OPTIMISATION OF OOCYTE
IN VITRO MATURATION USING
OOCYTE SECRETED FACTORS
Jaqueline Sudiman, MD, MRepSc
Robinson Institute
School of Paediatrics and Reproductive Health
Research Centre for Reproductive Health
Discipline of Obstetrics and Gynaecology
University of Adelaide, Adelaide
Australia
Thesis submitted to the University of Adelaide in total fulfilment of the
requirements for admission to the degree Doctor of Philosophy
October 2014
This is dedicated to my beloved father who taught me that
women have the right and equal opportunity
to an education as men
i
Abstract
During follicular development, oocyte and somatic cells communicate by producing
signals such as proteins, growth factors, hormones that interact in complex harmony.
The oocyte plays an active role in this communication by secreting soluble oocyte
secreted factors (OSFs) including growth differentiation factor 9 (GDF9) and bone
morphogenetic factor 15 (BMP15). Those two growth factors have been implicated in
follicular development and are related to female fertility. In vitro matured oocytes
have aberrant gene expression and altered matrix protein profiles in cumulus cells
compared to their in vivo mature counterparts, which leads to a decrease in oocyte
quality and embryo development post fertilisation.
The first aim of this study was to determine the effect of exogenous native OSFs from
denuded oocytes (DOs) on mouse in vitro maturation (IVM). In a series of
experiments, native OSFs were used in various ways to observe the role of FSH,
cumulus cells, and temporal effects, on production of native OSFs. Overall, co-
culture of COCs with DOs improved mouse embryo development. The highest
improvement in embryo development and embryo quality post co-culture of COCs
with DOs was when COCs were matured in separate IVM media in the presence of
FSH, then denuded at 3h to produce DOs, which were used in co-culture with COCs
for another 14-15 h. Unfortunately, it is not practical to generate such large numbers
of DOs in a clinical scenario. Therefore the next step was to find the functional
bioactive forms of pure recombinant proteins that could improve IVM outcomes.
The above concept led to the main hypothesis of this thesis that the developmental
competence of IVM oocytes can be improved by novel variants of purified
recombinant GDF9 and BMP15. In these studies, GDF9 and BMP15 from various
sources and forms were tested in cattle and mouse IVM. Two different forms of
proteins: pro-mature complex and mature domain of GDF9 and BMP15, were used in
cattle and mouse IVM. In this study, pro-mature complex of human BMP15
(hBMP15) improved cattle blastocyst development, which may, in part, be due to an
increased in nicotinamide adenine diphosphate [NAD(P)H] and reduced glutathione
(GSH) levels. Mature region of BMP15 had a moderate, albeit non-significant, effect
on cattle embryo developmental outcomes, whilst mature region of GDF9 was
ii
ineffective. Similar results were observed using pro-mature hBMP15, which improved
the developmental competence of in vitro matured mouse oocytes, where a
combination of mouse GDF9 (mGDF9) and hBMP15 (both in pro-mature complex
form) produces the highest blastocyst rate.
The work presented in this thesis has provided evidence that exogenous native
OSFs, and recombinant hBMP15 in its pro-mature complex form, are important for
oocyte developmental programming and prove useful for improving mouse and cattle
IVM oocyte developmental competence. Moreover, the source, doses and form of
recombinant proteins play an important role in improving developmental competence
of IVM oocytes. These results may contribute and translate to improve the success
rate of in vitro matured human oocytes.
iii
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 and, to the best of my
knowledge and belief, contains no material previously published or written by another
person, except where due reference has been 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.
Jaqueline Sudiman
September 2014
iv
Acknowledgements
I would like to express my sincere gratitude to my supervisors: Robert Gilchrist,
Jeremy Thompson and David Mottershead. Thank you for the opportunity to learn,
for the advice and guidance during my PhD candidature. The lessons you have
taught me, which are to be more critical, cautious and enthusiastic in science have
been tremendous help during my candidature and for my future career.
I must also give an enormous thank you to Lesley Ritter for assisting me with my
experiments and Melanie Sutton-McDowall for guidance in oocyte metabolism. My
very special thanks go to Satoshi Sugimura for helping me with everything and
making me believes in myself during my candidature.
Thanks to all the members of Gilchrist, Thompson and Mottershead groups. Post-
doc, fellow students and research assistants over the past 3 years: Laura Frank,
Dulama Richani, Xiaoqian Wang, Ryan Rose, Hannah Brown, Deanne Feil, Marie
Anastasi, Melissa White and Jun Yan Shi. Thank for all your friendship and supports.
I would also like to thank to all members in Robinson Institute that I can’t mention one
by one for their friendship especially for Izza Tan, Noor Lokman, Kay Govin, Linda
Liu, Tod Fullston, Hong Liu, Nicole Palmer, Lauren Sandeman, Wan Xian Kang and
Gary Heinemann. Also thanks to the animal house staff and Don for taking care of
mice and transporting the ovaries. I would also like send my thanks to Michelle
Lorimer for statistical support.
I must thank two of my very special friends during my “insane period of time”: Pranay
Sharma and Riksfardini Ermawar. Thank you for your friendships and continuous
support during my hard times and the tears.
My sincere thanks to Kencana Dharmapatni, Tony and Nes Lian-Llyod, Jane Dunn
and Bert, and Sandra and Wayne Turner to show me different side of Australia (My
cubicle and lab station are only a small part of Australia )
My PhD was financially supported by the International postgraduate research
scholarship. I would also like to acknowledge the financial support of Faculty of
v
Health Sciences and Robinson Institute for international and domestic travel
opportunities.
I would like to acknowledge my mum, brother, my very special sister, Josephine
Sudiman, for keeping me strong and supporting me with love and encouragement to
finish my PhD (no matter how hard it is!). Thanks to my grandma and dad for raising
me to believe that having education is a better way to grow up (I know you are
always here with me).
Finally, I would also like to say thanks to myself to be so strong and determine to
finish my PhD! Doing a PhD far away from your family is not an easy way but
Australia is the country where life seems easier and happier.
vi
Publications arising from this thesis
1. Sudiman, J, LJ Ritter, DK Feil, X Wang, K Chan, DG Mottershead, DM
Robertson, JG Thompson, and RB Gilchrist. (2014a) Effects of differing
oocyte-secreted factors during mouse in vitro maturation on subsequent
embryo and fetal development. J Assist Reprod Genet 31 295-306.
2. Sudiman, J, ML Sutton-McDowall, LJ Ritter, MA White, DG Mottershead, JG
Thompson, and RB Gilchrist. (2014b) Bone morphogenetic protein 15 in the
pro-mature complex form enhances bovine oocyte developmental
competence. PLoS One 9(7): e103563.
vii
Conference Proceedings
1. Sudiman J, Ritter LJ, Mottershead DG, Thompson JG and Gilchrist RB. The
Effect of Native and Recombinant Oocyte-Secreted Factors on In Vitro
Maturation. Poster was presented at Australian Society of Medical Research
in Adelaide (2011).
2. Sudiman J, Ritter LJ, Mottershead DG, Thompson JG and Gilchrist RB.
Oocyte Secreted Factors Improve the In Vitro Maturation of Mouse Oocytes
and Subsequent Embryo Development. Abstract was presented at Indonesian
Fertility and Endocrinology Society in Bali (2011).
3. Sudiman J, Ritter LJ, Feil DK, Wang X, Mottershead DG, Thompson JG and
Gilchrist RB. Effect of the Form and Temporal Addition of Oocyte-Secreted
Factors during In Vitro Maturation. Poster was presented at Robinson Institute
Research Symposium in Adelaide (2012), Ovarian Club in Prague (2012) and
Post graduate Research Conference in Adelaide (2013).
4. Sudiman J, Sutton-McDowall M, Ritter LJ, White MA, Mottershead DG,
Thompson JG and Gilchrist RB. Pro-mature Form of Bone Morphogenetic
Protein 15 (BMP15) in Oocyte Maturation Improves Subsequent Bovine
Embryo Development. Abstract was presented for Meat and Livestock finalist
award at Society for Reproductive Biology in Sydney (2013).
viii
Table of contents
Abstract………………………………………………………………………………………...i
Declaration……………………………………………………………………………………iii
Acknowledgements……………………………………………………………………….....iv
Publications arising from this thesis……………………………………………………….vi
Conference proceedings…………………………………………………………………...vii
Table of contents…………………………………………………………………………...viii
List of figures………………………………………………………………………………..xiii
List of tables………………………………………………………………………………....xv
Abbreviations……………………………………………………………………………….xvii
CHAPTER 1 LITERATURE REVIEW ......................................................................... 1
1.1 INTRODUCTION ...................................................................................... 2
1.2 NATURAL FOLLICULAR AND OOCYTE DEVELOPMENT ..................... 3
1.2.1 Maintenance and triggering oocyte nuclear maturation………….. . 5
1.2.2 Cytoplasmic maturation……………………………………………… . 7
1.2.3 Development of oocytes after fertilisation………………………….. 8
1.3 OOCYTE-SOMATIC CELL INTERACTIONS ........................................... 9
1.3.1 Gap Junction Signalling………………………………………………. 9
1.3.2 Paracrine Signalling…………………………………………………. 10
1.3.2.1 Promotion of Follicular Growth ........................................................... 10
1.3.2.2 Regulation of Granulosa and Cumulus Cell Proliferation and Cumulus
Cell Expansion ................................................................................................ 11
1.3.2.3 Prevention of Cumulus Cell Apoptosis ............................................... 12
1.3.2.4 Regulation of Steroidogenesis (Luteinization) and Differentiation of
Granulosa and Cumulus Cells ......................................................................... 12
1.4 TGF-Β SUPERFAMILY .......................................................................... 13
1.4.1 GDF9………………………………………………………………….. 13
ix
1.4.2 BMP15………………………………………………………………… 14
1.4.3 Mutation of GDF9 and BMP15……………………………………… 14
1.4.4 TGF-β Superfamily Signalling………………………………………. 15
1.4.5 Structure and Production of In vitro Recombinant GDF9 and BMP15………………………………………………………………………... 18
1.5 FIBROBLAST GROWTH FACTORS (FGFS) ......................................... 22
1.6 OOCYTE METABOLISM........................................................................ 23
1.6.1 Glutathione…………………………………………………………… 25
1.6.2 Role of OSFs in Cumulus and Oocyte Metabolism………………. 27
1.7 IN VITRO OOCYTE MATURATION ....................................................... 27
1.7.1 Selection of Oocytes for In Vitro Maturation……………………… 28
1.7.2 In Vitro Maturation vs In Vivo Maturation…………………………. 29
1.7.3 Ooctye Secreted Factors in the In Vitro Maturation of Oocytes… 29
1.8 CONCLUSION ....................................................................................... 31
1.9 HYPOTHESIS AND AIMS ...................................................................... 32
1.9.1 Hypothesis…………………………………………………………….. 32
1.9.2 Aims……………………………………………………………………. 32
CHAPTER 2 FACTORS AFFECTING THE CAPACITY OF EXOGENOUS OOCYTE SECRETED FACTORS TO IMPROVE MOUSE EMBRYO AND FETAL DEVELOPMENT POST-IVM ..................................................................................... 33
2.1 ABSTRACT ............................................................................................ 34
2.2 INTRODUCTION .................................................................................... 35
2.3 MATERIALS AND METHODS ............................................................... 38
2.3.1 Isolation and maturation of COCs…………………………………. 38
2.3.2 Generation of denuded oocytes as a source of native OSFs……38
2.3.3 Oocyte nuclear maturation…………………………………………. 41
2.3.4 In vitro fertilization…………………………………………………… 41
2.3.5 In vitro culture of embryos………………………………………….. 41
x
2.3.6 Differential staining of blastocysts…………………………………. 41
2.3.7 Vitrification and warming of blastocysts…………………………… 42
2.3.8 Embryo transfer and assessment of fetal outcomes……………...42
2.3.9 Immunodetection of oocyte GDF9…………………………………. 43
2.4 STATISTICAL ANALYSES ..................................................................... 44
2.5 RESULTS ............................................................................................... 44
2.5.1 Effect of co-culture of intact mouse COCs with native OSFs during IVM……………………………………………………………………………. 44
2.5.2 Temporal effect of co-culture of intact mouse COCs with native OSFs during IVM……………………………………………………………. 46
2.5.3 Effect of exposure of COCs to native OSFs on nuclear maturation ………………………………………………………………………….49
2.5.4 Temporal effects on oocyte GDF9 levels………………………….. 50
2.5.5 The role of FSH and cumulus cells in the production of native OSFs…………………………………………………………………………. 52
2.5.6 Effect of exposure of COCs to native OSFs during IVM on cryo-survival of blastocysts………………………………………………………. 55
2.5.7 Effect of exposure of COCs to native OSFs on pregnancy rates and fetal outcomes………………………………………………………….. 56
2.6 DISCUSSION ......................................................................................... 59
CHAPTER 3 BONE MORPHOGENETIC PROTEIN 15 IN THE PRO-MATURE COMPLEX FORM ENHANCES BOVINE OOCYTE DEVELOPMENTAL COMPETENCE ......................................................................................................... 63
3.1 ABSTRACT ............................................................................................ 64
3.2 INTRODUCTION .................................................................................... 65
3.3 MATERIALS AND METHODS ................................................................. 67
3.3.1 Collection of cells…………………………………………………….. 67
3.3.2 Granulosa cell thymidine assay…………………………………….. 68
3.3.3 Sources of BMP15 and GDF9 and treatment of COCs…………. 68
3.3.4 In vitro fertilization (IVF) and embryo culture………………………69
3.3.5 Blastocyst differential staining……………………………………… 70
xi
3.3.6 Glucose and lactate measurements……………………………….. 70
3.3.7 Autofluorescence of NAD(P)H and FAD++………………………… 71
3.3.8 Reduced glutathione (GSH)………………………………………… 71
3.4 STATISTICAL ANALYSIS ...................................................................... 71
3.5 RESULTS ............................................................................................... 72
3.5.1 Bioactivity of different BMP15 and GDF9 forms………………….. 72
3.5.2 Dose-response of pro-mature BMP15 on oocyte developmental competence………………………………………………………………….. 73
3.5.3 Comparison of pro-mature BMP15, mature BMP15 and mature GDF9 during IVM on subsequent embryo development………………... 74
3.5.4 Effect of pro-mature BMP15, mature BMP15 and mature GDF9 on COC glucose consumption and lactate production……………………… 78
3.5.5 Effect of pro-mature BMP15, mature BMP15 and mature GDF9 on oocyte FAD++ and NAD(P)H autofluorescence………………………….. 80
3.5.6 Effect of pro-mature BMP15, mature BMP15 and mature GDF9 on oocyte GSH………………………………………………………………….. 82
3.6 DISCUSSION ......................................................................................... 83
CHAPTER 4 EFFECT OF INTERACTIONS BETWEEN DIFFERING FORMS OF RECOMBINANT OOCYTE SECRETED FACTORS ON MOUSE OOCYTE DEVELOPMENTAL COMPETENCE ........................................................................ 87
4.1 ABSTRACT ............................................................................................ 88
4.2 INTRODUCTION .................................................................................... 89
4.3 MATERIAL AND METHODS .................................................................. 91
4.3.1 Isolation and maturation of COCs…………………………………. 91
4.3.2 Sources of BMP15 and GDF9 and treatment of COCs………….. 92
4.3.3 In vitro fertilization……………………………………………………. 93
4.3.4 In vitro culture of embryos…………………………………………… 94
4.3.5 Differential staining of blastocysts…………………………………. 94
4.4 RESULTS ............................................................................................... 95
xii
4.4.1 The effect of pro-mature mGDF9 and hBMP15 alone or in combination during IVM on subsequent embryo development………… 95
4.4.2 Interactions between GDF9 and BMP15: effect of differing forms during IVM on subsequent embryo development……………………….. 97
4.5 DISCUSSION ....................................................................................... 100
CHAPTER 5 GENERAL DISCUSSION .................................................................. 104
BIBLIOGRAPHY ..................................................................................................... 115
APPENDICES ......................................................................................................... 143
xiii
List of figures
Figure 1.1 Follicular development stages .................................................................. ..5
Figure 1.2 Oocyte-somatic cell interactions that regulate in vivo matured oocytes......7
Figure 1.3 Schematic representations of GDF9 and BMP15 homodimers and putative
GDF9:BMP15 heterodimers signalling in regulating cumulus and granulosa cell
functions.. .................................................................................................................. 18
Figure 1.4 The forms of mouse GDF9 and BMP15……………………………………..20
Figure 1.5 The structure of GDF9 and BMP15-post purification (pro-mature
complex/processed protein) which consist of dimers of mature regions and
associated pro-regions……………………………………………………………………..22
Figure 1.6 Glucose utilization in cumulus cells and oocytes…………………………..24
Figure 2.1 Schematic illustration of methods of producing native OSFs based on
pretreatment of oocytes with and without cumulus cells and FSH ............................ 40
Figure 2.2 GDF9 Western blot comparison of denuded oocyte (DO) extracts at 0h
and 3h (3h+FSH+CC) of pretreatment ...................................................................... 50
Figure 2.3 Quantitative analysis of Western blot denuded oocyte extracts at 0 and 3h
(3h+FSH+CC) of pretreatment……………………………………………………………51
Figure 2.4 Effect of treatment with native OSFs during IVM on subsequent outcomes
.................................................................................................................................. 57
Figure 3.1 Bioactivity of different BMP15 and GDF9 forms ....................................... 72
Figure 3.2 Glucose consumption and lactate production .......................................... 79
Figure 3.3 Intra-oocyte NAD(P)H, FAD ++, REDOX ratio ........................................... 81
Figure 3.4 Intra-oocyte GSH level ............................................................................. 82
Figure 4.1 In vitro maturation of mouse COCs in low-binding tube…………………...92
xiv
Figure 5.1 Different forms of GDF9 and BMP15 proteins ........................................ 111
xv
List of tables
Table 2.1 Effect of native OSFs during IVM on subsequent embryo development ... 45
Table 2.2 Effect of native OSFs during IVM on inner cell mass (ICM), trophectoderm
(TE) and total cell numbers (TCN) of day 6 blastocysts ............................................ 45
Table 2.3 Temporal effect of co-culture of intact mouse COCs with native OSFs on
subsequent embryo development ............................................................................. 47
Table 2.4 Temporal effect of co-culture of intact mouse COCs with native OSFs on
inner cell mass (ICM), trophectoderm (TE) and total cell numbers (TCN) of day 6
blastocysts ................................................................................................................ 48
Table 2.5 Effect of native OSFs added at 3 hours on oocyte nuclear maturation ..... 49
Table 2.6 Effects of cumulus cells and FSH on native OSFs efficacy at enhancing
post-IVM embryo development ................................................................................. 53
Table 2.7 Effects of cumulus cells and FSH on native OSFs on inner cell mass (ICM),
trophectoderm (TE) cells and total cell numbers (TCN) of day 6 blastocysts ............ 54
Table 2.8 Effect of native OSFs during IVM on the survival rate of post-warmed
blastocysts ................................................................................................................ 55
Table 2.9 Effect of native OSFs during IVM on subsequent fetal and placental weight
and crown-rump length ............................................................................................. 58
Table 3.1 Effect of graded doses of pro-mature BMP15 during IVM on oocyte
developmental competence ...................................................................................... 73
Table 3.2 Effect of supplementing COC cultures with different forms of BMP15
(mature BMP15 and pro-mature BMP15), mature GDF9 and FSH on oocyte
developmental competence ...................................................................................... 75
Table 3.3 Effect of supplementing COC cultures with different forms of BMP15
(mature BMP15 and pro-mature BMP15), mature GDF9 in the absence of FSH on
oocyte developmental competence ........................................................................... 76
xvi
Table 3.4 Number of total (TCN), inner cell mass (ICM) and trophectoderm (TE) cells
on day 8 expanded, hatching and hatched blastocysts following co-culture of COCs
with different forms of BMP15 (mature BMP15 and pro-mature BMP15), mature
GDF9 in the presence of FSH ................................................................................... 77
Table 3.5 Number of total (TCN), inner cell mass (ICM) and trophectoderm (TE) cells
on day 8 expanded, hatching and hatched blastocysts following co-culture of COCs
with different forms of BMP15 (mature BMP15 and pro-mature BMP15), mature
GDF9 in the absence of FSH .................................................................................... 77
Table 4.1 Effect of supplementing COC cultures with mGDF9, hBMP15 (pro-mature
complexes) and combination of both proteins on oocyte developmental
competence…………………………………………………………………………………96
Table 4.2 Effect of supplementing COC cultures with mGDF9, hBMP15 (pro-mature
complexes) and combination of both proteins on numbers of inner cell mass (ICM),
trophectoderm (TE) and total (TCN) cells in blastocysts ………….............................97
Table 4.3 Effect of supplementing COC cultures with a combination of hGDF9 and
hBMP15 (pro-mature complexes), a combination of mGDF9 and hBMP15 (isolated
mature region), or a hGDF9:hBMP15 heterodimer (isolated mature regions) on
oocyte developmental competence………………………………………………………98
Table 4.4 Effect of supplementing COC cultures with a combination of hGDF9 and
hBMP15 (pro-mature complexes), a combination of mGDF9 and hBMP15 (isolated
mature region), or a hGDF9:hBMP15 heterodimer (isolated mature regions) on
numbers of inner cell mass (ICM), trophectoderm (TE) and total (TCN) cells in
blastocysts ……………………………………………………………………………….....99
Table 5.1 Summary of developmental competency of mouse and cattle oocytes
undergoing IVM with the addition of native OSFs anddifferent forms and sources of
GDF9 and BMP15………………………………………………………………………...112
xvii
Abbreviations
ART assisted reproductive technology BSA bovine serum albumin BMP15 bone morphogenetic protein 15 hBMP15 human bone morphogenetic protein 15 BMPR2 bone morphogenetic protein receptor type II cAMP cyclic adenosine monophosphate COC cumulus oocyte complex DO denuded oocyte ECM extracellular matrix EGF epidermal growth factor FAD flavin adenine dinucleotide FAF fatty acid free FSH follicle stimulating hormone FGF fibroblast growth factor GC granulosa cell GDF9 growth differentiation factor 9 hGDF9 human growth differentiation factor 9 mGDF9 mouse growth differentiation factor 9 GSH reduced form of glutathione GV germinal vesicle GVBD germinal vesicle breakdown GJC gap junction communication HEK-293T human embryonic kidney cell line H-αMEM hepes-buffered minimum essential medium
xviii
H-TCM hepes-buffered tissue culture medium ICM inner cell mass IBMX 3-isobutyl-1-methylxanthine IVC in vitro culture IVF in vitro fertilization IVM in vitro maturation IVP in vitro production KL kit ligand LH luteinising hormone LHR luteinising hormone receptor MI metaphase I MII metaphase II NADPH nicotinamide adenine dinucleotide phosphate OOX oocytectomised complex OSF oocyte-secreted factor PMSG pregnant mare’s serum gondadotropin PPP pentose phosphate pathway TCM-199 tissue culture medium 199 TCN total cell number TE trophectoderm TGFβ transforming growth factor β TGFβR-II transforming growth factor β receptor type-II
Chapter 1: Literature Review
1
CHAPTER 1
LITERATURE REVIEW
Chapter 1: Literature Review
2
1.1 INTRODUCTION
The demand for assisted reproductive technology (ART) has increased over the last
few decades due to the rising rate of infertility among couples. This technology has
been applied successfully, with some IVF clinics achieving pregnancy rates of 30-
40% per embryo transfer. However, this technology has a drawback with the use of
gonadotropin hormones to generate large numbers of mature oocytes. These
hormones are expensive, and in countries where the cost of using ART comes from
the patient’s pocket, it is a substantial financial burden for the patients. The use of
these hormones is also associated with patient inconvenience due to the
administration by subcutaneous or intramuscular injection and the risk of ovarian
hyperstimulation syndrome (OHSS). In vitro maturation (IVM) of oocytes is an
emerging technology that eliminates or substantially reduces the use of gonadotropin
hormones. It is especially useful for women with polycystic ovary syndrome (PCOS)
who are less able to tolerate the side effects of these hormones. IVM also
significantly decreases the cost of ART which could allow more infertile couples from
low economic levels to access this technology. However, there is a discrepancy in
the success rate of embryos derived from in vitro matured oocytes compared to in
vivo matured oocytes, with the pregnancy rate post-IVM less than half of the
pregnancy rate post-IVF (Gremeau, et al. 2012). To date, many ingredients have
been added to IVM medium to nurture the immature oocytes in the Petri dish (Albuz,
et al. 2010, Hussein, et al. 2006, Richani, et al. 2013, Yeo, et al. 2008). However,
further development is needed to achieve an acceptable pregnancy success rate for
IVM-derived oocytes. Therefore, there is an urgent need to extend and develop new
techniques/media to increase the quality of in vitro matured oocytes. This can be
achieved by understanding the mechanisms and components which are responsible
for oocyte developmental competence.
Oogenesis and folliculogenesis involve cellular and molecular communication
between the oocyte and its surrounding somatic cells. Endocrine, paracrine and
autocrine signalling regulate this process to provide the oocyte the capacity to
undergo meiotic resumption and support subsequent embryo and fetal development.
There are two major oocyte secreted factors (OSFs) that regulate granulosa/cumulus
Chapter 1: Literature Review
3
cell function: bone morphogenetic protein 15 (BMP15), also known as GDF9b, and
growth differentiation factor 9 (GDF9) (Dube, et al. 1998, Nilsson and Skinner 2002).
Both of these proteins are expressed exclusively in oocytes from an early stage of
follicular development and are important in regulating female fertility. This review will
focuses on bi-directional communication between the oocyte and somatic cells
(granulosa cells and cumulus cells), particularly the transfer of proteins and
molecules that sustain the development of the somatic cells and the oocyte itself.
Secondly, it will focus on the role of OSFs, notably GDF9 and BMP15 in regulating
the function of somatic cells and the interaction and signalling which occurs between
these proteins and somatic cells. The function of GDF9 and BMP15 in reproduction
and the production of GDF9 and BMP15 in vitro will then be explained, followed by
the role of OSFs in oocyte metabolism. Finally, how OSFs improve the IVM system
will be discussed.
