THE IMPACT OF MATERNAL OVERNUTRITION DURING THE ... · THE IMPACT OF MATERNAL OVERNUTRITION DURING...

THE IMPACT OF MATERNAL OVERNUTRITION

DURING THE PERICONCEPTIONAL PERIOD ON

THE DEVELOPMENT OF POSTNATAL OBESITY IN

THE SHEEP

Leewen Rattanatray B. Biomedical Sc. (Hons)

Discipline of Physiology

School of Molecular and Biomedical Science

The University of Adelaide

A thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy to the University of Adelaide

May 2010

Table of Contents II

TABLE OF CONTENTS

TABLE OF CONTENTS ....................................................................................... II

ABSTRACT ........................................................................................................ VII

DECLARATION .................................................................................................. IX

ACKNOWLEDGEMENTS .................................................................................... X

RELATED PUBLICATIONS ............................................................................... XII

LIST OF FIGURES AND TABLES .................................................................... XIII

COMMONLY USED ABBREVIATIONS ........................................................... XVII

CHAPTER 1: LITERATURE REVIEW ................................................................. 2

1.1 INTRODUCTION ................................................................................... 2

1.2 FETAL ORIGINS OF ADULT DISEASE ................................................ 3

1.3 DEVELOPMENTAL PROGRAMMING OF THE METABOLIC

SYNDROME: EPIDEMIOLOGICAL EVIDENCE ............................................... 5

1.3.1 Maternal overnutrition and the developmental programming of obesity ....... 7

1.3.2 The obesity epidemic .................................................................................. 7

1.3.3 Maternal obesity .......................................................................................... 8

1.4 BENEFITS AND RISKS ASSOCIATED WITH DIETARY RESTRICTION

OF WOMEN WITH A HIGH BMI ...................................................................... 9

1.4.1 Benefits of dietary weight loss before entering pregnancy........................... 9

1.5 DEVELOPMENTAL PROGRAMMING OF OBESITY:

EPIDEMIOLOGICAL EVIDENCE ................................................................... 11

1.5.1 Relationship between maternal obesity and high birth weight ....................11

1.5.2 High maternal and fetal planes of nutrition result in a high birth weight infant

12

1.5.3 Maternal diabetes .......................................................................................12

1.5.4 High birth weight a predictor of adulthood adiposity, insulin resistance and

diabetes .................................................................................................................14

Table of Contents III

1.6 MATERNAL OVERNUTRITION AND OFFSPRING ADIPOSITY:

EXPERIMENTAL ANIMAL STUDIES ............................................................. 16

1.7 CRITICAL WINDOWS OF EARLY DEVELOPMENT: INTRA-UTERINE

GROWTH AND DEVELOPMENT .................................................................. 18

1.7.1 Follicular development and oogenesis .......................................................18

1.7.2 Fertilization and Embryogenesis.................................................................20

1.7.3 Gastrulation and the formation of the germ layers ......................................21

1.8 IMPACT OF NUTRITION ON THE DEVELOPMENT OF THE OOCYTE

AND EMBRYO................................................................................................ 22

1.8.1 Nutrition and gonadotropin secretion ..........................................................23

1.8.2 Nutrition and progesterone concentrations .................................................25

1.8.3 Nutrition and ovarian function .....................................................................25

1.8.4 Nutrition and embryo quality .......................................................................25

1.9 FUNCTION AND REGULATION OF ADIPOSE TISSUE ..................... 27

1.9.1 Function of adipose tissue during fetal development and later in life ..........27

1.9.2 Adipose tissue development .......................................................................29

1.9.3 Hormonal regulation of adipose tissue maturation ......................................30

1.9.4 Regulation of lipogenesis and lipolysis .......................................................32

1.9.5 Adipose derived molecules .........................................................................34

1.10 EXPERIMENTAL HYPOTHESES ........................................................ 37

CHAPTER 2: THE EFFECTS OF MATERNAL OVERNUTRITION AND/OR

DIETARY RESTRICTION DURING THE PERICONCEPTIONAL PERIOD ON

LAMB GROWTH AT BIRTH .............................................................................. 42

2.1 INTRODUCTION ................................................................................. 42

2.2 MATERIAL AND METHODS ............................................................... 45

2.2.1 Animals and nutritional feeding regime.......................................................45

2.2.2 Donor and recipient ewe synchronisation and pregnancy ...........................46

2.2.2.1 Super ovulation protocol .....................................................................46

2.2.2.2 Artificial insemination and embryo collection.......................................47

2.2.3 Determination of major transitions in structure of the embryo during early

embryogenesis ......................................................................................................48

2.2.4 Embryo transfer protocol ............................................................................48

2.2.5 Blood Sampling Regime .............................................................................49

2.2.5.1 Donor and recipient ewe blood sampling ............................................49

2.2.5.2 Lamb blood sampling .........................................................................52

Table of Contents IV

2.2.6 Lamb birth and growth measurements .......................................................52

2.2.7 Plasma non-esterified free fatty acid determination ....................................52

2.2.8 Plasma insulin determination ......................................................................52

2.2.9 Statistical Analyses ....................................................................................53

2.2.9.1 Ewe weights .......................................................................................53

2.2.9.2 Conception and pregnancy outcomes ................................................54

2.2.9.3 Plasma glucose, non-esterified fatty acid and insulin concentrations ..54

2.2.9.4 Lamb birth measures ..........................................................................54

2.3 RESULTS ............................................................................................ 55

2.3.1 Donor ewe weights .....................................................................................55

2.3.2 Donor ewe plasma glucose and insulin concentrations ..............................56

2.3.3 Periconceptional nutrition and embryo collection ........................................56

2.3.4 Body condition and weight of recipient ewes ..............................................56

2.3.5 Plasma glucose and insulin concentrations in recipient ewes .....................64


2.3.7 Lamb plasma glucose, insulin and free fatty acid concentrations ...............64

2.4 DISCUSSION ....................................................................................... 70

2.4.1 Weights of non-pregnant donor ewes and pregnant recipient ewes ...........70

2.4.2 Maternal plasma glucose and insulin concentrations ..................................71

2.4.3 Pregnancy outcomes..................................................................................72


2.5 SUMMARY ........................................................................................... 76

CHAPTER 3: THE EFFECTS OF MATERNAL OVERNUTRITION AND/OR

DIETARY RESTRICTION DURING THE PERICONCEPTIONAL PERIOD ON

LAMB GROWTH FROM BIRTH TO 4 MONTHS OF AGE ................................ 78

3.1 INTRODUCTION ................................................................................. 78

3.2 MATERIAL AND METHODS ............................................................... 79

3.2.1 Blood sampling regime ...............................................................................80


3.2.3 Glucose tolerance test................................................................................81

3.2.4 Plasma glucose, NEFA and insulin concentrations .....................................82

3.2.5 Post mortem and tissue collection ..............................................................82

3.2.6 Statistical Analyses ....................................................................................82

3.2.6.1 Plasma glucose, non-esterified fatty acid and insulin concentrations ..83

3.2.6.2 Lamb growth parameters and organ and tissue weights .....................83

Table of Contents V

3.3 RESULTS ............................................................................................ 84


3.3.2 Lamb plasma glucose, insulin and free fatty acid concentrations and

glucose tolerance test ............................................................................................84

3.3.3 Liver, kidney, pancreas, lung and brain weights .........................................85

3.3.4 Heart weight ...............................................................................................94

3.3.5 Adrenal weights ..........................................................................................94

3.3.6 Total adipose tissue weight ........................................................................97

3.3.7 Proportions of fat mass in the adipose tissue depots .................................97

3.4 DISCUSSION ..................................................................................... 111

3.4.1 Lamb growth measurements .................................................................... 111

3.4.2 Lamb plasma glucose, non-esterified fatty acid and insulin concentrations

112

3.4.3 Lamb weights ........................................................................................... 112

3.4.4 Lamb organ weights ................................................................................. 113

3.4.4.1 Lamb heart weights .......................................................................... 113

3.4.4.2 Lamb brain weights .......................................................................... 117

3.4.4.3 Lamb adrenal weights ...................................................................... 118

3.4.5 Adipose tissue weight ............................................................................... 120

3.5 SUMMARY ......................................................................................... 122

CHAPTER 4: IMPACT OF PERICONCEPTIONAL OVERNUTRITION AND/OR

DIETARY RESTRICTION ON THE EXPRESSION OF ADIPOGENIC AND

LIPOGENIC GENES IN THE POSTNATAL LAMB ......................................... 124

4.1 INTRODUCTION ............................................................................... 124

4.2 MATERIAL AND METHODS ............................................................. 126

4.2.1 Animals and nutritional feeding regime..................................................... 126

4.2.2 Post mortem and adipose tissue collection .............................................. 127

4.2.3 RNA extraction ......................................................................................... 128

4.2.4 Real Time Quantitative Reverse Transcription-PCR (qRT-PCR) .............. 128

4.2.5 Statistical Analysis .................................................................................... 131

4.2.5.1 Adipogenic and lipogenic gene expression ....................................... 131

4.3 RESULTS .......................................................................................... 132

4.3.1 Subcutaneous adipose tissue ................................................................... 132

4.3.2 Perirenal adipose tissue ........................................................................... 132

4.3.3 Omental adipose tissue ............................................................................ 133

Table of Contents VI

4.3.4 Comparison of adipogenic and lipogenic gene expression in all fat depots

133

4.4 DISCUSSION ..................................................................................... 148

4.5 SUMMARY ......................................................................................... 153

CHAPTER 5: SUMMARY AND CONCLUSIONS ........................................... 155

BIBLIOGRAPHY .............................................................................................. 164

Abstract VII

ABSTRACT

Women who enter pregnancy with an increased body weight have an increased

risk of developing gestational diabetes later in pregnancy and of having a baby

with a high birth weight and fat mass who is also at an increased risk of

becoming overweight or obese in childhood or later adult life. It is not known,

however, whether exposure of the oocyte and embryo during the

periconceptional period alone to maternal obesity is associated with an

increased risk of obesity in the offspring, and if so, whether the impact of

maternal obesity can be ameliorated by maternal weight loss immediately before

conception.

The present study has investigated in sheep, whether, a high plane of maternal

nutrition before and immediately after conception leads to the programming of an

increased expression of adipogenic and lipogenic genes and fat mass in the

offspring and whether a period of dietary restriction in overnourished mothers

reverses these changes. Non pregnant ewes (n=23) were randomly assigned to

one of four treatment groups, either control-control (CC) maintained at 100%

maintenance energy requirements (MER) for at least 5 months prior to

conception, control-restricted (CR) maintained at 100% MER for the first 4

months, then 1 month before conception were placed on a dietary restriction to

70% MER, high-high (HH) maintained ad libitum (170-190% MER) for 5 months

prior to conception or high-restricted (HR) maintained at ad libitum for 4 months,

and then 1 month before conception were placed on an energy-restricted diet

(70% MER). To determine the effect of overnutrition in the periconceptional

Abstract VIII

period only, single embryos were then transferred to recipient ewes which were

maintained on a control diet (100% MER) for the remainder of pregnancy. All

ewes were allowed to give birth naturally. At 4 months of age, lamb fat depots

were weighed and samples collected for the measurement of mRNA expression

for genes regulating adipogenesis and lipogenesis by quantitative real-time PCR.

The studies in this thesis have shown that periconceptional overnutrition

increased total fat mass in female lambs at 4 months of age. This change was

not associated with an increase in peroxisome proliferator-activated receptor γ,

leptin or adiponectin expression in the perirenal, omental and subcutaneous fat

depots. The period of dietary restriction in overnourished ewes ablated this

effect. These findings suggest that the effects of periconceptional overnutrition

on the oocyte or early embryo alters the subsequent development of adipose

tissue, and that the impact of periconceptional overnutrition may be reduced by

a period of dietary restriction prior to entering pregnancy.

IX

DECLARATION

This body of scientific work contains no material which has been accepted for the

award of any other degree or diploma in any university or other tertiary Institution

to Leewen Rattanatray 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 available for loan and photocopying, subject to the provisions of the

Copyright Act 1968.

I also give permission for the digital version of my thesis to be made available on

the web, via the University‟s digital research repository, the Library Catalogue,

the Australasian Digital Theses Program (ADTP) and also through web search

engines, unless permission has been granted by the University to restrict access

for a period of time.

Leewen Rattanatray

November 2009

X

ACKNOWLEDGEMENTS

I would like to thank all colleagues, students and supervisors who contributed to

this study and have shown great support throughout my Ph.D.

I am grateful to the members of the Early Origins of Adult Health Research

Group for their tremendous team effort and support and friendship during the

project. I would especially like to acknowledge the assistance of Dr. Severence

Mac Laughlin and Dr. Beverly Muhlhausler. Thank you both dearly for giving me

a “crash course” in running a large animal study, for your time, patience, advice

and guidance throughout my PhD Your unwavering support meant a lot to me

during the difficult moments during my PhD (and there were many).

Thank you also to those who assisted in blood sampling, glucose tolerance tests,

post-mortems and assays, especially Laura O‟Carroll, Melissa Walker, Bernard

Chuang, Pamela Sim, Petra Bos and the staff from Laboratory Animal Services.

This project would not have been possible without this great team behind me.

We are grateful to Dr. Dave Kleemann and Dr. Simon Walker for their

collaboration with the extensive animal protocol, and to the research staff at the

South Australian Research and Development Institute at Turretfield, including Dr.

Jen Kelly, and Skye Rudiger for their support during the preliminary stages of the

project. I would especially like to acknowledge the assistance of Dr. Dave

Kleemann and his expertise on nutrition and reproduction in the sheep and Dr.

Simon Walker for his assistance with the embryo transfer protocols.

XI

I wish to express my sincere gratitude to my wonderful family for their constant

love, encouragement and support throughout my life; I could not have achieved

this without the sacrifices that they have made in their lives for me. And to

Thuong, thank you for your constant love, understanding, encouragement,

unwavering support and your patience, thank you for being you.

Lastly, I would like to thank my supervisors Prof. Caroline McMillen and Assoc.

Prof. Jeff Schwartz. Thank you both for your guidance and support during the

start of my scientific career. I would especially wish to thank Caroline for her

vision, invaluable guidance and assistance in making this research possible. I am

greatly indebted to her for helping me to overcome obstacles in the process of

conducting the research and for the opportunities she has given me during my

PhD.

Related Publications XII

RELATED PUBLICATIONS

Rattanatray L, MacLaughlin SM, Kleemann DO, Walker SK, Muhlhausler BS and

McMillen IC (2010) Impact of Maternal Periconceptional Overnutrition on Fat

Mass and Expression of Adipogenic and Lipogenic Genes in Visceral and

Subcutaneous Fat Depots in the Postnatal Lamb. Submitted.

Zhang S, Rattanatray L, MacLaughlin SM, Cropley J, Suter C Molloy L,

Kleemann D, Walker S, Muhlhausler BS, Morrison JL and McMillen IC (2010)

Periconceptional Undernutrition in Normal and Overweight Ewes Leads to

Increased Adrenal Growth and Epigenetic Changes in Adrenal Igf2/H19 Gene in

Offspring. The FASEB Journal. In press.

