confined, translucent/slightly dark trophectoderm consisting of several-

many cells. Some cellular debris, consisting of not greater than 50% of cellular

material.

3 Poor. Major irregularities in shape of the embryonic mass or in size, color and

density of individual cells. Few cells in compartment, dark appearance, cellular debris

consisting of greater than 50% of the total embryonic mass.

4 Dead or degenerating. Granular appearance of cytoplasm, or fragmenting cells.

Non-viable.

Appendix 5: Published Version of Chapter II

‘Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle’

Maxime Sasseville, Firas K Albuz, Nancy Côté N, Christine Guillemette,Robert B Gilchrist, Francois J Richard.

Biology of Reproduction; 2009; 81; 415-425.

NOTE:

This article is available online to authorised users at:

http://dx.doi.org/10.1095/biolreprod.108.074450

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Participation in this publication

In this study, I participated in designing some experiments with the first author and the co-

authors. Moreover, I performed all the experiments related to oocyte meiotic maturation,

COC cAMP assay and finally oocyte developmental competence (IVF, embryo culture and

assessment).

I also participated in writing and editing the manuscript.

Induced IVM: A New Approach to Oocyte Maturation in vitro2.3.4 Oocyte nuclear maturation assessment...

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