1.2 NATURAL FOLLICULAR AND OOCYTE DEVELOPMENT
During fetal life the primordial germ cells (PGCs), which originate from epiblast cells
in the posterior region of the primitive streak, proliferate and migrate through the yolk
sac, hindgut endoderm, and the dorsal mesentery of the hindgut and finally rests in
the genital ridges at the ventral sides of the mesonephroi. The initiation signal for
epiblast cells to proliferate into PGSs is regulated in part by transforming growth
factor β (TGF-β) family members, bone morphogenetic protein 4 (BMP4), BMP8b
and BMP2 (Ying, et al. 2001). During the migration, the germ cells transform into an
irregular shape with protrusions and pseudopodia, with the formitself crucial for the
migration act (Lin, et al. 1982). In the mouse, c-kit receptor tyrosine kinase, kit-ligand
(KL) or stem-cell factor (SCF), TIAR 1 and leukemia inhibitory factor (LIF) are factors
that are required for proliferation and migration of germ cells (Cheng, et al. 1994,
Godin, et al. 1991, Matsui, et al. 1990). In the genital ridges, the PGCs differentiate
into either as oogonia or spermatogonia (Picton, et al. 1998). In the ovary, oogonia
undergo mitotic divisions and connect with each other by intercellular cytoplasmic
bridges which are known as germ cell clusters or cortical cords (van den Hurk and
Zhao 2005). In the human ovary, the population of oogonia is approximately 600,000
at 8 weeks of intrauterine life and can reach a peak of 6-7 million by 20 weeks of
Chapter 1: Literature Review
4
gestation (Strauss III and Barbieri 2009). Those oogonia then undergo further DNA
replication and enter the first meiosis divisions: leptotene, zygotene and pachytene
stages of meiotic prophase I before arresting at the diplotene stage (van den Hurk
and Zhao 2005). At the same time the meiotic process begins, the oogonia, which
are now called primary oocytes, will invest themselves with one layer of squamous
somatic cells to form primordial follicles. Oogonia undergo apoptosis if those germ
cells do not enter meiosis by the seventh month of gestation (Strauss III and Barbieri
2009). At the end of gestation, only approximately 700,000 primordial follicles are
found in human ovaries (Strauss III and Barbieri 2009). This number further reduces
by approximately 300,000 in puberty and only 400-500 grow into mature antral
follicles during a woman’s reproductive life (Strauss III and Barbieri 2009).
The development of primordial follicles to primary follicles is marked by the changing
of squamous cells of granulosa cells into a single layer of cuboidal cells. The single
layer of cuboidal cells develops into several layers of cuboidal granulosa cells
surrounding primary oocytes to form secondary follicles, and those cells start to
develop follicle stimulating hormone (FSH), estrogen and androgen receptors
(Gosden 2002, Lintern-Moore, et al. 1974). Blood vessels develop to supply the
follicle, and the zona pellucida becomes more highly structured (Strauss III and
Barbieri 2009). During the proliferation of granulosa cells in secondary follicles,
oocytes grow in diameter and volume extensively (Braw-Tal 2002) followed by the
development of theca layers from interstitial stroma. Refer to Fig. 1.1. The theca has
two layers: the theca interna is adjacent to the basal lamina of the follicle and
consists of steroidogenic cells and is highly vascularised, whilst, the theca externa
consists of non-steroidogenic cells (Magoffin 2005). Both granulosa and theca cells
are required for the production of steroids, via what is known as the two cell-two
gonadotropin concept of follicle steroid biosynthesis. Luteinizing hormone (LH)
stimulates theca cells to express cholesterol side-chain cleavage cytochrome P450
(CYP11A), 3-hydroxysteroid dehydrogenase (3β-HSD), and 17α-hydroxylase/C17-20
lyase cytochrome P450 (CYP17) enzymes, which are used to synthesise
androstenedione from cholesterol. FSH stimulates the expression of aromatase
cytochrome P450 (CYP19) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in the
Chapter 1: Literature Review
5
granulosa cells. Those enzymes metabolise androstenedione to estradiol (van den
Hurk and Zhao 2005). The small follicle has a low ratio of estrogen to androgen, but
this ratio is slowly reversed during follicular growth. The dominant follicle can be
distinguished from other follicles by an increased level of FSH in the antral fluid
(Sullivan, et al. 1999), high concentrations of estradiol (van Dessel, et al. 1996), low
levels of androgen (McNatty, et al. 1975), a high mitotic index of the granulosa cells
(Strauss III and Barbieri 2009) and a transfer of dependency from FSH to LH (van
den Hurk and Zhao 2005). Estradiol levels reach their peak in the ovulatory follicle,
and the pituitary ovulatory LH surge, leads to production of hyaluronic acid by the
cumulus cells and down regulation of CYP17 in theca cells to switch them from
androgen-producing to progesterone-producing cells (Magoffin 2005). Most follicles
do not develop to ovulatory follicle. In monoovular animals such as sheep, cattle and
humans, only one preovulatory follicle is ovulated each cycle to produce a mature
oocyte. On the other hand, polyovulatory animals (e.g. mouse) develop many
preovulatory follicles during each ovarian cycle.
Figure 1.1 Follicular development stages (taken from
https://www.med.unsw.edu.au)
1.2.1 Maintenance and triggering oocyte nuclear maturation
In the primary follicle, the oocyte remains arrested at prophase of the first meiotic
division (GV stage) by cyclic adenosine monophosphate (cAMP)-dependent protein
kinase A (PKA). cAMP is synthesized from G proteins (Gs) in the oocyte membrane
(Mehlmann, et al. 2002), as well as supplied by somatic cells, and enters the oocyte
through gap junctions (Anderson and Albertini 1976). Somatic cells also generate
cyclic guanosine monophosphate (cGMP) that also passes through gap junctions into
the oocyte, inhibiting the degradation of cAMP and thus blocking meiotic progression
Chapter 1: Literature Review
6
(Norris, et al. 2009). In the event of an LH surge, the oocyte phosphodiesterase type
3 (PDE3) enzyme is activated degrading cAMP which induces the maturation
promoting factors (MPF), causing the oocyte to resume meiotic maturation (Tsafriri,
et al. 1996). This event also leads to disconnection of the gap junctions and loss of
receptivity of the oocyte receptors for cAMP and cGMP and lead to releasing the first
polar body of the oocyte (Norris, et al. 2008, Norris, et al. 2009, Sela-Abramovich, et
al. 2005, Sela-Abramovich, et al. 2006). Refer to Fig. 1.2.
The preovulatory LH and FSH surges also induce expression of the epidermal growth
factor (EGF)-like peptides: amphiregulin (AREG), epiregulin (EREG) and β-cellulin
(BTC) (Park, et al. 2004). Activation of the EGF receptor (EGFR) in granulosa and
cumulus cells through the ERK1/2 pathways is followed by the production of a
hyaluronan-rich matrix. Hyaluron synthase 2 (Has2), tumor necrosis factor (TNF)-α-
induced protein 6 (Tnfaip6), pentraxin (Ptx3), prostaglandin-endoperoxide synthase 2
(Ptgs2) (Park, et al. 2004, Russell and Robker 2007) are all involved in granulosa
and cumulus cell expansion. OSFs such as GDF9 and BMP15 are also known to be
involved in this process, through their receptors (bone morphogenetic protein
receptor type-II and activin receptor-like kinases) which activate the Sma and Mad
related (SMAD) intracellular signals to stimulate expansion. The signalling from OSF
receptors to granulosa and cumulus cells activates many functions of these cells,
such as proliferation, expansion, luteinisation, mucification and metabolism (Refer to
section 1.3, Fig. 1.2). Interestingly, during oocyte maturation, signals from both the
EGF and OSF receptors interact with each other in the cumulus cells, making a
complex harmony (Gilchrist 2011).
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7
Figure 1.2 Oocyte-somatic cell interactions that regulate in vivo matured
oocytes. There are three processes involved in this pathway: interaction of
GDF9/BMP15 through SMAD2/3 signalling and EGFR; LH and FSH-induction of the
EGF-like peptides in granulosa and cumulus cells; and the role of cGMP, PDE and
cAMP regulating oocyte meiosis. Adapted and modified from (Gilchrist 2011). GJC=
gap junction communication.
1.2.2 Cytoplasmic maturation
Maturation of oocytes within selected follicles is accompanied by cytoplasmic
maturation. The process of cytoplasmic maturation is less obvious compared to
nuclear maturation. In mature human oocytes, actin cytoskeleton is moved and
organized in the cortex therefore oocytes become highly asymmetric to maintain
organelles, proteins and RNAs within the ooplasm (Coticchio, et al. 2014, Levi, et al.
2013). Moreover cortical granules, endoplasmic reticulum and mitochondria also
move toward the oocyte cortex (Strauss III and Barbieri 2009, Tian, et al. 2007).
Chapter 1: Literature Review
8
Recent evidence shows Fyn kinase and thick filamentous microtubules in the human
ooplasm are important for successful maturation-competence of oocytes (Levi, et al.
2013).
Oocyte cytoplasmic maturation is important for the ability of the oocyte to be fertilised
and to develop into a zygote. It has also been shown that there is a strong correlation
between oocyte cytoplasmic maturation and the size of follicles. Mature oocytes
which are retrieved from large antral follicles have the capability to develop to the
blastocyst stage when compared to MII oocytes which are obtained from small antral
follicles (Leibfried and First 1979). Moreover, oocytes from pre-pubertal animals have
poorer cytoplasmic maturity, as evidenced by more abnormalities in their
ultrastructure, alterations in metabolic activity and lower embryo developmental
potential compared to those from adults (Leibfried and First 1979, O'Brien, et al.
1996, Succu, et al. 2007)
1.2.3 Development of oocytes after fertilisation
If there is a fertilisation event, the oocyte resumes the second meiotic division and
extrudes the second polar body. In cattle oocytes, male and female pronuclei
develop around 9-14 hours after insemination. The pronuclei enlarge and migrate
centrally, then fuse and undergo syngamy at around 17 hours after insemination. The
earliest cleavage in bovine can be seen at 24 hours after fertilisation and reaches its
maximum rate at 30 hours after fertilisation (Rizos, et al. 2002). Blastocyst formation
in bovine embryos starts at day 6 after insemination (Rizos, et al. 2002). In mouse,
these events occur faster. The formation and migration of the male and female
pronuclei are completed within 5 hours after insemination and the first cleavage of
mouse embryo takes place around 17-19 hours after insemination (Witkowska 1981).
The blastocyst formation in mouse starts at 4.5 days after insemination. This
embryonic structure consists of two different cell types: inner cell mass (ICM) and
trophectoderm (TE) cells (Thouas, et al. 2001). The inner cell mass (ICM) cell
number is a reasonable measure of blastocyst quality and probability of implantation
as the ICM is the portion of the blastocyst that derives the fetus. It has been shown
that there is a strong correlation between an increase in ICM cell numbers and
Chapter 1: Literature Review
9
successful implantation and fetal development (Sudiman, et al. 2014a, Yeo, et al.
2008).
1.3 OOCYTE-SOMATIC CELL INTERACTIONS
There are important and multiple modes of communication between theca cells,
granulosa cells and the oocyte, required for follicular development. Communication
between the oocyte and somatic cells is mediated by gap junction and paracrine
signalling. Theca cells secrete factors such as transforming growth factor alpha
(TGF-α), and beta (TGF-β), keratinocyte-derived growth factor (KGF) and hepatocyte
growth factor (HGF) to regulate the development of granulosa cells (Nilsson and
Skinner 2001, Parrott, et al. 1994). The granulosa cells produce KL to promote theca
cell organization (Parrott and Skinner 1997). Oocytes actively regulate follicular
development by producing oocyte derived factors such as GDF9, BMP15, bone
morphogenetic protein 6 (BMP6) and fibroblast growth factors (FGFs) which are
transmitted to the granulosa cells via paracrine signalling. This bi-directional
communication is important to transfer molecules from the oocyte to somatic cells,
and vice versa to sustain granulosa and cumulus cell health and oocyte development
itself.
1.3.1 Gap Junction Signalling
Oocyte and cumulus cells are seperated from each other by the zona pellucida. To
allow small molecules, amino acids and cAMP to penetrate from the cumulus cells
into the oocyte and other cumulus cells, they develop trans-zonal cytoplasmic
projections that go through the zona pellucida and abut the oocyte membrane
(Albertini, et al. 2001). This type of channel is important in order to achieve the
development of follicles and oocytes. Cumulus cells perfuse small molecules such as
purines, linoleic acid, ions, cAMP and other low molecular weight peptides into the
oocyte to maintain meiotic arrest, induce meiotic resumption and support cytoplasmic
maturation of the oocyte (Tanghe, et al. 2002). This gap junctional communication
also occurs at all close cell apposition such as at the surface of oocyte microvilli,
between cumulus-cumulus cells and cumulus-granulosa cells (Kidder and Mhawi
2002).
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10
Gap junction channels are composed of connexons, a cylindrical organelle that forms
an intercellar channel. Each connexon is made up from connexins, a hexamer of
protein subunits. Gap junctions allow inorganic ions, second messengers and small
metabolites (< 1 kDa) to pass from cell to cell (Kidder and Mhawi 2002). There are
many different kind of connexins, however, there are two well-known connexins
which are important in follicular and oocyte development: connexin-37 and connexin-
43 (Ackert, et al. 2001, Simon, et al. 1997). Homozygous mutant connexin-43 mice
show abnormalities in follicular development and retarded oocytes which failed to be
fertilized (Ackert, et al. 2001). Oocytes in mice deficient in connexin-37 arrest before
they reach meiotic completion (Simon, et al. 1997).
In oocytes, there is a strong correlation between the loss of gap junction
communication and the resumption of meiosis (Larsen, et al. 1986, Thomas, et al.
2004). It has been demonstrated that the gap junction communication between the
oocyte and cumulus cells is essential to maintaining meiotic arrest, especially in early
oocyte maturation (Thomas, et al. 2004). In bovine oocytes, gap junction
communication levels fall progressively from 0-9 hours of maturation (Thomas, et al.
2004).
1.3.2 Paracrine Signalling
Another type of communication between the oocyte and its somatic cells (granulosa
and cumulus cells) is paracrine signalling. Soluble growth factors secreted by the
oocyte to cumulus cells via paracrine signalling. Below the mechanism of OSFs
impacts on ovarian folliculogenesis and cumulus cells functions.
1.3.2.1 Promotion of Follicular Growth
OSFs such as GDF9 and BMP15 are important during follicular development. A
deficiency of GDF9 and/or BMP15 can reduce fertility due to follicular development
retardation (see section 1.4.3). In in vitro culture, addition of GDF9 or BMP15 alone
promotes growth of pre-antral follicles of the mouse only during the first 24 hours;
interestingly, after 24 hours in culture, BMP15 causes shrinkage and atresia of
follicles and increases apoptosis of the granulosa cells (Fenwick, et al. 2013).
However, a combination of both proteins induces the development of the follice
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11
beyond 24 hours of culture (Fenwick, et al. 2013). The situation is different in bovine
in vitro cultured pre-antral follicles, where the addition of either BMP15 or a
combination of FSH and BMP15 enhances the growth of secondary follicles (Passos,
et al. 2013). These results confirm that GDF9 and BMP15 have different roles in
different species in term of promoting follicular development.
1.3.2.2 Regulation of Granulosa and Cumulus Cell Proliferation and Cumulus
Cell Expansion
An increase in granulosa and cumulus cell proliferation in response to OSFs can be
observed by [3H] thymidine uptake as an indicator of DNA synthesis in cells (Li, et al.
2000, Vanderhyden, et al. 1992). It has been shown that recombinant GDF9 or native
OSFs; which are produced by culturing denuded oocytes (DOs) with somatic cells,
play a significant role in creating extracellular matrix (ECM) for cumulus cell
expansion by inducing Has2, Tnfalp6 , Ptgs2 and Ptx3 mice gene expression
(Dragovic, et al. 2005, Dragovic, et al. 2007).
Mouse cumulus expansion depends on the presence of the oocyte (Buccione, et al.
1990, Salustri, et al. 1990). Oocytectomised complexes (OOXs) are cumulus oocyte
complexes (COCs) which have undergone microsurgical removal of the oocyte.
Mouse OOXs are unable to undergo cumulus expansion even in the presence of
FSH (Buccione, et al. 1990, Salustri, et al. 1990). FSH induced the synthesis of
hyaluronic acid, the protein that is required for cumulus expansion and stabilility of
the extracellular matrix, in the COC group but not in the OOX group (Buccione, et al.
1990). Interestingly, cumulus cell expansion of OOXs can be induced in the presence
of FSH by co-culturing OOXs with DOs or culturing the OOXs in the oocyte-
conditioned medium (medium produced by co-culturing the OOXs with DOs,
granulosa cells with DOs or COCs with DOs) (Buccione, et al. 1990, Gilchrist, et al.
2008, Salustri, et al. 1990). Therefore, the presence of FSH is important, but not
sufficient, to induce the cumulus expansion of mouse OOX (Salustri, et al. 1990).
Interestingly, in the pig and cow, expansion of the cumulus cells does not depend on
the presence of oocytes, even though they secrete OSFs (Gilchrist and Ritter 2011,
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12
Prochazka, et al. 1991, Ralph, et al. 1995, Singh, et al. 1993, Vanderhyden 1993). It
has been shown that oocyte-conditioned medium from pig oocytes can induce the
cumulus expansion of mouse and rat OOX (Prochazka, et al. 1998, Vanderhyden
1993). Vanderhyden et al., (1993) also demonstrated that oocytes from different
species secrete these soluble factors at different times. In vitro matured mouse
oocytes are capable of secreting OSFs from the time they resume the first meiotic
divison until metaphase II of the second meiosis, whilst in pig, the oocyte secretes
OSFs only during the transition between GV and metaphase I (Nagyova, et al. 2000,
Vanderhyden 1993). In general, the capability of OSFs to regulate the functions of
cumulus and granulosa cells occurs mostly in fully grown oocytes in the antral phase,
declining after the LH surge with re-initiation of meiosis (Gilchrist, et al. 2008).
1.3.2.3 Prevention of Cumulus Cell Apoptosis
Hussein et al (2005) demonstrated for the first time how OSFs can prevent cumulus
cell apoptosis by co-culturing OOXs with denuded oocytes (Hussein, et al. 2005). In
this elegant experiment, the OOXs were exposed to DOs in a dose dependent
manner by incubating OOXs with increasing numbers of oocytes. The apoptosis level
reduced linearly with the increasing number of oocytes. The results also showed that
recombinant BMP15 and BMP6 are potent anti-apoptotic factors, but suprisingly,
recombinant GDF9 had little effect. Interestingly, the incidence of apoptosis in COCs
was higher in the outer layer of the cumulus cells whilst in OOXs+DOs, it was the
inner layer (Hussein, et al. 2005). This effect may, in part, be due to OSFs promoting
the expression of anti-apoptotic Bcl-2 and supressing pro-apoptotic Bax in cumulus
cells.
1.3.2.4 Regulation of Steroidogenesis (Luteinization) and Differentiation of
Granulosa and Cumulus Cells
There are two somatic cell populations in the ovarian follicle: those that attach
directly to the oocytes (cumulus cells) and the cells that line the follicle wall (mural
granulosa cells). Both populations are differentiated during the antral phase of
follicular development. LH receptors (LHR) and LHR mRNA are detected abundantly
in mural granulosa cells but only at low levels in cumulus cells; thus this is an
important marker that distinguishes granulosa and cumulus cells (Eppig, et al. 1997).
Chapter 1: Literature Review
13
Since the production of progesterone triggers the luteinization of follicles, OSFs
suppress elevation of LHR mRNA in mural granulosa cells and cumulus cells.
However, if oocytes are microsurgically removed from COCs (OOX), the cumulus
cells express higher levels of LHR mRNA (Eppig, et al. 1997). Oocytes at GV stage
are more effective in suppressing expression of LHR mRNA in cumulus cells
compared to those at metaphase II (Eppig, et al. 1997). The same result was also
obtained by Li et al (2000) showing that OSFs inhibit the production of progesterone
in cumulus and granulosa cells. Moreover, recombinant GDF9 and BMP15 together
are able to inhibit FSH-stimulated progesterone production by granulosa cells
(McNatty, et al. 2005a). In contrast with progesterone production, mouse OSFs
induce the production of estradiol by granulosa cells (Vanderhyden, et al. 1993),
although this is not the case in bovine where OSFs suppress estradiol production
(Glister, et al. 2003).
1.4 TGF-β SUPERFAMILY
The TGF-β superfamily proteins are involved in the proliferation, differentiation and
death of cells. This superfamily is present in a diverse range of species, including
humans, and it consists of six sub-groups: TGF-β 1, 2, 3, Anti-Mȕllerian hormone
(AMH), inhibin A and B, activin A, B and AB, 20 different BMPs and 9 GDFs (Burt
and Law 1994, Ghiglieri, et al. 1995). GDF9 and BMP15 are members of the TGF-β
superfamily and important in female fertility.
1.4.1 GDF9
In 1993, McPherron and Lee discovered for the first time GDF9 in the mouse ovary
(McPherron and Lee 1993). In polyovulatory animals, the expression of GDF9 starts
from the primary follicle stage (Jaatinen, et al. 1999, Laitinen, et al. 1998), whilst in
monoovular animals such as cow, sheep and humans, the expression of GDF9 is
first observed in primordial follicles and is observed thereafter in the oocyte
(Aaltonen, et al. 1999, Bodensteiner, et al. 1999, Eckery, et al. 2002). GDF9 mRNA
expression has also been reported in non-ovarian tissue such as spermatocytes,
spermatids, sertoli cell cytoplasm, uterus and bone marrow (Fitzpatrick, et al. 1998).
Chapter 1: Literature Review
14
Recombinant GDF9 can mimic the effect of OSFs such as a cumulus expansion
enabling factor in mouse oocytes (Dragovic, et al. 2005, Vanderhyden, et al. 2003).
Recombinant GDF9 stimulates Has2, cyclooxygenase 2 (Cox-2) and steroidogenic
acute regulator protein (StAR) mRNA sythesis, and suppresses urokinase
plasminogen activator (uPA) and LHR mRNA in granulosa and cumulus cells, thus
playing an important role in inducing cumulus cell expansion and differentiation
(Elvin, et al. 1999a). However, Dragovic et al (2005) showed that other proteins must
also be involved in mouse cumulus cell expansion, since a GDF9 antagonist is not
sufficient to completely neutralize oocyte-induced expansion.
1.4.2 BMP15
Dube and co-workers were the first group to discover BMP15, also referred to as
GDF9B, in mouse oocytes (Dube, et al. 1998). The first expression of BMP15
appears at a later developmental stage compared to GDF9. In mice the expression of
BMP15 is expressed in the oocyte beginning at the one-layer primary follicle stage
and continuing through ovulation (Dube, et al. 1998). Studies in human and sheep
oocytes have shown that the expression of BMP15 is at the late primary follicle stage
(Aaltonen, et al. 1999, Galloway, et al. 2000).
Similar to GDF9, BMP15 regulates rat cumulus and granulosa cell proliferation and
this effect is FSH-independent (Otsuka, et al. 2000). However, BMP15 is a selective
modulator of FSH action as it decreases FSH-induced progesterone production but
has no effect on estradiol production; therefore, BMP15 is a candidate for a potent
granulosa cell luteinization inhibitor (Otsuka, et al. 2000). BMP15 inhibits StAR, P450
aromatase, LHR and inhibin/activin subunit mRNA in rat granulosa cells (Otsuka, et
al. 2001b) but stimulates the expression of KL (Otsuka and Shimasaki 2002).
1.4.3 Mutation of GDF9 and BMP15
Unlike most other members of the TGF-β superfamily, the function and expression of
both GDF9 and BMP15 differ markedly among species. The ratio of GDF9 and
BMP15 in oocytes is species-specific. In the oocytes of polyovulatory animals such
as mouse and rat, the ratio of GDF9:BMP15 is around 5:1, with the exception of pig
oocytes, where it is much lower (0.5:1) (Crawford and McNatty 2012). In monoovular
Chapter 1: Literature Review
15
animals such as cow and sheep, the level of BMP15 mRNA in oocytes is much
higher or at least equal to GDF9 (Crawford and McNatty 2012). Homozygous mutant
GDF9 mice are sterile due to a block in follicular development beyond the primary
follicle stage (Dong, et al. 1996), and display 2-3 fold increased in the levels of LH
and FSH (Erickson and Shimasaki 2000). The size of oocytes inside the primary
follicles of these mutant mice is much larger than control mice (Erickson and
Shimasaki 2000). Moreover, in the granulosa cells there is a lack of FSH receptors, a
decrease in mitosis, overexpression of KL and no apoptosis (Elvin, et al. 1999b).
However, BMP15 is not essential for mouse fertility. BMP15 null mice demonstrate
only a mild reduction in ovulation and fertilization rates (Yan, et al. 2001). Double
homozygote mice (BMP15 -/-, GDF9 -/-) have the same ovarian phenotype with
homozygous mutant GDF9 mice (Yan, et al. 2001).
On the other hand, GDF9 and BMP15 deficient sheep (through passive immunization
or homozyogus for naturally occuring mutations) are sterile (Galloway, et al. 2000,
Hanrahan, et al. 2004). Interestingly, heterozygous GDF9 and BMP15 sheep have an
increased incidence of mutiple pregnancies and ovulation due to increased LH
sensitivity in secondary follicles that leads to a rise in the number of antral follicles
(Galloway, et al. 2000, Hanrahan, et al. 2004, Juengel, et al. 2004). In humans, rare
mutations and genetic variations in GDF9 may contribute to PCOS (Laissue, et al.
2006, Wang, et al. 2010) and dizygotic twinning (Montgomery, et al. 2004, Palmer, et
al. 2006), and low ovarian reserve (Dixit, et al. 2005, Wang, et al. 2013), whilst
mutations in BMP15 lead to premature ovarian failure (Di Pasquale, et al. 2004). In
contrast, BMP6 has shown no major effect on ovarian function and follicular
development. Homozygous null BMP6 mice are fertile with normal offspring
(Solloway, et al. 1998), but BMP6 plays a role in regulating steroidogenesis by
reducing the FSH-stimulated progesterone level without affecting FSH-induced
estradiol synthesis (Otsuka, et al. 2001a).