McMillen IC, Rattanatray L, Duffield JA, Morrison JL, MacLaughlin SM, Gentili S,

Muhlhausler BS. (2009) The Early Origins of Later Obesity: Pathways and

Mechanisms. Advances in Experimental Medicine and Biology Volume 646: 71-

81

McMillen IC, Rattanatray L, Duffield JA, Morrison JL, MacLaughlin SM, Gentili S,

Muhlhausler BS. (2009) The Early Origins of Later Obesity: Pathways and

Mechanisms. In B Koletzko,D Molnár, T Decsi and A de la Hunty (Eds) Early

Nutrition Programming and Health Outcomes in Later Life. Obesity and Beyond,

71-82

XIII

LIST OF FIGURES AND TABLES

Figure 1.1 The intergenerational cycle of obesity 15

Figure 1.2 A schematic diagram showing the sequence of events in origin, growth and rupture of the tertiary follicle and degradation of the corpus luteum 24

Figure 2.2.1 Nutritional protocol 50

Figure 2.2.2 The major transitions in structure of the bovine embryo during early embryogenesis 51

Figure 2.2.1 Blood sampling regime in the donor and recipient ewes 57

Figure 2.3.1 Effect of periconceptional overnutrition and/or dietary restriction on the weight of donor ewes during the nutritional feeding protocol from 35 weeks before conception to 1 week after conception 58

Figure 2.3.2 Effect of periconceptional overnutrition and/or dietary restriction on the change in the weight of the non-pregnant donor ewes between 25 weeks before conception to the day of embryo transfer (day 6-7 pregnancy) 59

Table 2.3.1 Donor ewe body condition scores between 5 weeks prior to conception and conception 60 Table 2.3.2 Plasma glucose concentrations in donor ewes at 5, 4 and 1 week prior to conception and at conception 61 Figure 2.3.3 Plasma insulin concentration in donor ewes between 5 weeks before conception and conception 62 Table 2.3.3 Periconceptional nutrition and/or dietary restriction and the stage of embryo development 63 Table 2.3.4 Plasma glucose and insulin concentrations in recipient ewes at 5 weeks before and 7 weeks after conception 65 Table 2.3.5 Periconceptional nutrition and pregnancy outcomes and lamb survival 66 Table 2.3.6 Effect of periconceptional overnutrition and/or dietary restriction on growth parameters at birth 67

XIV

Figure 2.3.4 Effect of periconceptional overnutrition and/or dietary restriction on birth weight in male and female lambs 68 Table 2.3.7 Effect of periconceptional overnutrition and/or dietary restriction on plasma glucose, non-esterified free fatty acid and insulin concentrations at birth in the offspring 69 Figure 3.3.1 The growth rate of lambs from the 4 nutritional treatment groups between 1 and 16 weeks of age 86 Table 3.3.1 The effect of periconceptional overnutrition and/or dietary restriction on growth parameters at 4 months of age 87 . Figure 3.3.2 Effect of periconceptional overnutrition and/or dietary restriction on male and female lamb weight at 4 months of age 88 Figure 3.3.3 Effect of periconceptional overnutrition and/or dietary restriction on plasma glucose concentration between birth and 4 months of age in lambs 89 Figure 3.3.4 Effect of periconceptional overnutrition and dietary restriction on the plasma non-esterified free fatty acid concentration from birth to 4 months of age in lambs 90 Figure 3.3.5 Effect of periconceptional overnutrition and/or dietary restriction on the plasma insulin concentration from birth to 4 months of age in lambs 91 Figure 3.3.6 Plasma glucose and insulin concentrations responses to intravenous glucose challenge at 3 months of age in lambs 92 Figure 3.3.7 Area under the glucose and insulin response curves after the intravenous glucose challenge 93 Figure 3.3.8 Effect of periconceptional nutrition on absolute organ weights and relative to body weight of 4 month old lambs 95 Figure 3.3.9a Effect of periconceptional overnutrition and dietary restriction on the absolute brain weight in female and male lambs at 4 months of age 98 Figure 3.3.9b Effect of periconceptional overnutrition and dietary restriction on the brain weight relative to body weight in female and male lambs at 4 months of age 99 Figure 3.3.10a Effect of periconceptional overnutrition and dietary restriction on absolute heart of 4 month old male and female lambs 100

*

XV

Figure 3.3.10b Effect of periconceptional overnutrition and dietary restriction on heart weight relative to body weight of 4 month old male and female lambs 101 Figure 3.3.11 Effect of periconceptional overnutrition and/or dietary restriction on absolute adrenal weight and relative to body weight of 4 month old lambs 102 Figure 3.3.12 Effect of periconceptional overnutrition and/or dietary restriction on total fat mass of male and female lambs at 4 months of age 103 Figure 3.3.13 Relationship between total fat mass in 4 month old female offspring and the weight of the donor ewe at conception 104 Figure 3.3.14 Effect of periconceptional overnutrition and/or dietary restriction on omental adipose tissue depot weight of 4 months old male and female lambs 105 Figure 3.3.15 Effect of periconceptional overnutrition and/or dietary restriction on perirenal adipose tissue depot weight of 4 months old male and female lambs 106 Figure 3.3.16 Effect of periconceptional overnutrition and/or dietary restriction on subcutaneous adipose tissue depot weight of 4 months old male and female lambs 107 Figure 3.3.17 The proportion of the total fat mass in the different adipose depots in female lambs at 4months of age 108 Figure 3.3.18 The proportion of the total fat mass in the different adipose depots in male lambs at 4 months of age 109 Table 3.3.2 Differences between the proportion of the total adipose tissue in each fat depot between male and female lambs at 4 months of age 110 Table 4.2.1 Sequences of real time PCR primers for adipogenic, lipogenic and reference genes 130 Figure 4.3.1 Normalised expression of adipogenic and lipogenic genes in the subcutaneous adipose tissue depot of male and female lambs at 4 months of age 135 Figure 4.3.2 Normalised expression of adipogenic and lipogenic genes in the perirenal adipose tissue depot of male and female lambs at 4 months of age 137 Figure 4.3.3 Normalised expression of adipogenic and lipogenic genes in the omental adipose tissue depot of male and female lambs at 4 months of age 139

XVI

Figure 4.3.4 Correlation between donor ewe weight at conception and perirenal G3PDH mRNA expression in female lambs at 4 months of age 141 Table 4.3.1 Correlations between the relative mRNA expression of PPARγ and other adipogenic and lipogenic genes in the subcutaneous, perirenal and omental adipose tissue depots of lambs at 4 months of age 142 Figure 4.3.5 Relative adipose tissue depot specific expression of PPARγ mRNA of male and female lambs at 4 months of age 143 Figure 4.3.6 Relative adipose tissue depot specific expression of G3PDH mRNA of male and female lambs at 4 months of age 144 Figure 4.3.7 Relative adipose tissue depot specific expression of LPL mRNA of male and female lambs at 4 months of age 145 Figure 4.3.8 Relative adipose tissue depot specific expression of leptin mRNA of male and female lambs at 4 months of age 146 Figure 4.3.9 Relative adipose tissue depot specific expression of adiponectin mRNA of male and female lambs at 4 months of age 147 Figure 5.1 Proposed mechanism for the impact of periconceptional overnutrition on the intergenerational cycle of obesity 159 Figure 5.2 Proposed mechanism for the impact of late gestational overnutrition on the intergenerational cycle of obesity 161 Figure 5.3 Proposed mechanism for the impact of periconceptional overnutrition and late gestational overnutrition on the intergenerational cycle of obesity 162

XVII

COMMONLY USED ABBREVIATIONS

A B C

ACE angiotensin-converting enzyme ACTH adrenocorticotrophic hormone Ad libitum to any desired extent ANOVA analysis of variance BAT brown adipose tissue BCS body condition score BMI body mass index bp base pairs cAMP cyclic adenosine monophosphate CC control-control C/EBP CCAAT enhancer binding protein CR control-restricted D E F G

d days DNA deoxyribonucleic acid FFA free fatty acids FSH follicle stimulating hormone GDM gestational diabetes mellitus GLUT glucose transporter G3PDH glycerol-3-phoshate dehydrogenase H I J K L

h hours HFD high fat diet hGh human growth hormone HH high-high HPA hypothalamic-pituitary-adrenal axis HR high-restricted ICM inner cell mass IGF insulin-like growth factor IGFR insulin-like growth factor receptor I.V intravenous LGA large for gestational age LH luteinizing hormone LPL lipoprotein lipase

XVIII

M N O

MER maintenance energy requirements min minute(s) mRNA messenger ribonucleic acid n number NEFA non-esterified free fatty acids P Q R S

PCON periconceptional overnutrition PCR polymerase chain reaction PCUN periconceptional undernutrition PPARγ peroxisome proliferator-activated receptor γ PPRE peroxisome proliferator response elements qRT-PCR real-time quantitative reverse transcription

polymerase chain reaction RAS renin-angiotensin system RNA ribonucleic acid RPLP0 ribosomal protein large subunit P0 RT-PCR reverse transcription polymerase chain reaction RxR retinoid x receptor SEM standard error of the mean SPSS statistical package for the social sciences

T U V W X Y Z

TZD thiazolidinediones WAT white adipose tissue

I

CHAPTER 1

2

CHAPTER 1: LITERATURE REVIEW

1.1 INTRODUCTION

There is a current global increase in the prevalence of obesity (body mass index

(BMI) ≥ 30kg/m2), with a disproportionate number of young women being classed

as obese. Studies suggest that more women are now entering pregnancy with a

higher BMI than a decade ago, with deleterious consequences not only for the

health of the mother, but for the long-term health of her child. Furthermore,

obesity during pregnancy is also associated with adverse health risks for the

mother, pregnancy complications and long-term health implications for the

offspring (Garbaciak 1985; Cnattingius 1998; Cogswell, Perry et al. 2001; Jolly

2003; Kristensen 2005; McMillen, Rattanatray et al. 2009). More importantly, a

high maternal BMI and gestational diabetes are each associated with an

increase in fetal birth weight and adiposity. It has also been shown that a high

birth weight is also associated with an increased risk of being obese throughout

childhood to adulthood. Many attempts have been made to investigate the

mechanisms underlying these associations in animal models in order to

understand how an increase in early nutritional supply results in the emergence

of an increased risk of metabolic disease in the offspring. Previous studies

however, have not been able to ascertain whether maternal obesity before

pregnancy is directly involved in the programming of obesity in the offspring or

whether maternal weight gain and obesity during pregnancy plays the more

critical role in the programming of obesity. It is important to differentiate between

the effects of a nutrient rich embryonic environment and a nutrient rich fetal

3

environment on the programming of offspring obesity in later life. The timing of

maternal obesity and its consequences on the development of offspring obesity

will inform novel approaches to implementing interventions for overweight

women of child bearing age wishing to conceive, and potentially reduce the

incidence of the “intergenerational cycle of obesity”.

This review will begin by briefly summarizing human epidemiological studies

focusing on maternal obesity, diabetes and weight gain before and during

pregnancy and the association with later onset of adult obesity. The embryonic

stages of development and the impact of early nutrition on the biology of the

developing adipocyte will be addressed. In addition the effects of maternal

overnutrition at different stages of gestation, as well as the little that is known

about the effect of nutrition during the periconceptional period will be discussed,

highlighting areas which are novel and require further investigation.

1.2 FETAL ORIGINS OF ADULT DISEASE

The developmental origins of adult health hypothesis proposed that

environmental factors particularly suboptimal intra-uterine environments such as

poor maternal nutrition act in early life to result in altered fetal growth and

development. A suboptimal intra-uterine environment subsequently predisposes

the fetus to an increased risk for developing adverse health outcomes in later

adult life, including cardiovascular disease, obesity and the metabolic syndrome

(Barker, Hales et al. 1993; Barker 2007). A number of factors may induce a

4

suboptimal intra-uterine environment including maternal nutrient restriction, poor

maternal-fetal nutrient transfer by the placenta and maternal drug use, including

smoking. A suboptimal intra-uterine environment results in adaptions by the

embryo and fetus to the environment, including morphological changes to the

development of fetal tissues, changes to hormone concentrations and/or

changes to the sensitivity to the action of these hormones on target tissues. The

concept of adaptive responses in embryonic and fetal development to a

suboptimal environment, leading to an altered “setting” of a physiological system

occurring during a sensitive period of development is coined “programming”.

Whilst earlier adaptions may confer early advantages for fetal survival in utero it

is hypothesized that these “trade-offs” may incur delayed adverse health

outcomes leading to the development of diseases in later life (Lucas 1998;

Mcmillen and Robinson 2005).

In 1992, Hales and Barker coined the “thrifty phenotype” hypothesis (see (Hales

and Barker 2001) to explain the relationship between poor fetal environment,

infant growth and the increased risk of developing diseases in later life, including

impaired glucose tolerance (Hales and Barker 1992) the metabolic syndrome

(Barker, Hales et al. 1993) and cardiovascular disease (Barker and Osmond

1986). The pivotal elements of this hypothesis suggest that under the conditions

of a suboptimal fetal environment an adaptive response occurs which favours the

optimal growth of key body organs including the brain and heart to the detriment

of other organs and tissues including the musculoskeletal system. These

adaptive responses subsequently lead to a postnatal metabolism which is

designed to improve postnatal survival during critical periods of intermittent or

5

poor nutrient availability. However it is proposed that these altered physiological

adaptations become deleterious when the nutritional abundance in the postnatal

environment exceeds that of the prenatal environment. The “thrifty phenotype”

proposal and the “predictive adaptive response” concept hypothesize that the

disease manifests only when the postnatal nutrient environment is considerably

different from the predicted nutritional environment in utero (Hales and Barker

2001; Gluckman and Hanson 2004).

1.3 DEVELOPMENTAL PROGRAMMING OF THE METABOLIC

SYNDROME: EPIDEMIOLOGICAL EVIDENCE

The “fetal origins of adult disease” hypothesis was first derived from

epidemiological studies of fetal programming which focused on the relationship

between infant birth weight and the incidence of adult disease including

metabolic abnormalities associated with the metabolic syndrome including

hypertension (Barker, Dull et al. 1990), insulin resistance (Phillips, Barker et al.

1994), obesity (Yajnik 2002) and dyslipidemia (Barker, Hales et al. 1993).

Epidemiological data found that in localised areas of Britain there were high

infant mortality rates between 1921-1925. These localised areas were similar to

those which experienced high incidences of ischemic heart disease between

1968-1978 (Barker and Osmond 1986). It has been well established that a low

birth weight is a reflection of a suboptimal intra-uterine environment and

therefore poor fetal growth and is associated with an increased incidence of fetal

morbidity and mortality. The importance of maternal nutrient deprivation on birth

6

weight and subsequent adulthood disease has been investigated in a series of

epidemiological studies most notably, the Dutch Hunger Winter Famine study, in

which pregnant women experienced 5 months of famine during the winter of

1944-1945 in Amsterdam, the Netherlands. Exposure to a restricted caloric

intake during early gestation, as an embryo or fetus in the first trimester, resulted

in individuals with an increased incidence of coronary heart disease,

hypertension, increased body mass index and glucose intolerance and these

associations were independent of size at birth (Ravelli, Stein et al. 1976; Ravelli,

van der Meulen et al. 1999; Roseboom, van der Meulen et al. 2000; Roseboom,

van der Meulen et al. 2001). Exposure to the famine in late gestation, however,

was associated with an increased incidence of adulthood obesity and glucose

intolerance (Ravelli, Stein et al. 1976; Ravelli, van der Meulen et al. 1999).

Furthermore babies born small after exposure to the famine at any time in

gestation, particularly female babies were more susceptible to developing adult

onset diabetes (Roseboom, van der Meulen et al. 2001). However, great

controversy surrounds the relationship between birth weight and adulthood

metabolic or cardiovascular disease (Huxley, Neil et al. 2002). The siege of

Leningrad, in Russia from 1941-1943 (in which caloric intake was restricted)

found no apparent relationship between birth weight and adult glucose

homeostasis (Stanner, Bulmer et al. 1997). The “thrifty phenotype” and

“predictive adaptive response” hypotheses may provide an explanation for this

observation, since a poor nutritional environment preceded and followed the

Leningrad siege. Therefore the adaptive fetal response may have been suitable

for the nutrient-poor postnatal environment. In contrast, nutrient availability

following the Dutch winter famine was relatively abundant and therefore the

7

postnatal plane of nutrition was greater than that was predicted by the adaptive

response and therefore an increase incidence of adult disease was the result.

1.3.1 MATERNAL OVERNUTRITION AND THE DEVELOPMENTAL PROGRAMMING OF

OBESITY

Previous studies have focused on the impact of maternal nutrient detriment in

the context of “early origins of adult disease” over the past decade, however

maternal nutrient abundance and an increase in maternal body mass is

becoming more prevalent in both developed and developing countries. The effect

of maternal overnutrition and thus embryonic and/or fetal overnutrition on the

developmental origins of adult health and disease is an emerging area of

research interest. Early exposure to overnutrition during critical developmental

windows alters the development of body systems including central and peripheral

neuroendocrine responses which affect the developmental programming of fat

cells and the appetite regulatory system, altering whole body metabolism.

Subsequently maternal overnutrition in early fetal life results in the emergence of

poor health for the offspring in childhood and adulthood.