1.4.4 TGF-β Superfamily Signalling
The TGF-β superfamily proteins bind as homo or heterodimers to their type-I or type-
II receptors. These receptors have a serine/theronine protein kinase domain which
bind together and act as a dimeric assembly factor. Phosphorylated intracellular
Chapter 1: Literature Review
16
receptor-regulated signal transducers, known as SMADs, transport the signals into
the nucleus of the cells (Massague 2000). SMADs are nuclear proteins and interact
with transcription factors. They control the expression of genes, either by activation
or repression of gene expression.
Both BMP15 and GDF9 activate the type-II receptor (BMPR2) before they trigger the
SMADs. BMP15 also activates the ALK 6 receptor whilst GDF9 activates the ALK 5
receptor (Kaivo-Oja, et al. 2005, Vitt, et al. 2002). The activation of BMPR2 and ALK6
by BMP15 phosphorylates SMAD 1/5/8 which assembles with co-SMAD 4 (Moore, et
al. 2003), whilst GDF9 triggers the activation of SMAD 2/3 which cooperates with co-
SMAD 4 (Kaivo-Oja, et al. 2005, Mazerbourg, et al. 2004), before they translocate to
the nucleus to regulate specific genes (Fig. 1.3). Moreover, the TGF-β family and
BMPs may also utilize the MAPK (mitogen-activated protein kinase) pathway (Moore,
et al. 2003). It has been reported that BMP2 activates p38 MAPK, c-Jun N-terminal
kinase (JNK) and triggers the activation of alkaline phosphatase activity (Nohe, et al.
2002). Both BMP2 and BMP4 also activate the nuclear factor-kB (NFKB) pathway
(Mohan, et al. 1998).
There is a strong synergistic effect between GDF9 and BMP15 on granulosa cell
proliferation, inhibition of FSH-stimulated progesterone production and stimulation of
inhibin production (McNatty, et al. 2005a). The combination of mature regions of
mouse GDF9 and human BMP15 at a low dose induces a synergistic interaction
which is mediated by SMAD 2/3 signalling (Mottershead, et al. 2012). This
synergisitic interaction also involves the EGF receptor-activated ERK1/2 (also known
as MAPK3/1) signalling pathway (Mottershead, et al. 2012). In sheep, the synergistic
effect of ovine GDF9 and BMP15 involves an initial interaction with BMPR2, and
BMPR1 is either recruited later or is already associated with BMPR2, to induce this
effect (Edwards, et al. 2008).
Interestingly, there are species differences in physiological functions and signalling of
GDF9 and BMP15. It has been shown that both ovine and murine GDF9+BMP15
have a synergistic effect on [3H] thymidine uptake on rat granulosa cells through
SMAD 2/3 (Reader, et al. 2011). However, there is a divergency in non-SMAD
Chapter 1: Literature Review
17
signalling between these two species. Inhibitors of NFKB and p38-MAPK pathways
had no effect on murine GDF9+BMP15 but had a significant effect on ovine
GDF9+BMP15 stimulation of [3H] thymidine uptake (Reader, et al. 2011). Whilst,
addition of the JNK pathway inhibitor has no effect on ovine GDF9+BMP15 it has a
significant effect on murine GDF9+BMP15 on [3H] thymidine uptake (Reader, et al.
2011). In addition, murine GDF9+BMP15, but not ovine GDF9+BMP15, also utilise
the ERK-MAPK signalling pathway (Reader, et al. 2011).
Another combination of GDF9 and BMP15 is in the heterodimers form. Heterodimers
of GDF9:BMP15 use the BMPR2 in order to activate and signal through
phosphorylation of SMAD2/3 (Peng, et al. 2013), athough the existence and function
of a GDF9:BMP15 heterodimers is controversial (Mottershead, et al. 2013) (See
section 1.4.5 for GDF9 and BMP15 heterodimer structure). Heterodimers of
GDF9:BMP15 activate BMPR2 and ALK4/5/7 to trigger SMAD2/3 phosphorylation
with ALK6 co-receptor in mouse and human granulosa cells (Peng, et al. 2013).
Putative heterodimers of recombinant human or mouse GDF9:BMP15 enhance
granulosa cell activation ~3000 fold compared to BMP15 homodimers, and ~10-30
fold compared to GDF9 homodimers, as well as increase the expression of genes
such as Ptx3, Ptgs2 and Has2 in the ECM, compared to GDF9 and BMP15
homodimers alone or in combination (Peng, et al. 2013).
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18
Figure 1.3 Schematic representations of GDF9 and BMP15 homodimers and
putative GDF9:BMP15 heterodimers signalling in regulating cumulus and
granulosa cell functions. Adapted and modified from (Peng, et al. 2013).
1.4.5 Structure and Production of In vitro Recombinant GDF9 and
BMP15
The most common method to date of treating COCs with OSFs is by co-culturing
them with DOs. However, it is impractical to use this procedure in human IVF clinics
due to the limited number of DOs available. Therefore, it is important to produce the
bioactive recombinant GDF9 and BMP15 proteins to be used practically.
Chapter 1: Literature Review
19
GDF9 and BMP15 both consist of a pro-region and mature region (Fig. 1.4). The pro-
region is important for activities such as folding, formation of the disulfide bond and
regulation of protein bioactivity (Gray and Mason 1990, Harrison, et al. 2011).
To date, it is still unclear what forms of GDF9 and BMP15 are secreted by the oocyte
into the cumulus cells, and whether the form produced by oocytes matured in vitro is
similar to what may occur in vivo. In sheep follicular fluid, the majority of GDF9 and
BMP15 is detected as pro-protein (McNatty, et al. 2006), whereas sheep oocytes
secrete the mature domains of GDF9 and BMP15 during IVM (Lin, et al. 2012).
Mouse in vitro matured oocytes secrete GDF9 as a mixture of the unprocessed pro-
protein and mature domain (Gilchrist, et al. 2004b). Rat IVM oocytes secrete the
mature domains of GDF9 and BMP15 (Lin, et al. 2012).
Both GDF9 and BMP15 proteins lack a conserved cysteine residue found within the
mature region of other members of the TGF-β superfamily, and therefore the
dimerization of mature regions occurs non-covalently and lacks the intersubunit
disulphide bond (McPherron and Lee 1993). In theory, this may readily facilitate the
formation of GDF9:BMP15 heterodimers.
Chapter 1: Literature Review
20
Figure 1.4 The forms of mouse GDF9 and BMP15. Un-processed protein/pro-
protein of GDF9 and BMP15 consist of two structures; the pro-region and mature
region. The molecular scale of un-processed GDF9 and BMP15 is ~65 KDa. During
processing, the pro and mature regions are separated at their protease processing
site. Processed mouse GDF9 from transfected 293H cells is generally secreted in the
media as a dimer of a pro-mature complex. The molecular size of the pro-region of
GDF9 is ~42 KDa and mature region ~17 KDa. Mouse BMP15 from transfected 293
cells is secreted as monomers of the pro-region (~40 kDa) or mature region (~28 kDa
for glycosylated mature region and ~24 KDa for un-glycosylated) or pro-protein (~65
kDa). Since the regions are attached only by electrostatic interactions (non-covalent
bonding), GDF9 and BMP15 may easily interact with each other, making
heterodimers. Adapted and modified from (McIntosh, et al. 2008).
Chapter 1: Literature Review
21
In general, recombinant GDF9 and BMP15 are produced in vitro using a human
embryonic kidney cell line (HEK-293 T) as host cells. Either mouse, sheep or human
GDF9 and BMP15 full length cDNA is cloned into the pEFIRES-P expression vector
and transfected into the host cells using fugene 6 transfection reagent. The cells that
express high levels of the recombinant protein (assessed by increasing the
concentration of puromycin) are selected and the levels of GDF9 and BMP15 are
measured in comparison with a standard curve of Escherichia coli expressed oGDF9
or oBMP15 (Kaivo-Oja, et al. 2003, McNatty, et al. 2005a). This is the methodology
that was used over the previous decade, however, the GDF9 and BMP15 produced
by this method are not pure. Nevertheless, this partially purified GDF9 and BMP15
has been used in cattle and mouse IVM media, and they improved the quality of in-
vitro matured oocytes and thus subsequent embryo development and fetal survival
rate (Hussein, et al. 2011, Hussein, et al. 2006, Yeo, et al. 2008).
In the present study, novel purified recombinant human and mouse GDF9 and
recombinant human BMP15 was used (produced in-house by Drs D.Mottershead and
Assoc Prof R. Gilchrist) Briefly, BMP15 and GDF9 plasmids were transfected into
host cells (HEK-293T cell lines) which were cultured in Dulbecco’s modified eagles
medium (DMEM-Hams F12). An 8-histidine tag (His8-tag) and a strep II epitope tag
at the N terminus of the pro-region were used for purification of the pro-mature
complex (Fig. 1.5). The resultant BMP15 protein was secreted into the serum-free
media with a processing efficiency of the pro and mature regions of over 90%. The
BMP15 or GDF9-containing production media was then subjected to immobilized
Ni2+ based ion-affinity purification targeting the His-tag. This methodology generates
highly purified GDF9 and BMP15 preparation in the form of pro-mature complex (Fig.
1.5) (Pulkki, et al. 2011).
Chapter 1: Literature Review
22
Figure 1.5 The structure of GDF9 and BMP15-post purification (pro-mature
complex/processed protein) which consist of dimers of mature regions and
associated pro-regions. SS: Strep-tag. (David Mottershead: personal
communication)
1.5 FIBROBLAST GROWTH FACTORS (FGFS)
Another family of proteins that are secreted by the oocyte are the fibroblast growth
factors (FGFs). FGFs consist of 22 members grouped into 11 subfamilies, and have
five known FGF receptor genes (Sleeman, et al. 2001). The expression of FGFs
differs among species. FGF2 is synthesized by immature oocytes in bovine
primordial follicles, and activates follicle growth via stimulation of granulosa cell
proliferation and follicular basement membrane synthesis (van Wezel, et al. 1995).
Whilst the expression of FGF2 starts in early gonad development in the rat (Koike
and Noumura 1994), FGF8 is expressed in mouse antral follicles but was not
detected in the fetal rat (Valve et al., 1997) or in adult human ovary (Valve et al.,
2000). However, the expression FGF8 is detected in bovine preantral follicles
Chapter 1: Literature Review
23
(Buratini, et al. 2005a, Buratini, et al. 2005b). FGF17 is expressed in primordial,
primary and antral follicles and acts as a potential mediator of granulosa cell
differentiation by suppressing follicular steroidogenesis (Machado, et al. 2009).
Interestingly, FGF17 was also found abundantly in the granulosa and theca cells of
atretic follicles, indicating that it may be involved in the process of follicle atresia
(Machado, et al. 2009).
Recently, it has been demonstrated that FGF8 in combination with BMP15 enhances
the metabolism of glucose (glycolysis) in mouse cumulus cells (Sugiura, et al. 2007).
The products of glycolysis, such as pyruvate, are important in oocyte development
(Sugiura, et al. 2007). Another member of the FGF family, FGF10, improves the
blastocyst rate in cattle when added during the IVM phase (Zhang, et al. 2010). The
addition of exogenous FGF10 also increases BMP15 mRNA in cultured bovine
oocytes (Zhang, et al. 2010).
1.6 OOCYTE METABOLISM
The cumulus and granulosa cells surrounding the oocyte are essential to support
oocyte metabolism and its capacity to undergo maturation, fertilization and further
development. In the absence of cumulus cells, the immature oocyte demonstrates
undetected levels of glycolytic activity (Zuelke and Brackett 1992) due to being
incapable to utilize glucose (Biggers, et al. 1967). The oocye depends on somatic
cells to uptake amino acids and transport them through gap junctional channels to
the oocyte (Colonna and Mangia 1983). Only pyruvate and oxaloacetate are able to
be used by oocytes if no cumulus cells are present (Biggers, et al. 1967).
During maturation, glucose activity increases linearly with oocyte maturation. Mature
oocytes have significantly higher glycolytic activity compared to immature oocytes
(Spindler, et al. 2000). During fertilization (fusion and decondensation) the glycolytic
and pentose phosphate pathway (PPP) activities increase significantly (Urner and
Sakkas 1999).
Chapter 1: Literature Review
24
Glucose
ROS
GSH GSSG
TCA
Cycle
ATP
Oxidative
phosphorylation
Pyruvate
(Sutton-McDowall et al 2010 Reprod)
PPP
NADPH
NADPH
PRPP
Meiotic
regulation
Glucose
PPP
Glucose
Oocyte
Cumulus CellGlycolysis
Pyruvate
Pyruvate
Lactate
Lactate
+ FSH
HBP ECM
expansion
O-linked
glycosylation
Post-translation
modifications
+ FSH
Figure 1.6 Glucose utilization in cumulus cells and oocytes. Glucose can be
utilized by cumulus cells via glycolysis, the pentose phosphate pathway (PPP), and
the hexosamine pathway (HBP). The end product of glucose utilization via glycolysis
is lactate or pyruvate. Pyruvate is converted to Acetyl CoA and further oxidised by
the TCA cycle in mitochondria, followed by oxidative phosphorylation in the
intermembrane of mitochondria to produce adenosine triphosphate (ATP) via
electron transfer. The final product of the HBP is hyaluronic acid for extracellular
matrix expansion of CCs. The PPP utilizes glucose to produce glyceraldehydehyde
3-P and fructose 6-P, which can be re-utilized in glycolysis. One of the most
important products of the PPP is phosphoribosylpyrophosphate (PRPP) which is
used for purine synthesis and is involved in oocyte nuclear maturation. Another
important product of the PPP is the reducing agent nicotinamideadenine dinucleotide
phosphate(NADPH), involved in glutathione metabolism for homeostasis in cells.
Adapted and modified from (Sutton-McDowall, et al. 2010). FAD= flavin adenine
dinucleotide; GSG= reduced glutathione; GSSG= oxidised glutathione.
NAD=Nicotinamide adenine dinucleotide.
Chapter 1: Literature Review
25
Glycolytic activity contributes to ATP production since in this pathway, glucose is
converted into pyruvate as a source of Acetyl CoA for the TCA cycle where it is
further metabolised in the mitochondria by oxidative phosphorylation (Downs and
Utecht 1999, Dumollard, et al. 2007). In COCs, phosphofructokinase, as a key
enzyme for glycolytic activity, has the highest activity in glucose metabolism followed
by glucose-6-phosphate dehydrogenase (for PPP). It has been shown that there is a
correlation between low glycolytic activity and developmental competence of COCs.
The glycolytic activity is much lower in DOs compared to COCs (Downs and Utecht
1999). Oocytes derived from pre-pubertal cattle have lower blastocyst development
rates and also show a reduction and delay of glycolytic activity during maturation
compared to those derived from adults (Steeves and Gardner 1999). Moreover, in
vitro matured cat oocytes have significantly lower glycolytic activity and glucose
oxidation compared to in vivo counterparts (Spindler, et al. 2000). It has also been
shown that oocytes that utilize more glucose (≥ 2 pmol/oocyte/hr) during maturation
have a significantly higher blastocyst development rate compared to those which
utilize less glucose (0.1-2 pmol/oocyte/hr) (Spindler, et al. 2000).
The major product of the hexosamine pathway is hyaluronic acid which is important
to build ECM in cooperation with FSH-induced cumulus expansion (Sutton-McDowall,
et al. 2004). Supplementing IVM medium with glucosamine as an alternative hexose
substrate reduces significantly pig and cattle embryo development. However, the
effect of glucosamine induced reduction in developmental competence can be
reversed by using the inhibitor of o-linked glycosylation, as one of hexosamine
pathway substrates (Sutton-McDowall, et al. 2006). The pentose phosphate pathway
(PPP) is another pathway involved in glucose metabolism, and produces the
reducing agent NADPH which is a key regulator of glutathione metabolism (see
below). The PPP is also involved in meiotic maturation, as it synthesises
phosphoribosyl pyrophosphate (PRPP) (Downs, et al. 1996, Downs and Utecht
1999). Refer to Fig. 1.6.
1.6.1 Glutathione
Glutathione has an important role in the cell as a defence against oxidative stress.
Glutathione has two forms: the reduced form (GSH) and the oxidized form (GSSG).
Chapter 1: Literature Review
26
The thiol groups of GSH consist of cysteine, proline and glutamine which can react
with hydrogen peroxide and be oxidised by glutathione oxidase into GSSG. In turn,
GSSG in the presence of NADPH can be converted by glutathione reductase back
into GSH (Meister and Anderson 1983). A direct correlation does not always appear
between GSH and GSSG concentrations. For example, a reduction in the
concentration of GSH is not always correlated with an increase in GSSG, because
the thiol groups can also bond with other molecules.
2GSH+H2O2 GSSG+ 2H2O Glutathione oxidase GSSG+NADPH+H+ 2GSH+NADP+ Glutathione reductase
GSH concentration reaches a peak in mature oocytes, with the concentration in MII
mouse oocytes approximately double the concentration of GV stage oocytes
(Perreault, et al. 1988, Yoshida, et al. 1993). The quality of oocytes is also related to
the concentration of GSH. It has been demonstrated that in vivo matured oocytes
contain more GSH compared to in vitro matured oocytes, which may be due to the
higher concentration of oxygen during IVM (Brad, et al. 2003, Luberda 2005).
Another function of GSH during oocyte maturation is to maintain meiotic spindle
structure to ensure the normal development of zygotes (Yamauchi and Nagai 1999,
Yoshida, et al. 1993). Exposure of matured oocytes during fertilization to diamide
(specific GSH oxidant) produces abnormal female pronuclei (Zuelke, et al. 1997).
Several low molecular weight thiol compounds such as cysteamine (2-
mercaptoethylamine, MEA) and N-acetylcysteine (NAC) promote cystine, which is
the only source of mammalian cells for GSH synthesis uptake. Thus these
compounds increase intracellular GSH biosynthesis (Issels, et al. 1988).
Supplementing IVM medium with cysteamine increases the intracellular
concentration of GSH in the oocyte and improves blastocyst development in cattle
(de Matos, et al. 1995), buffalo (Anand, et al. 2008, Gasparrini, et al. 2003), sheep
(de Matos, et al. 2002) and goat (Romaguera, et al. 2010).
Chapter 1: Literature Review
27
1.6.2 Role of OSFs in Cumulus and Oocyte Metabolism
During maturation there are several genes involved in glycolysis such as Eno1,
Pkm1, Tpi, Aldoa, Ldh1 and Pfkp, which are upregulated predominantly in the
cumulus cells near the antrum (Sugiura, et al. 2005). Mouse OOXs display
decreased expression of cumulus cell genes encoding glycolytic enzymes; however,
if the OOXs are co-cultured with fully grown oocytes from antral follicles, the activity
is restored (Sugiura, et al. 2005). In contrast in the presence of FSH, there is no
difference in glycolytic activity in cumulus cells among intact bovine COCs, OOXs or
OOXs co-cultured with DOs (Sutton, et al. 2003b).
Co-culture of COCs with DOs increases GPX1 gene expression (related to the
production of glutathione peroxidise as an antioxidant in oocytes), and enhances the
expression of StAR, which contributes to nucleus and cytoplasmic maturation (Dey,
et al. 2012). Bovine oocytes matured in the presence of the mature form of BMP15
showed a significant increase in glucose uptake and in mRNA encoding GLUT1 and
GFPT1 at 22 hours, and GFPT2 at 12 hours and 22 hours culture, but interestingly
did not increase lactate production (Caixeta, et al. 2013). Whilst oocytes matured
with the pro-mature complex form of BMP15 showed that even though there is no
increase in glucose uptake, there is a significant increase in oocyte NAD(P)H and
FAD++ autofluorescence which is related to increased ATP production (Sutton-
McDowall, et al. 2012) structure and production of in vitro recombinant GDF9 and
BMP15.
1.7 IN VITRO OOCYTE MATURATION
In vitro maturation of cattle oocytes is used to reduce the costs of commercial
embryo production and transfer (Brackett, et al. 1982), decrease the generation
interval, increase the quality of offspring and further embryogenesis and assisted
reproduction research (Hoshi 2003). In 2005, the annual number of viable cattle
offspring derived from IVM was about 133 000 (Thibier 2006). In contrast, in humans,
the number of babies born from IVM is reported in the literature at only around 1300
births (Suikkari 2008).
Chapter 1: Literature Review
28
Compared to conventional IVF, implantation, clinical pregnancy and live birth rates
are significantly lower using IVM (Gremeau, et al. 2012). In all studies reported so
far, babies born through IVM have normal body weight, height and head
circumference compared to IVF counterparts (Cha, et al. 2005) and the general
population (Soderstrom-Anttila, et al. 2006). However, some studies have also found
that 19% of IVM babies have minor neuropsychological developmental delays at the
age of 12 months, but by 2 years of age the development was within the normal
range compared to natural born children (Soderstrom-Anttila, et al. 2006). Looking at
obstetric complications, around 30-40% of pregnancies from IVM cycles ended with
Caesarean section (Soderstrom-Anttila, et al. 2006). However, it is difficult to
speculate whether these complications are due directly to IVM, since preterm
delivery, placenta praevia, gestational diabetes and pre-eclampsia also occur
significantly more often in IVF pregnancies compared to natural pregnancies
(Jackson, et al. 2004).
1.7.1 Selection of Oocytes for In Vitro Maturation
In animal embryo production, the diameter of the follicle and oocyte, the presence of
cumulus cells, the homogeneity of the cytoplasm and maternal age all affect the
maturation of oocytes (Leibfried and First 1979). Romaguera et al demonstrated that
oocytes which are generated from small follicles (<3mm) in prepubertal goats
produce significantly lower blastocyst rates following fertilization compared to oocytes
from follicles with a diameter greater than 3 mm (Romaguera, et al. 2010). In cattle
and sheep embryo production, it has been reported that oocytes aspirated from
prepubescent animals have poor cytoplasmic maturation, abnormalities in the
ultrastructure, alterations in metabolic activity and slower embyro development rates
compared to adult counterparts (Succu, et al. 2007). On the other hand,
prepubescent mice have a better response to gonadotrophin stimulation than adults
and these immature oocytes readily undergo IVM and develop blastocysts.
Developmental competence post-fertilization also has a positive correlation with the
size of oocytes (Otoi, et al. 1997). This study found that bovine oocytes at a diameter
of 115 microns have full meiotic competence but have not acquired full
developmental competence. They aquire this capibility to develop into blastocysts
Chapter 1: Literature Review
29
when they reach a diameter of 120 microns. Leibfried and First (1979) reported that
there was a strong correlation between homogeneous cytoplasm and an intact
cumulus layer and a higher proportion of oocytes with homogeneous cytoplasm and
intact cumulus layers reach maturity (Leibfried and First 1979).
1.7.2 In Vitro Maturation vs In Vivo Maturation
There is clear evidence that oocytes aspirated from large antral follicles resume
spontaneous maturation in vitro (Pincus and Enzmann 1935). In in vivo matured
oocytes, the follicular environment is responsible for meiotic arrest and meiotic
resumption of the oocyte.
In vitro maturation of oocytes usually leads to the spontaneous maturation of the
nucleus, a lack of synchronization between nuclear and cytoplasmic maturation
(Succu, et al. 2007), abberant gene and protein expression in granulosa and cumulus
cells compared to in vivo matured oocytes (Dunning, et al. 2007, Kind, et al. 2013,
Richani, et al. 2013), and disregulation of oocyte-cumulus cell gap-junctional
communication (Thomas, et al. 2004). It has also been reported that mouse in vitro
matured oocytes have a different position, organization and distribution of the meiotic
spindle and cytoplasmic microtubules compared to in vivo counterparts (Sanfins, et
al. 2003). Using terminal deoxynucleotidyltransferase 3’nick end labelling (TUNEL), it
has been shown that in vitro derived porcine embryos express more TUNEL positive
cells compared to their in vivo counterparts, suggesting that the in vitro culture
system generates DNA fragmentation (Long, et al. 1998). All these factors lead to
reduced oocyte developmental competence of in vitro matured oocytes. Even though
the rate of nuclear maturation of in vitro matured oocytes is similar to that of in vivo
matured oocytes, the blastocyst rates of embryos derived from in vitro matured cattle
and sheep oocytes is less than half of in vivo matured oocytes (Leibfried-Rutledge, et
al. 1987, Thompson, et al. 1995).
1.7.3 Ooctye Secreted Factors in the In Vitro Maturation of Oocytes
It has been hypothesised that IVM oocytes may have decreased expression and
signalling of GDF9 and BMP15. Mester et al (2013) showed no expression of BMP15
protein in in vitro matured mouse oocytes. By contrast, in mouse oocytes undergoing
Chapter 1: Literature Review
30
in vivo maturation, the mature form of BMP15 is visible from 6 hours post-hCG
administration and further up-regulated at 9 hours post-hCG, but this protein is not
detected in in vitro matured oocytes (Mester, et al. 2013). Another study showed that
the stimulating effect of BMP15 on cumulus expansion occurs only after the LH/hCG
surge (Yoshino, et al. 2006), whilst in vitro matured mouse oocytes post-PMSG are
unable to replicate this effect (Yoshino, et al. 2006).
To overcome the deficiency of GDF9 and BMP15 in in vitro matured oocytes,
previous studies have used DOs as a source of native exogenous OSFs, and co-
cultured these with immature COCs. As anticipated, DOs increased oocyte
developmental competence in animals such as cow (Dey, et al. 2012, Hussein, et al.
2011, Hussein, et al. 2006), goat (Romaguera, et al. 2010) and pig (Gomez, et al.
2012). The important part of the co-culture of COCs with DOs is the ratio and volume
of medium used. Hussein et al (2011) used a ratio of 1 COC: 5 DO with the volume
0.5 DO/µl medium in order to increase bovine blastocyst rate up to 20% higher
compared to controls. Gomez et al (2012) showed that the ratio of COCs to DOs in
pig should be at least 1:4, with 0.2 DO/µl to observe the beneficial effect of native
OSFs on pig oocytes. Moreover, DOs from small follicles also have a positive effect
on development competence of IVM oocytes as well as DOs from large follicles,
indicating that DOs from small follicles are also capable of producing OSFs
(Romaguera, et al. 2010). Other than follicle size, another factor that affects the
capacity of exogenous OSFs to improve developmental competence is the timing of
addition of the DOs (Hussein, et al. 2011).
Maturing COCs in the presence of exogenous recombinant BMP15 and GDF9
significantly increased bovine blastocyst development (Hussein, et al. 2011, Hussein,
et al. 2006). Even though mouse COCs treated with GDF9 did not show an
improvement in blastocyst rate, there is a significant increase in the inner cell mass
number which led to an improvement in the fetal survival rate (Yeo, et al. 2008). The
improved blastocyst rate can be reduced in the presence of SB431542 and follistatin
which are antagonists of GDF9 and BMP15, respectively (Hussein, et al. 2006,
Romaguera, et al. 2010).
Chapter 1: Literature Review
31
1.8 CONCLUSION
It is becoming increasingly important to reduce the cost of ART, as well as to
eliminate the side effects of gonadotropin hormones. In vitro maturation of oocytes
has the potential to alleviate these problems, as it is a less invasive and cheaper
method compared to IVF. However, the success rate of in vitro matured oocytes as
measured by embryo development is much lower compared to in vivo matured
oocytes. Understanding the mechanisms that improve the quality of the in vitro
matured oocyte could improve the success rate post IVM.
It is now a well-known concept that there is an intimate communication between
oocyte and somatic cells. The oocyte secretes OSFs into the cumulus cells to
regulate the functions of cumulus cells, and in return the cumulus cells perfuse small
molecules and ions through gap junctions into the oocyte to maintain the functional
activity and developmental competence of the oocyte. Previous studies showed co-
culture of COCs with Dos, as a source of native OSFs, improved developmental
competence of in vitro matured oocyte, however, application of this method to
generate such a huge numbers of DOs is not practical in human IVF and animal
production. Therefore, the next step is to find the proteins/molecules in OSFs which
are functionally active to improve the quality of in vitro matured oocytes.
The preparations of GDF9 and BMP15 available commercially (R&D Systems)
contain different structure of proteins compared to native proteins, therefore their
roles in in vitro maturation are likely to be different. This thesis will examine the
influence of native OSFs on mouse in vitro matured oocytes and observe the
difference between commercially available GDF9 and BMP15 (R&D Systems), which
contain only the mature region of these proteins, to the pro-mature complex forms,
on in vitro matured cattle and mouse oocytes and subsequent embryo development.
This work will provide a new perspective on the importance of bi-directional
communication between oocytes and somatic cells and its implications for
improvement of in vitro matured oocytes and embryo production in domestic species
and human infertility.
Chapter 1: Literature Review
32
1.9 HYPOTHESIS AND AIMS
1.9.1 Hypothesis
Based on the literature review above, the following hypothesis is proposed:
developmental competence of in vitro matured murine and bovine oocytes can be
improved by exposure to either native OSFs, added at differing time points during in
vitro maturation, or to novel purified recombinant GDF9 and BMP15 on their pro-
mature complex forms.
1.9.2 Aims
There are three main aims of this project.
1. To examine factors affecting the capacity of exogenous oocyte secreted
factors to improve mouse embryo and fetal development post-IVM.
2. To examine the effect of a novel variant of hBMP15 (pro-mature complex vs
mature domain) compared with the mature domain of mGDF9 on metabolic
activity in oocytes and development of embryos after IVM in cattle.
3. To examine the effect of a novel variant of GDF9 and BMP15 (both in pro-
mature complex forms) and a combination of different forms (pro-mature
complex vs mature domain) and sources (mouse and human) of GDF9 and
BMP15 on developmental competence of IVM mouse oocytes.
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
33
CHAPTER 2
FACTORS AFFECTING THE CAPACITY OF
EXOGENOUS OOCYTE SECRETED FACTORS TO
IMPROVE MOUSE EMBRYO AND FETAL
DEVELOPMENT POST-IVM
(Sudiman, et al. 2014a)
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
34
2.1 ABSTRACT
The aim of this study was to determine factors affecting the efficiency of exogenous
oocyte-secreted factors (OSFs) in improving the developmental competence of
mouse in vitro matured oocytes. We hypothesised that varying native OSF exposure
would have differential effects on post-in vitro maturation (IVM) embryo and fetal
development due to the different forms that are secreted by denuded oocytes (DOs)
under different conditions. Mouse cumulus oocyte complexes (COCs) were co-
cultured with DOs from 0h or 3h of IVM. DOs were matured for 3h as either intact
COCs+/-FSH before denuding, or as DOs+FSH. COCs were fertilised and blastocyst
development was assessed on days 5 and 6, and were then either differentially
stained to determine ICM numbers or vitrified/warmed embryos were transferred to
recipients. Embryos derived from COCs+DOs had significantly improved blastocyst
rates and ICM numbers compared to controls (P<0.05). The highest response was
obtained when DOs were first added to COCs at 3h of IVM, after being pre-treated
(0-3h) as COCs+FSH. Compared to control, co-culture with DOs from 3h did not
affect implantation rates but more than doubled fetal yield (21% vs 48%; P<0.05).
GDF9 Western blot analysis was unable to detect any differences in quantity or form
of GDF9 (17 and 65 kDa) in extracts of DO at 0h or 3h. This study provides new
knowledge on means to improve oocyte quality in vitro which has the potential to
significantly aid human infertility treatment and animal embryo production
technologies.
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
35
2.2 INTRODUCTION
In vitro maturation (IVM) of oocytes is an alternative system used in assisted
reproductive technology for generating embryos in vitro, which reduces or eliminates
the need to administer gonadotropins to patients. In addition, IVM is widely used in
the domestic animal sector to further advanced-breeding technologies. However,
there is a discrepancy in the success rate of conventional in vitro matured oocytes
compared to in vivo matured oocytes. In women, it is reported that the pregnancy
rate post-IVM is less than half of the pregnancy rate post-IVF (in vitro fertilisation)
(Gremeau, et al. 2012). To overcome this challenge, it is important to understand
factors that regulate the maturation and development of oocytes which can be
implemented in clinical and veterinary scenarios to improve the success rate of IVM
(Gilchrist 2011).
Oocytes acquire developmental competence in the ovarian follicle. There are
numerous molecules, proteins and cellular processes involved in the bi-directional
communication axis between the oocyte and follicular somatic cells (Gilchrist, et al.
2004a). The oocyte regulates this communication network to a significant degree.
The oocyte secretes soluble factors (OSFs) which are transmitted to cumulus cells
via paracrine signalling to regulate a multitude of important cumulus cell processes,
including; proliferation (Gilchrist, et al. 2006, Vanderhyden, et al. 1992), apoptosis
(Hussein, et al. 2005), differentiation (Li, et al. 2000), luteinisation (Salustri, et al.
1990), metabolism (Caixeta, et al. 2013, Su, et al. 2008, Sugiura, et al. 2007, Sutton-
McDowall, et al. 2012) and expansion (Dragovic, et al. 2005, Dragovic, et al. 2007).
Another type of communication between the oocyte and cumulus cells is via gap
junctional signalling, mediated by cumulus trans-zonal cytoplasmic projections that
abut the oocyte membrane, enabling transport of cAMP, purines/pyrimidines, amino
acids and other small regulatory molecules (Eppig and Downs 1984, Salustri and
Siracusa 1983, Salustri, et al. 1989, Thomas, et al. 2004). Cumulus cells provide
signals and molecules to the oocyte to regulate meiosis and promote developmental
competence (Dey, et al. 2012, Gilchrist, et al. 2008). Removal of cumulus cells during
maturation has a significant detrimental effect on nuclear maturation, normal
fertilization and developmental competence of oocytes (Wongsrikeao, et al. 2005,
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
36
Zhang, et al. 1995). Follicle-stimulating hormone (FSH) is typically added to IVM
medium to stimulate cumulus expansion (Salustri, et al. 1989) and increase normal
fertilization rates and fetal development of IVM oocytes (Ali and Sirard 2005,
Merriman, et al. 1998).
Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15)
are two well-known soluble growth factors derived from oocytes that are important for
normal cumulus cell function and hence normal oocyte development (Gilchrist, et al.
2008). In mouse oocytes, the expression level of GDF9 mRNA is much higher
compared to BMP15 (Crawford and McNatty 2012), reflecting the relative importance
of their roles in folliculogenesis. Homozygous GDF9 mutant mice are sterile due to a
block in follicular development beyond the primary follicle stage (Dong, et al. 1996),
whereas BMP15 null mice demonstrate only a mild reduction in ovulation and
fertilization rates (Yan, et al. 2001). Homozygous GDF9 or BMP15 mutant sheep
derived either by natural mutation or active immunization are sterile, however,
heterozygous carriers of mutations in either GDF9 or BMP15 have an increased
ovulation rate and multiple pregnancies (Galloway, et al. 2000, Hanrahan, et al.
2004, Juengel, et al. 2004). GDF9 and BMP15 also play an important role in human
fertility, where several mutations of GDF9 may be potentially associated with
polycystic ovary syndrome (Wang, et al. 2010) and rare variants of GDF9 contribute
to dizygotic twinning (Palmer, et al. 2006). Moreover, rare mutations of GDF9 and
BMP15 are associated with premature ovarian failure (Chand, et al. 2006, Dixit, et al.
2005, Wang, et al. 2013).
GDF9 and BMP15 in the proteolytically processed form consist of two structural
parts: the pro-region and the mature region (McIntosh, et al. 2008). GDF9 and
BMP15 are processed and secreted as non-covalent complexes of the pro- and
mature regions, the mature region being the bioactive receptor binding region,
whereby the pro-region plays a vital and species-specific role in regulating bioactivity
(Al-Musawi, et al. 2013, Mottershead, et al. 2008, Simpson, et al. 2012). Immunizing
mice against the pro-regions of GDF9 or BMP15 caused ovary abnormalities and
smaller litter sizes (McIntosh, et al. 2012).
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
37
However, the exact forms of GDF9 and BMP15 secreted by oocytes in vivo and in
vitro are unclear. Mouse in vitro matured oocytes secrete GDF9 as a mixture of the
unprocessed pro-protein and mature domain (Gilchrist, et al. 2004b). Rat IVM
oocytes secrete the mature domains of GDF9 and BMP15 (Lin, et al. 2012). Sheep
follicular fluid contains GDF9 and BMP15 proteins in the unprocessed pro-protein
form only (McNatty, et al. 2006), whereas sheep oocytes secrete the mature domains
of GDF9 and BMP15 during IVM (Lin, et al. 2012). To our knowledge, the only
commercially available forms of GDF9 and BMP15 produced in a recombinant
mammalian cell expression system are from R&D Systems and these consist of
purified mature domains only.
IVM COCs have aberrant gene expression and altered matrix protein profiles in
cumulus cells compared to in vivo matured oocytes (Dunning, et al. 2007, Kind, et al.
2013, Richani, et al. 2013), and in vitro matured oocytes also have notably
decreased BMP15 protein compared to in vivo matured oocytes (Mester, et al. 2013).
Currently, studies show that addition of exogenous native OSFs during IVM
significantly improves subsequent goat (Romaguera, et al. 2010), cow (Dey, et al.
2012, Hussein, et al. 2011, Hussein, et al. 2006) and pig (Gomez, et al. 2012)
embryo development, and recombinant GDF9 and BMP15 in their pro-mature form
improve in vitro matured cattle (Hussein, et al. 2011, Hussein, et al. 2006, Sutton-
McDowall, et al. 2012) and mouse oocytes (Yeo, et al. 2008). In addition, differing
degrees of improved bovine oocyte competence were observed depending on the
temporal pattern of addition of OSFs during IVM (Hussein, et al. 2011). In the current
study we examine factors that affect the production of native OSFs by mouse
oocytes, such as the timing of addition of denuded oocytes (DOs), the presence of
cumulus cells and FSH before denuding COCs and addition to IVM, and whether
these factors affect the form of GDF9. Outcomes examined were embryo
development, cryotolerance of blastocysts and fetal measures following embryo
transfer.
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
38
2.3 MATERIALS AND METHODS
All chemicals and media were purchased from Sigma Chemical (St Louis, MO, USA)
unless indicated otherwise.
2.3.1 Isolation and maturation of COCs
Mice used in this study were maintained in the Animal House, Medical School,
University of Adelaide. This study was approved by the local animal ethics committee
and conducted in accordance with the Australian Code of Practice for the Care and
Use of Animals for Scientific Purposes.
Twenty-one to twenty-eight day-old SV129 mice were injected intraperitoneally with 5
IU equine chorionic gonadotropin (eCG; Folligon, Intervet, Castle Hill, Australia), and
ovaries were collected 46-48 hours (h) later. Ovaries were cleaned of any connective
tissues and placed in HEPES-buffered αMEM (handling medium, GIBCO, USA)
supplemented with 3 mg/ml fatty-acid free bovine serum albumin (FAF BSA, MP
Biomedicals, USA), 1 mg/ml fetuin and 50 µmol 3-isobutyl-1-methylxanthine (IBMX).
Antral follicles were punctured with 27-gauge needles and immature COCs collected
in handling medium, then washed twice with handling medium minus IBMX. Only
COCs with compact cumulus cells (CCs) were taken. The rationale behind the use of
IBMX was to maintain all COCs in the same nuclear stage before transfer into
maturation medium or being denuded. For the control group, 20 COCs were cultured
in 50 µl IVM drops [(αMEM supplemented with 3 mg/ml FAF BSA, 1 mg/ml fetuin and
50 mIU/ml FSH (Puregon, Organon, Oss, Netherlands)] overlaid with mineral oil in 60
mm Petri dishes (Falcon, Becton Dickinson, USA) for 17-18 h at 370C in a humidified
atmosphere of 5% CO2 in air. For treatment groups, 20 COCs were cultured with 50
DOs in each 50 µl IVM drop (1 DO/µl).
2.3.2 Generation of denuded oocytes as a source of native OSFs
DOs were generated by vortexing COCs for ~2 minutes in αMEM handling medium in
order to remove the CC.
The DOs were then subjected to three processing methods used to produce native
OSFs before co-culture with immature COCs (refer to Fig. 2.1):
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
39
1. COCs were denuded at 0 h before co-culture of 50 DOs with 20 COCs in IVM
medium containing FSH. For COCs+DOs, IBMX was not present in the
handling medium, and therefore these DOs were at the GVBD stage. This
group was utilized to assess the benefits of DOs as a source of native OSFs
and will be referred to as COCs+DOs. By contrast, for COCs+DOs (0h [GV]),
IBMX was present in the handling medium and therefore these DOs were at
the GV stage.
2. COCs were matured first for 3 h in separate IVM medium containing FSH.
After 3 h of IVM, COCs were denuded and 50 DOs were co-cultured with 20
COCs for another 14-15 h in IVM medium containing FSH (Fig. 2.1 A). This
group is based on the design by Hussein et al (2011), and was utilized to
examine temporal, cumulus cell and FSH effects on the production of native
OSFs. This group is referred to as COCs+DOs at 3h+FSH+CC.
3. Intact COCs were matured first for 3 h in separate IVM medium without FSH
(Fig. 2.1 B). After 3 h of IVM, COCs were denuded and 50 DOs were co-
cultured with 20 COCs for another 14-15 h in IVM medium containing FSH.
This group assessed the production of native OSFs in the absence of FSH
during the first 3 h of maturation and is referred to as COCs+DOs at 3h-
FSH+CC.
4. COCs were denuded at 0 h and then matured for 3 h as DOs in separate IVM
medium containing FSH, then 50 of these DOs were co-cultured with 20
COCs for another 14-15h in IVM medium containing FSH (Fig. 2.1 C). This
group is referred to as COCs+DOs at 3h+FSH-CC and was designed to
assess the effect of CCs on production of native OSFs.
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
40
Figure 2.1 Schematic illustration of methods of producing native OSFs based
on pretreatment of oocytes with and without cumulus cells and FSH. A. Intact
COCs cultured for 3 h in separate IVM medium containing FSH. After 3 h, COCs
were denuded of CCs and the resultant DOs were then co-cultured with a separate
cohort of COCs + FSH for 14-15 h, before the COCs underwent standard IVF and
embryo culture (3h+FSH+CC). B. COCs pre-cultured for 3 h in separate IVM medium
without FSH, before denuding and co-culture (3h-FSH+CC). C. COCs were denuded
at 0 h and then pre-cultured for 3 h in separate IVM medium containing FSH, then
these DOs were co-cultured with COCs for 14-15h (3h+FSH-CC).
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
41
2.3.3 Oocyte nuclear maturation
An experiment was performed to assess whether adding native OSFs affects the
timing of oocyte nuclear maturation. At 12 h and 15 h of IVM, COCs were denuded
and fixed in paraformaldehyde for 30 minutes, then stained with DAPI. The presence
of the first polar body was determined using an upright microscope (Nikon, TE 2000-
E) under UV light.
2.3.4 In vitro fertilization
Sperm from CC x Bagg, A strain (CBA) F1 male mice aged 6-24 weeks and of
proven fertility were incubated for 1 hour in IVF medium (COOK® IVF, Research
medium, Catalogue No: K-RVWA- 50, Australia) under 5% CO2 at 370C for
capacitation. Post-IVM, 5-10 COCs were transferred into a 90 µl equilibrated IVF
drop overlaid with mineral oil and then 10 µl of capacitated sperm were added into
the drops. Three hours post-fertilization under 5% CO2 at 370C, the presumptive
zygotes were denuded of sperm and cumulus cells. Insemination day was counted
as day 1 (0 h).
2.3.5 In vitro culture of embryos
Five to ten presumptive zygotes were cultured in 20 µl drops of equilibrated culture
media (COOK® IVC, Research medium, Catalogue No: K-RVCL-50, Australia)
overlaid with mineral oil under 6% CO2, 5% O2 and 89% N2. Five to ten embryos
were cultured for six days, with cleavage rates assessed on day 2 (24 h post-
fertilization) and blastocysts assessed on day 5 (96-100 h post-fertilization) and day 6
(120-24 h post-fertilization).
2.3.6 Differential staining of blastocysts
Differential staining was performed on day 6 blastocysts to assess inner cell mass
(ICM) and trophectoderm (TE) cells. Briefly, expanded, hatching and hatched
blastocysts were placed into pronase (5 mg/ml) at 370C until the zona dissolved. The
zona-free blastocysts were incubated in 10 mM trinitrobenzene sulfonic acid (TNBS)
in 0.4% polyvinyl alcohol (PVA) in phosphate-buffered saline (PBS) at 40C for 10
minutes then washed twice before being transferred into 0.1 mg/ml anti dinitrophenol-
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
42
BSA antibody at 370C for 10 minutes. Embryos were then incubated in 10 µg/ml
propidium iodide for 5-10 minutes at 370C, followed by 4 µg/ml Hoechst 33342 in
96% ethanol at 40C overnight. The blastocysts were transferred onto a glass
microscope side, covered by a cover slip and assessed immediately under UV
fluorescence using an upright microscope (Nikon, TE 2000-E, excitation, 340-380
nm; emission, 440-480 nm), where ICM cells appeared blue and TE cells appeared
pink.
2.3.7 Vitrification and warming of blastocysts
Hatching and expanded blastocysts on day 6 were vitrified and then within 1-2 weeks
those blastocysts were warmed and assessed for cryotolerance, prior to embryo
transfer. Vitrification and warming solutions were prepared in HEPES-buffered
αMEM. The vitrification solution consisted of 2.3 M dimethylsulfoxide (DMSO) + 3 M
ethylene glycol supplemented with 0.75 M sucrose. The corresponding equilibration
solution contained half the concentration of cryoprotectants of the vitrification solution
and no sucrose. Vitrification steps were performed at 370C. Blastocysts were placed
in equilibration solution for 3 minutes then transferred to vitrification solution, with
minimal transfer of equilibration solution (less than 3 µl), placed on a fibre-plugTM
(CVM kit, Cryologic, Victoria, Australia) and touched on a pre-cooled steel block in
liquid nitrogen, and then sealed in a pre-cooled straw (CVM kit, Cryologic) for storage
in liquid nitrogen. The time taken from leaving equilibration solution to vitrification on
the block was 30 - 40 sec. Vitrified blastocysts were warmed at 370C and sequentially
passed through 3 solutions of sucrose (0.3 M, 0.25 M and 0.15 M) in HEPES-
buffered αMEM for 5 minutes each. Blastocysts were held in culture medium for 2-3 h
and then blastocyst morphology was assessed before being transferred into foster
mothers. The criteria used for blastocyst cryosurvival were as follows; normal:
blastocysts expanded with no signs of lysis, collapse or degeneration; abnormal:
blastocysts with signs of lysis, collapse and degeneration (Gautam, et al. 2008).
2.3.8 Embryo transfer and assessment of fetal outcomes
Vitrified/warmed blastocysts assessed as normal were transferred into pseudo
pregnant recipients (Swiss female mice aged 12-20 weeks, mated with vasectomised
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
43
F1 CBA males). On day 3.5 of pseudo pregnancy, recipients were anaesthetized with
2% Avertin (0.015 ml/g body weight) prior to embryo transfer. For each recipient, six
normal expanded or hatching blastocysts post-vitrification/warming were transferred
to each uterine horn, with control embryos in one horn and treatment embryos in the
other. Eight replicates were performed with a total of 96 blastocysts transferred into 8
recipients. The number of implantation sites, fetuses, fetal and placental weights and
fetal crown to rump lengths were assessed on day 17 post-embryo transfer.
2.3.9 Immunodetection of oocyte GDF9
DOs and oocyte-conditioned media were collected for western blot analysis of the
form and quantity of GDF9 protein. Briefly, COCs were collected and denuded at 0 h
and suspended at a ratio of 8 DOs/µl in IVM medium in Eppendorf tubes. DOs were
matured for 17-18 h under 5% CO2 at 370C. Post-IVM, DOs were pelleted by
centrifugation and oocyte-conditioned medium was transferred to a separate tube
and snap frozen in PBS. Oocytes were washed once in PBS, after which the PBS
was removed and the cells snap frozen. Oocytes and oocyte-conditioned medium
were also collected from the 3h+FSH+CC group (Fig. 2.1 A). COCs were matured
first for 3 h in IVM medium, then denuded and matured for an extra 14-15 h in fresh
IVM medium. Post-IVM, oocytes and oocyte-conditioned medium were processed as
above. Samples (30-60 DOs or media equivalent) were boiled for 3 minutes in 1xLDS
reducing buffer [15ul, 1 x NovexNuPAGE LDS sample buffer (NP0009)] with β-
mercaptoethanol (NovexNuPAGE NP0007, Carlsbad, USA) and centrifuged. The
supernatant was fractionated by SDS-PAGE for 1h at RT on 4-12% Bis-Tris pre-cast
gel (BioRad, Hercules, Ca, USA, Cat#345-0125) in MES buffer (Novex, NuPAGE,
NP0002) followed by electrotransfer at 30V overnight at 40C onto a nitrocellulose
membrane (Hybond-C Extra, GE Healthcare Life Sciences, Bucks, UK). The
membrane was incubated with biotinylated GDF9- monoclonal antibody 53 (Oxford
Brookes University, Oxford, UK) at 1:2,000 for 1hr at RT then washed, followed by a
further incubation with horseradish peroxidase-conjugated anti-mouse IgG
(SNN2004, Lot No 892329B, Biosource), at 1:10,000 for 1h at RT. Immunoreactive
proteins were detected using Lumilightchemiluminesence reagents (Roche
Diagnostics, GmbH Mannheim, Germany). Membranes were scanned on the
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
44
BioRadChemiDoc MP system and the images analysed using Image J software
(Biorad). Recombinant mGDF9 (17k) was used as a reference protein. Biotinylated
molecular weight protein standards (Cat No. MW001) were obtained from R&D
Systems, (Minneapolis, USA). Parallel controls (blanks) were undertaken with oocyte
extracts in the absence of biotinylated antibody but in the presence of HRP-IgG
antibody. Five replicates were performed to detect the form and quantity of GDF9
protein.
2.4 STATISTICAL ANALYSES
All proportional data for embryo development were arcsine transformed and analysed
using either an independent t-test or multivariate ANOVA with least significant
difference (LSD) post-hoc tests. Log transformation was performed for blastocyst cell
number when data were not normally distributed, and analysed using an independent
t-test or one way ANOVA with either LSD or Dunnet’s T3 post-hoc tests, depending
on whether the data did or did not pass the homogeneity of variance test,
respectively. Fetal measurements were analysed with independent t-tests with LSD
post-hoc tests. Oocyte nuclear maturation, post-warm survival rate of blastocysts,
and post-embryo transfer data for fetal survival and implantation rates were assessed
using a Chi-square test. All statistical analyses were performed using SPSS version
13 (SPSS Inc, Chicago, IL, USA). Differences were considered statistically significant
at P < 0.05. Data are expressed as means ± SEM.
2.5 RESULTS
2.5.1 Effect of co-culture of intact mouse COCs with native OSFs
during IVM
Exposure of intact COCs to exogenous native OSFs from DOs throughout IVM
significantly (P < 0.05) increased the cleavage rate compared to COCs cultured
alone. ICM cell numbers were also significantly increased in blastocysts derived from
COCs exposed to DOs, whereas TE and total cell numbers were not affected (Tables
2.1 and 2.2).
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
45
Table 2.1 Effect of native OSFs during IVM on subsequent embryo development
Values with different superscript within a column are statistically different (P < 0.05).
Data are presented as means ± SEM of 4 replicate experiments. Each replicate
experiment consisted of 53-60 oocytes.
1Percentage of cleaved embryos per total oocytes
2Percentage of blastocysts generated 96-100 h post-fertilization per cleaved embryo
3Percentage of blastocysts generated 100-125 h post-fertilization per cleaved embryo
4Percentage of hatching blastocysts generated 100-125 h post fertilization per
cleaved embryo
5 COCs were cultured without DOs
6GVBD-stage denuded oocytes (DO) added from t= 0h of IVM
Table 2.2 Effect of native OSFs during IVM on inner cell mass (ICM), trophectoderm
(TE) and total cell numbers (TCN) of day 6 blastocysts
Treatments ICM TE TCN
Control1
COC+DO2
18.5±0.8a
25.3±1.5b
55.8±2.1
55.5±2.5
74.3±2.6
80.8±3.5
Values with different superscripts within a column are statistically different (P < 0.05).