1.3.2 THE OBESITY EPIDEMIC

There has been a marked increase in the global prevalence of obesity in the past

two decades. Currently more than 50% of all American, British and Australian

adults are classed as overweight (i.e. have a body mass index (BMI) ≥ 25 kg/m2)

and more than 24% of adults in the United States (U.S) are classified as obese

(BMI ≥ 30 kg/m2) (LaCoursiere, Bloebaum et al. 2005). There has also been a

8

marked increase in the proportion of children who are overweight or obese, with

more than 15% of U.S children aged 6-19 being classed as overweight (Ogden,

Flegal et al. 2002). The prevalence of both adulthood and childhood obesity

continues to increase. Despite the increase in overweight and obesity trends in

adults particularly women of child-bearing age, who are at a higher risk of being

overweight or obese (Cogswell, Perry et al. 2001), there is less attention paid to

the trends of increased BMI specifically among pregnant women (LaCoursiere,

Bloebaum et al. 2005).

1.3.3 MATERNAL OBESITY

More women are now entering pregnancy with a higher BMI than a decade ago.

A recent study by La Coursiere and colleagues (LaCoursiere, Bloebaum et al.

2005) showed that pre-pregnancy overweight and obesity increased from 25.1%

in 1991 to 35.2% in 2001, whereas maternal obesity at delivery rose from 28.7%

to 39.1% during the same period. This increased incidence in the proportion of

women entering pregnancy with a high BMI is concerning, since there are many

adverse risks associated with being overweight or obese entering pregnancy or

during pregnancy.

Entering pregnancy obese is associated with an increased risk of impaired

reproductive function and infertility, which may be attributed to the increase in

insulin resistance (Clark, Thornley et al. 1998; Bellver, Rossal et al. 2003; Bellver

and Pellicer 2004; Rhind 2004). Being obese during pregnancy increases the risk

of health complications for the mother, leading to a higher risk of developing

hypertension, preeclampsia and gestational diabetes mellitus (GDM) during

9

pregnancy (Cogswell, Perry et al. 2001). There are also health consequences for

the children born to obese mothers. There is an increased risk for the

development of congenital abnormalities and perinatal mortality (Garbaciak

1985; Cnattingius 1998; Cogswell, Perry et al. 2001; Kristensen 2005). Maternal

obesity also increases the risk of giving birth to a large for gestational age (LGA)

infant (i.e. birth weight ≥ 95th percentile of infants of the same age) (Garbaciak

1985; Frisancho 2000; Laitinen 2001; Parsons, Power et al. 2001; Pietiläinen

2001; Jensen 2003; Jolly 2003; Kristensen 2005; LaCoursiere, Bloebaum et al.

2005) which may contribute to the increased risk in the development of

complications during labor, still birth or early neonatal death (Garbaciak 1985;

Jolly 2003; Kristensen 2005).

1.4 BENEFITS AND RISKS ASSOCIATED WITH DIETARY

RESTRICTION OF WOMEN WITH A HIGH BMI

1.4.1 BENEFITS OF DIETARY WEIGHT LOSS BEFORE ENTERING PREGNANCY

Obesity is more prevalent in women, affecting young women disproportionately

and is therefore of epidemiological importance. The “intergenerational cycle of

obesity” suggests that maternal obesity programs fetal, neonatal, childhood and

adulthood obesity in the offspring, and affects maternal health and pregnancy

outcomes. Obesity and its co morbidities, type 2 diabetes, coronary heart

disease, hypertension and dyslipidemia are easily diagnosed; however it is the

case that little progress has been made in obesity prevention in the last decade

resulting in an increasing burden of this disease in the developed world.

10

Previous studies suggest that childhood obesity may be prevented by

normalizing body composition and nutrition and improving the general health of

young women of childbearing age before becoming pregnant, thereby preventing

the prevalence of the “intergenerational cycle of obesity” and the serious co-

morbidities associated with obesity (Kral 2004). Weight loss by dieting and

exercise has been recommended to overweight and obese patients who are at

risk of cardiovascular disease (Yu-Poth, Zhao et al. 1999; Mertens and Van Gaal

2000) and women who are infertile and wishing to conceive (Clark, Thornley et

al. 1998; Norman, Noakes et al. 2004).

Studies have shown that a modest weight loss of 5-10% of baseline weight in

overweight and obese patients results in a lowered blood pressure in both

hypertensive and non-hypertensive patients (Mertens and Van Gaal 2000).

Dietary intervention of reduced dietary fats and cholesterol improved lipid

profiles, significantly decreasing plasma lipids and lipoproteins and total

cholesterol (Yu-Poth, Zhao et al. 1999). Dieting and weight loss in overweight

and obese people has multiple beneficial effects on important cardiovascular

disease risk factors.

Lifestyle changes in relation to exercise and diet for 6 months in a group of

obese anovulatory women resulted in significant weight loss (10.2 kg/m2) with

90% of these women resuming spontaneous ovulation and 77% achieving

pregnancy, resulting in a significant increase in the number of successful live

births. The incidence of miscarriage also decreased as a result of weight loss

11

from 75% prior to the commencement of the program to 18% (Clark, Thornley et

al. 1998). The improvement in ovulation, pregnancy rates and outcomes may be

attributed to the increase in maternal insulin sensitivity, as a direct result of the

weight loss program. The effects of dietary restriction, particularly early in

pregnancy in overweight and obese mothers, on the development of offspring

have not been investigated.

1.5 DEVELOPMENTAL PROGRAMMING OF OBESITY:

EPIDEMIOLOGICAL EVIDENCE

1.5.1 RELATIONSHIP BETWEEN MATERNAL OBESITY AND HIGH BIRTH WEIGHT

Many epidemiological studies have suggested a role of a high maternal BMI in

the programming of offspring obesity. It has been demonstrated that maternal

weight, maternal BMI entering pregnancy and maternal weight gain during

pregnancy are positively associated with the birth weight of the offspring

(Frisancho 2000; Laitinen 2001; Parsons, Power et al. 2001; Pietiläinen 2001;

Jensen 2003; Jolly 2003). This relationship suggests that heavier mothers tend

to give birth to heavier babies with high adiposity; and being born LGA is

associated with long-term adverse consequences for the offspring.

12

1.5.2 HIGH MATERNAL AND FETAL PLANES OF NUTRITION RESULT IN A HIGH BIRTH

WEIGHT INFANT

It has been proposed that the relationship between a high maternal BMI and

LGA infant may be attributed to the sustained high plane of nutrition consumed

by overweight and obese women before entering or during pregnancy. An

increase in nutritional consumption leads to maternal metabolic and placental

adaptations, resulting in an increase in maternal insulin resistance and hence

fetal nutrient supply. Since the fetus is unable to alter its nutrient intake, there is

a promotion of fetal overgrowth and fatness (Pedersen 1954; Pedersen 1954;

Pedersen 1954; Catalano 2003). It has been found that direct measures of

fatness such as skin folds are also greater in newborn infants of obese mothers

(Frisancho 2000). Furthermore, many studies have supported the role of

maternal hyperglycemia and hyperinsulinemia in the programming of high birth

weight and adiposity in the offspring (Alexander and Bell 1990; Catalano 2003;

Boney 2005). These studies therefore suggest that the effects of the nutrient

environment persist into postnatal life.

1.5.3 MATERNAL DIABETES

Pregnancies complicated by maternal diabetes or the development of gestational

diabetes later in pregnancy are associated with increased birth weight and

adiposity in the offspring (Whitaker 1998; Dabelea 2000; Jolly 2003; Ehrenberg,

Mercer et al. 2004). Altered metabolism in a pregnancy complicated by diabetes

increases infant fatness at birth. It has been suggested that the increase in

maternal glucose and amino acids transferred to the fetus induces pancreatic β-

cell hyperplasia and hyperinsulinemia in the offspring, which in turn promotes the

13

development of excess adipose tissue mass by hyperplasia and hypertrophy of

adipocytes (Pedersen 1954; Pedersen 1954; Pedersen 1954). It is difficult

however, to determine whether maternal obesity and/ or diabetes, or GDM are

independently associated with, or synergistically contributes to the increased risk

of having a LGA infant, since diabetes and GDM are each a consequence of

being overweight or obese.

Studies show that gestational diabetes and maternal obesity are independently

associated with an increased risk of giving birth to LGA infants, with maternal

BMI influencing the LGA infant phenotype more than diabetes (46.7% vs. 4.1%)

(Ehrenberg, Mercer et al. 2004). The study by Dabelea and colleagues (2000)

showed that Pima Indian siblings born after the mother was diagnosed with

diabetes were at a higher risk of developing diabetes themselves and had a BMI

2.6 kg/m2 higher than siblings that were born before the mother developed

diabetes. However Pima Indians, in general, have a reported higher incidence of

diabetes and obesity (Dabelea 2005). This strongly suggests that the

predisposition to develop diabetes may be contributed partly to an enhanced

genetic predisposition by may be exacerbated by the high nutrient environment

in utero. Infants of pregnancies compromised by diabetes are also at increased

risk of developing obesity later life. Offspring born to mothers who developed

GDM during pregnancy were at 17.1% risk of being overweight and 9.7% were

overweight in adolescence (Frisancho 2000).

14

1.5.4 HIGH BIRTH WEIGHT A PREDICTOR OF ADULTHOOD ADIPOSITY, INSULIN

RESISTANCE AND DIABETES

Epidemiological studies discussed previously showed that maternal weight, BMI,

pregnancy weight gain and maternal diabetes results in increased birth weight

and adiposity in the offspring. Studies also show that there are long-term

consequences associated with being born LGA. Extensive studies demonstrate a

J or U shaped relationship between birth weight and adult fat mass. A higher

prevalence of adulthood obesity occurs with birth weight at both the lower and

higher ends of the birth weight spectrum (Fall, Osmond et al. 1995; Curhan,

Chertow et al. 1996; Parsons, Power et al. 2001). A longitudinal study by

Parsons and colleagues (Parsons, Power et al. 2001) examined the relationship

between birth weight and adult BMI at the age of 33 in a British cohort involving

10,683 participants. This study shows a strong correlation between high birth

weight and high adult BMI in this population, independent of growth rate during

childhood. This association is attributed to maternal weight and BMI,

independent of confounding factors such as maternal height, paternal height,

socio-economic status or smoking habits. This study suggests that infant birth

weight predicts adult BMI and thus heavier mothers have heavier babies and

these babies have a high BMI in adult life.

Previous studies have shown that GDM and mildly impaired glucose tolerance

increases the risk of obesity and glucose intolerance in the offspring in later life

(Silverman, Rizzo et al. 1991; Gillman 2003). The study by Gillman and

colleagues (2003) shows that offspring born to mothers who developed GDM

during pregnancy were at a higher risk of being overweight in adolescence (odds

15

Pre-pregnancy

obesity

↑ Risk of heavy infant

↑ Risk of adulthood

obesity

↑ Risk of gestational

diabetes

↑ Risk of childhood

obesity

ratio of 1.4) and this was associated with each 1kg increment of birth weight.

Multivariate analyses however, showed that maternal BMI attenuated the

association between GDM and the increased risk of becoming overweight in the

adolescent offspring (Gillman 2003).

Tracking of adolescent fatness is clearly shown in the U.S population, in the

study by Frisancho and colleagues (Frisancho 2000), such that heavier

newborns become heavier or fatter adolescents, only when the mother had a

high BMI. This study demonstrates the potential of an “ intergenerational cycle of

obesity”, in which maternal obesity increases the risk of developing GDM, which

contributes to the biological programming of increased fat mass at birth, and

being born with a high birth weight, which programs for an increased risk of

childhood obesity, which in turn, is a good predictor of adulthood obesity.

Figure 1.1 The intergenerational cycle of obesity

16

1.6 MATERNAL OVERNUTRITION AND OFFSPRING ADIPOSITY:

EXPERIMENTAL ANIMAL STUDIES

Consistent with many human epidemiological observations, experimental studies

in animal models such as the rodent and the sheep show that the nutrient

environment to which the developing fetus is exposed, can alter the growth

trajectory of the offspring. Experimental models of maternal obesity developed by

overfeeding dams (Shankar, Harrell et al. 2008) determined that in utero

exposure to maternal obesity increased the risk of obesity in the offspring. This

study also showed that offspring of mothers fed a high fat diet had significantly

increased body weight as well as body fat percentage than the offspring of

control fed dams. Similar observations were observed in larger animal models of

maternal overnutrition. It has been shown that maternal diabetes increased the

number and size of lipid containing adipocytes in the fetal adipose tissue of the

pig leading to an increase in adiposity in the offspring (Hausman, Kasser et al.

1982) and increased lipogenesis in the fetal adipose tissue (Kasser, Gahagan et

al. 1982). It is suggested that maternal diabetes stimulates de novo fatty acid

synthesis in the fetal adipose tissue which increases lipid accumulation in the

fetal adipocytes. Glucose infused sheep fetuses had higher serum insulin levels

and were significantly heavier with increased adiposity than saline infused sheep

fetuses (Stevens, Alexander et al. 1990). This study suggests that fetal growth

and adiposity are responsive to an increased glucose supply and that these

effects may be mediated through the actions of fetal insulin. Muhlhausler and

colleagues previously showed that exposure to maternal overnutrition (150%

metabolisable energy requirements) during the last 30-40 days of gestation in

17

the pregnant ewe resulted in an increase in fasting plasma glucose

concentrations, in the absence of an increase in plasma insulin concentrations

during the first month of postnatal life in the lamb. Furthermore exposure to

maternal overnutrition during late gestation also resulted in an increase in body

fat deposition in the lambs at 30d of age (Muhlhausler, Duffield et al. 2007). Guo

and colleagues (Guo and Jen 1995) reported increased bodyweight and fat

composition in adult offspring of dams fed 40% fat during pregnancy in the rat.

Consistent with these findings, Khan and colleagues (Khan, Dekou et al. 2004;

Khan, Dekou et al. 2005) showed that offspring of dams fed a 24% fat diet during

pregnancy and suckling and reared on a normal diet were significantly heavier

and had more visceral fat deposits than offspring of control fed dams. Offspring

of fat fed mothers who were then reared on a fat rich diet postnatally became

markedly obese.

These studies indicate that the increased predisposition of disease such as

obesity which manifest in adult life may be acquired very early in development

through both inappropriate and excessive nutrition in embryonic or fetal life. The

development of many organ systems may be compromised during critical

windows of development, permanently impairing their function, predisposing to

an increased risk of developing diseases in later life, such as type-2 diabetes

and cardiovascular disease. Many animal studies have been informative in

understanding timings of exposure, critical developmental windows, mechanisms

and long-term outcomes of developmental programming. Pathways of glucose

and fat metabolism are particularly susceptible to alterations in the maternal

environment including maternal nutrient imbalance and maternal body

18

composition. However these studies undertaken in the developmental

programming of offspring obesity and diabetes, to date, have focused mainly on

perturbations associated with maternal and fetal undernutrition, rather than

maternal obesity, overnutrition and diabetes which is more socially relevant in

society today, with the increase prevalence of obesity in many developed and

developing countries. Furthermore experimental models of maternal overnutrition

have focused mainly on the exposure of the fetus to a high nutrient environment

mid to late gestation and not the effects of exposure during earlier stages of

development, which may be critical in the programming of offspring obesity in

later life.

1.7 CRITICAL WINDOWS OF EARLY DEVELOPMENT: INTRA-

UTERINE GROWTH AND DEVELOPMENT

1.7.1 FOLLICULAR DEVELOPMENT AND OOGENESIS

At birth the human ovary contains approximately one million oocytes which are

unable to be replenished and which continually degenerate. Before birth, germ

cells from the mesoderm migrate to the ovary and proliferate and differentiate

into primordial follicles. Primordial follicles are characterized by an incomplete

layer of flattened granulose cells surrounding the oocyte and the granulosa cells

and oocyte complex is referred to as a follicle. These primordial follicles mature

further into primary follicles, characterized by a complete single layer of follicular

cells, in which they remain growth arrested until puberty, unless they degrade

first.

19

Maturation into a secondary follicle occurs when menstruation occurs. The

follicle begins to produce the zone pelludica, a translucent non cellular

membrane, located between the oocyte and granulosa cells. A fluid filled space

begins to form within the multilayer granulosa cells known as the antrum and the

outer cellular layer begins to form ovarian connective tissue.

There is a final selection of one dominant mature follicle with a high receptivity to

FSH, which leads to a dramatic growth in follicle size and increased antrum,

which will ultimately undergo ovulation. Prior to ovulation the mature tertiary

follicle is no longer growth arrested and is able to undergo its first mitotic division.