Data are presented as means ± SEM. Mean blastocyst cell numbers following
differential staining of 16-18 blastocysts.
1COCs were cultured without DOs
2GVBD-stage denuded oocytes (DO) added from t= 0h of IVM
Treatments No. of
Oocytes
Cleavage1
(%)
Blastocyst
on day 52
Blastoycst
on day 63
Hatching
Blastocyst
on day 64
Control5
COC+DO6
224
233
90.6±1.8a
96.1±0.8b
52.8±8.9
60.1±4.9
77.3±4.6
74.3±2.4
58.8±8.6
68.0±1.9
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
46
2.5.2 Temporal effect of co-culture of intact mouse COCs with
native OSFs during IVM
A previous study by Hussein, et al. 2011 demonstrated that there is a temporal effect
of native OSFs on bovine oocyte developmental competence. Based on this result,
we assessed mouse oocyte developmental competence following co-culture of COCs
with DOs in a temporal manner. COCs were co-cultured with DOs from 0 h (0h [GV]
stage) or with DOs precultured as COCs for 3h+FSH+CC (refer to Fig. 2.1 A).
Following co-culture of COCs with DOs from 0-17 h (0h [GV] stage), a significant
increase was observed in cleavage rate compared to controls (Table 2.3), however
there were no significant differences in blastocyst rates or ICM number (Table 2.4).
By contrast, treating COCs with DOs from 3h (3h+FSH+CC) significantly (P < 0.05)
increased blastocyst and hatching blastocyst rates measured at day 6 and
subsequent ICM cell numbers, compared to the control group (Tables 2.3 and 2.4).
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
47
Table 2.3 Temporal effect of co-culture of intact mouse COCs with native OSFs on subsequent embryo development
Values with different superscripts within a column are statistically different (P < 0.05).
Data are presented as means ± SEM of 4 replicates experiments. Each replicate experiment consisted of 37-40 oocytes.
1Refer to Fig. 2.1 for explanation of experimental design
2 Percentage of cleaved embryos per total oocytes
3Percentage of blastocysts generated 96-100 h post-fertilization per cleaved embryo
4Percentage of blastocysts generated 100-125 h post-fertilization per cleaved embryo
5Percentage of hatching blastocysts generated 100-125 h post fertilization per cleaved embryo
6COCs were cultured without DOs
Treatments Pre-treatment of
OSFs1
No. of
Oocytes
Cleavage2
(%)
Blastocyst
on day 53
Blastoycst
on day 64
Hatching
Blastocyst
on day 65
Control6
COC+DO
COC+DO
N/A
0h [GV]
3h+FSH+CCs
152
155
153
87.5±1.6a
95.5±2.2b
94.8±1.1a,b
52.2±7.2
57.5±2.6
65.5±1.7
67.4±6.0a
77.2±4.4a,b
83.4±2.6b
53.3±5.6a
66.9±2.8a,b
74.5±2.8b
Chapter 2: Factors affecting the capacity of exogenous oocyte secreted factor
to improve mouse
48
Table 2.4 Temporal effect of co-culture of intact mouse COCs with native OSFs on
inner cell mass (ICM), trophectoderm (TE) and total cell numbers (TCN) of day 6
blastocysts
Values with different superscripts within a column are statistically different (P < 0.05).
Data are presented as means ± SEM. Mean blastocyst cell numbers following
differential staining of 13-22 blastocysts.
1Refer to Fig. 2.1 for explanation of experimental design
2COCs were cultured without DOs
Treatments Pre-treatment of
OSFs1
ICM TE TCN
Control2
COC+DO
COC+DO
N/A
0h [GV]
3h+FSH+CCs
18.7±1.9a
22.0±1.7a,b
24.8±1.3b
51.1±3.4
52.4±2.4
55.7±2.7
69.8±4.3
74.4±3.5
80.5±3.4
Chapter 2: Factors affecting the capacity of exogenous oocyte secreted factor
to improve mouse
49
2.5.3 Effect of exposure of COCs to native OSFs on nuclear
maturation
Native OSFs regulate CC cGMP levels (Wigglesworth, et al. 2013) which affects
meiosis and, according to Dey, et al. 2012, co-culture of COCs with DOs increased
nuclear maturation rate in bovine oocytes. Since the highest competence treatment
group from the previous experiment was COCs co-cultured with DOs from 3h
(3h+FSH+CC), we hypothesised that an effect on oocyte nuclear maturation could
account for this enhanced oocyte quality in mice. However, in the present
experiment, there was no significant difference in the maturation rates of control
oocytes or COCs exposed to DOs (3h+FSH+CC) at 12h or 15h of IVM (Table 2.5).
Table 2.5 Effect of native OSFs added at 3 hours on oocyte nuclear maturation
Treatments Pre-treatment of
OSFs1
Maturation time
(h)
N MII oocytes
(%)
Control2
COC + DO
N/A
3h+FSH+CCs
12
15
12
15
111
74
113
78
92.8
91.9
93.8
93.6
Data are presented as means of 3 replicate experiments. Each replicate experiment
consisted of 33-39 oocytes.
1Refer to Fig. 2.1 for explanation of experimental design
2COCs were cultured without DOs
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
50
2.5.4 Temporal effects on oocyte GDF9 levels
Given the beneficial effects of pre-culture of oocytes with their CCs for 3h with FSH
on OSF-stimulated oocyte developmental competence (Table 2.4), we attempted to
quantify the amount and form of GDF9 expressed within and secreted by oocytes.
The band for the un-processed pro-protein was observed at ~65 kDa and the mature
protein at ~17 kDa, with the mature protein less abundant in oocyte extracts
compared to that observed in the recombinant mGDF9 standard (Fig. 2.2). Three
hours of pre-culture (3h+FSH+CC) had no effect on the total quantity or the
proportion of pro-versus mature-GDF9 in oocytes (Fig. 2.3). No GDF9 bands were
observed in the DO conditioned media.
Figure 2.2 GDF9 Western blot comparison of denuded oocyte (DO) extracts at
0h and 3h (3h+FSH+CC) of pretreatment. mGDF9 refers to recombinant mGDF9
standard. Blank refers to DO sample in the absence of the GDF9 primary antibody.
The 65kDa band corresponds to the unprocessed GDF9 pro-protein while the 17kDa
band corresponds to the mature domain. The 90 kDa-180 kDa bands seen in all
samples are attributed to the non-specific presence of biotinylated proteins in the cell
extracts.
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
51
Figure 2.3 Quantitative analysis of Western blot denuded oocyte extracts at 0
and 3h (3h+FSH+CC) of pretreatment. Data from 5 separate experiments. The
Western blot image was quantified using BioRadChemiDoc MP system and analysed
using Image J software (Biorad)
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
52
2.5.5 The role of FSH and cumulus cells in the production of native
OSFs
We hypothesized that FSH and cumulus cells are two factors that affect the
production of native OSFs. In this experiment, DOs were generated using different
methods (refer to Fig. 2.1). Following co-culture of COCs with precultured DOs
(3h+FSH+CC; refer to Fig. 2.1 A), a significantly increased blastocyst rate on day 5
was observed compared to control, which contributed to an improvement in ICM
number compared to control (Tables 2.6 and 2.7). DOs that were precultured as
intact COCs but without FSH (3h-FSH+CC, refer to Fig. 2.1 B) did not stimulate
development competence in their co-cultured COCs; these exhibiting embryo
developmental parameters equivalent to the control and ICM numbers significantly
lower than those stimulated with FSH (3h+FSH+CC). Interestingly, there was a
significant improvement in blastocyst rate on day 6 from COCs co-cultured with DOs
that were without CCs from 0-3h (3h+FSH-CC, refer to Fig. 2.1 C) compared to the
control. Overall, all treatment groups co-cultured with DOs tended to show an
improvement in embryo development.
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
53
Table 2.6 Effects of cumulus cells and FSH on native OSFs efficacy at enhancing post-IVM embryo development
Values with different superscripts within a column are statistically different (P < 0.05)
Data are presented as means ± SEM of 5 replicate experiments. Each replicate experiment consisted of 36-39 oocytes.
1Refer to Fig. 2.1 for explanation of experimental design
2Percentage of cleaved embryos per total oocytes
3Percentage of blastocysts generated 96-100 h post-fertilization per cleaved embryo
4Percentage of blastocysts generated 100-125 h post-fertilization per cleaved embryo
5Percentage of hatching blastocysts generated 100-125 h post fertilization per cleaved embryo
7COCs were cultured without DOs
Treatments Pre-treatment of
OSFs1
No. of
Oocytes
Cleavage2
(%)
Blastocyst
on day 53
Blastoycst
on day 64
Hatching
Blastocyst
on day 65
Control7
COC+DO
COC+DO
COC+DO
N/A
3h+FSH+CCs
3h-FSH+CCs
3h+FSH-CCs
192
191
190
191
92.7±2.9
93.6±1.9
94.7±1.9
93.7±2.5
52.5±3.4a
65.2±5.6b
61.0±3.6a,b
63.8±3.5a,b
76.6±3.0a
83.2±2.0a,b
79.5±2.5a,b
86.2±2.9b
60.8±3.3
69.0±3.4
66.7±4.0
70.4±2.7
Chapter 2: Factors affecting the capacity of exogenous oocyte secreted factor
to improve mouse and fetal development post-IVM
54
Table 2.7 Effects of cumulus cells and FSH on native OSFs on inner cell mass (ICM),
trophectoderm (TE) cells and total cell number (TCN) of day 6 blastocysts
Values with different superscripts within a column are statistically different (P < 0.05).
Data are presented as means ± SEM. Mean blastocyst cell numbers following
differential staining of 33-42 blastocysts.
1Refer to Fig. 2.1 for explanation of experimental design
2COCs were cultured without DOs
Treatments Pre-treatment of
OSFs1
ICM TE TCN
Control2
COC+DO
COC+DO
COC+DO
N/A
3h+FSH+CCs
3h-FSH+CCs
3h+FSH-CCs
11.9±0.6a
13.8±0.5b
11.7±0.5a
12.2±0.7a,b
43.1±2.2
43.6±1.5
46.4±1.9
44.1±2.0
55.0±2.4
57.4±1.6
58.2±2.0
56.3±2.4
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
55
2.5.6 Effect of exposure of COCs to native OSFs during IVM on
cryo-survival of blastocysts
With respect to cryopreserved blastocysts, the blastocyst re-expansion rate post-
vitrification/warming (normal morphology of blastocysts) in blastocysts derived from
COCs exposed to native OSFs (COCs+DOs at 3h+FSH+CC) did not differ
significantly from controls (Table 2.8). Therefore exposure of COCs to native OSFs
did not measurably alter the cryotolerance of blastocysts.
Table 2.8 Effect of native OSFs during IVM on the survival rate of post-warmed
blastocysts
Data are presented as means of 6 replicate experiments. Each replicate experiment
consisted of 10-19 blastocysts.
1Refer to Fig. 2.1 for explanation of experimental design
2COCs were cultured without DOs
Treatments Pre-treatment of
OSFs1
No of
Blastocysts
Normal
(%)
Abnormal
(%)
Control2
COC+DO
N/A
3h+FSH+CCs
90
92
73 (81.1)
74 (80.4)
17 (18.9)
18 (19.6)
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
56
2.5.7 Effect of exposure of COCs to native OSFs on pregnancy rates
and fetal outcomes
After 2 h of warming, the morphologically normal blastocysts were transferred into
pseudo pregnant recipients. There was no difference in implantation rates between
the control and treatment group (COCs co-cultured with DOs [3h+FSH+CC]).
However, the proportion of fetuses that developed to day 17 per implantation site,
from COCs co-cultured with native OSFs, was more than double that of the control
(21% vs 48%, P < 0.05, Fig. 2.4). Moreover, based on gross morphological criteria,
those fetuses were normal, with no significant differences between treatments in fetal
or placental weight or fetal crown-to-rump length (Table 2.9).
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
57
Figure 2.4 Effect of treatment with native OSFs during IVM on subsequent
pregnancy outcomes. Blastocysts were derived from standard IVM (control) or from
COCs exposed to native OSFs during IVM (COC+DO; 3h+FSH+CC pretreatment).
Control and treatment blastocysts were vitrified on day 6 and then later warmed and
transferred to pseudo pregnant recipients. Six control and six treatment blastocysts
were transferred into each uterine horn of a recipient (n=8). Pregnancy outcomes
were analysed on day 17. Implantation rate; implantation sites/embryos transferred
(n = 48 embryos transferred/treatment). Fetal yield; day 17 fetuses/implantation sites
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
58
Table 2.9 Effect of native OSFs during IVM on subsequent fetal and placental weight
and crown-rump length
Data are presented as means ± SEM of 6 and 13 fetuses (control and treatment,
respectively) from 8 recipients.
1Refer to Figure 2.1 for explanation of experimental design
2COCs were cultured without DOs
Treatments Pre-treatment
OSFs1
Placental
weight (g)
Fetal weight
(g)
Crown to
rump length
(mm)
Control2 N/A 0.2 ± 0.01 1.8 ± 0.1 24.5 ± 1.9
COC + DO 3h+FSH+CC 0.2 ± 0.01 1.7 ± 0.1 25.6 ± 0.8
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
59
2.6 DISCUSSION
Our results show that treatment of COCs during IVM with native OSFs, enhances
oocyte quality, as measured by subsequent embryo cleavage, blastocyst and fetal
survival rates. These results are supported by previous studies which have shown
that exogenous native OSFs (obtained by co-culture with DOs), when used as
supplements in oocyte IVM, significantly improve embryo development in cattle (Dey,
et al. 2012, Hussein, et al. 2011, Hussein, et al. 2006), pigs (Gomez, et al. 2012) and
goats (Romaguera, et al. 2010). In contrast, addition of recombinant mature
homodimers of GDF9 and/or BMP15 (R&D Systems) in IVM media did not improve
mouse embryo development (Sudiman, et al. 2014a). However, previous results from
our laboratory using a complex of pro- and mature-domains of GDF9 and BMP15
(pro-mature protein) showed a positive effect on bovine (Hussein, et al. 2011,
Hussein, et al. 2006, Sutton-McDowall, et al. 2012) as well as mouse (Yeo, et al.
2008) oocyte developmental competence.
From this study, we suggest that there are a number of factors which influence the
capacity of exogenous OSFs to improve mouse embryo and fetal development post-
IVM namely; the capacity of cumulus cells and FSH to regulate native OSF
production, and the temporally regulated potency of the native OSF pool.
Supplementation of IVM with OSFs has the potential to dramatically improve the
efficiency of IVM and therefore its applicability in human and veterinary clinics
(Gilchrist 2011), as we saw more than a doubling in fetal yield using this approach.
However, co-culture of COCs with denuded oocytes is not practical in a clinical
scenario. Even though the exact identity of growth factors secreted by denuded
oocytes in vitro remains poorly characterised, the principal OSFs are thought to be
GDF9, BMP15, fibroblast growth factor 8 (FGF8) and FGF10, with widely differing
roles for individual growth factors between species (Dong, et al. 1996, Galloway, et
al. 2000, Gilchrist, et al. 2004b, Hanrahan, et al. 2004, Juengel, et al. 2004, Lin, et al.
2012, Sugiura, et al. 2007, Zhang, et al. 2010). Notably, the only commercially
available recombinant preparations of GDF9 and BMP15 produced in mammalian
cells, had no effect on post-IVM embryo development, despite the extensive dose
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
60
range tested (Sudiman, et al. 2014a). By contrast, recombinant FGF10 added during
bovine IVM enhances subsequent embryo development (Zhang, et al. 2010).
When full length recombinant GDF9 and BMP15 are expressed in mammalian cells
such as human embryonic kidney 293H cells, the proteins are processed (pro and
mature domains are proteolytically cleaved) and secreted into the medium as dimers
or monomers of pro- and mature regions, that are non-covalently bound as pro-
mature complexes (McIntosh, et al. 2008). Our in-house produced GDF9 and BMP15
preparations (Gilchrist, et al. 2004b, Hickey, et al. 2005), which contain both the pro-
and mature domains, when added to IVM, significantly improve the quality of the
blastocysts generated (as measured by ICM cell number) and thus improve fetal
survival rates in mouse (Yeo, et al. 2008), as well as increase embryo development
in cattle (Hussein, et al. 2011, Hussein, et al. 2006, Sutton-McDowall, et al. 2012).
The current results demonstrated that blastocyst development and fetal survival rates
were further improved when COCs were co-cultured with DOs from 3 h (versus
control), if those DOs were matured as COCs for the first 3 hours in the presence of
FSH, then subsequently denuded and added to IVM. This result supports previous
findings in bovine IVM oocytes, which suggested a temporal effect on secretion of
OSFs (Hussein, et al. 2011). It has been demonstrated that even a short exposure of
COCs to FSH significantly increases subsequent bovine blastocyst development, via
protein kinase C activation (Ali and Sirard 2005). Hence, the FSH-treated CCs may in
some manner improve the quantity and/or quality of factors which are secreted by the
resultant DO. During the 3 h maturation, the oocyte and cumulus cells communicate
with each other through gap junctional communication and via paracrine factors,
which presumably provides enough time for the oocyte to obtain the beneficial effect
of FSH through the receptors in cumulus cells. This effect may be mediated more by
paracrine factors rather than gap junctional communication. After meiotic resumption,
gap junctional communication between the oocyte and cumulus cells is lost (Thomas,
et al. 2004). The action of OSFs can be observed even without direct contact
between cumulus cells and oocytes, suggesting that they are acting in a paracrine
manner (Hussein, et al. 2005). In cattle, the developmental competence of DOs co-
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
61
cultured with COCs is significantly improved compared to DOs cultured alone (Dey,
et al. 2012, Hussein, et al. 2006).
Based on the notable improvement in oocyte developmental competence from the
3h+FSH+CC group, we anticipated that COCs matured first for 3 h in medium
containing FSH would produce different quantities and/or forms of GDF9, compared
to oocytes denuded at 0 h. However, unfortunately we were unable to detect any
GDF9 in oocyte-conditioned medium, unlike the recent study by Lin et al (2012). This
may be due to our oocyte-conditioning medium procedures or the western blot being
insufficiently sensitive to detect actual secreted forms. Also, we could not observe
any effect of the 3 h pre-culture with FSH treated cumulus cells on the GDF9
produced within the oocyte. It showed that there was no difference in the amount or
the proportion of either the pro-protein or the mature form of GDF9 in DOs at 0 h or 3
h. While GDF9 is the principal OSF produced by mouse oocytes (Crawford and
McNatty 2012, Gilchrist, et al. 2004b), other OSFs such as BMP15 (Yoshino, et al.
2006), FGF10 (Zhang, et al. 2010) and FGF8 (Sugiura, et al. 2007) may be
processed and interact differently in DOs at 0 h and 3 h.
Overall, we have shown that there is an obvious advantage to treatment of COCs
with exogenous OSFs during IVM. Mouse exogenous OSFs did not affect oocyte
nuclear maturation, in contrast with bovine oocytes matured in vitro, where co-culture
with DOs improves nuclear maturation (Dey, et al. 2012). Clearly exogenous OSFs
have major effects on key aspects governing the developmental program of oocytes,
although it is not yet clear how and by which exact mechanisms this is achieved.
Other groups have shown that native OSFs play a significant role in; the activity of
hyaluronic acid synthase 2 (HAS2), which correlates with mucification and expansion
of CCs (Dragovic, et al. 2005), increased GPX1 gene expression (related to the
production of glutathione peroxidise as an antioxidant in oocytes), and enhanced
expression of steroidogenic acute regulatory protein (StAR), which contributes to
oocyte nuclear and cytoplasmic maturation (Dey, et al. 2012). Bovine oocytes
matured with the mature form of BMP15 showed a significant increase in glucose
uptake (Caixeta, et al. 2013), and those matured with pro-mature BMP15 exhibited
Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor
to Improve Mouse and Fetal Development Post-IVM
62
increased oxidative phosphorylation activity in the oocyte (Sutton-McDowall, et al.
2012).
The results of this current study confirm that OSFs notably improve embryo
development and fetal survival in mice, and extend this knowledge by demonstrating
for the first time that the effect of OSFs on mouse oocytes depends on timing,
presence of FSH and cumulus cells. The beneficial effect of native OSFs on oocyte
quality and subsequent embryo development can be applied in the veterinary clinic;
however, application of co-culture of COCs with DOs is not possible in human IVF
clinics since it is unethical and not feasible to generate supernumerary DOs simply
for OSF supplementation of media. Thus, the next step is to identify and purify the
proteins that are secreted by oocytes, such as GDF9 and BMP15, into a functional
form that can be added into IVM medium, to improve the quality of IVM oocytes and
subsequent embryo development and pregnancy rates.
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
Enhances Bovine Oocyte Developmental Competence
63
CHAPTER 3
BONE MORPHOGENETIC PROTEIN 15 IN THE
PRO-MATURE COMPLEX FORM ENHANCES
BOVINE OOCYTE DEVELOPMENTAL
COMPETENCE
(Sudiman, et al. 2014b)
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
Enhances Bovine Oocyte Developmental Competence
64
3.1 ABSTRACT
Developmental competence of in vitro matured (IVM) oocytes needs to be improved
and this can potentially be achieved by adding recombinant bone morphogenetic
protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM media. The aim of
this study was to determine the effect of a purified pro-mature complex form of
recombinant human BMP15 versus the commercially available bioactive forms of
BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo
development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were
matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15,
mature BMP15 or mature GDF9; each in the absence or presence of FSH. Metabolic
measures of glucose uptake and lactate production from COCs and autofluorescence
of NAD(P)H, FAD++ and GSH were measured in oocytes after IVM. Following in vitro
fertilisation and embryo culture, day 8 blastocysts were stained to determine the cell
numbers. COCs matured in medium +/- FSH containing pro-mature BMP15
displayed significantly improved blastocyst development (57.7±3.9%, 43.5 ± 4.2%)
compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH
(36.1±3.0%), respectively. The mature form of BMP15 produced intermediate levels
of blastocyst development; not significantly different to control or pro-mature BMP15
levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione
(GSH) levels were increased by both forms of BMP15 in the absence of FSH.
Exogenous BMP15 in its pro-mature form during IVM provides a functional source of
oocyte-secreted factors to improve bovine blastocyst development. This form of
BMP15 may prove useful for improving cattle and human artificial reproductive
technologies.
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
Enhances Bovine Oocyte Developmental Competence
65
3.2 INTRODUCTION
The oocyte and the somatic cells surrounding the oocyte (cumulus cells in antral
follicles) communicate via a complex bi-directional signalling axis mediated via
paracrine and gap junctional means (Gilchrist, et al. 2008). This communication is
important for transporting molecules such as growth factors, cyclic nucleotides,
amino acids and other small regulatory molecules from cumulus cells into oocytes,
and vice versa, in order to sustain cumulus cell health and oocyte development
(Gilchrist, et al. 2008, Herlands and Schultz 1984, Ka, et al. 1997, Zhang, et al.
1995). Oocyte-secreted growth factors (OSFs) act on cumulus cells to regulate
multiple functions, including; differentiation of the cumulus cell lineage (Li, et al.
2000), the proliferation and expansion of cumulus cells (Dragovic, et al. 2005,
Dragovic, et al. 2007, Gilchrist, et al. 2006, Vanderhyden, et al. 1992), prevention of
luteinisation (Li, et al. 2000, Vanderhyden, et al. 1993) and apoptosis (Hussein, et al.
2005) and regulation of cumulus cell metabolism (Eppig 2005, Sugiura, et al. 2005,
Sutton-McDowall, et al. 2012). Healthy cumulus cells are vitally important for the
development of oocytes through the provision of substrates and regulatory
molecules, required for the oocyte to undergo appropriate developmental
programming in order to support early embryo development (Gilchrist 2011).
Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15)
are two major and well-known OSFs, required for follicular development and
ovulation (Dong, et al. 1996, Galloway, et al. 2000, Hanrahan, et al. 2004, Juengel, et
al. 2002). The function and expression of both proteins differs markedly between
species (Crawford and McNatty 2012). In polyovular animals, oocyte expression of
GDF9 is notably higher than BMP15, suggesting a minor role for BMP15 in the
regulation of their reproduction. Consistent with this notion, homozygous mutant
GDF9 mice are sterile due to a block in follicular development beyond the primary
follicle stage (Dong, et al. 1996), whereas BMP15 homozygous mutant mice
demonstrate only a mild reduction in fertility (Yan, et al. 2001). In contrast, in
monoovulatory animals, the ratio of oocyte GDF9 to BMP15 is nearly equal
(Crawford and McNatty 2012), reflecting the importance of both of these growth
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
Enhances Bovine Oocyte Developmental Competence
66
factors in follicular development in these species. Sheep homozyous for certain
naturally occuring mutations in either GDF9 or BMP15 are sterile (Galloway, et al.
2000, Hanrahan, et al. 2004). Interestingly, sheep heterozygous for these mutant
forms of GDF9 or BMP15 have an increased ovulation rate and incidence of mutiple
pregnancies due to increased LH sensitivity in secondary follicles that leads to a rise
in the number of antral follicles (Galloway, et al. 2000, Hanrahan, et al. 2004,
Juengel, et al. 2004). Furthermore, in humans, rare mutations and genetic variance
of GDF9 and BMP15 are associated with polycystic ovary syndrome (Wang, et al.
2010), dizygotic twinning (Palmer, et al. 2006) and premature ovarian failure (Chand,
et al. 2006, Dixit, et al. 2005, Wang, et al. 2013).
Like other members of the TGF-β superfamily, the pro-proteins (un-processed
proteins) of GDF9 and BMP15 are composed of a pro-region and a mature region
(McMahon, et al. 2008). The pro-regions of these proteins can only be dissociated
from the corresponding mature regions after processing at their protease processing
site, after which they form a pro-mature complex held together by non-covalent
interactions (McIntosh, et al. 2008). There still remains considerable uncertainty
exactly which forms of GDF9 and BMP15 are secreted by the oocyte to act on the
surrounding cumulus cells, and whether the form of these proteins produced by
oocytes matured in vitro is similar to what may occur in vivo. For example, sheep
follicular fluid contains GDF9 and BMP15 proteins only in the unprocessed pro-
protein form (McNatty, et al. 2006), whereas sheep oocytes secrete the mature
domains of GDF9 and BMP15 during IVM (Lin, et al. 2012). In vitro matured mouse
oocytes secrete GDF9 as a mixture of the pro-protein and mature domain (Gilchrist,
et al. 2004a), whilst in vitro matured rat oocytes secrete mature domain GDF9 only
(Lin, et al. 2012). Commercially available GDF9 and BMP15 proteins from the one
supplier of these peptides (R&D Systems) contain only the mature regions of these
proteins; however, our novel BMP15 contains both pro and mature regions, as a
processed pro-mature complex. Consistent with the role of pro-regions of other TGF-
β superfamily members, it has now been demonstrated that the pro-regions of GDF9
(Watson, et al. 2012) and BMP15 interact with granulosa and cumulus cells (Mester,
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
Enhances Bovine Oocyte Developmental Competence
67
et al. 2013). Hence, we hypothesised that the presence of the pro-region is important
to retain the full function of BMP15 or GDF9 and may affect oocyte developmental
competence.
Our group has previously shown that addition of partially purified GDF9 or BMP15,
which contains a mixture of proteins including the pro-mature complex form, to in
vitro maturation (IVM) medium improves mouse and cattle blastocyst development
(Hussein, et al. 2011, Hussein, et al. 2006, Yeo, et al. 2008). Moreover, addition of a
purified BMP15 pro-mature complex to IVM medium increases bovine embryo
development (Sutton-McDowall, et al. 2012). No studies to date have examined the
effect of the form of GDF9 or BMP15 added during IVM on embryo development. In
this study, we examined the effect of the mature (R&D Systems) and pro-mature (in-
house) forms of human BMP15, as well as the mature form of mouse GDF9 (R&D
Systems), on bovine oocyte developmental competence following IVM. We examined
the metabolic activity of COCs, including glucose uptake and lactate production, as
well as mitochondrial and antioxidant activity that occurred during the maturation of
bovine oocytes.
3.3 MATERIALS AND METHODS
Unless otherwise specified, all chemicals and reagents were purchased from Sigma
(St Louis, MO).
3.3.1 Collection of cells
Bovine ovaries were collected from a local abattoir and transported to the laboratory
in 30-350C saline. Cumulus-oocyte complexes (COCs) and mural granulosa cells
(GCs) were aspirated from 3-8 mm antral follicles using an 18-gauge needle and a
10 ml syringe. The follicular fluid containing COCs was transferred into a 10 ml
Falcon tube at 390C. The follicular aspirate was allowed to sediment and the cellular
pellet was washed with handling medium (HEPES-buffered tissue cultured medium-
199; TCM-199, MP Biomedicals, Solon, OH, USA) supplemented with 4 mg/ml fatty-
acid-free bovine serum albumin (FAF-BSA; ICPbio Ltd, Auckland, NZ). Intact COCs
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
Enhances Bovine Oocyte Developmental Competence
68
with compact cumulus cells, >3 cell layers and with an evenly pigmented oocyte
cytoplasm, were selected under a dissecting microscope and washed twice in
handling medium.
3.3.2 Granulosa cell thymidine assay
To confirm that the forms of recombinant GDF9 and BMP15 used in this study were
bioactive on bovine ovarian cells, a standard granulosa cell tritiated thymidine
incorporation bioassay was performed, as previously described (Gilchrist, et al. 2003,
Gilchrist, et al. 2006). In brief, GCs were collected from 3-8 mm follicles. The GCs
were washed twice in protein-free culture medium (bicarbonate-buffered TCM-199).
Cells were added to the wells of 96-well plates (Falcon) at a final concentration of 50
x 104 cells/ml, and cultured at 370C, 96% with 5% CO2 in humidified air for 18 hours,
followed by a further 6 hour pulse of 15.4 kBq tritiated thymidine ([3H]-thymidine, MP
Biomedicals) under the same conditions. Following culture, GCs were harvested and
incorporated [3H]-thymidine was quantified using a scintillation counter as an
indicator of GC DNA synthesis. Each treatment was performed in duplicate and the
assay was repeated five times.
3.3.3 Sources of BMP15 and GDF9 and treatment of COCs
COCs were matured in base IVM medium [bicarbonate-buffered TCM 199 + 4 mg/ml
FAF BSA +/- 0.1 IU/ml FSH (Puregon, Organon, Oss, Netherlands)] in a 4-well Nunc
dish (Nunclon, Denmark) and cultured for 23 hours at 390C with 5% CO2 in humidified
air. Recombinant mouse mature region GDF9 (Cat No: 739-G9) and human mature
region BMP15 (Cat No: 5096-BM) were purchased from R&D Systems (Minnaepolis,
MN, USA).
The recombinant human pro-mature complex of BMP15 was produced from a stable
human embryonic kidney (HEK)-293T cell line generated in our laboratory. Briefly, an
expression cassette encoding the human BMP15 DNA sequence was synthesized
(Genscript USA, Inc., Piscataway, NJ) incorporating the rat serum albumin signal
sequence at the 5’ end followed by a His8 tag and a Strep II epitope tag at the N-
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
Enhances Bovine Oocyte Developmental Competence
69
terminus of the BMP15 pro-region. This expression cassette was transferred to the
pEF-IRES expression vector, as previously described (Hobbs, et al. 1998) and stably
transfected HEK-293T cell lines producing human BMP15 were established via
puromycin selection. For production of the BMP15 protein, confluent monolayers
were transferred to serum-free media (DMEM/F12 + 0.1 mg/ml BSA + 125 IU/ml
heparin) for a 48 hour collection period. The resultant BMP15 protein was secreted
into the serum-free media with a processing efficiency of cleavage of the pro and
mature regions of over 90%. The BMP15 containing production media was subjected
to immobilized Ni2+ based ion-affinity purification targeting the His-tag as described
previously (Pulkki, et al. 2011). Samples were tested for the presence of pro-mature
BMP15 protein by silver staining. The dose of processed mature region was
quantified using Western blotting with the mab28 monoclonal antibody (Pulkki, et al.
2011) and the commercially available mature region of hBMP15 (R&D Systems) as a
standard.
The effects of graded doses of the purified pro-mature BMP15 were tested at 10, 100
and 200 ng/ml. Four replicate experiments were performed with 27-55 COCs per
dose per replicate. The concentration of pro-mature BMP15 was chosen based on
this dose-response assessment of embryo development. For all further experiments
the treatment media contained pro-mature BMP15, mature BMP15 or mature GDF9
at 100 ng/ml +/- 0.1 IU/ml FSH. Six replicate experiments were performed with 20-46
COCs per treatment per replicate.
3.3.4 In vitro fertilization (IVF) and embryo culture
In vitro production of embryos was undertaken using defined, serum-free media
(Bovine Vitro series of media, IVF Vet Solutions, Adelaide, Australia), as previously
described (Hussein, et al. 2006). 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 (GE Health Care, Australia) in a 12 ml Falcon tube
(Becton Dickinson, Franklin Lakes, NJ, USA) and centrifuged for 20-25 minutes at
271xg. The supernatant was removed and the pellet resuspended in wash medium
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
Enhances Bovine Oocyte Developmental Competence
70
[VitroWash (IVF Vet Solutions) + 4 mg/ml FAF BSA)] and centrifuged for 5 minutes at
56 xg. The supernatant was removed and the pellet re-suspended in IVF medium
[VitroFert (IVF Vet Solutions) + 4 mg/ml FAF BSA + 10 IU/ml heparin] with a final
concentration of 1x106 spermatozoa/ml. COCs were inseminated in pre-equilibrated
drops of IVF medium for 23 h at 390C in 6% CO2 in humidified air. Post-insemination,
cumulus cells were removed by manual pipetting with a 1000 µl Gilson pipette. Five
presumptive zygotes were cultured in 20 µl drops of pre-equilibrated cleavage
medium [VitroCleave (IVF Vet Solutions) + 4mg/ml BSA + 1 mM L-carnitin] overlaid
with mineral oil at 390C in 5% O2, 6% CO2 and 89% N2, for 5 days. On day 5,
embryos were transferred into pre-equilibrated blastocyst medium [VitroBlast (IVF
Vet Solutions) + 4mg/ml BSA + 1 mM L-carnitin] and cultured for another 3 days.
Blastocysts were assessed on day 7 and day 8.
3.3.5 Blastocyst differential staining
Differential staining was performed using a modified method as previously described
by Thouas, et al. 2001. Expanded, hatching and hatched blastocysts were placed
into 1% (v/v) Triton X-100 containing 1 mg/ml propium iodide for 20-30 seconds.
Blastocysts were washed in absolute ethanol before incubation in 2.5 mg/ml Hoechst
33342 solution for 2 minutes, then mounted in a drop of glycerol in PBS on
microscope slides and covered with a cover slip. Embryos were examined under a
fluorescence microscope (Nikon, TE 2000-E) at 200x equipped with an ultraviolet
filter (excitation, 340-380 nm; emission, 440-480 nm). The inner cell mass (ICM)
appeared blue and trophectoderm (TE) cells nuclei stained pink.
3.3.6 Glucose and lactate measurements
Groups of 10 COCs were matured in 100 µl of IVM medium. After 23 h maturation,
spent media were analysed for glucose and lactate levels using a Hitachi 912
chemical analyser (F. Hoffmann-La Roche Ltd; Basel, Switzerland). Glucose uptake
and lactate production are expressed as pmol/COC/h. Twelve replicate experiments
were performed with 10 COCs per treatment, per replicate.
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
Enhances Bovine Oocyte Developmental Competence
71
3.3.7 Autofluorescence of NAD(P)H and FAD++
Ten COCs were matured in 100 µl of IVM medium. At 23 h maturation, COCs were
denuded and oocytes were transferred into 10 µl drops of handling medium overlaid
with mineral oil in glass-bottomed confocal dishes (Cell E&G; Houston, TX, USA).
The autofluorescent intensity of FAD++ was observed using a blue laser (excitation,
473 nm; emission, 490-520 nm), and NAD(P)H using a violet laser (excitation; 405
nm, emission: 420-520 nm) with a confocal microscope (Olympus; Fluoview FV10i).
The fluorescence from the oocytes was normalized using a fluorescence standard
(Inspeck; Molecular Probes). Laser power, gain and pin-hole settings were kept
consistent. Four replicates were performed with 10 oocytes per replicate per
treatment group.
3.3.8 Reduced glutathione (GSH)
At 23 h maturation, COCs were denuded and washed in handling medium. Denuded
oocytes were incubated for 30 minutes in 12.5 µM monochlorobimane (MCB, Catt
No: 69899) and transferred into 10 µl drops of handling medium overlaid with mineral
oil in glass-bottomed confocal dishes (Cell E&G; Houston, TX, USA). The fluorescent
intensity was observed using a DAPI filter (excitation, 358 nm; emission, 461 nm)
with a confocal microscope (Olympus; Fluoview FV10i). Ten COCs were measured
per treatment group per replicate and each experiment was replicated 3 times.
3.4 STATISTICAL ANALYSIS
All proportional data for embryo development were arcsine transformed prior to
analysis using multivariate analysis of variance (ANOVA). Log transformation was
performed if the data were not normally distributed, and analysed using one-way
ANOVA for blastoycst cell numbers, glucose production and lactate consumption.
Autofluorescence of NADP(H), FAD++ and GSH were analysed by two-way ANOVA.
In all cases there was no main effect of FSH (P > 0.05), hence subsequently the form
of GDF9/BMP15 was tested within +/- FSH treatment by one-way-ANOVA followed
by a least-significant difference (LSD) post hoc test. All analysis was performed with
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
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SPSS version 13 (SPSS Inc, Chicago, IL, USA). Differences were considered
significant at P < 0.05. Data are expressed as means ± SEM.
3.5 RESULTS
3.5.1 Bioactivity of different BMP15 and GDF9 forms
All forms of the three recombinant proteins (pro-mature BMP15, mature BMP15 and
mature GDF9) were bioactive on bovine GC, as evidenced by significant stimulation
of [3H]-thymidine incorporation compared to the control (Fig. 3.1). The mature region
of GDF9 protein tended to cause the highest level of GC [3H]-thymidine incorporation
and pro-mature BMP15 the least.
Figure 3.1 Bioactivity of different BMP15 and GDF9 forms. Granulosa cell tritiated
thymidine incorporation following exposure to mature GDF9, mature BMP15 or pro-
mature BMP15 at 100 ng/ml. Bars represent means ± SEM and different lowercase
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
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letters indicate a statistically significant difference (P<0.05). Data were derived from 5
independent replicates.
3.5.2 Dose-response of pro-mature BMP15 on oocyte
developmental competence
Supplementation of IVM media with pro-mature BMP15 had no effect on subsequent
embryo cleavages rates; however, significantly increased blastocyst development on
day 8 at each dose examined, compared to control and buffer groups (P < 0.05;
Table 3.1). There was no observable dose-response to BMP15 ranging from the
lowest dose (10 ng/ml) to the highest dose (200 ng/ml); however, 200 ng/ml
produced a high blastocyst rate of 60%; a 1.5-fold higher embryo yield than under
control IVM conditions. No adverse effect of elution buffer (PBS/500 mM imidazole
buffer) during IVM on either cleavage or blastocyst development was observed
(Table 3.1).
Table 3.1 Effect of graded doses of pro-mature BMP15 during IVM on oocyte
developmental competence
Values with different superscript within a column are statistically different (P < 0.05).
Data are presented as means ± SEM of 6 replicate experiments. Each replicate
experiment consisted of 27-55 oocytes.
Treatments No. of oocytes Cleavage
(%)
Blastocyst
day 8 per cleaved
(%)
Control
Buffer (Imidazole) 500 mM
BMP15 10 ng/ml
BMP15 100 ng/ml
BMP15 200 ng/ml
151
159
151
150
156
76.5±3.3
82.2±6.3
87.4±5.1
83.6±4.9
85.8±1.3
39.9±2.3a
40.9±4.6a
54.0±3.3b
55.5±6.0b
59.7±4.4b
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3.5.3 Comparison of pro-mature BMP15, mature BMP15 and mature
GDF9 during IVM on subsequent embryo development
In the presence of FSH, addition of pro-mature BMP15 to IVM medium significantly
increased blastocyst development on day 7 compared to control (Table 3.2).
Irrespective of treatment, there were no significant differences in cleavage rates, day
8 blastocyst rates, inner cell mass, trophectoderm or total cell numbers (Tables 3.2
and 3.4). There was a trend towards an improvement in blastocyst development on
day 8 from pro-mature BMP15 compared to control, but this was not significant (p =
0.07). The mature form of BMP15 produced intermediate levels of blastocyst
development which were not significantly different to pro-mature BMP15 or controls.
The mature region of GDF9 did not improve any embryo parameter and blastocyst
development on day 7 was significantly lower than both forms of BMP15 (Table 3.2).
As expected, in the absence of FSH, all embryo development and embryo quality
measures were generally lower than in the presence of FSH (Tables 3.3 and 3.5). In
the absence of FSH, blastocyst development with pro-mature BMP15 was also
significantly higher than the control on day 7 (P < 0.05). The trend for improved
blastocyst development with mature BMP15 in the presence of FSH was not evident
in the absence of FSH. Mature GDF9 without FSH did not improve embryo
development or quality.
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Table 3.2 Effect of supplementing COC cultures with different forms of BMP15 (mature BMP15 and pro-mature BMP15),
mature GDF9 and FSH on oocyte developmental competence
Values with different superscript within a column are statistically different (P < 0.05).
Data are presented as means ± SEM of 6 replicate experiments. Each replicate experiment consisted of 20-46 oocytes.
Treatments No. of
Oocytes
Cleavage
(%)
Blastocyst
day 7
per cleaved
(%)
Blastocyst
Day 8
per cleaved
(%)
Control
GDF9 mature 100 ng/ml
BMP15 mature 100 ng/ml
BMP15 pro-mature 100 ng/ml
214
195
207
210
94.2±1.4
88.8±3.1
92.1±1.8
89.1±1.6
43.3±2.4a
36.1±3.0a
50.1±2.6b
57.7±3.9b
52.8±3.9
52.0±4.1
60.0±3.8
61.7±3.5
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Table 3.3 Effect of supplementing COC cultures with different forms of BMP15 (mature BMP15 and pro-mature BMP15),
mature GDF9 in the absence of FSH on oocyte developmental competence
Values with different superscript within a column are statistically different (P < 0.05). Data are presented as means ± SEM of
6 replicate experiments. Each replicate experiment consisted of 27-40 oocytes.
Treatments No. of
Oocytes
Cleavage
(%)
Blastocyst
day 7
per cleaved
(%)
Blastocyst
Day 8
per cleaved
(%)
Control
GDF9 mature 100 ng/ml
BMP15 mature 100 ng/ml
BMP15 pro-mature 100 ng/ml
191
189
195
188
76.4±4.8
85.5±3.1
84.9±7.3
81.6±4.3
28.9±3.7a
33.1±9.5a,b
32.3±4.5a,b
43.5±4.2b
39.3±3.2
38.4±4.1
38.5±3.7
48.7±3.8
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Table 3.4 Number of total (TCN), inner cell mass (ICM), trophectoderm (TE) cells in
day 8 expanded, hatching and hatched blastocysts following co-culture of COCs with
different forms of BMP15 (mature BMP15 and pro-mature BMP15), mature GDF9 in
the presence of FSH
Treatments ICM TE TCN
Control
GDF9 mature 100 ng/ml
BMP15 mature 100 ng/ml
BMP15 pro-mature 100 ng/ml
30.5±1.6
28.7±1.9
31.8±3.0
34.6±1.8
59.7±3.5
69.7±5.0
68.4±4.4
63.4±2.6
90.2±4.7
98.4±6.0
100.2±6.2
97.9±3.6
Data are presented as means ± SEM. Mean blastocyst cell numbers following
differential staining of 28-38 blastocysts.
Table 3.5 Number of total (TCN), inner cell mass (ICM), trophectoderm (TE) cells in
day 8 expanded, hatching and hatched blastocysts following co-culture of COCs with
different forms of BMP15 (mature BMP15 and pro-mature BMP15), mature GDF9 in
the absence of FSH
Data are presented as means ± SEM. Mean blastocyst cell numbers following
differential staining of 22-37 blastocysts.
Treatments ICM TE TCN
Control
GDF9 mature 100 ng/ml
BMP15 mature 100 ng/ml
BMP15 pro-mature 100 ng/ml
24.5±1.9
27.4±2.0
26.5±1.8
29.1±1.8
77.3±5.8
73.8±4.6
72.1±6.7
78.6±6.3
101.8±6.3
101.3±5.2
98.6±7.9
107.7±7.1
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3.5.4 Effect of pro-mature BMP15, mature BMP15 and mature GDF9
on COC glucose consumption and lactate production
There was an increase in COC glucose uptake and lactate production (almost 2-fold)
in response to FSH, across all BMP15/GDF9 treatment groups and controls (Fig.
3.2). Similar results have been reported that FSH significantly increases glucose
uptake and lactate production (Roberts, et al. 2004). None of the BMP15 or GDF9
proteins tested altered COC glucose consumption or lactate production, regardless of
FSH treatment.
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Figure 3.2 Glucose consumption and lactate production. Post 23 h maturation of
COCs after treatment with different forms of BMP15 (mature BMP15 and pro-mature
BMP15) or mature GDF9 at 100 ng/ml, in the absence or presence of FSH, spent
IVM medium were analysed for glucose and lactate levels. A. Glucose uptake. B.
Lactate production. Bars represent the means ± SEM. Data were derived from 12
independent replicates for each treatment.
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3.5.5 Effect of pro-mature BMP15, mature BMP15 and mature GDF9
on oocyte FAD++ and NAD(P)H autofluorescence
Across all treatments, FSH had no effect on intra-oocyte NAD(P)H, FAD++, or the
REDOX ratio, as assessed by confocal microscopy and analysed by 2-way ANOVA
(main effect FSH; P > 0.05). In the absence of FSH, there was significantly higher
autofluorescence of NAD(P)H in the oocytes treated with pro-mature BMP15
compared to controls (P < 0.05, Fig. 3.3 A). However, in the presence of FSH, the
NAD(P)H level in controls increased to the same level as pro-mature BMP15. There
was a significantly lower level of NAD(P)H autofluorescence in oocytes matured in
the presence of GDF9 compared to control and pro-mature BMP15 groups in the
presence of FSH. No increase in autofluorescence of FAD++ (Fig. 3.3 B) or change in
REDOX ratio (Fig. 3.3 C) in any treatment groups was observed compared to
controls.
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Figure 3.3. Intra-oocyte NAD(P)H, FAD++, REDOX ratio. Effect of treatment of
intact COCs with different forms of BMP15 (mature BMP15 and pro-mature BMP15)
or mature GDF9 at 100 ng/ml, +/- FSH on intra-oocyte metabolic activity. A.
Autofluorescence of NAD(P)H. B. Autofluorescence of FAD++. C. REDOX ratio
(FAD++/NAD(P)H). Bars represent the means ± SEM. Data were derived from 4
independent replicates for autofluorescence on intra-oocyte NAD(P)H, FAD++, and
REDOX ratio. Columns with different superscripts are significantly different (P <
0.05); a,bminus FSH, x-yplus FSH.
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3.5.6 Effect of pro-mature BMP15, mature BMP15 and mature GDF9
on oocyte GSH
Since a significant increase in NAD(P)H levels in oocytes treated with pro-mature
BMP15 (-FSH) was detected, we then examined if this was associated with an
increase in oocyte GSH, on the basis that if NADPH increased, then the capacity to
regenerate reduced GSH was a likely consequence. In the absence of FSH, both
forms of BMP15 significantly increased oocyte GSH levels compared to the controls
(Fig. 3.4). Mature GDF9 produced an intermediate level of GSH. However, in the
presence of FSH, the GSH level of controls was comparable with all other
treatments. There was no overall effect of FSH on oocyte GSH levels (main effect
FSH; P > 0.05).
Figure 3.4 Intra-oocyte GSH level. Effect of treatment of intact COCs with different
forms of BMP15 (mature BMP15 and pro-mature BMP15) or mature GDF9 at 100
ng/ml, +/- FSH on intra-oocyte metabolic activity. Bars represent the means ± SEM.
Data were derived from 3 independent replicates. Columns with different superscripts
are significantly different (P < 0.05)
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3.6 DISCUSSION
In this present study, we compared the effects of different forms of BMP15 (pro-
mature and mature) and the mature form of GDF9 during IVM on bovine embryo
development. The pro-mature form of BMP15 was the only form of OSF that
significantly increased subsequent embryo development relative to the control.
Mature domain GDF9 was ineffective and mature domain BMP15 lead to a
moderate, albeit non-significant, improvement in embryo yield. We have also
produced pro-mature human GDF9; however, the utilization of this protein is
problematic in culture, as we have found that it has a very high binding affinity for
plastic culture-ware (Mottershead DG, unpublished data) and we have not yet
determined a suitable culture environment.
Oocyte-secreted growth factors have been studied extensively either in their native
form (i.e. as secreted by oocytes) or as recombinant growth factors (Gilchrist, et al.
2008). It is now a well-known concept that co-culture of denuded oocytes (as a
source of native exogenous OSFs) with COCs improves developmental competence
of pig (Gomez, et al. 2012), cattle (Dey, et al. 2012, Hussein, et al. 2011, Hussein, et
al. 2006), goat (Romaguera, et al. 2010) and mouse (Sudiman, et al. 2014a) IVM
oocytes. However, a full characterisation of the forms and indeed the complete
identities of native OSFs have not been undertaken. Previous results showed that
addition of either non-purified BMP15 or GDF9 (containing pro-mature complexes)
improved cattle blastocyst development, and pro-mature mouse GDF9 improved
mouse fetal yield (Hussein, et al. 2011, Hussein, et al. 2006, Yeo, et al. 2008).
However, so far, there is no published report of any significant improvements after
addition of the only commercially available mammalian cell expressed mature
regions of GDF9 or BMP15. Our previous findings (Sudiman, et al. 2014a) indicated
that addition of the mature regions of GDF9 or BMP15 (R&D Systems) did not
improve the developmental competence of mouse IVM oocytes.
Although the form of GDF9 and BMP15 which are produced by the oocyte and
released into the somatic cell compartment of the follicle remains elusive, it is known
that both the pro-region and mature region play important roles in the function of
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
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these proteins. The mature domain of TGF-β superfamily proteins is the bioactive
receptor binding region, and while the mature domain is complexed with the pro-
region after processing to form the pro-mature complex, this form can either be latent
or active depending on the particular superfamily member (Brown, et al. 2005, Gray
and Mason 1990, Wakefield, et al. 1988). In the case of human GDF9, the
association of the pro-region with its mature region confers latency (Mottershead, et
al. 2008, Simpson, et al. 2012). Further, the pro-region has important functions in
protein folding, formation of the disulfide bonds and regulation of bioactivity (Gray
and Mason 1990, Harrison, et al. 2011). It seems plausible that the pro-domain of
BMP15 may interact with the extracellular matrix of cumulus cells during maturation
and facilitate presentation of the mature domain to cumulus cell receptors. In support
of this concept, it has been shown that the GDF9 pro-mature complex binds strongly
to heparin sepharose, suggesting that heparan sulfate proteoglycans on cumulus
cells may act as co-receptors mediating oocyte secreted factor signalling (Watson, et
al. 2012). Moreover, the pro-region of GDF9 and BMP15 interact with mural
granulosa cells (Mester, et al. 2013). Collectively, these results indicate that the pro-
domains of GDF9 and BMP15 exhibit important interactions with ovarian somatic
cells, and may explain why the isolated mature regions of GDF9 and BMP15 appear
to have little effect on oocyte developmental competence.
In vitro matured COCs have aberrant expression of genes and proteins compared to
COCs matured in vivo (Dunning, et al. 2007, Kind, et al. 2013, Richani, et al. 2013).