The follicle is now ready to respond to the pre-ovulatory FSH and LH surge and

complete the first stage of its cycle by releasing the ovum into the peritoneal

cavity. The follicle swells due to the increased accumulation of follicular fluid and

increases in size and ruptures and releases the ovum and antral fluid. The

remaining cells (stratum granulosa and theca interna cells) develop into the

corpus luteum, which grows rapidly and functions as an endocrine organ,

secreting progesterone important in preparing the lining of the uterus for the

implantation of the fertilized ovum. The formation and maintenance of the corpus

luteum is dependent upon the secretion of LH from the pituitary. If pregnancy

occurs, the placenta takes over as the primary progesterone producer and the

corpus luteum degrades and becomes the corpus albicans (Figure 1.2).

20

1.7.2 FERTILIZATION AND EMBRYOGENESIS

Fertilization occurs in the upper uterine fallopian tubes. The spermatozoa must

penetrate the corona radiata and zona pellucida before it can make contact with

the plasma membrane of the ovum. Once the spermatozoon has entered the

fluid filled space and the plasma membrane of the ovum, the plasma membranes

of the ovum and spermatozoon fuse and signals the second mitotic division of

the ovum resulting in a mature ovum. There is a breakdown of the nuclear

membrane of the spermatozoa and an interaction between the components of

the spermatozoa and the cytoplasm of the ovum. The male and female genetic

material fuse and thus, the first mitosis of the union of the spermatozoa and the

ovum is the actual fusion of their chromosomes, leading to the first cleavage

division.

Following the first cleavage division, the zygote undergoes a series of cellular

divisions, with cells from the first few cleavages being metabolically

unspecialized. Compaction occurs in the 8 cell stage, the blastomeres flatten and

become tightly joined and intracellular connections are formed. When the cells

resemble a mulberry, the embryo is known as a morula and consists of

approximately 16 blastomeres. A fluid filled central cavity, the blastocoel,

develops and a blastocyst is formed. Blastocysts are characterized by an

enlargement of the blastocoel cavity as well as an emergence of distinctively

different cells within the embryo. Different cytoplasmic areas of the early embryo

act on different cleavage nuclei in different ways initiating the development of

different cell lines. The formation of the blastocyst occurs between 7-8d in the

21

sheep and continues to elongate before implantation into the uterus at 15-16d.

Implantation requires the interaction between the blastocyst and uterus.

The blastocyst has two population of cells; trophoblasts and inner cell mass

(ICM) cells. Trophoblasts are the cells of the outer wall of the blastocyst, one of

their main functions is to pump fluid and induce changes in the uterine lining

upon implantation. Trophoblast cells eventually develop into the placenta

enabling maternal-fetal nutrient exchange. Inner cell mass cells are located in

the inner surface of the trophoblasts and are joined by gap junctions. The cells of

the inner cell mass form the embryo and membranes. A formation of a layer of

cells beneath the inner cell mass known as the primitive endoderm contributes to

the formation of the yolk sac and provides early nourishment for the developing

embryo.

1.7.3 GASTRULATION AND THE FORMATION OF THE GERM LAYERS

The blastocyst undergoes the process of gastrulation, the process involving the

well ordered rearrangements of the cells in the embryo. The cells undergo

morphogenetic movements, via cell migration and regrouping leading to the

reorganization of cells into 3 germ layers; ectoderm (outer cell layer), mesoderm

(middle cell layer) and endoderm (inner cell layer). Cells segregate out from the

inner cell mass and form a thin layer of hypoblast cells. The hypoblast forms the

primitive endoderm which lines the yolk sac and the remainder of the ICM cells

becomes epiblast cells. The ICM cells become more regularly arranged and are

called the embryonic disk. Thickening of the embryonic disk occurs at what is to

become the caudal end of the embryo. Expansion of cells in the embryonic disk

leads to the formation of the primitive streak, in which epiblast cells are able to

22

migrate through. The epiblast cells form ectodermal cells and contain cells which

will migrate through the primitive streak to form endodermal or mesodermal germ

layers.

The ectodermal cells become embryonic stem cells which are involved in the

development of the nervous system including the brain and the spinal cord. The

mesodermal cells become pluripotent embryonic stem cells giving rise to the

development of the cardiovascular and muscular system cellular lineages. The

endodermal cells become pluripotent embryonic stem cells giving rise to the

development of the digestive, respiratory, urinary and reproductive systems

cellular lineages.

1.8 IMPACT OF NUTRITION ON THE DEVELOPMENT OF THE

OOCYTE AND EMBRYO

It has been well demonstrated that early embryo mortality is a significant cause

of reproductive failure in ruminants and part of this may be attributed to

nutritional influences around the time of mating (Dunne, Diskin et al. 1999).

However it is not clear as to whether nutrition affects the development of the

embryo via a direct change in the follicular environment, an altered

developmental capacity of the oocyte or through early developmental changes to

the embryo. Nutrition can impact on the reproductive function of ruminants. High

protein diets in cattle, leading to high urea concentrations have been associated

23

with decreased fertility rates and reduced embryo quality. It has been proposed

that this is a consequence of an altered uterine environment.

1.8.1 NUTRITION AND GONADOTROPIN SECRETION

Nutrient restriction in sheep has resulted in lower ovulation rates associated with

a decreased LH (luteinizing hormone) pulse frequency as a result of inadequate

hypothalamic gonadotrophin secretion (Rhind, Rae et al. 2001; Rhind 2004).

Long term nutrient restriction has been shown to reduce circulating LH to

inadequate levels leading to the development of anestrous in cattle (Rhodes,

Fitzpatrick et al. 1995).

24

Figure 1.2 A schematic diagram showing the sequence of events in origin,

growth and rupture of the tertiary follicle and degradation of the corpus

luteum

Follow clockwise starting at primordial germ cell. Adapted from Patten‟s

Foundations of Embryology 6th Edition, Bruce M. Carlson (1996).

a1172507

Text Box

NOTE: This figure is included on page 24 of the print copy of the thesis held in the University of Adelaide Library.

25

1.8.2 NUTRITION AND PROGESTERONE CONCENTRATIONS

Feed intake can also influence the concentration of progesterone. Progesterone

can affect the LH pulse frequency though its negative feedback system.

Furthermore progesterone is thought to play a role in the maturation of the

oocyte and embryo development (Kleemann, Walker et al. 1994). In sheep, ad

libitum feed intake reduces circulating progesterone concentrations, reducing

pregnancy rates (Parr, Davis et al. 1987; Parr 1992), the rate of development of

the embryo as well as their viability (Creed, McEvoy et al. 1994). It has been

shown that the peripheral concentrations of progesterone on days 0 and 1 after

the LH peak are particularly important for the survival of the sheep embryo, by

modifying follicular maturation and oocyte quality (Ashworth, Wilmut et al. 1987).

1.8.3 NUTRITION AND OVARIAN FUNCTION

High energy and high protein diets have been shown to increase the ovulation

rates in ewes, presumably by an increase in glucose entry rates into the cell.

Similarly glucose infusion studies have shown increased ovulation rates

(Downing, Joss et al. 1995). It has been suggested that short term increases in

energy intake may directly affect follicle recruitment and growth.

1.8.4 NUTRITION AND EMBRYO QUALITY

It has been previously reported that ewes fed a low energy diet produced a high

proportion of viable ova when compared to those produced from ewes fed a high

energy diet (McEvoy, Robinson et al. 1995). This suggests that oocyte quality is

26

influenced by dietary intake and that a critical window in which nutrition can

affect oocyte quality exists prior to conception.

In a recent study by Minge and colleagues (Minge, Bennett et al. 2008) murine

oocytes from rats fed a high fat diet (HFD) for 16 weeks prior to conception to

day 8 of gestation resulted in poor oocyte quality. It was observed that there was

a decline in the number of embryos undergoing further differentiation into 4 to 8

cell blastocyst stage, morula/ blastocyst stage and expanded blastocyst/

hatching stage compared to embryos produced by the control diet dams.

Furthermore, HFD exposure led to the abnormal localization of blastomeres in

the inner cell mass and an increase in the proportion of cells in the

trophectoderm. Currently no postnatal studies have been performed to determine

the impact of a high fat diet on the abnormal localization of cells within the ICM

and trophectoderm. This altered allocation of cells in the ICM may alter the

growth and development of the fetus. Interestingly embryos produced by dams

exposed to a HFD prior to conception supplemented with rosiglitazone, an insulin

sensitizing agent, improved the developmental potential of the embryos and

significantly reduced the number of cells within the trophectoderm layer, when

compared to HFD and vehicle.

It has been previously discussed that direct glucose infusion increases ovulation

rates, but embryo quality reduces dramatically and is associated with reduced

pregnancy rates in ewes (Yaakub, Williams et al. 1997). The mechanisms

underlying the impact of glucose on embryo development are unclear. However it

has been suggested that high glucose concentrations may interfere with the

27

cellular signaling during follicular, oocyte and early embryo growth and

development (Boland, Lonergan et al. 2001).

It is proposed that a high energy environment may alter the glucose-insulin,

insulin-like growth factor-1 (IGF-1) axis which may impact on early embryo

development. There is an increasing metabolic reliance on glucose between the

zygote and blastocyst stage, in which both IGF-1 and insulin are able to stimulate

glucose uptake. It has been suggested that the glucose transporter, GLUT-1, is

the possible transporter increasing glucose uptake, initiated by insulin stimulated

recruitment of IGF-1 (Pantaleon and Kaye 1998).

It is clear that nutrition of the early embryo can have a profound effect on the

subsequent development of the oocyte, embryo and reproductive potential. It is

evident that effects on fetal development is likely to be a consequence of the

nutritional environment, which can be programmed very early in development

and even prior to conception.

1.9 FUNCTION AND REGULATION OF ADIPOSE TISSUE

1.9.1 FUNCTION OF ADIPOSE TISSUE DURING FETAL DEVELOPMENT AND LATER IN LIFE

Obesity occurs when there is a long-term excess in the amount of energy

consumed over that expended by an individual. When this exceeds the amount

of energy expended by the body, excess energy is stored as triacylglycerols in

the adipose stores (Rosen and Spiegelman 2000; Sherwood 2004; Bruce and

28

Byrne 2009). The function of adipose tissue can be identified by the adipose cell

type. Adipose tissue development occurs before birth in humans (Merklin 1973)

and in larger animals such as the pig (Hausman, Kasser et al. 1982) and the

sheep (Alexander 1978). During development, adipose tissue depots begin as a

small cluster of lipid droplets surrounded by extensive stroma and a network of

capillaries. As development progresses the amount of lipid accumulates in the

clusters and increases in size, however the number of clusters remain relatively

constant (Aihaud, Grimaldi et al. 1992). Adipose tissue comprises of either

multilocular cells with multiple small lipid locules and an abundance of

mitochondria, characteristic of brown adipose tissue (BAT) or unilocular cells

comprising of a dominant lipid locule, characteristic of white adipose tissue

(WAT) (Gemmell and Alexander 1978; Rosen and Spiegelman 2000;

Muhlhausler 2002). The physiological role of BAT is distinct to that of WAT. The

primary role of BAT is of thermogenesis, by dissipating energy rather than storing

it and therefore is a defense against cold. Fetal adipose tissue is primarily

characterized as BAT, whereas in adulthood WAT is more abundant with less

obvious BAT depots (Rosen and Spiegelman 2000). The traditional role of WAT

is typically regarded to be that of insulation, protection and long term energy

storage, in which fatty acids may be mobilized for oxidation in other organs

during periods of food deprivation (Kersten 2001; Trayhurn, Bing et al. 2006).

Therefore the size of adipose tissue stores corresponds to the energy balance of

the individual, which results in larger adipose tissue stores in periods of positive

energy balance and depleted adipose tissue stores in periods of negative energy

balance. Adipose tissue however, has been increasingly recognized to function

as an endocrine tissue, secreting adipokines which signal energy balance and

29

consequently plays a primary role in maintaining normal glucose homeostasis

(Ahima 2006). Therefore despite excess adipose tissue accumulation being

associated with a range of co-morbidities, adipose tissue plays an important role

in the regulation and balance of energy homeostasis.

In large animal species such as the human (Merklin 1973) and sheep (Alexander

1978) adipose tissue depots are present before birth, however there are

differences in the distribution of adipose tissue before birth in these species. In

the human infant, visceral adipose tissue depots consist of mostly brown adipose

tissue, whereas the subcutaneous adipose tissue depot consists predominantly

of white adipose tissue. Similarly in the fetal sheep the visceral adipose tissue

consists of predominantly multilocular, brown adipocytes (Gemmell and

Alexander 1978). In fetal sheep, preadipocytes consisting of fewer mitochondria

begin to accumulate lipid at 70d gestation in the visceral and subcutaneous

adipose tissue sites. Mitochondria within the perirenal preadipocytes proliferate

at 80-90d gestation and the perirenal adipose tissue develops into brown fat. The

subcutaneous adipose tissue however develops into white fat with fewer

mitochondria (Gemmell and Alexander 1978). The adipose tissue mass

continues to increase relative to body weight up until 110d gestation (term~150d)

(Alexander 1978).

1.9.2 ADIPOSE TISSUE DEVELOPMENT

Adipogenesis is a complex process which involves the commitment of multi-

potent embryonic stem cells derived from the mesenchymal stroma to the

adipocyte cell lineage. Preadipocyte cells undergo a series of intermediary

30

stages of differentiation including clonal expansion and further differentiation to

form mature adipocytes upon stimulation of appropriate inducers of

differentiation (Klaus 1997; Gregoire 1998; Gregoire 2001).

1.9.3 HORMONAL REGULATION OF ADIPOSE TISSUE MATURATION

The progression of adipocyte maturation following the commitment of multi-

potent stem cells to the adipocyte lineage is regulated by autocrine and

paracrine factors (Hwang, Loftus et al. 1997). It has been established that

paracrine signals provide primary cues for cellular commitment of stem cells in

early embryonic development. In adult life autocrine signals monitor energy

homeostasis and signal the need to increase or decrease adipose tissue storage

capacity accordingly, by increasing adipocyte number and size (Bray, York et al.

1989). Presumably the synergy of both autocrine and paracrine factors

determines whether further multi-potent cells are committed to undergoing

differentiation into the adipocyte cell lineage. The principal factors which induce

adipocyte differentiation in many cell systems include insulin, insulin like growth

factor-1 (IGF-1), glucocorticoids, thyroid hormone and cyclic adenosine

monophosphate (cAMP) (Spiegelman and Green 1980; Ntambi and Young-

Cheul 2000).

IGF-1 is a ~7.6 kDa polypeptide with a highly conserved sequence across

species (Tavakkol, Simmen et al. 1988). IGF-1 is secreted by the liver and

adipose tissue in a region specific manner (Villafuerte, Fine et al. 2000).

Previous studies have shown that human and porcine preadipocytes in primary

cell culture also express and secrete IGF-1 (Hausman, Wright et al. 1993;

31

Wabitsch, Hauner et al. 1995). The IGF-1 receptor (IGF-1R) is expressed

abundantly on the surface of preadipocytes and the addition of IGF-1 in serum

free media is sufficient to induce adipocyte maturation (Smith 1988). Insulin also

exerts its effects through binding to the IGF-1 receptor. Insulin however, binds to

IGF-1R with a low affinity and therefore relatively high concentrations of insulin

are required to initiate adipocyte maturation. During adipocyte maturation

however, the number of insulin receptors increases during differentiation and the

adipocytes become increasingly sensitive to insulin at physiological

concentrations.

Hydrocortisone is a major fetal adipogenic hormone. In fetal pigs,

dexamethasone, a glucocorticoid analogue, induced more cytodifferentiation in

cultured cells from 75d and 105d old fetuses (Hausman 1992). It has also been

shown that insulin increases the number and binding of glucocorticoid receptors

in preadipocytes as well as enhanced preadipocyte differentiation in cell cultures

treated with glucocorticoid or their analogues (Gregoire, Genart et al. 1991;

Chen, White et al. 1995). This may suggest that glucocorticoids are involved in

regulating insulin action on adipogenesis in vitro.

In vitro studies involving the treatment of primary cultures of stromal-vascular

cells from porcine adipose tissue with human growth hormone (hGh) showed a

reduced number and size of fat cell clusters and a decrease in preadipocyte

differentiation (Hausman 1992). This study suggests that hGh impedes

preadipocyte differentiation and may reduce adipose tissue growth in later life.