Notably, it has been shown that mouse COCs express processed BMP15 protein
during in vivo oocyte maturation, but not during IVM (Mester, et al. 2013). From our
data, it is clear that BMP15 improved bovine embryo development compared to
GDF9. However, GDF9 may have the capability to improve oocyte quality during IVM
if it were in a different form; e.g. as a pro-mature complex or if bovine or human
GDF9 were used instead of mouse GDF9. Different species origins of recombinant
OSFs have different effects on granulosa cell sterol biosynthesis and bioactivity
(McNatty, et al. 2005a, b). Interestingly, mouse GDF9 in its mature form is
significantly more bioactive on bovine granulosa cells compared to BMP15 (both
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
Enhances Bovine Oocyte Developmental Competence
85
forms; current study). Hence, the lack of effect of mouse GDF9 on oocyte
competence was not due to lack of bioactivity of this preparation on bovine cells.
The improvement of embryo development after addition of pro-mature BMP15 was
accompanied by an increase in oocyte autofluorescence of NAD(P)H. However,
there was no increase in oocyte FAD++ or change in REDOX status. These results
differ slightly from our previous published data which showed that the addition of pro-
mature BMP15 increased not only NAD(P)H but also increased FAD++ levels (Sutton-
McDowall, et al. 2012). Unfortunately, we cannot distinguish between
autofluorescence from NADPH derived from the pentose phosphate pathway (PPP)
or isocitrate dehydrogenase activity, and autofluorescence from NADH, which is a
product of several metabolic pathways including glycolysis, and the tricarboxylic acid
(TCA cycle). If the increasing level of NAD(P)H in the oocyte after treatment with pro-
mature BMP15 is in part due to increased NADPH, then this may also relate to the
higher levels of GSH in oocytes, as NADPH is required for reduction of oxidised
glutathione via the enzyme glutathione reductase. Glutathione is an antioxidant
molecule in the cell and acts as a defence mechanism in oxidative stress (Luberda
2005, Meister and Anderson 1983). Reactive oxygen species (ROS), such as
superoxide anion, react with the thiol of GSH and oxidise glutathione (GSSG)
(Luberda 2005). As a reducing agent, NADPH aids in the conversion of GSSG into
GSH. Raising the level of GSH during IVM is well documented to improve oocyte
quality during bovine oocyte maturation (de Matos and Furnus 2000, de Matos, et al.
1997). Interestingly, even though there was no increase in NAD(P)H levels in COCs
matured in the presence of mature BMP15, the level of GSH was significantly
increased compared to control and GDF9 mature treatments. This is perhaps due to
lower levels of ROS production in this group, something that could be confirmed in
future experiments.
The aim of testing the medium in the absence of FSH was to observe the function of
exogenous growth factors on IVM oocytes without any influence of metabolic
activators. Cumulus cell function during IVM is profoundly altered by FSH treatment,
including cumulus cell energy metabolism (Sutton, et al. 2003b). The present study
shows that in the absence or presence of FSH, the addition of pro-mature BMP15 to
Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form
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IVM increases significantly embryo development on day 7 and yielded more
blastocysts on day 8 compared to any other group. However, in the presence of FSH,
the influence of recombinant OSFs on NAD(P)H and GSH levels were diminished.
These results are similar with the previous reports that showed FSH can mask the
influence of OSFs on cumulus cell metabolism (Sutton-McDowall, et al. 2012, Sutton,
et al. 2003a). Moreover, it has been shown that in the absence of alteration of
metabolism by FSH, the influence of OSFs on glycolytic activity leads to the
activation of oxidative phosphorylation in mitochondria to produce ATP, rather than
lactate production (Sugiura, et al. 2005, Sutton-McDowall, et al. 2012). Our
collaborators have shown that addition of mature BMP15 increases glucose uptake
but has no effect on lactate production by Nellore (Bosindicus) bovine COCs, under
slightly different culture conditions (Caixeta, et al. 2013). We did not observe any
increases in glucose uptake in any OSF treatment group, including with mature
BMP15, in the presence or absence of FSH. This result is supported by our previous
study (Sutton-McDowall, et al. 2012).
In conclusion, we found that the form of the oocyte-secreted growth factors GDF9
and BMP15 is an important determinant of their function during oocyte IVM, and that
it affects subsequent embryo development. Only the pro-mature form of BMP15
added in IVM medium significantly improved oocyte developmental competence, and
importantly from a practical perspective, there was no evidence that the only
commercially available mammalian forms of GDF9 and BMP15 could increase cattle
embryo production. Improvement of bovine IVM oocyte competence, may be, in part,
due to an increase in oocyte NAD(P)H and GSH.
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
87
CHAPTER 4
EFFECT OF INTERACTIONS BETWEEN DIFFERING
FORMS OF RECOMBINANT OOCYTE SECRETED
FACTORS ON MOUSE OOCYTE DEVELOPMENTAL
COMPETENCE
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
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4.1 ABSTRACT
The aim of this study was to determine the effect of various sources and forms of
recombinant growth differentiation factor 9 (GDF9) and bone morphogenetic protein
15 (BMP15) during in vitro maturation on embryo development, including
investigation of any additive effects of the two proteins. Cumulus oocyte complexes
(COCs) were collected from mice 24-25 h post-eCG (low developmental competence
model) and were matured in vitro in basic in vitro maturation medium (αMEM+FSH)
or treated with pro-mature human BMP15 (hBMP15), pro-mature mouse GDF9
(mGDF9) and combinations of different forms (pro-mature complex vs mature
domain) and sources (mouse and human) of GDF9 and BMP15. Following in vitro
fertilisation and embryo culture, day 6 blastocysts were stained to determine the cell
numbers. COCs matured in medium containing pro-mature hBMP15 or a
combination of hBMP15 and mGDF9 at 50 ng/ml each (both in pro-mature form)
displayed significantly improved blastocyst development compared to controls and
pro-mature mGDF9 (58.1±3.0%, 68.4±4.5% vs 37.6±4.9% and 40.4±6.8%
respectively, P < 0.05). In contrast, no improvement in developmental competence
was observed after addition of a combination of the mature domains of hBMP15 and
mGDF9, or heterodimers of human GDF9 (hGDF9) and hBMP15 (consisting of
mature domains only). These results show that hBMP15 in pro-mature complex form
improves mouse blastocyst developmental competence. Mature domain proteins did
not affect development suggesting that GDF9 and BMP15 pro-domains play an
important role in oocyte developmental competence.
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
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4.2 INTRODUCTION
Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15)
are two major proteins that are secreted from oocytes. These proteins are important
in ovarian folliculogenesis and required for fertility in a species-specific manner
(Dong et al., 1996, Dube et al., 1998, Galloway et al., 2000). Unlike most of the TGF-
β superfamily members, the expression and bioactivity of GDF9 and BMP15 differ
markedly among species (Crawford and McNatty, 2012). In polyovular animals such
as mouse and rat, the expression of GDF9 is higher compared to BMP15, and
mouse GDF9 (mGDF9) is readily bioactive, hence GDF9 is more important than
BMP15 for fertility in these species (Dong et al., 1996, Yan et al., 2001). In
monoovulatory animals such as cattle and sheep, the ratio of GDF9 to BMP15
expression is closer to 1:1 (Crawford and McNatty, 2012), therefore BMP15 plays a
more prominent role, although both factors are needed.
GDF9 and BMP15 are part of the TGF-β superfamily, and consist of two regions: the
pro-region, and the mature region, which together are known as a pro-protein (un-
processed protein). After cleavage at their protease processing site, the pro-region is
held together with the mature region by non-covalent interactions, creating the pro-
mature complex form (McIntosh et al., 2008, Mottershead et al., 2008). The wild type
human GDF9 (hGDF9) is secreted in a latent complex as pro and mature regions,
whilst mouse GDF9 is secreted in an active form (Mottershead et al., 2008).
Sequence-wise, 64% of the amino acids in the pro-region of hGDF9 are identical to
those of mGDF9, and the mature domains of mouse and human GDF9 share 90%
amino acid homology (Simpson et al., 2012). Separation of the mature region of
hGDF9 from its pro-region activates the protein, allowing it to stimulate the SMAD3
signalling pathway, indicating that the pro-domain contributes to the latency of the
hGDF9 pro-mature complex (Mottershead et al., 2008). In contrast to hGDF9, human
BMP15 (hBMP15) is a potent stimulator of rat (Otsuka et al., 2000) and human
granulosa cell proliferation (Di Pasquale et al., 2004). Ovine pro-mature GDF9
(oGDF9), ovine BMP15 (oBMP15) and mouse BMP15 (mBMP15) are also probably
secreted in latent forms (McNatty et al., 2005). However, a combination of oBMP15
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
90
with either mGDF9 or oGDF9 increases granulosa cell bioactivity by 3-fold compared
to addition of each growth factor alone (McNatty et al., 2005). Hence, GDF9 and
BMP15 presumably interact with each other, which in some cases lead to the
activation of otherwise latent proteins.
After processing, the mature region of these proteins is non-covalently associated
with the pro-region, therefore the mature or pro-regions of GDF9 can interact with
mature or pro-regions of BMP15 and create heterodimers (McIntosh, et al. 2008).
Recently, Peng et al (2013) claimed to have produced a recombinant GDF9:BMP15
heterodimer, and showed that it is a potent activator of mouse and human granulosa
cell proliferation, and also increases expression of cumulus cell expansion genes,
such as Ptx3, Has2, and Ptgs2. However, the molecular composition of this
heterodimer is controversial (Mottershead et al., 2013). While it is not always the
case that GDF9 or BMP15-stimulated granulosa cell proliferation translates into an
improvement in embryo development following in vitro maturation (IVM), some
studies have demonstrated that high expression of cumulus expansion-related
genes, which are downstream of GDF9, is correlated with improvement in oocyte
quality and embryo development (Anderson et al., 2009, McKenzie et al., 2004).
Together, this information shows that compared to most members of the TGFβ
superfamily, a particularly complex level of species-specific control of protein
bioactivity exists in the case of GDF9 and BMP15. This means it is not necessarily
obvious which form of these oocyte-secreted factors (OSF) should be added to in
vitro maturation (IVM) medium. Furthermore, to date, little is known about the form
these proteins are secreted in, and how they interact with receptors. The only form of
GDF9 and BMP15 that can be detected in sheep follicular fluid is the unprocessed
pro-protein (McNatty et al., 2006), whereas in vitro sheep oocytes secrete the mature
domains of GDF9 and BMP15 (Lin et al., 2012). Mouse IVM oocytes secrete GDF9
as a mixture of the unprocessed pro-protein and mature domain (Gilchrist et al.,
2004, Lin et al., 2012), whilst rat IVM oocytes secrete the mature domains of GDF9
and BMP15 (Lin et al., 2012).
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Secreted Factors on Mouse Oocyte Developmental Competence
91
Supplementation of cattle IVM oocytes with recombinant pro-mature oBMP15 or
hBMP15 is more effective at increasing subsequent developmental competence than
the mature region of hBMP15 or the mature of region mGDF9 (Hussein et al., 2006,
Hussein et al., 2011, Sudiman et al., 2014b, Chapter 3). The only commercially
available forms of GDF9 and BMP15 from R&D Systems consist of only the mature
region of these proteins, and although very bioactive, these proteins are ineffective in
mouse IVM (Sudiman et al., 2014a). Therefore the objective of this study was to
examine the effects of different forms (pro-mature complex and mature), different
sources (mouse and human), and putative interactions between GDF9 and BMP15
(heterodimers GDF9:BMP15), during mouse IVM on subsequent embryo
development.
4.3 MATERIAL AND METHODS
All chemicals and media were purchased from Sigma Chemical (St Louis, MO, USA)
unless indicated otherwise.
4.3.1 Isolation and maturation of COCs
Mice used in this study were maintained in the Animal House, Medical School,
Adelaide University. The study was 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.
Twenty-one to twenty-eight day-old SV129 mice were injected intraperitoneally with 5
IU equine chorionic gonadotrophin (eCG; Folligon, Intervet, Castle Hill, Australia),
and ovaries were collected 24 h later. This in vivo priming protocol was chosen as it
produces oocytes that are inherently less developmentally competent than after 48 h
of eCG (Nogueira et al., 2003). Ovaries were cleaned of any connective tissue and
placed in HEPES-buffered αMEM (handling medium, Life Technologies)
supplemented with 3 mg/ml fatty-acid free bovine serum albumin (FAF BSA, MP
Biomedicals, LLC) and 1 mg/ml fetuin. Antral follicles were punctured with 27-gauge
needles and immature COCs collected in handling medium. Only COCs with
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
92
compact cumulus cells were taken. Twenty COCs were cultured in 200 µl IVM
medium [αMEM supplemented with 3 mg/ml FAF BSA, 1 mg/ml fetuin and 50 mIU/ml
FSH (Puregon, Organon, Oss, Netherlands)], in a low-binding Eppendorf tube
(LoBind Tube 0.5 ml, Eppendorf AG) overlaid with 100 µl mineral oil (Fig. 4.1). The
aim to use the Eppenderf tube was to minimize interactions between proteins of
interest and the plastic. Ten small holes were made in the lid to ensure gas
equilibration of IVM medium. The COCs were matured for 17-18 h at 370C in a
humidified atmosphere of 5% CO2 in air.
Figure 4.1 In vitro maturation of mouse COCs in low-binding tube
4.3.2 Sources of BMP15 and GDF9 and treatment of COCs
Recombinant mouse mature region GDF9 (catalogue No: 739-G9) and human
mature region BMP15 (catalogue No: 5096-BM) were purchased from R&D Systems
(Minnaepolis, MN, USA). Recombinant human pro-mature complexes of GDF9 and
BMP15 were produced in-house as described in Chapter 3 using a stable human
embryonic kidney (HEK)-293T cell line. The GDF9:BMP15 covalent heterodimer was
produced by David Mottershead (University of Adelaide) by co-expressing mutated
human BMP15 and GDF9 DNA sequences in HEK-293T cells. Both of the respective
COCs
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
93
DNA sequences had a Ser within the mature region mutated to a Cys residue, to
produce proteins with the intact “Cys-knot” structure characteristic of members of the
TGF-β superfamily. This also enabled production of mature regions of GDF9 and
BMP15 capable of forming covalent dimers. When these mutated DNA sequences
are co-expressed, the cells produce a mixture of covalent homodimers and
heterodimers. Conditioned medium containing the secreted GDF9 (Ser-Cys) and
BMP15 (Ser-Cys) mutant proteins was purified using nickel immobilised metal affinity
chromatography (IMAC) targeting the His-tag on the N-terminus of the pro-regions,
as described previously (Pulkki et al., 2011). After IMAC, the mature regions were
separated from the pro-regions by using rpHPLC (reverse phase high performance
liquid chromatography). This purification was performed by Adelaide Proteomics
Centre using Jupiter 5u C4 300A HPLC columns with a Widepore C4 4x3.0 mm
SecurityGuard Cartridge (Phenomenex, Lane Cove, Australia). One column was
dedicated for heterodimers of GDF9:BMP15 and eluted fractions were collected from
this column. The GDF9:BMP15 heterodimer preparation consists of only the mature
regions of hGDF9 and hBMP15 linked together by a disulphide bridge. The dose of
processed mature region was quantified using Western blotting with the mab28
monoclonal antibody (Pulkki, et al. 2011) and standardised with commercially
available mature region of GDF9 and BMP15 (R&D Systems).
For the first experiment, pro-mature mGDF9 or pro-mature hBMP15, each at 100
ng/ml, or a combination of these proteins at 50 ng/ml each, were added to IVM media
to observe mouse embryo development and quality. For the second experiment, the
effect of the combinations of pro-mature hGDF9 and pro-mature hBMP15 at 100
ng/ml each, mature mGDF9 and mature hBMP15 (R&D Systems) at 100 ng/ml each,
or heterodimers of mature region hGDF9:hBMP15 at 100 ng/ml were tested. Three
replicate experiments were performed with ~30 COCs per treatment per replicate.
4.3.3 In vitro fertilization
Sperm from CBA F1 male mice aged 6-24 weeks and of proven fertility were
incubated for 1 hour in IVF medium (COOK® IVF, Research medium, Catt No: K-
RVWA- 50) under 5% CO2 at 370C for capacitation. Post-maturation, 5-10 COCs
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
94
were underwent IVF into a 90 µl equilibrated IVF drop overlaid with mineral oil, and
10 µl capacitated sperm was added to each drop. After three hours of incubation
under 5% CO2 at 370C, the presumptive zygotes were denuded of sperm and
cumulus cells and placed into culture drops. Insemination day was counted as day 1
(0 h).
4.3.4 In vitro culture of embryos
Five to ten presumptive zygotes were cultured per 20 µl equilibrated culture drop
(COOK® IVC, Research medium, Cat. No: K-RVCL-50) overlaid mineral oilat 370C in
5% O2, 6% CO2 and 89% N2. Five to ten embryos were cultured for six days, with
cleavage rates assessed on day 2 (24 h post-fertilization) and blastocysts assessed
on day 5 (96-100 h post-fertilization) and day 6 (120-24 h post-fertilization).
4.3.5 Differential staining of blastocysts
Differential staining was performed on day 6 blastocysts to assess inner cell mass
(ICM) and trophectoderm (TE) cells counts. Briefly, expanded, hatching and hatched
blastocysts were placed into pronase (5 mg/ml) at 370C until the zona dissolved. The
zona-free blastocysts were incubated in 10 mM trinitrobenzene sulfonic acid (TNBS)
in 0.4% polyvinyl alchol (PVA) in phosphate-buffered saline (PBS) at 40C for 10
minutes, then washed twice in PBS before being transferred into 0.1 mg/ml anti
dinitrophenol-BSA antibody at 370C for 10 minutes. Embryos were then incubated in
10 µg/ml propidium iodide for 5-10 minutes at 370C, followed by 4 µg/ml Hoechst
33342 in 96% ethanol at 40C overnight. The blastocysts were then transferred onto a
glass microscope side, covered by a cover slip and assessed immediately under UV
fluorescence using an upright microscope (Nikon, TE 2000-E, excitation, 340-380
nm; emission, 440-480 nm), where ICM cells appeared blue and TE cells appeared
pink.
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
95
4.4 RESULTS
4.4.1 The effect of pro-mature mGDF9 and hBMP15 alone or in
combination during IVM on subsequent embryo development
Addition of pro-mature hBMP15 alone or in combination with pro-mature mGDF9 to
IVM medium, led to a substantial, almost 2-3-fold, increase in blastocyst
development on days 5 (P < 0.01) and 6 (P < 0.05), compared to controls and pro-
mature mGDF9 alone (Table 4.1). There were no significant differences in cleavage
rates or inner cell mass, trophectoderm cells or total cell numbers across all
treatment groups and controls (Tables 4.1, 4.2). The highest blastocyst rate was
derived from COCs exposed to a combination of these two proteins, however it did
not significantly differ from those exposed to hBMP15 alone.
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
96
Table 4.1 Effect of supplementing COC cultures with mGDF9, hBMP15 (pro-mature complexes) and combination of both
proteins on oocyte developmental competence
Treatments No. of
Oocytes
Cleavage
(%)1
Blastocyst
on day 52
Blastocyst
on day 63
Hatching
Blastocyst
on day 64
Control
mGDF9 pro-mature 100 ng/ml
hBMP15 pro-mature 100 ng/ml
mGDF9 pro-mature 50 ng/ml + hBMP15 pro-mature 50 ng/ml
94
86
92
92
63.1±6.2
60.7±3.0
73.2±6.5
58.6±12.8
17.2±1.0a
19.4±5.4a
41.7±7.0b*
45.4±4.3b*
37.5±5.0a
40.4±6.8a
58.1±3.0b
68.4±4.5b*
20.3±4.1
27.0±7.2
46.2±6.9
42.2±8.1
Values with different superscripts within a column are statistically different (P < 0.05), * = P < 0.01 compared to control and
mGDF9
Data are presented as means ± SEM of 3 replicate experiments. Each replicate experiment consisted of 27-35 oocytes.
1Percentage of cleaved embryos per total oocytes
2Percentage of blastocysts generated 96-100h post-fertilization per cleaved embryo
3Percentage of blastocysts generated 100-125h post-fertilization per cleaved embryo
4Percentage of hatching blastocysts generated 100-125 h post fertilization per cleaved embryo
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
97
Table 4.2 Effect of supplementing COC cultures with mGDF9, hBMP15 (pro-mature
complexes) and combination of both proteins on numbers of inner cell mass (ICM),
trophectoderm (TE) and total (TCN) cells in blastocysts
Data are presented as means ± SEM. Mean blastocyst cell numbers following
differential staining of 10-15 blastocysts.
4.4.2 Interactions between GDF9 and BMP15: effect of differing
forms during IVM on subsequent embryo development
While there was a slight improvement in blastocyst development with a combination
of pro-mature hGDF9 and hBMP15, this was not significantly different compared to
controls. No significant differences in embryo quality were found among treatment
groups compared to controls (Tables 4.3, 4.4).
Treatments ICM TE TCN
Control
mGDF9 pro-mature 100 ng/ml
hBMP15 pro-mature 100 ng/ml
mGDF9 pro-mature 50 ng/ml +
hBMP15 pro-mature 50 ng/ml
13.7±1.3
12.0±1.0
14.8±1.5
15.3±1.7
30.0±3.1
28.5±2.9
41.9±4.2
34.0±4.6
45.0±3.3
40.5±3.4
56.7±5.2
49.3±5.0
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
98
Table 4.3 Effect of supplementing COC cultures with a combination of hGDF9 and hBMP15 (pro-mature complexes), a
combination of mGDF9 and hBMP15 (isolated mature regions), or a hGDF9:hBMP15 heterodimer (isolated mature regions) on
oocyte developmental competence.
Treatments Form No. of
Oocytes
Cleavage
(%)1
Blastocyst
on day 52
Blastocyst
on day 63
Hatching
Blastocyst
on day 64
Control
hGDF9 100 ng/ml + hBMP15 100 ng/ml
mGDF9 100 ng/ml + hBMP15 100 ng/ml
hGDF9:hBMP15 heterodimer 100 ng/ml
N/A
Pro-mature
Mature
Mature
94
86
92
92
64.2±9.9
71.8±1.6
67.4±5.0
72.6±5.8
26.1±10.0
32.6±6.2
17.8±7.8
20.0±9.1
49.5±10.7
55.5±0.5
46.0±4.5
44.4±6.6
29.8±7.7
33.2±8.5
25.5±11.3
21.8±5.1
Data are presented as means ± SEM of 3 replicate experiments. Each replicate experiment consisted of 24-35 oocytes.
1Percentage of cleaved embryos per total oocytes
2Percentage of blastocysts generated 96-100h post-fertilization per cleaved embryo
3Percentage of blastocysts generated 100-125h post-fertilization per cleaved embryo
4Percentage of hatching blastocysts generated 100-125 h post fertilization per cleaved embryo
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
99
Table 4.4 Effect of supplementing COC cultures with a combination of hGDF9 and hBMP15 (pro-mature complexes), a
combination of mGDF9 and hBMP15 (isolated mature regions), or a hGDF9:hBMP15 heterodimer (isolated mature regions) on
numbers of inner cell mass (ICM), trophectoderm (TE) and total (TCN) cells in blastocysts
Data are presented as means ± SEM. Mean blastocyst cell numbers following differential staining of 8-10 blastocysts.
Treatments Form ICM TE TCN
Control
hGDF9 100 ng/ml + hBMP15 100 ng/ml
mGDF9 100 ng/ml + hBMP15 100 ng/ml
hGDF9:hBMP15 heterodimer 100 ng/ml
N/A
Pro-mature
Mature
Mature
14.2±1.8
17.5±1.6
15.7±2.4
13.7±1.9
40.9±4.1
36.9±4.7
39.4±5.7
28.2±3.5
55.1±3.9
54.5±5.7
55.1±7.3
41.8±5.2
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
100
4.5 DISCUSSION
In this study, mouse oocytes were matured in vitro in low-binding Eppendorf tubes in
order to minimize interactions between the proteins of interest and the plastic of Petri
dishes. In previous studies, it was found that the pro-mature hGDF9 protein has a
very high binding affinity for plastic culture-ware (Mottershead DG, unpublished
data). However, by using low-binding Eppendorf tubes, we minimized the interaction
between pro-mature hGDF9 and the tube. Preliminary experiments showed that
there was no adverse effect on subsequent embryo development after maturation in
low-binding Eppendorf tubes compared to Petri dishes (see Appendix 3).
Based on the previous work by Nogueira et al (2003), we also used immature COCs
collected 24-25 h post-PMSG injection, instead of 46-48 h post-injection, in order to
create a COC IVM system which does not have unnaturally elevated oocyte
developmental competence. This proved effective as control blastocysts rates were
approximately 30-40% lower than in Chapter 2, and hence enabled us to detect any
beneficial effects on embryo development from treatments.
In the present study, pro-mature hBMP15 alone and the combination of mGDF9 and
hBMP15, in their pro-mature forms, substantially improved embryo development
compared to the control group and mGDF9 alone. The improvement in embryo
development observed was enhanced further with the combination of both proteins.
This supports similar results from Chapter 3, which showed that pro-mature hBMP15
significantly increased bovine embryo development at 100 ng/ml. Recently, it has
been demonstrated that in vitro matured oocytes are deficient in BMP15 protein
expression, compared to their in vivo counterparts (Mester, et al. 2013), thus addition
of exogenous pro-mature hBMP15 in IVM medium improves in vitro matured oocyte
developmental competence. Interestingly, even though the expression of GDF9 is
notably higher than BMP15 in polyovular animals (Crawford and McNatty 2012), and
pro-mature mGDF9 is a potent activator in granulosa cell proliferation assays
(Mottershead, et al. 2008), it did not improve embryo development post-IVM in the
current study. Likewise, mature region mGDF9 did not improve the development of
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
101
mouse IVM oocytes (Sudiman, et al. 2014a). In contrast, addition of partially purified
pro-mature mGDF9 to in vitro maturing bovine COCs increased blastocyst
development, and in mouse improved fetal survival rates (Hussein, et al. 2005,
Hussein, et al. 2011, Yeo, et al. 2008). The differences in results are likely to be due
to the different purification methods used to produce proteins. Previous studies have
used unpurified or partially-purified recombinant mGDF9 preparations, which contain
other proteins that are produced by the host cells (Kaivo-Oja, et al. 2003, McNatty, et
al. 2005a). GDF9 clearly benefits from the presence of BMP15, and it may also
benefit from interactions with other proteins, which may have been present in the
preparations used in previous studies.
The second experiment further examined interactions between GDF9 and BMP15
proteins and their effects on oocyte development in vitro. Even though there was a
slight improvement in blastocyst rate with the combination of pro-mature hGDF9 and
pro-mature hBMP15, it did not significantly differ compared to the control group. Pro-
mature hGDF9 alone is latent and is unable to induce granulosa cell proliferation
(Mottershead, et al. 2008); however, the combination of hGDF9 and hBMP15 is
capable of inducing granulosa cell proliferation (Mottershead, unpublished data).
Hence, the lack of effect of hGDF9 + hBMP15 on competence is interesting, in the
context of the positive effect of mGDF9 + hBMP15. This suggests tightly regulated
species-specific OSF control of oocyte competence. Consistent with this, pro-mature
hBMP15 interacts differently with pro-mature GDF9 sourced from mouse versus
human (McNatty, et al. 2005a).
It has been shown that the maximum synergistic effect of GDF9 and BMP15 in
stimulating granulosa cell proliferation is obtained at a dose of 12.5 ng/ml each
(Mottershead, et al. 2012). In general terms, COCs are less sensitive than isolated
granulosa cells to GDF9 and BMP15, and usually higher doses are used in IVM
(Dragovic, et al. 2005). Nonetheless, it is possible that using the higher doses of
these proteins induced an inhibitory, rather than stimulatory effect, on cumulus
expansion and competence. Unfortunately, we are unable to score the cumulus cell
expansion due to the COCs forming clumps during maturation in the low-binding
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
102
Eppendorf tubes. No positive effect on embryo development could be observed from
a combination of mature region mGDF9 and hBMP15 (R&D Systems), a result which
supports previous data from our lab showing that in combination, doses from 50
ng/ml up to 200 ng/ml did not improve mouse oocyte developmental competence
(Sudiman, et al. 2014a).
The first records of production of a recombinant GDF9:BMP15 heterodimer were in
2013, and this is the first attempt at using these preparations in oocyte IVM in any
species. The construct used in this study has a mutated cysteine in the mature region
and was purified by IMAC followed by HPLC chromatography, to generate an
isolated covalent heterodimer of the mature regions of hGDF9 and hBMP15
(Mottershead, unpublished data). This preparation is exceptionally potent on isolated
mouse granulosa cells (Mottershead, unpublished data). Nonetheless, the
heterodimer had no effect on mouse oocyte developmental competence (current
study). Since the heterodimer consists only of the mature regions of hGDF9 and
hBMP15, we hypothesize that the pro-regions may also be important for heterodimer-
mediated effects, as they are for homodimer-mediated effects, on developmental
competence of oocytes. Since heterodimers of GDF9:BMP15 use both receptors
from GDF9 and BMP15 (BMPR2 + ALK 4/5/7:ALK6) before triggering
phosphorylation of SMAD 2/3 in cumulus cells (Kaivo-Oja, et al. 2005, Peng, et al.
2013), we believe that this form has important implications for female fertility.
However, further studies are required to examine this hypothesis and other effects of
a heterodimer on oocyte function and the physiological dose that is required to
improve oocyte developmental competence.
The present results clearly show that hBMP15 in pro-mature complex form improves
mouse oocyte developmental competence. However, no effect could be observed of
the mature domain of these proteins, alone or in combination, on mouse IVM
oocytes. This suggests the pro-mature complex is the functional form required to
improve the developmental competence of IVM oocytes. Moreover, since there was a
trend toward further improvement with a combination of the pro-mature forms of
mGDF9 and hBMP15, it suggests that these proteins interact with each other.
Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte
Secreted Factors on Mouse Oocyte Developmental Competence
103
Nevertheless, since different species have different requirements for GDF9 and
BMP15, and the source of these recombinant proteins (mouse, human, sheep)
affects their interaction with granulosa and cumulus cells, further studies are required
to observe the species-specific dose and form requirements of these proteins for
optimal embryo development.
Chapter 5: General Discussion
104
CHAPTER 5
GENERAL DISCUSSION
Chapter 5: General Discussion
105
In vitro maturation (IVM) of oocytes has been successfully used in laboratory/small
animals and in the livestock industry for the production of embryos in vitro (Thibier
2006). In contrast, since the IVM procedure was introduced into human assisted
reproductive technology (ART), only 1300 live births have been produced by this
method (Suikkari 2008). Even though IVM in human ART has so far yielded limited
success, this emerging technology has the potential to substitute for standard human
in vitro fertilisation (IVF) protocols, or become an adjuvant to IVF for many reasons.
This method does not require the use of gonadotropin hormones (used in standard
IVF), therefore the side effects associated with gonadotropin use, such as ovarian
hyperstimulation syndrome (OHSS), and patient inconvenience, can be avoided. It
also reduces the cost of ART which makes this technology available for patients with
lower economic status. To date, several studies have demonstrated improvements to
the IVM process via different methods, such as administration of lower doses of
gonadotropin hormone (Jurema and Nogueira 2006), modification of the maturation
medium by adding exogenous ATP (Xue, et al. 2004), EGF-like peptides (Richani, et
al. 2013), or cAMP modulators (Albuz, et al. 2010), or prolonging gap junction
communication between the oocyte and cumulus cells (Albuz, et al. 2010, Thomas,
et al. 2004). Recent studies have shown that co-culture of denuded oocytes (DOs)
with cumulus oocyte complexes (COCs) improves embryo development derived from
in vitro matured oocytes in the cow (Dey, et al. 2012, Hussein, et al. 2011, Hussein,
et al. 2006), goat (Romaguera, et al. 2010) and pig (Gomez, et al. 2012). It has been
demonstrated that the oocyte actively secretes soluble paracrine factors (oocyte
secreted factors; OSFs) which act on the surrounding somatic cells and allow the
oocyte to control its own microenvironment (Gilchrist et al., 2008). Lower embryo
development post-IVM is at least in part due to aberrant gene and protein expression
(including BMP15), in in vitro matured oocytes compared to in vivo counterparts
(Dunning, et al. 2007, Mester, et al. 2014). Therefore the addition of native
exogenous OSFs derived from denuded oocytes, or exogenous recombinant proteins
such as GDF9 and BMP15, can be used to improve the developmental competence
of in vitro matured oocytes.
Chapter 5: General Discussion
106
This study focused on OSFs and recombinant proteins (GDF9 and BMP15) and their
capacity to improve IVM systems. Results from Chapter 2 showed that exogenous
native OSFs improved the developmental competence of mouse IVM oocytes, and
led to a doubling in fetal yield. Moreover, this study gives an insight into factors
affecting the capacity of these native OSFs to improve mouse IVM, such as the
presence of cumulus cells and FSH, and the temporal regulation of the native OSF
pool. However, even though co-culture of COCs with DOs can be used effectively in
cattle and animal reproduction, it is not possible to collect the necessary numbers of
DOs for use in clinical human ART. Therefore, the next step was to examine the
effect of recombinant proteins that can be used to mimic the beneficial effects of
exogenous native OSFs.
GDF9 and BMP15 are two major growth factors secreted by oocytes, and are critical
for female fertility. Homozygous mutations in genes encoding these proteins
contribute to reduced fertility in mouse and sheep (Dong, et al. 1996, Galloway, et al.
2000, Hanrahan, et al. 2004, Juengel, et al. 2002). In humans, genetic variance and
rare mutations of GDF9 and BMP15 contribute to PCOS, dizygotic twinning and
premature ovarian failure (Chand, et al. 2006, Dixit, et al. 2005, Palmer, et al. 2006,
Wang, et al. 2013). Like all members of the TGF-β family, GDF9 and BMP15 consist
of pro and mature regions (together called the pro-protein). After processing, the pro
and mature regions of these proteins are held together by non-covalent bonding. To
date, it is still unclear which forms of GDF9 and BMP15 are secreted by in vivo and in
vitro matured oocytes, and there appears to be important species differences
(Gilchrist, et al. 2006, Lin, et al. 2012, McNatty, et al. 2006). The only commercially
available mammalian cell derived GDF9 and BMP15 is sold by R&D Systems, and
consist only of the mature domains of these proteins (refer to Figs. 5.1 C and D).
Previous studies from Hussein et al (2006, 2011) and Yeo (2008) using partially-
purified recombinant mouse GDF9 and sheep BMP15, which contain pro and mature
regions of these proteins, in IVM showed an improvement in mouse and cattle
blastocyst development and fetal yield. So far, there are no other studies showing
improvement in embryo development after addition of the mature domains of GDF9
and BMP15.
Chapter 5: General Discussion
107
We suspected that the pro-region of these proteins plays an important role in
determining oocyte developmental competence. Therefore, the main aim of chapter 4
was to observe the effect of different forms of GDF9 and BMP15 (pro-mature and
mature domain only) from difference sources (mouse and human). We used purified
recombinant proteins from different sources that were purified using a His-tag
purification method (Pulkki, et al. 2011). Refer to Figs. 5.1 A and B. Even though
Western blot results from Chapter 2 were unable to detect any differences in the
quantity and form of GDF9 (17 and 65 kDa) from extracts of DO at 0h or 3h, we
found that human BMP15 (hBMP15) in its pro-mature complex form was the most
effective at increasing developmental competence of in vitro matured cattle and
mouse oocytes (Chapters 3 and 4, Table 5.1). We also demonstrated that pro-mature
hBMP15 increases oocyte metabolism during maturation (Chapter 3). However, the
mechanisms by which pro-mature complex proteins interact with the cumulus cell
matrix and the receptors, and what the exact function of the pro-region is during
oocyte maturation, are still unclear. In future experiments, different forms and
sources of recombinant proteins in IVM medium could be used and observed on
measures such as the activation of the Smad signalling in cumulus cells and any
differences in the production of diffusible factors such as cAMP and cGMP by the
cumulus cells. It would be also interesting to examine differences in meiotic activity
and the developmental programming of oocytes in response to these different protein
forms and sources.
In vivo matured oocytes from polyovular animals express GDF9 as the major
component of OSFs (Crawford and McNatty 2012). However in this present study,
mouse GDF9 (mGDF9) in pro-mature complex form did not improve mouse embryo
development (Chapter 4, Table 5.1). Similar to results from our previous study, no
improvement was observed after addition of mGDF9 and hBMP15 (both mature
domains only) or a combination of both proteins at a range of doses (without the
presence of the pro-domain, Sudiman et al. 2014a, Chapter 4, Table 5.1).
Nevertheless, when pro-mature mGDF9 was combined with pro-mature hBMP15 at a
dose of 50 ng/ml each, there was a significant improvement in mouse embryo
development (Chapter 4, Table 5.1). Moreover, results from our previous study
showed that the mature region of BMP15 combined with fibroblast growth factor 17
Chapter 5: General Discussion
108
(FGF17) increased the number of inner cell mass cells in cattle blastocysts
(Appendix 2). Therefore, it remains to be investigated whether BMP15 or any other
OSFs such as BMP6 (Solloway, et al. 1998), FGF10 (Zhang, et al. 2010), FGF11
(Sugiura, et al. 2007) are required for GDF9 in order to have the optimal impact on
developmental competence of IVM oocytes. The challenge is to find the optimal dose
of these recombinant proteins which is effective in improving in vitro matured oocyte
developmental competence. Results from Chapter 4 showed that a combination of
pro-mature hGDF9 and hBMP15 at 100 ng/ml each had a small positive impact on
mouse embryo development (refer to Table 5.1). Therefore the effect of recombinant
proteins depends on the dose, and the source, of proteins and this suggests tightly
regulated species-specific OSF control of oocyte competence.
Even though this study provides evidence that the pro-mature form of hBMP15, and
a combination of pro-mature mGDF9 and hBMP15, are effective, it may be
problematic to apply these results to human ART. GDF9 and BMP15 are biologically
active substrates, and it could be argued that the only source which should be used
for human ART should be the human recombinant proteins. Unfortunately, wild type
hGDF9, which consists of pro and mature regions, is secreted in a latent complex
(Mottershead, et al. 2008). In the future, it will be interesting to observe the effect of
bioactive mutants of pro-mature hGDF9, produced by substituting the Gly residue
with aArg residue at position 391, on human IVM oocytes (Simpson, et al. 2012).
Moreover, in monovular animals such as cattle, the ratio of GDF9 and BMP15 is
nearly equal (Crawford and McNatty 2012) and a combination of pro-mature hGDF9
and pro-mature hBMP15 is bioactive (Mottershead, unpublished data). Therefore, we
propose that a combination of hGDF9 and hBMP15 in their pro-mature complex
forms is also a promising area to investigate for the purpose of improving human IVM
success.
The choice of animal experimental model is an important issue for future research in
this area. In Chapter 4 we used the approach of different timing for the collection of
COCs (24-25 h post-PMSG), since the conventional model using immature COCs
collected 46-48 h post-PMSG from pre-pubertal mice, produces a very high level of
blastocyst development (Chapter 2). It can be argued that this model has
Chapter 5: General Discussion
109
questionable physiological relevance to the types of oocytes used in human or
veterinary IVM. The low-developmental competence model allows us to better detect
an actual improvement resulting from different treatments. Animal models such as
pig (Coy and Romar 2002), buffalo (Gautam, et al. 2008) or goat (Romaguera, et al.
2010), which have lower developmental competence, perhaps are more useful and a
better model for human applications, since IVM may be of particular importance for
polycystic ovary syndrome (PCOS) patients whose oocytes have compromised
developmental competence. For future experiments, it will also be interesting to
observe the effect of native OSFs or pro-mature GDF9 and BMP15 on oocytes
derived from small follicles and pre-pubertal animals. Commonly, researchers use
oocytes from large follicles (Leibfried and First 1979) and derived from adult animals
(Damiani, et al. 1996), due to better embryo development. Previous studies have
shown that co-culture of COCs derived from small follicles with DOs from either large
or small follicles increased goat blastocyst development (Romaguera, et al. 2010).
However, whether recombinant pro-mature GDF9 and BMP15 also have the same
positive effect on COCs from small follicles is still unknown.
Future directions from this study may also include design of functional heterodimers
of OSFs; notably of GDF9 and BMP15. Even though the concept of heterodimers is
well described (McIntosh, et al. 2008), the production of the putative protein was only
first described in 2013 (Peng, et al. 2013). However, the validity of a heterodimer of
GDF9:BMP15 in this study is still controversial since there is a lack of evidence to
show the molecular composition of this preparation, which may contain a mix of
dimers of homodimers of GDF9 and BMP15 (Mottershead, et al. 2013). This current
study is the first to use a heterodimer of GDF9:BMP15 which has a mutated cysteine
in the mature region and forms covalent heterodimers of the mature regions
(Mottershead, unpublished data; see Chapter 4 for production of this protein). It has
been shown that GDF9 pro-mature complex binds strongly to heparin sepharose
(Watson, et al. 2012), and the pro-regions of GDF9 and BMP15 interact with isolated
mural granulosa cells (Mester, et al. 2014). We suggested that the pro-region of
BMP15 may interact with the extracellular matrix of cumulus cells during maturation
and facilitate presentation of the mature domain to cumulus cell receptors. Therefore,
by creating heterodimers with a covalent bond, we hypothesised that the dimers of
Chapter 5: General Discussion
110
the mature regions of GDF9:BMP15 interact with the receptors in cumulus cells
instead of extracellular matrix of cumulus cells (refer to Fig. 5.1 E). However, results
from Chapter 4 showed that heterodimers of GDF9:BMP15 mature regions do not
improve oocyte developmental competence (refer to Table 5.1). It seems probable
that the pro-mature complex form of this protein is also required. Therefore for future
experiments, the effect of heterodimers of GDF9:BMP15 which contain both the pro
and mature regions, on embryo development should be observed (refer to Fig. 5.1
F). Another direction would be to investigate the signalling pathway of heterodimers,
specifically in cumulus cells.
Chapter 5: General Discussion
111
Figure 5.1 Different forms of GDF9 and BMP15 proteins. A and B. The pro-mature
complex forms of GDF9 and BMP15 consist of homodimers of mature regions and
their associated pro-regions. C and D. The commercially available GDF9 and BMP15
consist of homodimers of mature regions only. E. Heterodimers of GDF9:BMP15
consist of dimers of GDF9 and BMP15 mature regions with a covalent bond, as used
in Chapter 4. F. Our proposed heterodimers of GDF9:BMP15 which consist of dimers
of pro and mature regions of GDF9 and BMP15 for future experiments.
Chapter 5: General Discussion
112
Table 5.1 Summary of developmental competency of mouse and cattle oocytes undergoing IVM with the addition of native
OSFs and different forms and sources of GDF9 and BMP15
Treatments Form Embryo development
Mouse IVM oocytes Cattle IVM oocytes
Native OSFs Possibly mix of pro-mature and mature domains of GDF9 and BMP15
↑↑ (Chapter 2, Sudiman et al. 2014a)
↑↑ (Dey, et al. 2012, Hussein, et al. 2011, Hussein, et al. 2006)
mGDF9 Partially purified (Pro-mature) ↑↑ (Yeo, et al. 2008)
↑ (Hussein, et al. 2011, Hussein, et al. 2006)
oBMP15 Partially purified (Pro-mature) N/A ↑↑ (Hussein, et al. 2011, Hussein, et al. 2006)
mGDF9 + oBMP15 Partially purified (Pro-mature) N/A ↑↑ (Hussein, et al. 2006)
mGDF9 Pro-mature - (Chapter 4)
N/A
hBMP15 Pro-mature ↑↑ (Chapter 4)
↑↑ (Chapter 3, Sudiman et al. 2014b)
Chapter 5: General Discussion
113
↑↑ = large increase, ↑ = increase, - = no change, N/A= not available
Treatments Form Embryo development
Mouse IVM oocytes Cattle IVM oocytes
mGDF9 Mature - (Sudiman et al., 2014a)
- (Chapter 3, Sudiman et al., 2014b)
hBMP15 Mature - (Sudiman et al., 2014a)
↑ (Chapter 3, Sudiman et al., 2014b)
mGDF9 + hBMP15 Mature - (Sudiman et al., 2014a)
N/A
mGDF9 + hBMP15 Pro-mature ↑↑ (Chapter 4)
N/A
hGDF9 + hBMP15 Pro-mature ↑ (Chapter 4)
N/A
hGDF9:hBMP15 Heterodimers - (Chapter 4)
N/A
Chapter 5: General Discussion
114
To date, there are no definitive and non-invasive technologies available to measure
developmental competence of immature oocytes. This study measures various
metabolic activities of oocytes matured in the presence of different recombinant
proteins. However, the common use of FSH, a metabolic activator, in IVM medium
(including human) masks these measurements (Chapter 3, Sudiman et al. 2014b).
Therefore, in future experiments, we need to investigate measurements which are
not affected by metabolic activators, such as; analysis of gene arrays in cumulus
cells, substrates released or energy production by the oocyte during maturation,
meiotic competence and so on.
There is still a long way to go to determine the optimal IVM conditions for human
oocytes, to improve the success rate to a level comparable to that of traditional IVF.
However, implementing IVM systems in IVF practice is important since this
technology is cheaper and safer in some patients. This study contributed to IVM
system development by showing that native oocyte secreted factors improve in vitro
matured oocyte developmental competence, and investigated factors that affect
production of OSFs. Furthermore, it demonstrated that the beneficial effect of native
OSFs can be mimicked by adding recombinant proteins (GDF9 and BMP15) in pro-
mature complex form. However, testing the effect of various forms, combinations,
sources and doses of BMP15 and GDF9 during IVM, on a wide range of outcome
measures for different species, including human, are important future steps in this
process.
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Appendices
143
APPENDICES
Appendices
144
Appendix 1: The effect of native pig OSFs on mouse
IVM oocyte developmental competence
This experiment was performed to observe the effect of pig OSFs on mouse in vitro
matured oocytes. Mouse COCs were cultured in 50 µl drops of base medium (α MEM
supplemented with 3 mg/ml fatty-acid free bovine serum, 1 mg/ml fetuin and 50
mIU/ml FSH). Mouse COCs were distributed into 4 treatment groups: (1) 20 mouse
COCs were cultured in 50 µl IVM drop (control), (2) 20 mouse COCs were co-
cultured with 25 pig COCs, (3) 20 mouse COCs were co-cultured with 50 pig COCs,
(4) 20 mouse COCs were co-cultured with 25 pig DOs, (5) 20 mouse COCs were co-
cultured with 50 pig DOs. After 17 h in maturation medium, mouse COCs were
inseminated and blastocyst development was assessed on days 5 and 6.
Result:
As shown in Table 1, there was no difference in blastocyst development after
addition of pig denuded oocytes or cumulus oocyte complexes on mouse embryo
development. Co-culture of mouse COCs with 50 pig DOs significantly decreased
cleavage rate compared to control group.
Appendices
145
Table 1 Effect of native pig OSFs on mouse IVM oocytes on subsequent embryo
development
Values with different superscript within a column are statistically different (P < 0.05).
Data are presented as means ± SEM of 4 replicate experiments. Each replicate
experiment consisted of 19-40 oocytes.
1Percentage of cleaved embryos per total oocytes
2Percentage of blastocysts generated 96-100h post-fertilization per cleaved embryo
3Percentage of blastocysts generated 100-125h post-fertilization per cleaved embryo
4Percentage of hatching blastocysts generated 100-125 h post fertilization per
cleaved embryo
Treatments No. of
Oocytes
Cleavage2
(%)
Blastocyst
on day 53
Blastocyst
on day 64
Hatching
Blastocyst
on day 65
Control
COC + 25 DO
COC + 50 DO
COC + 25 COC
COC + 50 COC
156
132
133
140
124
91.1±3.8a
82.1±3.2a,b
79.8±5.2b
84.1±4.1a,b
88.4±2.5a,b
37.5±5.5
48.8±7.8
49.0±10.8
54.2±5.5
42.2±6.3
62.3±6.1
68.2±4.0
74.0±8.9
76.1±2.9
61.8±2.9
43.1±3.9
51.7±8.6
59.5±8.7
61.1±6.8
48.3±3.9
Appendices
146
Appendix 2: The effect of FGF17 and BMP15 on
bovine in vitro matured oocytes and subsequent
embryo development
The aim of this study was to determine interactive effects between FGF17 and
BMP15 on bovine in vitro matured oocytes and subsequent embryo development.
COCs were cultured in base medium (bicarbonate-buffered TCM 199 + 4 mg/ml FAF
BSA + 0.1 IU/ml FSH), with the following additional supplements: (1) none (control),
(2) 100 ng/ml FGF 17 (R&D System), 100 ng/ml BMP15 (R&D System) and (4) 100
ng/ml FGF17 + 100 ng/ml BMP15 (both R&D System).
Results:
There was no significant difference on bovine embryo development between control
and treatment groups, however, there was a significant improvement on inner cell
mass number in the combined of FGF17 and BMP15 group compared to the control
group which suggests an interaction of these combined proteins on embryo quality.
Appendices
147
Table 2 Effect of supplementing COC cultures with BMP15, FGF17 and combination
both proteins at 100 ng/ml dose on bovine oocyte developmental competence
Data are presented as mean ± SEM of 4 replicate experiments. Each replicate
experiment consisted of 36-41 oocytes.
Table 3 Effect of supplementing COC cultures with BMP15, FGF17 and combination
both proteins at 100 ng/ml on numbers of inner cell mass (ICM), trophectoderm (TE)
cells and total cell numbers (TCN) of blastocyst on day 6
Treatments ICM TE TCN
Control
FGF17
BMP15
FGF17 + BMP15
25.3±1.6a
27.7±1.7a,b
26.9±1.4a,b
31.4±2.0b
80.9±4.2
76.6±3.9
81.4±3.2
83.2±5.2
105.9±4.2
104.3±4.4
108.3±3.3
114.7±5.9
Values with different superscript within a column are statistically different (P < 0.05).
Data are presented as mean ± SEM of 4 replicates. Mean blastocyst cell numbers
following differential staining of 29-40 blastocysts.
Treatments No. of
Oocytes
Cleavage
(%)
Blastocyst
on day 7
Blastocyst
on day 8
Hatching
Blastocyst
on day 8
Control
FGF17
BMP15
FGF17 + BMP15
156
157
153
154
84.6±3.8
91.7±1.9
88.9±4.4
88.3±2.4
49.2±5.7
44.4±4.6
49.3±1.1
50.7±3.1
61.4±5.4
55.6±6.7
59.6±2.7
61.8±3.8
50.6±5.6
40.0±10.4
46.9±5.5
44.0±1.8
Appendices
148
Appendix 3: The effect of Petri Dish and Eppendorf
Tube on Mouse IVM oocytes
This prelimenary experiment for chapter 4 was performed to observe the effect of
Eppendorf tube compared to Petri dish as a conventional compartment to culture IVM
oocytes.
COCs were collected from 3-4 wks and were injected intraperitoneally with 5 IU
equine chorionic gonadotrophin (eCG; Folligon, Intervet, Castle Hill, Australia), and
ovaries were collected 24-25 h later. COCs then were matured in vitro in basic in
vitro maturation medium (α MEM supplemented with 3 mg/ml fatty-acid free bovine
serum, 1 mg/ml fetuin and 50 mIU/ml FSH) in Petri dish or in Eppendorf tube (Figure
4.1). Twenty COCs were matured in either a 50 µl IVM drop overlaid with mineral oil
on Petri dish or in a 200 µl drop overlaid with mineral oil in an Eppendorf tube.
As shown in Table 4, there was no obvious difference in embryo development after
cultured in Petri dish or Eppendor tube.
Table 4 Effect of maturation of mouse COCs in Petri dish or Eppendorf tube on
mouse embryo development
Data are presented as means ± SEM of 1 replicate experiment.
Treatments No. of
Oocytes
Cleavage
(%)
Blastocyst
on day 5
Blastocyst
on day 6
Hatching
Blastocyst
on day 6
Petri dish
Eppendorf tube
20
23
14 (70%)
20 (87%)
4 (29%)
6 (30%)
8 (57.1%)
13 (65%)
5 (62.5%)
10 (76.9%)
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