32

Following the commitment of multipotent embryonic stem cells, preadipocytes

undergo clonal expansion and further differentiation to form adipocytes, signaled

by a range of transcription factors. There is considerable evidence that the

CCAAT/enhancer binding protein β (C/EBPβ) promotes the expression of critical

adipogenic transcription factors including C/EBPα and peroxisome proliferator-

activated receptor γ (PPARγ) genes. C/EBPα and PPARγ serve as pleiotropic

transcriptional activators that coordinately induce the expression of a suite of

adipocyte specific genes and lipid accumulation involved in adipocyte

differentiation (Gregoire 1998; Ntambi and Young-Cheul 2000; Rosen and

Spiegelman 2000; Gregoire 2001; Rosen, Hsu et al. 2002). Many studies have

shown that PPARγ is critical in the development of adipose tissue. The

expression of PPARγ in non-adipogenic, fibroblastic and myogenic cell lines is

capable of conversion of these cell types to adipocytes (Tontonoz 1994; Hu,

Tontonoz et al. 1995). Further studies involving chimeric mice (fusion of mutant

PPARγ and normal embryonic stem cells) showed that the neonates lacking

significant brown fat depots died as a result shortly after term (Rosen 1999).

1.9.4 REGULATION OF LIPOGENESIS AND LIPOLYSIS

PPARγ is a member of the nuclear receptor family, activated by the binding of

natural or synthetic ligands, such as Thiazolidinediones (TZDs) which enhance

insulin sensitivity (Rosen 1999; Lazar 2004). Once PPARγ heterodimerises with

retinoid X receptor (RxR) it functions as an adipogenic transcription factor and

the PPARγ-RxR heterodimer is able to bind to specific sequences of DNA,

peroxisome proliferator response elements (PPRE) (Auwerx 1999; Picard and

Auwerx 2002; Lazar 2004). PPARγ transcriptionally regulates the differentiation

33

of a suite of genes involved in the mediation of lipogenesis and to enhance

insulin sensitivity (Lazar 2004, Rosen 1999). Three isoforms of PPARγ (γ1, γ2,

γ3) have been identified in humans (Picard and Auwerx 2002).

PPARγ agonists promote adipogenesis and post prandial fatty acid/ triglyceride

storage within adipocytes and consequently both increase adipose tissue mass

but improves insulin sensitivity (Picard and Auwerx 2002). Thiazolidinedione

(TZD), a novel drug developed for the treatment for diabetes, has provided

evidence of the role of PPARγ in enhanced insulin sensitivity in humans

(Lehmann, Moore et al. 1995).

TZD treatment improves glucose tolerance, enhances the expression of genes

encoding for fatty acid binding protein and lipoprotein lipase (LPL). LPL

increases the local generation of free fatty acids from lipoprotein particles within

adipose tissue, thereby contributing to the enhanced insulin sensitivity by

decreasing circulating free fatty acids (Lazar 2004; von Eynatten, Schneider et

al. 2004). PPARγ also increases the expression of the insulin sensitizing

hormone, adiponectin within the adipose tissue. Adiponectin also actively

influences insulin sensitivity by increasing fatty acid oxidation in the muscle and

decreases gluconeogenic enzymes in the liver (Iwaki, Matsuda et al. 2003;

Kajantie, Hytinantti et al. 2004; Kadowaki and Yamauchi 2005). In later life,

PPARγ can act to suppress genes which induce lipolysis and the release of fatty

acids, such as leptin and the β3 adrenergic receptor. An increase in white

adipose tissue mass, consisting of more small newly differentiated adipocytes

34

and fewer larger mature adipocytes accompanies these biological effects (Picard

and Auwerx 2002).

PPARγ, therefore plays a central role in the regulation and maintenance of

adipogenesis, lipogenesis, lipid storage and energy homeostasis in adipose

tissue. Whilst there is a possible role of PPARγ expression in the pathogenesis

of altered adipocyte development in adult obesity, it is unknown however,

whether overnutrition followed by energy restriction during the periconceptional

period alters the expression of PPARγ or other genes involved in the regulation

of adipose tissue development before birth. Alterations in the expression of key

adipogenic and lipogenic genes and/or their regulation may contribute to an

increased susceptibility to the development of obesity and insulin resistance in

later life.

1.9.5 ADIPOSE DERIVED MOLECULES

White adipose tissue is able to produce and secrete a large number of

hormones, cytokines and proteins which influence energy homeostasis. The

circulating levels, or the expression of molecules derived from adipose tissue,

modulated by PPARγ, may affect insulin action in fat and/ or other tissues

including skeletal muscle and the liver, these include free fatty acids, leptin and

adiponectin.

Free fatty acids (FFA) are the major secretory product of WAT. The sympathetic

nervous system is a key regulator of the stimulation of lipolysis, regulating the

release of fatty acids from the breakdown of triacylglycerol. PPARγ activation in

35

the adipose tissue decreases circulating FFA and there is partitioning of fatty

acids towards the adipose tissue, rather than skeletal muscle (Picard and

Auwerx 2002) resulting in enhanced insulin sensitivity, since fatty acid

accumulation in the skeletal muscle would result in insulin resistance.

Leptin is a hormone predominantly synthesized and secreted by adipose tissue,

including WAT, BAT, as well as the stomach, placenta, mammary glands,

ovarian follicules and certain fetal organs (Trayhurn, Hoggard et al. 1999;

Trayhurn and Beattie 2001; Trayhurn, Bing et al. 2006). WAT, however is the

principal site of leptin synthesis and is the primary contributor to circulating levels

of the hormone and its expression is influenced by the status of energy stores in

the adipose tissue. Leptin acts at the hypothalamic centre in the brain which is

involved in regulating feeding behavior and energy balance. Leptin is decreased

in low insulin states, resulting in a reduced signal of fat mass to the brain and

periphery to maintain an increase in fat mass (Ahima and Flier 2000; Ahima and

Flier 2000; Ahima, Saper et al. 2000).

It is evident from both human and experimental animal models that circulating

plasma leptin levels correlate with body fatness and BMI and may mediate the

effects of PPARγ on insulin sensitivity (Considine, Sinha et al. 1996; Havel,

Kasim-Karakas et al. 1996; Blache, Tellam et al. 2000). Glucose infusion

administered late in gestation in the sheep resulted in chronic fetal

hyperglycemia and hyperinsulinemia, an increase in fat mass (Alexander and

Bell 1990) and increased expression of leptin mRNA in the perirenal adipose

tissue (Devaskar, Anthony et al. 2002). Interestingly leptin mRNA expression

36

was up-regulated following an experimental increase in fetal insulin

concentrations with maintained euglycemia (Devaskar, Anthony et al. 2002).

These studies suggest that insulin is a key mediator of fetal leptin synthesis in

the adipose tissue. In a model of maternal overnutrition late gestation in the fetal

sheep, it was shown that there was a positive correlation between relative mass

of the unilocular component of the perirenal and interscapular fat and circulating

leptin concentrations in these fetal sheep (Muhlhausler 2002). This suggests that

in fetal life, there is a relationship between the amount of lipid stored in the

adipocytes and the synthesis of leptin. More importantly this relationship

demonstrates that circulating leptin concentrations may be a signal of fat mass

even before birth.

Although leptin is expressed in all WAT depots it has been suggested that there

are substantial differences between the level of mRNA expression of the leptin

gene in different adipose tissue sites, as well as differences in expression

according to developmental age and inter-species differences. It has been

shown that in humans the leptin gene is expressed at higher levels in the

subcutaneous adipose tissue than the omental adipose tissue (Hube, Lietz et al.

1996; Montague, Prins et al. 1997).

Adiponectin is another hormone synthesized and secreted by adipose tissue,

however its production is inversely proportional to the amount of fat mass in the

body (Maeda, Takahashi et al. 2001). Adiponectin is an insulin sensitizing

hormone and its concentrations are found to be decreased in obesity related

metabolic disorders and insulin resistance (Fasshauer, Paschke et al. 2004), and

37

increased when insulin sensitivity is improved by weight loss (Yang, Lee et al.

2001).

The cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase (G3PDH)

enzymes are involved in glycerol phosphate shuttle in glycolysis, the process

which derives energy from the oxidation of glucose. The cytosolic G3PDH

enzyme regenerates oxidized nicotinamide adenine dinucleotide (NAD+) from its

reduced form NADH, and the mitochondrial G3PDH regenerates oxidized flavin

adenine dinucleotide (FAD) from its reduced form FADH2 which is produced

during glycolysis (Voet 2004). Glycerol-3-phosphate is also utilised in intracellular

triacylglycerol synthesis.

1.10 EXPERIMENTAL HYPOTHESES

The prevalence of obesity is increasing globally and more women are entering

pregnancy either overweight or obese. Obese women are at greater risk of poor

fertility rates, gestational diabetes during pregnancy and adverse pregnancy

outcomes. Maternal obesity also increases the risk of giving birth to a heavier

term infant which has increased fat mass at birth. Being born heavier and fatter

also results in an increased risk of becoming overweight or obese in childhood

and adulthood. Many epidemiological studies have determined that maternal

obesity both before entering pregnancy and maternal weight gain during

pregnancy is associated with an increased risk of developing obesity in later life

in the offspring. These studies and experimental models of maternal obesity,

however, have not been able to elucidate whether maternal periconceptional

38

overnutrition alone programs an increased risk of developing obesity in later life

in the offspring and the underlying physiological mechanisms are not clearly

understood. It is not known whether it is exposure of the oocyte and embryo to

maternal obesity at the start of pregnancy or exposure of the fetus to maternal

weight gain during pregnancy that is associated with an increased risk of obesity

in the offspring. It is also not clear whether maternal weight loss by dieting before

becoming pregnant can improve the long term health of children.

We have developed a “novel” model of maternal obesity in the sheep in which

we transferred embryos from overnourished sheep at one week after conception

to ewes of normal body weight.

In Chapter 2, I hypothesized that maternal overnutrition during the

periconceptional period from at least 5 months prior to conception until 7 days

after conception will significantly increase maternal weight and produce an obese

phenotype, increasing maternal plasma insulin concentrations prior to

conception. A dietary restriction period imposed one month prior to conception to

one week post conception in the periconceptionally overnourished ewes would

reduce the obese phenotype to an average body condition comparable to the

control ewes. I proposed that the transfer of the periconceptionally overnourished

and/or dietary restricted embryos to control fed recipient ewes would result in an

increased body weight, increased insulin and glucose concentrations at birth

when compared to offspring exposed to either a control maternal diet or a period

of dietary restriction in the periconceptional period.

39

No current study has investigated the long term effects of periconceptional

overnutrition, experienced solely during the period before and in the

preimplantation period on later body composition in the offspring.

Periconceptional overnutrition has been shown to alter the formation of the

developing blastocyst, however the long term consequences on the development

of the offspring have not been established. We have therefore investigated the

impact of maternal periconceptional overnutrition and/or dietary restriction, from

5 months prior to conception to one week post conception, on the growth and

development of the postnatal lamb from birth until 4 months of age.

In Chapter 3, I hypothesized that maternal periconceptional overnutrition would

result in a heavier offspring at birth and at 4 months of age, with increased

plasma insulin and glucose concentrations in postnatal life. I further

hypothesized that periconceptional overnutrition would increase fat mass in the

offspring at 4 months of age and that a period of dietary restriction prior to

conception would ablate this effect.

Many experimental models of individuals exposed to maternal nutrient excess,

maternal obesity or pregnancies complicated by gestational diabetes or mild

glucose intolerance increase the risk of adiposity in the offspring. It has been

previously shown that exposure to maternal nutrient excess during critical stages

of fetal development, alters adipogenic and lipogenic gene expression. We

therefore investigated the impact of maternal nutrient excess during critical

windows of embryo development on the programming of adipogenic and

lipogenic gene expression in the postnatal lambs at 4 months of age.

40

In Chapter 4, I hypothesized that lambs exposed to periconceptional

overnutrition would show an up-regulation of adipogenic and lipogenic gene

expression in adipose tissue depots and an increase in the lipogenic capacity of

the adipocytes in postnatal life.

41

CHAPTER 2

42

CHAPTER 2: THE EFFECTS OF MATERNAL OVERNUTRITION

AND/OR DIETARY RESTRICTION DURING THE

PERICONCEPTIONAL PERIOD ON LAMB GROWTH AT BIRTH

2.1 INTRODUCTION

As discussed in Chapter 1 during the past two decades there has been an

increase in the prevalence of overweight or obese women of child bearing age in

the western world (LaCoursiere, Bloebaum et al. 2005). A range of studies have

found that women entering pregnancy with a higher BMI are more at risk of

developing clinical complications during pregnancy and of adverse health

outcomes for their offspring (Garbaciack 1985; Cnattingius 1998; Cogswell, Perry

et al. 2001; Jolly 2003; Kristensen 2005).

Maternal weight at the start of pregnancy, maternal weight gain during pregnancy

and the development of GDM are all positively associated with birth weight of the

offspring (Frisancho 2000; Jensen 2003; Jolly 2003). Furthermore offspring with

a high birth weight are, in turn, at an increased risk of being overweight or obese

throughout childhood and adulthood (Silverman, Rizzo et al. 1991; Rasmussen

and Johansson 1998; Frisancho 2000; Parsons, Power et al. 2001; Oken and

Gillman 2003). As discussed in Chapter 1, a range of experimental studies in

animal models have attempted to investigate the relationship between maternal

obesity in pregnancy and adverse metabolic outcomes for her offspring. These

previous studies, however, have not been able to dissociate the effects of

maternal obesity at the start of pregnancy from the effects of maternal weight

43

and weight gain during pregnancy on the programming of obesity in the offspring.

There have been no studies which have differentiated between the effects of a

nutrient rich environment experienced by the embryo in the periconceptional,

preimplantation period and a nutrient rich environment experienced by the fetus

after placentation, on the risk of being obese after birth.

A number of experimental studies have investigated the effects of maternal

nutrient restriction during the early stages of pregnancy, when nutrient demands

on the early conceptus are minimal. It has been reported that maternal

undernutrition during the periconceptional period has specific effects on the

cardiovascular, metabolic and endocrine systems of the fetus and adult (Kwong,

Wild et al. 2000; Edwards, Bryce et al. 2002; Bloomfield, Oliver et al. 2003;

Gardner, Pearce et al. 2004). Thus the embryo may make adaptive changes to

the level of maternal nutrition experienced before implantation and these

changes can result in an altered developmental trajectory for organ systems both

before and after birth.

Currently no study has investigated the long term consequences of

periconceptional overnutrition, experienced during the period before conception

and in the preimplantation period. Minge and colleagues determined the effect of

diet induced obesity on the development of the embryo and oocyte quality in

mice fed a high fat diet from 16 weeks prior to conception through to conception.

In this study, embryo development was also monitored in vitro 2-5 days after

conception. A high fat diet resulted in oocytes of poor quality, and a decrease in

blastocyte survival. Furthermore abnormal cellular localization of germ cells was

44

apparent in embryos exposed to a high fat diet (Minge, Bennett et al. 2008). The

long term effects of this altered embryo development on fetal growth and

development in later life has not yet been investigated. Similarly no experimental

studies have investigated whether interventions, such as a period of dietary

restriction before conception in an overweight or obese mother can alter the

outcomes for her offspring.

We have therefore investigated the impact of maternal overnutrition during the


after conception, on the growth and development of the postnatal lamb at birth.

In order to determine the impact of the plane of nutrition experienced during the

periconceptional period, single embryos were transferred from donor ewes,

which underwent a nutritional protocol to recipient ewes, which were previously

maintained on a control diet. These recipient ewes were maintained on a control

diet throughout pregnancy and allowed to give birth naturally. We determined the

impact of periconceptional overnutrition and/or dietary restriction on maternal

weight, plasma insulin and glucose concentrations of the donor ewes and on

lamb weight, plasma insulin, glucose and free fatty acid concentrations at birth.

45

2.2 MATERIAL AND METHODS

2.2.1 ANIMALS AND NUTRITIONAL FEEDING REGIME

All procedures were approved by the University of Adelaide Animal Ethics

Committee. Eighty-three South Australian Merino ewes were used in this study.

Donor ewes (n=23) were moved into an enclosed shed and housed in pens 20

days before the start of the feeding regime. All ewes were weighed and a body

condition score (BCS) was assessed by an experienced assessor employing a

scale of 1-5 with intervals or 0.25 (Russell et al. 1969). Using this scale, a body

condition score of 1 represents an extremely emaciated animal and a body

condition score of 5 represents a morbidly obese animal. During this 20 day

period, ewes were acclimatized to a pelleted diet containing cereal hay, lucerne

hay, barley, oats, almond shells, lupines, oat bran, lime and molasses (Johnsons

& Sons Pty Ltd, Kapunda, South Australia, Australia). The pellets provided 9.5

MJ kg-1 of metabolisable energy (ME) and 120 gkg-1 of crude protein and

contained 90.6% dry matter. All ewes received 100% of metabolisable energy

requirements (MER) for the maintenance of a non-pregnant ewe according to

weight, as defined by the Ministry of Agriculture (Ministry of Agriculture 1984).

At the end of this acclimatization period, donor ewes were randomly assigned to

one of four treatment groups, either control-control (CC), control-restricted (CR),

high-high (HH) or high-restricted (HR) nutritional treatment groups as described

below:

46

CC ewes (n=6) were a control group maintained at 100% MER for 5

months prior to conception with a BCS of 3.0-3.5 (Figure 2.2.1).

CR ewes (n=6) were maintained at 100% MER for the first 4 months,

then 1 month prior to conception were placed on a dietary restriction of

70% MER (Figure 2.2.1).

HH ewes (n=6) were maintained at a high BCS of 4.0-4.5, and were fed

an ad libitum diet (170-190% MER) for 5 months prior to conception

(Figure 2.2.1).

HR ewes were maintained at a high body condition, and were fed an ad

libitum diet (170-190% MER) for 4 months, and then 1 month prior to

conception were placed on an energy restricted diet of 70% MER (Figure

2.2.1).

Throughout the protocol all ewes had ad libitum access to water. Donor ewes

were weighed and their body condition score assessed and scored

approximately every two weeks after commencing the feeding regime until 7

days gestation.

2.2.2 DONOR AND RECIPIENT EWE SYNCHRONISATION AND PREGNANCY

2.2.2.1 Super ovulation protocol

The reproductive cycles of all experimental ewes were synchronized and super

ovulation was induced by the administration of an intravaginal progestagen

pessary (45mg flugestone acetate; Intervet, Paris, France) for 12 days, followed

by the administration of follicle stimulating hormone (FSH, Folltropin, equivalent

47

to NIH-FSH-P1 standard) to the donor ewes, administered in 6 equal injections

given over 3 days, twice daily, commencing 48h before pessary removal. Each

donor ewe also received 500IU eCG (Pregnecol) at the time of the first FSH

injection and another ovarian stimulant drug, GnRH (fertagyl) was administered

to the donor ewes following pessary removal.

2.2.2.2 Artificial insemination and embryo collection

Fresh semen was collected from a ram of proven fertility (using an artificial

vagina) in a protocol specified by Kakar and colleagues (Kakar, Maddocks et al.

2005). The donor ewes were inseminated by laparoscopy with approximately 20

million spermatozoa being placed directly into the lumen of each uterine horn

36h after pessary withdrawal. During the insemination procedure, ewes were

lightly sedated with I.V. administration of Rompum (Xylazine; 0.1ml) and local

administration of Lignocane.

Donor ewes were fully anaesthetized 6-7 days following insemination and

embryos were collected by laparoscopy by flushing the oviducts with saline. After

embryo collection the ewes were allowed to recover and then were returned to

pasture. The recovered embryos were classified according to their structure

during early embryogenesis.

48

2.2.3 DETERMINATION OF MAJOR TRANSITIONS IN STRUCTURE OF THE EMBRYO DURING

EARLY EMBRYOGENESIS

Following the flushing of the ewe‟s oviducts with saline, embryos were collected

into culture dishes. They were counted and scored based on the major

transitions in structure of the embryo during early embryogenesis, as either

compact morula, early blastocyst, blastocyst or expanded blastocyst. Compact

morula were defined by the formation of tight junctions between the blastomeres

following the development of an 8 or 16 cell embryo, leading to deformation of

their round shape and the compaction of cells (Figure 2.2.2A). Early blastocysts

were defined by the formation of junctional complexes between blastomeres and

a small amount of blastocoelic fluid accumulation inside the embryo (Figure

2.2.2B).

2.2.4 EMBRYO TRANSFER PROTOCOL

Donor embryos of good quality (8 cells and acceptable morphology) were

transferred to adult recipient ewes maintained on a control diet (100% MER) at

embryonic day 6-7 by laparoscopy. The laparoscopy procedure and sedation

level was similar to that described for the insemination procedure. One embryo

was transferred laparoscopically to the uterine horn. Each ewe only received

embryos originating from ewes in only one nutritional group. Sixty-three ewes

underwent embryo transfer to ensure the birth of at least 8 offspring per

treatment group, with a maximum of 16 offspring per treatment group. These

recipient ewes were maintained on a diet calculated to provide 100% energy

requirements for the maintenance of a pregnant ewe bearing a singleton fetus,

49

as specified by the U.K Ministry of Agriculture, Fisheries and Food (Ministry of

Agriculture 1984).

Pregnancy was confirmed by ultrasonography at day 49 of gestation when 47

singleton pregnancies were confirmed (CC n=9, CR n=12, HH n=13, HR n=13).

The pregnant ewes were allowed to give birth naturally in a lambing pen

(term=150 ± 3 days).

2.2.5 BLOOD SAMPLING REGIME

2.2.5.1 Donor and recipient ewe blood sampling

Blood samples (10ml) were collected by jugular venepuncture from the donor

ewes into heparinised tubes containing serum separating gel and kept on ice

until centrifuged. Baseline samples were collected from all donor ewes at one

month prior to the start of the dietary intervention period and another sample was

taken 2 weeks before the commencement of the dietary intervention period

(Figure 2.2.3). Donor ewe blood samples were then collected at 2 weeks and 4

weeks into the dietary intervention period.

Baseline blood samples were also taken from all recipient ewes prior to the

embryo transfer and at day 49 of gestation. Blood samples were centrifuged at

1500g for 10min at 4oC and plasma was stored at -20oC for subsequent

determination of glucose and insulin concentrations.

50

Figure 2.2.1 Nutritional protocol

51

Figure 2.2.2 The major transitions in structure of the bovine embryo during

early embryogenesis. Examples derived from Hypertext for Biomedical

Sciences: Cleavage and Blastocyst formation (2000) Bowen, R

http://www.vivo.colostate.edu/hbooks/index.html

a1172507

Text Box

NOTE: This figure is included on page 51 of the print copy of the thesis held in the University of Adelaide Library.

52

2.2.5.2 Lamb blood sampling

Blood samples (5ml) were collected by jugular venepuncture at birth into

heparinised vacutainer tubes containing serum separating gel and kept on ice

until centrifuged. Blood samples were centrifuged at 1500g for 10min at 4oC and

plasma was stored at -20oC for subsequent determination of glucose, insulin and

non-esterified free fatty acid concentrations.

2.2.6 LAMB BIRTH AND GROWTH MEASUREMENTS

Lamb birth weight, crown-rump length, shoulder height, abdominal circumference

and temperature were taken within the first 12 h of birth.

2.2.7 PLASMA NON-ESTERIFIED FREE FATTY ACID DETERMINATION

Plasma non-esterified free fatty acid concentrations were determined

spectrophotometrically using a Konelab 20XTi automated sample analyser

(Konelab 20, Program Version 6.0 automated analysis system, Thermo Fisher

Scientific, Suwanee, USA by standard enzymatic methods) (Duffield, Vuocolo et

al. 2009) The intra and inter assay coefficients of variance were both < 10%.

2.2.8 PLASMA INSULIN DETERMINATION

Plasma insulin concentrations were measured using a radioimmunoassay (Rat

insulin kit, Linco Research, Inc., Missouri, USA), which was validated for use

with sheep plasma. This assay has previously been shown to have a cross-

reactivity of 100% with sheep insulin and no detectable cross-reactivity with

53

related proteins (C-peptide, glucagon, somatostatin, pancreatic polypeptide or

IGF-1) (Linco Research, Inc., Missouri, USA). The recovery of exogenous

insulin added to lamb plasma was 96.5 ± 3.7 % there was a linear relationship

between the amount of insulin observed compared to that expected. When

increasing volumes of lamb plasma were added into the assay, the

displacement curve was parallel to the assay standard curve. Samples (10 µl)

were assayed in duplicate and added to borosilicate glass tubes with 100 µl of

hydrated 125I-Insulin and guinea-pig anti-rat insulin antibody and incubated

overnight at 4C. Precipitating reagent (1ml) was added and tubes were

centrifuged for 25 min at 2000g, then aspirated and total counts measured by

gamma counter (Muhlhausler 2002). The sensitivity of the assay was 0.01 ng/ml

and the intra and inter assay coefficients of variance were both <10%.

2.2.9 STATISTICAL ANALYSES

All data are presented as mean ± standard error of the mean (SEM). Data were

analysed using the Statistical Package for the Social Sciences version 17.0

(SPSS Inc., Illinois, USA) and STATA10: Data analysis and Statistical Software

for repeated measures (Stata Corp. LD., Texas, USA).

2.2.9.1 Ewe weights

The weights of the donor ewes assigned to one of the four treatment groups

during the periconceptional period were compared during the nutritional regime

using two way Analysis of Variance (ANOVA) with repeated measures.

54

Specified factors for the ANOVA included group (CC, CR, HH, and HR) and

time.

2.2.9.2 Conception and pregnancy outcomes

The effects of periconceptional nutrition on both pregnancy rates and lamb

survival rates were compared using one way ANOVA.

2.2.9.3 Plasma glucose, non-esterified fatty acid and insulin

concentrations

The effects of nutritional treatment on maternal or lamb plasma concentrations

of glucose, non-esterified fatty acid and insulin were compared using

multifactorial ANOVA with repeated methods. Specified factors for the ANOVA

included group (CC, CR, HH or HR), time and lamb gender.

2.2.9.4 Lamb birth measures

The effects of periconceptional nutrition on birth weight, crown rump length,

abdominal circumference and shoulder height were analyzed using

multifactorial ANOVA where the specified factors included group (CC, CR, HH

or HR) and lamb gender.

When a significant interaction between major factors was identified, the data

were split on the basis of the interacting factors and reanalyzed. The Bonferroni

post hoc test was used to identify significant differences between mean values

and a probability level of 5% (P<0.05) was taken as significant.

55

2.3 RESULTS

2.3.1 DONOR EWE WEIGHTS

The weights of the non-pregnant donor ewes assigned to CC (54.5 ± 1.53kg,

n=7), CR (54.5 ± 1.04kg, n=10), HH (56.3 ± 3.24kg, n=12) or HR (56.0 ± 2.00kg,

n=12) groups were not different before the start of the feeding regime (32 weeks

before conception) (Figure 2.3.1).

The weights of the HH and HR ewes between 25 weeks prior to conception and

embryo transfer were significantly higher compared to the CC and CR groups. At

4 weeks before conception, after a minimum of 4 months on the feeding regime,

there was a significant difference in the weights of the donor ewes in the different

treatment groups. The ewes which were overnourished were heavier (HH; 73.4 ±

2.8kg, n=12 and HR; 74.1 ± 1.5kg, n=12) than the ewes on the control level of

nutrition (CC; 59.0 ± 1.1kg, n=7 and CR; 60.0 ± 0.9kg, n=10) (P<0.001) (Figure

2.3.1). The donor body condition scores from 5 weeks prior to conception until

conception were also greater in the HH and HR groups when compared to the

CC and CR groups (P<0.0001) (Table 2.3.1).

The period of dietary restriction in the CR group during the 4 weeks prior to

conception to embryo transfer (d 6-7) resulted in less weight gain in this group

during this period (3.33 ± 1.36kg, n=10) compared to the CC group (6.80 ±

0.85kg, n=7) and similarly the weight gain was less in the HR group (12.36 ±

56

0.82kg, n=12) compared to the HH group (16.52 ± 1.04kg, n=12) (P<0.001)

(Figure 2.3.2).

2.3.2 DONOR EWE PLASMA GLUCOSE AND INSULIN CONCENTRATIONS

There was no effect of periconceptional overnutrition on the plasma glucose

concentrations in ewes at 5, 4 or 1 week before conception (Table 2.3.2).

Plasma insulin concentrations were significantly higher in the HR group and

tended to be higher in the HH group when compared with the CC and CR and

groups (Figure 2.3.3, P<0.05).

2.3.3 PERICONCEPTIONAL NUTRITION AND EMBRYO COLLECTION

Periconceptional overnutrition and/or dietary restriction did not significantly affect

the developmental stage of the embryo at d 6-7 pregnancy (Table 2.3.3).

2.3.4 BODY CONDITION AND WEIGHT OF RECIPIENT EWES

Recipient ewes were maintained at a normal body condition score (3.00 ± 0.03,

n=48) from the start of the donor ewe feeding regime to the time of conception

(3.30 ± 0.03, n=48). There was no difference between the weights of the

recipient ewes allocated to carry the CC, CR, HH or HR embryos one week

before embryo transfer (CC, 62.2 ± 0.02kg, n=7; CR, 64.8 ± 0.01kg, n=11; HH,

58.8 ± 0.01kg, n=12; HR, 61.1 ± 0.01kg, n=12).

57

Figure 2.2.3 Blood sampling regime in the donor and recipient ewes

58

Figure 2.3.1 Effect of periconceptional overnutrition and/or dietary

restriction on the weight of donor ewes during the nutritional feeding

protocol from 35 weeks before conception to 1 week after conception

The mean weights of the donor ewes from 35 weeks before conception (-35

weeks) to one week after conception (+1 week) in the CC (dark blue circles,

n=6), CR (light blue circles, n=6), HH (purple triangles, n=6), HR (pink triangles,

n=5) groups.

* denotes a significant difference between the weight of the donor ewes in the

HH and HR groups compared to the CC and CR groups (P<0.05).

†

*

*

*

* * * * * * * * * * * * * * * * * * * * * * *

59


restriction on the change in the weight of the non-pregnant donor ewes

between 25 weeks before conception to the day of embryo transfer (day 6-7

pregnancy)

Periconceptional dietary restriction in the CR (n=6) and HR (n=5) resulted in less

weight gain when compared to the CC (n=6) and HH groups (n=6) respectively.

Mean values with different superscripts are significantly different (P< 0.001).

Treatment group

CC CR HH HR

Cha

nge

in w

eigh

t (kg

)

0

2

4

6

8

10

12

14

16

18

20

c

d

a

b

60

Donor ewe body

condition score

-5 weeks -4 weeks -1 week conception

CC (n=6)

3.13 ± 0.09 3.08 ± 0.05 2.92 ± 0.05 3.04 ± 0.04

CR (n=6)

3.00 ± 0.06 3.04 ± 0.08 2.92 ± 0.05 3.04 ± 0.08

HH (n=6)

4.08 ± 0.08 * 4.17 ± 0.08 * 4.29 ± 0.08 * 4.25 ± 0.09 *

HR (n=5)

4.35 ± 0.10 * 4.35 ± 0.10 * 4.00 ± 0.11 * 4.05 ± 0.15 *

Table 2.3.1 Donor ewe body condition scores between 5 weeks prior to

conception and conception

* denotes a significant difference between the donor body condition scores of the

HH and HR ewes and the CC and CR ewes (P<0.0001).

61

Plasma glucose

concentration (mmol/l)

-5 weeks -4 weeks -1 week conception

CC (n=7)

3.28 ± 0.20 3.80 ± 0.57 4.24 ±0.25 3.76 ± 0.15

CR (n=10)

3.32 ± 0.26 3.13 ± 0.60 3.82 ±0.27 3.73 ± 0.26

HH (n=12)

4.21 ± 0.61 4.07 ± 0.37 4.00 ± 0.22 3.66 ± 0.13

HR (n=12)

3.36 ± 0.23 3.53 ± 0.09 3.68± 0.18 3.53 ± 0.14

Table 2.3.2 Plasma glucose concentrations in donor ewes at 5, 4 and 1

week prior to conception and at conception

There was no effect of periconceptional overnutrition and/or dietary restriction on

the plasma glucose concentrations at 5, 4 and 1 week prior to conception and

conception.

62

Figure 2.3.3 Plasma insulin concentration in donor ewes between 5 weeks

before conception and conception

Mean values with different superscripts are significantly different (P<0.05)

Treatment group

CC CR HH HR

Plas

ma

insu

lin c

once

ntra

tion

(ng/

ml)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

a a

a b

b

63

Compact morula

Early blastocyst Blastocyst

Expanded blastocyst

CC (n=55)

54.5%

9.1% 20% 16.4%

CR (n=45)

57.8% 15.5% 20% 6.7%

HH (n=42)

42.9% 7.1% 47.6% 2.4%

HR (n=55)

45.4% 10.9% 27.3% 16.4%

Table 2.3.3 Periconceptional nutrition and/or dietary restriction and the

stage of embryo development

The number of embryos recovered at embryo collection and the proportion of

embryos observed in the compact morula, early blastocyst, blastocyst and

expanded blastocyst stage.

64

2.3.5 PLASMA GLUCOSE AND INSULIN CONCENTRATIONS IN RECIPIENT EWES

There was no effect of periconceptional nutrition on the plasma glucose and

insulin concentrations of the recipient ewes between 5 weeks before and 7

weeks after conception (Table 2.3.4)


There was no significant effect of either PCON and/or dietary restriction on lamb

weight, crown rump length, abdominal circumference or shoulder height at either

birth (Table 2.3.6). Male lambs were significantly heavier at birth, however, than

females (Figure 2.3.4, P< 0.05).

2.3.7 LAMB PLASMA GLUCOSE, INSULIN AND FREE FATTY ACID CONCENTRATIONS

There was no effect of PCON and/ or restriction on the plasma glucose, insulin

and non-esterified free fatty acid concentrations (Table 2.3.7) at birth. However

plasma insulin concentrations of male lambs were significantly higher when

compared to female lambs at birth (4.91 ± 1.30 ng/ml vs. 2.17 ± 0.56 ng/ml,

P=0.018).

65

Table 2.3.4 Plasma glucose and insulin concentrations in recipient ewes at

5 weeks before and 7 weeks after conception

Nutritional treatment group of embryo

transferred

Plasma glucose concentration

(mmol/l)

Plasma insulin concentration

(ng/ml)

-5 weeks +7 weeks -5 weeks +7 weeks

CC (n=9)

4.79 ± 0.45 3.38 ± 0.18 0.28 ± 0.07 0.32 ± 0.05

CR (n=12)

4.74 ± 0.24 3.81 ± 0.19 0.31 ± 0.05 0.32 ± 0.05

HH (n=14)

5.53 ± 0.32 3.83 ± 0.22 0.32 ± 0.05 0.40 ± 0.05

HR (n=12)

4.75 ± 0.30 3.51 ± 0.09 0.28 ± 0.04 0.35 ± 0.05

66

Table 2.3.5 Periconceptional nutrition and pregnancy outcomes and lamb

survival

Embryos

transferred

Number of

positive pregnancy

scans

Pregnancy

rate

Gestation

length (days) Live births

Lambs which survived to 4

months of age

Male Female

CC

13 9 69% 150.0± 0.65 8 2 5

CR

16 12 75% 150.1± 0.43 10 7 3

HH

17 13 76% 150.8 ± 0.30 12 7 5

HR

16 13 75% 150.5 ± 0.58 13 5 7

67

Table 2.3.6 Effect of periconceptional overnutrition and/or dietary

restriction on growth parameters at birth

Growth measures at birth

CC (n=7)

CR (n=10)

HH (n=12)

HR (n=12)

Birth weight (kg) 5.59 ± 0.18 5.98 ± 0.27 5.63 ± 0.18 5.48 ± 0.21

Crown rump length (cm)

55.14 ± 0.53 54.75 ± 1.21 55.45 ±0.63 56.08 ± 1.14

Abdominal

circumference (cm)

40.79 ± 1.00 41.17 ± 1.04 40.51 ±0.63 40.84 ± 0.69

Shoulder height

(cm) 43.86 ± 1.15 43.40 ± 1.35 43.79 ±0.65 43.38 ± 0.52

68


restriction on birth weight in male and female lambs

Periconceptional overnutrition and/or dietary restriction did not affect lamb birth

weight. Male lambs (closed bars) were significantly heavier than females (striped

bars) in all treatment groups at birth (* denotes P<0.05).

* * * *

* * * *

69

Table 2.3.7 Effect of periconceptional overnutrition and/or dietary

restriction on plasma glucose, non-esterified free fatty acid and insulin

concentrations at birth in the offspring


the plasma glucose, NEFA and insulin concentrations at birth. * denotes a

significant difference between the plasma insulin concentrations between male

and female lambs at 4 months of age (P=0.018).

Treatment

group

Plasma glucose concentration

(mmol/l)

Plasma NEFA

concentration (mEq/l)

Plasma insulin concentration

(ng/ml)

Males Females Males Females Males Females

CC (n=7) 7.35±4.02 3.74±0.92 0.48±0.08 0.66±0.08 10.2±7.99* 1.94±1.48

CR (n=10) 4.87±1.19 5.09±1.17 0.87±0.21 0.99±0.25 7.50±3.00* 2.70±1.67

HH (n=12) 4.96±0.87 6.06±0.25 0.83±0.09 0.96±0.12 2.34±1.03* 3.00±0.84

HR (n=12) 6.19±1.59 5.63±1.88 0.67±0.09 0.70±0.28 3.29±1.34* 1.25±0.79

70

2.4 DISCUSSION

The objective of this study was to investigate whether the plane of nutrition of the

ewe before conception and during early embryo development altered fetal

growth.

2.4.1 WEIGHTS OF NON-PREGNANT DONOR EWES AND PREGNANT RECIPIENT EWES

Periconceptional overnutrition resulted in a greater weight gain in the donor ewes

compared to ewes fed a control plane of nutrition by 4 weeks before conception.

The periconceptionally overnourished ewes were fed an ad libitum diet of

approximately 170-190% MER for at least 5 months prior to conception, during

which they achieved a body condition score of 4.25 ± 0.09 at conception. The

body condition scores of the ewes in the HH group were consistent with an

obese phenotype (Russell 1969). In the control (CC) group, ewes were fed a diet

calculated to provide 100% ME for at least 5 months prior to conception and the

mean body condition score of the CC group at conception was 3.04 ± 0.04,

consistent with a normal phenotype (Russel, Doney et al. 1969). The imposition

of a period of dietary restriction resulted in a lower weight gain in the CR group,

compared to the CC group, and in the HR group compared to the HH group,

although the HR ewes remained heavier than both the CC and CR ewes. This is

consistent with the objectives of the study which were to use a prolonged period

of overnutrition to generate an obese maternal phenotype before imposing a

period of moderate dietary restriction in a sub group of the obese animals before

conception.

71

In this study the effects of a moderate dietary restriction for 4 weeks resulted in a

significant but modest decrease in ewe weight. Donor weight loss from 5 weeks

prior to conception to conception was 5.45 ± 0.63kg in the CR group and 7.9 ±

0.52kg in the HR group. Previous studies have shown that more severe levels of

nutrient restriction (50% MER) during the periconceptional period resulted in

significant adverse effects on pregnancy and survival rates (O'Callaghan and

Boland 1999; O‟Callaghan, Yaakub et al. 2000; Lozano, Lonergan et al. 2003).

Interestingly in the present study there was no impact of either prolonged

overnutrition or dietary restriction on the rates of embryo development or on

pregnancy rates after embryo transfer.

In the present study, recipient ewes were successfully maintained at a body

weight and a body condition score of 3-3.5 for at least 4 months prior to

conception. This is consistent with the objectives of the study, which were to

determine the effect of transfer of an embryo from a donor ewe which had

experienced periconceptional overnutrition with or without dietary restriction to a

recipient ewe of a normal body condition. The recipient ewes were also fed a

control diet calculated to provide metabolisable energy requirements for a ewe

carrying a singleton fetus throughout gestation through to weaning to ensure a

nutritionally controlled gestational environment.

2.4.2 MATERNAL PLASMA GLUCOSE AND INSULIN CONCENTRATIONS

There was no effect of periconceptional overnutrition or dietary restriction on

plasma glucose concentrations in non-pregnant donor ewes at 5, 4 and 1 week

before conception and at conception. This is not consistent with previous studies

72

which have demonstrated that maternal plasma glucose concentrations are

dependent upon current maternal dietary intake both before and after a change

in nutritional status (Wallace, Bourke et al. 1999; Muhlhausler, Adam et al.

2005). Maternal plasma insulin concentrations tended to be higher, however, in

the HH ewes and were significantly higher in the HR ewes when compared to the

CC and CR ewes. This may suggest that periconceptional overnutrition is

associated with a degree of maternal insulin resistance in the HH and HR groups

in the present study.

As expected there was no difference in the plasma glucose and insulin

concentrations in the recipient ewes allocated to receive the embryos from the

different nutritional treatment groups at 5 weeks prior to conception. There was

also no difference between plasma glucose and insulin concentrations in the

recipient ewes carrying the embryos from the 4 different nutritional treatment

groups at 7 weeks after conception. This is consistent with the objectives of this

study, which was to provide a controlled nutrient environment for the transferred

embryos.

2.4.3 PREGNANCY OUTCOMES

Body condition and plane of nutrition experienced during pregnancy has been

previously shown to affect oocyte quality and pregnancy outcomes in sheep and

cattle (Boland, Lonergan et al. 2001; Adamiak, Mackie et al. 2005; Chagas, Bass

et al. 2007; Leroy, Van Soom et al. 2007). In the present study however,

periconceptional overnutrition or restriction did not significantly affect pregnancy

or survival rates to 4 months of age.

73

Few studies have reported effects of maternal nutrition on oocyte quality in

detail. It has been shown that a higher proportion of viable ova are produced

from ewes fed a low energy diet than those ewes fed a high energy diet

(McEvoy, Mayne et al. 1995; McEvoy, Robinson et al. 1995). Kendrick and

colleagues (Kendrick, Bailey et al. 1999) examined the effects of high energy

diets and post partum interval in lactating dairy cows. It was observed that high-

energy fed cows had smaller follicles but produced more good quality oocytes

than low-energy fed cows. This suggests that oocyte quality is not only

dependent upon nutritional plane, but also the physiological state of the animal.

In contrast short-term restriction of dietary intake has been shown to increase

subsequent pregnancy rates in cattle (Dunne, Diskin et al. 1999).

It is known that high glucose concentrations present in in vitro culture media are

deleterious to embryo development (Fraser, Waite et al. 2007; Minge, Bennett et

al. 2008). The underlying mechanisms by which glucose has an adverse effect

on embryo development are unknown. However it has been suggested that

glucose may interfere with cellular signalling at different stages of follicular,

oocyte or early embryo development (Fraser, Waite et al. 2007; Martin,

Vonnahme et al. 2007).

The formation of the blastocyst is a well recognized key developmental event in

embryo development. The blastocoel cavity forms as a consequence of fluid

transport across the trophectoderm. In a recent study by Minge and colleagues

(Minge, Bennett et al. 2008) murine oocytes exposed to a high fat diet for 16

74

weeks prior to conception resulted in poor oocyte quality with a decrease in the

proportion of blastocyte survival and abnormal cellular differentiation of the

embryo. It was observed that there was a decline in the number of embryos

undergoing further differentiation into 4 to 8 cell blastocyts, as well as the

abnormal localization of blastomeres in the inner cell mass and an increase in

the proportion of cells located in the trophectoderm.

In the present study, the effects of periconceptional overnutrition and/or

restriction on embryo development were examined. We did not, however,

observe any differences between the stagings of the developing embryo from

compact morula, early blastocyst, blastocyst to expanded blastocyst stage at

embryonic day 6-7.

Further evidence from other animal models has shown that maternal

periconceptional undernutrition alters the proportion of cells between the inner

cell mass and trophectoderm (Erwich and Robinson 1997; Kwong, Wild et al.

2000). Therefore the effect of periconceptional nutrition on the alteration of the

proportion of cells allocated to the inner call mass or trophectoderm in the

developing embryo may be an important mechanism by which periconceptional

overnutrition and/or restriction may alter the growth and development of the

offspring.

75


There was no effect of periconceptional overnutrition and/or restriction on weight,

crown rump length, abdominal circumference or shoulder height of lambs at

birth. No previous study has determined the effect of periconceptional

overnutrition on birth parameters. However epidemiological studies would

suggest that maternal obesity entering pregnancy increases the risk of giving

birth to a heavier infant (Garbaciack 1985; Frisancho 2000; Laitinen 2001;

Parsons, Power et al. 2001; Pietiläinen 2001; Jensen 2003; Jolly 2003;

Kristensen 2005; LaCoursiere, Bloebaum et al. 2005). However under these

circumstances it is impossible to differentiate between the effects of maternal

obesity, maternal weight gain or the subsequent development of gestational

diabetes to the contribution of having a larger infant. The present study suggests

that a high maternal weight and/or maternal insulin resistance on entering

pregnancy does not result in an increase in birth weight of the offspring, as

occurs with maternal obesity in human pregnancy. One possibility is that it is

exposure of the embryo or fetus to maternal obesity at some point after day 7 of

gestation that contributes to an increase in fetal body growth and an increase in

birth weight.

76

2.5 SUMMARY

We have developed a novel model for investigating the effects of maternal

periconceptional overnutrition in the sheep. An ad libitum diet, providing 170-

190% of metabolisable energy requirements, for a period of at least 5 months

prior to conception resulted in an obese phenotype in the non-pregnant ewe. We

have shown that there were significant differences in maternal weight gain

between the HH ewes fed a high plane of nutrition compared to CC ewes fed a

control plane of nutrition during the periconceptional period. Plasma insulin

concentrations were also higher in the HH and HR ewes. Furthermore we have

successfully developed a model of maternal dietary restriction in the obese ewe

with significant differences in the weight gain during the restriction period

between the CC and CR group and the HH and HR groups. This is the first

study to determine the impact of overnutrition during the periconceptional period

separate from the remainder of pregnancy. There was no effect of

periconceptional overnutrition and/or dietary restriction on the weight, growth

measures, plasma glucose, free fatty acid and insulin concentrations of the

lambs at birth. There may be however, longer term consequences of the

exposure of the developing embryo to nutrient excess and/or dietary restriction.

This will be discussed further in Chapter 3, which reports the outcomes for the

lambs in this study at 4 months of age.

77

CHAPTER 3

78

CHAPTER 3: THE EFFECTS OF MATERNAL OVERNUTRITION

AND/OR DIETARY RESTRICTION DURING THE

PERICONCEPTIONAL PERIOD ON LAMB GROWTH FROM BIRTH

TO 4 MONTHS OF AGE

3.1 INTRODUCTION

In Chapter 2 it was established that there was no effect of maternal overnutrition

and/or dietary restriction during the periconceptional period on the weight or

growth measures or on the circulating concentrations of glucose, NEFAs or

insulin in the offspring measured within 24h of birth. As discussed in Chapter 1,

however, a range of epidemiological, clinical and experimental studies have

shown a clear association between maternal obesity and the risk of obesity and

related metabolic disorders for their offspring in later life (Parsons, Power et al.

2001; Pietiläinen 2001; Yajnik 2002). Furthermore pregnancies complicated by

maternal diabetes, gestational diabetes and, or mild glucose intolerance during

pregnancy results in an increased risk for obesity and type 2 diabetes in the

offspring (Silverman, Rizzo et al. 1998).

Currently no study has investigated the long term consequences of

periconceptional overnutrition, experienced solely during the period before and in

the preimplantation period on later body composition in the offspring. We have

therefore investigated the impact of maternal overnutrition during the


after conception, on the growth and development of the postnatal lamb. As

79

described in Chapter 2, in order to determine the impact of the plane of nutrition

experienced during the periconceptional period, single embryos were transferred

from donor ewes in each of 4 nutritional treatment groups, to recipient ewes,

which were maintained on a control diet before and throughout pregnancy. We

have measured the effects of maternal PCON and/ or dietary restriction on lamb

growth rates, plasma glucose, NEFA and insulin concentrations and organ

weights at 4 months of age. We have also investigated the growth of the major

adipocyte depots i.e., subcutaneous, perirenal and omental depots at this

postnatal age.


Animals and nutritional feeding regime

As described in Chapter 2, donor ewes (n=23) were randomly assigned to one of

four treatment groups, either control-control (CC), control-restricted (CR), high-

high (HH) or high-restricted (HR) and fed according to the nutritional protocol for

at least 5 months prior to conception. The reproductive cycles of all experimental

ewes were synchronized and super ovulation was induced. Fresh semen was

collected from a ram of proven fertility and the donor ewes were inseminated by

laparoscopy. Following insemination embryos were collected and single embryos

were transferred to adult recipient ewes maintained on a control diet (100%

MER) at embryonic day 6-7 by laparoscopy.

80

These recipient ewes were maintained on a diet calculated to provide 100%

energy requirements for the maintenance of a pregnant ewe bearing a singleton

fetus, as specified by the U.K Ministry of Agriculture, Fisheries and Food

(Ministry of Agriculture 1984).

Pregnancy was confirmed by ultrasonography at day 49 of gestation. The

pregnant ewes were allowed to give birth naturally in a lambing pen (term=150 ±

3 days) (see Chapter 2). Lambs were weaned at 3 months of age and were

individually pen fed diet calculated to provide 100% energy requirements for a

growing lamb, as specified by U.K Ministry of Agriculture, Fisheried and Food


3.2.1 BLOOD SAMPLING REGIME

Blood samples (5ml) from the lambs were collected by jugular venepuncture at

birth, then daily for the first 5 days following birth, then twice a week for the first

month of life, then once a week until 3 months of age and then on the day of

post-mortem following an overnight fast into heparinised tubes containing serum

separating gel and kept on ice until centrifuged. Blood samples were centrifuged

at 1500g for 10min at 4oC and plasma was stored at -20oC for subsequent

determination of glucose and insulin and non-esterified free fatty acid

concentrations.

81


Lamb birth weight, crown-rump length, shoulder height, abdominal circumference

and temperature were taken within the first 12 h of birth. Growth measurements

were made every day for the first 5 days, then twice a week for the first month of

birth, and then every week until 3 months of age.

3.2.3 GLUCOSE TOLERANCE TEST

At 3 months of age, 29 lambs (CC n=7, CR n=7, HH n=8, HR n=7) underwent

glucose tolerance tests following a 12 hour overnight fast, (lambs were allowed

free access to water). Angiocath intravenous catheters (Becton Dickinson,

Sandy, UT, USA) were inserted into either the left or right jugular vein. The

catheterised vein was flushed with 5ml of heparinised saline (Baxter, Old

Toongabbie, NSW, Australia). The lamb was allowed at least 30 min to recover

from the catheterisation before the first blood sample was collected at 30 min

and 15 min before infusion of glucose. Baseline blood samples (5ml) were

collected into heparinised tubes containing serum separating gel and kept on ice

until centrifuged. A blood sample immediately before glucose infusion was taken

and the start of glucose infusion was designated as time 0. Glucose (50%;

0.5g/kg Baxter, Old Toongabbie, NSW, Australia) was infused at a rate of

30g/min. Blood samples were collected at 2, 5, 10, 15, 20, 30, 45, 60, 90, 120,

180 min (Sloboda, Moss et al. 2005). Blood samples were centrifuged at

4000rpm for 10min at 4oC and plasma was stored at -20oC for subsequent

determination of glucose and insulin concentrations.

82

3.2.4 PLASMA GLUCOSE, NEFA AND INSULIN CONCENTRATIONS

Plasma glucose, NEFA and insulin concentrations in the lamb plasma were

analysed as described in Chapter 2.

3.2.5 POST MORTEM AND TISSUE COLLECTION

At 4 months of age, lambs were killed with an overdose of sodium

pentobarbitone (Virbac Pty Ltd, Peakhurst, NSW, Australia). Samples of

omental, perirenal and subcutaneous adipose tissue were immediately collected,

weighed and snap frozen with liquid nitrogen and subsequently stored at -80oC.

All organs were dissected out and individually weighed, including the liver,

kidneys, pancreas, lungs, brain, heart and adrenals. All adipose tissue stores

from the omental, perirenal, subcutaneous, pericardial, epididymal/ parametrial

and axillary fat depots were carefully excised and weighed.

3.2.6 STATISTICAL ANALYSES

All data are presented as mean ± standard error of the mean (SEM). Data were

analysed using the Statistical Package for the Social Sciences version 17.0

(SPSS Inc., Illinois, USA) and STATA10: Data analysis and Statistical Software

for repeated measures (Stata Corp. LD., Texas, USA).

83

3.2.6.1 Plasma glucose, non-esterified fatty acid and insulin

concentrations

The effects of nutritional treatment on lamb plasma concentrations of glucose,

NEFA and insulin were compared using multifactorial ANOVA with repeated

measures. Specified factors for the ANOVA included group (CC, CR, HH or HR)

time and lamb gender.

3.2.6.2 Lamb growth parameters and organ and tissue weights

The effects of periconceptional nutrition on growth parameters measured from

birth to 4 months of age (weight, crown-rump length, abdominal circumference

and shoulder height) were compared using multifactorial ANOVA with repeated

measures. Organ and tissue weights, expressed in absolute terms and relative

to body weight, were compared using a two way ANOVA. Specified factors for

the ANOVA included treatment group (CC, CR, HH or HR) and gender.

When a significant interaction between major factors was identified, the data

were split on the basis of the interacting factors and reanalyzed. The Duncans

multiple range test or Bonferroni post hoc test was used to identify significant

differences between mean values and a probability level of 5% (P<0.05) was

taken as significant.

84

3.3 RESULTS


There was no effect of either periconceptional overnutrition or dietary restriction

on lamb growth (Figure 3.3.1), crown rump length, abdominal circumference or

shoulder height of lambs at 4 months of age (Table 3.3.1). Male lambs had a

significantly longer crown-rump length (106.89 ± 1.35cm vs. 101.39 ± 145cm, n=

22, P<0.05) and greater shoulder height than female lambs at 4 months of age

(68.52 ± 0.97cm vs. 63.79 ± 0.59cm, n=19, Table 3.3.1, P<0.05). Male lambs

were also significantly heavier at 4 months of age than females (Figure 3.3.2,

P<0.05).

3.3.2 LAMB PLASMA GLUCOSE, INSULIN AND FREE FATTY ACID CONCENTRATIONS AND

GLUCOSE TOLERANCE TEST


the plasma glucose (Figure 3.3.3), NEFA (Figure 3.3.4) and insulin

concentrations (Figure 3.3.5) between birth and 4 months of age. Plasma

glucose and insulin concentrations before and after the administration of a

0.5g/kg bolus of glucose were not different between groups. There was,

however, a significant effect of time on both plasma glucose and insulin

concentrations in all treatment groups (Figure 3.3.6). Areas under the glucose

and insulin response curves were not different between groups (Figure 3.3.7).

85

3.3.3 LIVER, KIDNEY, PANCREAS, LUNG AND BRAIN WEIGHTS

There was no significant effect of periconceptional overnutrition and/or dietary

restriction on the relative weights of the lamb liver, kidney, pancreas, lung or

brain at 4 months of age (Figure 3.3.8). There was an interaction between the

effects of periconceptional nutrition and gender on the absolute brain weights of

lambs at 4 months of age (Figure 3.3.9a). Periconceptional dietary restriction

(CR) resulted in an increase in absolute brain weight in the female lambs

compared to all other treatment groups at 4 months of age (Figure 3.3.9a).

Female lambs also had larger brains relative to body weight compared to male

lambs at 4 months (Figure 3.3.9b).

86

Figure 3.3.1 The growth rate of lambs from the 4 nutritional treatment

groups between 1 and 16 weeks of age

* denotes a significant difference of lamb weight at weeks 2 to 16 compared to

week 1 of postnatal life, P<0.0001.

* *

* *

* *

* *

* *

*

Weeks After Birth1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Wei

ght (

kg)

0

5

10

15

20

25

30

35

40

CC CR HH HR

* *

* *

* *

* *

* * *

*

87

Table 3.3.1 Effect of periconceptional overnutrition and/or dietary restriction on growth measures at 4 months of age

* denotes a significant difference between the growth measures of the male and female lambs at 4 months of age, P<0.05.

Lamb growth measures at 4 months of age

CC CR HH HR

Male Female Male Female Male Female Male Female

Weight (kg)

34.6±2.2* 29.2±0.9 32.9±1.2* 29.7±2.2 35.1±1.6* 30.8±1.0 30.92±1.3* 31.1±0.9

Crown rump length (cm)

110.0±4.0* 102.0±3.6 106.3±2.6* 101.5±4.3 109.6±2.2* 96.60±2.5 103.4±2.5* 104.8±1.0

Abdominal circumference

(cm) 99.5±3.5 93.8±1.3 97.6±1.7 93.3±5.2 97.4±1.9 98.2±3.3 95.7±1.8 97.0±1.8

Shoulder height (cm)

70.2±0.7* 64.9±1.0 69.3±1.4* 63.0±1.7 70.6±1.5* 61.8±1.0 64.7±2.0* 64.9±0.9

88


restriction on the weight of male and female lambs at 4 months of age

Periconceptional overnutrition and/or dietary restriction did not affect lamb weight

at 4 months of age. Male lambs (closed bars) were significantly heavier than

female lambs (striped bars) in all treatment groups at birth.

* denotes a significant difference between birth weights in male and female

lambs, P<0.05.

Treatment group

CC CR HH HR

Wei

ght (

kg)

0

10

20

30

40

* * * *

89

Age (days)

0 20 30 40 50 60 70 80 90 100 110 120

Plas

ma

gluc

ose

conc

entr

atio

ns (m

ol/l)

0

2

4

6

8 CC CR HH HR


restriction on plasma glucose concentration between birth and 4 months of

age in lambs

There was no effect of periconceptional nutrition on plasma glucose

concentrations from birth (day 0) to 4 months of age. * denotes a significant

difference in plasma glucose concentration when compared to 1-42d of age

across all treatment groups, P<0.05.

5 4 3 2 1

*

* *

90

Age (days)

0 20 30 40 50 60 70 80 90 100 110 120

Plas

ma

NEF

A c

once

ntra

tions

(mEq

/l)

0.0

0.2

0.4

0.6

0.8

1.0

1.2 CC CR HH HR


restriction on the plasma non-esterified free fatty acid concentration from

birth to 4 months of age in lambs

There was no effect of periconceptional nutrition on the plasma non-esterified

free fatty acid (NEFA) concentration from birth (day 0) to 4 months of age.

*denotes a significant difference in plasma NEFA concentration when compared

to 1-2d and 112d of age across all treatment groups, P<0.05.

*

*

*

*

5 4 3 2 1

* * *

91

Age (days)

0 20 30 40 50 60 70 80 90 100 110 120

Plas

ma

insu

lin c

once

ntra

tions

(ng/

ml)

0

2

4

6

8

10

CC CR HH HR


restriction on the plasma insulin concentration from birth to 4 months of

age in lambs

There was no effect of periconceptional nutrition on the plasma insulin

concentration from birth (day 0) to 4 months of age. * denotes a significant

difference in plasma insulin concentrations when compared to 1-3d of age

across all treatment groups, P<0.05.

*

5 4 3 2 1

* *

92

Figure 3.3.6 Plasma glucose and insulin concentrations responses to

intravenous glucose challenge at 3 months of age in the lambs

Different superscripts (e.g. a,b) denotes a significant effect of time across all

treatment groups, P<0.05.

Time (minutes)

-15 0 15 30 45 60 75 90 105 120 135 150 165 180

Gluc

ose

conc

entra

tion

(mm

ol/L

)

0

2

4

6

8

10

12

14

16

18

20CC CRHHHR

Time (minutes)

-15 0 15 30 45 60 75 90 105 120 135 150 165 180

Insu

lin c

once

ntra

tion

(ng/

ml)

0

1

2

3

4

5

a a a

a

b b

bc

c

c c

c

a

a a a

a a

b

bc

bc bc

bc

c c

c

a

a

93

Area under the glucose response curve

Area under the insulin response curve

Figure 3.3.7 Area under the glucose and insulin response curves after the

intravenous glucose challenge

Treatment group

CC CR HH HR

mm

ol m

in L

-1

0

200

400

600

800

1000

1200

1400

CCCRHHHR

Treatment group

CC CR HH HR

ng m

in m

l -1

0

100

200

300

400

94

3.3.4 LIVER, KIDNEY, PANCREAS AND LUNG WEIGHT


the absolute or relative liver, kidney, pancreas and lung weight at 4 months of

age.

3.3.5 HEART WEIGHT

There was an interaction between the effects of periconceptional nutrition and

gender on the relative heart weight at 4 months of age (Fig 3.3.10 a,b, P<0.05).

There was no effect of periconceptional nutrition and/or dietary restriction on the

relative heart weight in male lambs but in female lambs, heart weight was

relatively higher in the CR group when compared to all other treatment groups

and was not different from the CC group.

3.3.6 ADRENAL WEIGHTS

Periconceptional dietary restriction in the CR and HR groups resulted in a higher

absolute and relative adrenal weights at 4 months of age when compared with

the CC (Figure 3.3.11a) and CC and HH (Figure 3.3.11b) respectively.

95

Treatment group

CC CR HH HR

Abs

olut

e ki

dney

wei

ght (

g)

0

20

40

60

80

100

120

140

Treatment group

CC CR HH HR

Rel

ativ

e to

tal k

idn

ey w

eig

ht

(g/k

g)

0

1

2

3

4

Treatment group

CC CR HH HR

Abs

olut

e liv

er w

eigh

t (g)

0

100

200

300

400

500

600

Treatment group

CC CR HH HR

Rel

ativ

e liv

er w

eig

ht

(g/k

g)

0

2

4

6

8

10

12

14

16

18

20

96

Treatment group

CC CR HH HR

Ab

solu

te lu

ng

wei

gh

t (g

)

0

100

200

300

400

500

600

Treatment group

CC CR HH HR

Ab

solu

te p

ancr

eas

wei

gh

t (g

)

0

2

4

6

8

10

12

14

Figure 3.3.8 Effect of periconceptional nutrition on absolute organ weights

and relative to body weight of 4 month old lambs

There was no significant difference in the absolute or relative weights of the liver,

kidney, pancreas or the lung between the treatment groups at 4 months of age.

Treatment group

CC CR HH HR

Rel

ativ

e p

ancr

eas

wei

gh

t (g

/kg

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Treatment group

CC CR HH HR

Rel

ativ

e lu

ng

wei

gh

t (g

/kg

)

0

2

4

6

8

10

12

14

16

18

20

97

3.3.7 TOTAL ADIPOSE TISSUE WEIGHT

When total adipose tissue mass was analysed there was a significant interaction

between treatment group and gender (P=0.056) and therefore the data were split

according to gender. Periconceptional overnutrition alone (HH group) resulted in

a higher total fat mass in female lambs compared to lambs in the CC and CR

groups (Figure 3.3.12). In the HR group, total fat mass in female lambs was not

different from either the HH group, or the CC and CR groups. There was no

significant effect of periconceptional overnutrition and/or dietary restriction,

however, on the total fat mass in male lambs at 4 months of age. There was a

significant relationship between the weight of the donor ewe at conception and

the total fat mass of female, but not male lambs at 4 months of age (Figure

3.3.13).

3.3.8 PROPORTIONS OF FAT MASS IN THE ADIPOSE TISSUE DEPOTS


the proportion of fat mass in the different adipose tissue depots (Figures 3.3.17

and 3.3.18). Female lambs however, had a significantly larger proportion of

omental and visceral (i.e. the sum of omental and perirenal fat) fat mass than

the male lambs (Table 3.3.2).

98

Treatment group

CC CR HH HR

Abs

olut

e br

ain

wei

ghts

(g)

0

20

40

60

80

100

120

Treatment group

CC CR HH HR

Abs

olut

e br

ain

wei

ght (

g)

0

20

40

60

80

100

120

Figure 3.3.9a Effect of periconceptional overnutrition and/or dietary restriction on the absolute brain weight in female

and male lambs at 4 months of age

The effect of periconceptional overnutrition and dietary restriction on the absolute brain weight in male (blue striped bars) and

female (red striped bars) lambs at 4 months of age. There was an interaction between the effects of sex and treatment on the

absolute brain weight of the lambs (P=0.07). Dietary restriction in the control ewes (CR) resulted in higher absolute brain

weights in the female offspring (red striped bars) when compared to CC, HH and HR groups. Different superscripts (e.g. a,b)

denotes significant differences between mean values.

a b

a a

99

Figure 3.3.9b Effect of periconceptional overnutrition and/or dietary restriction on the brain weight relative to body

weight in female and male lambs at 4 months of age

The effect of periconceptional overnutrition and dietary restriction on the brain weight relative to body weight in male (blue

striped bars) and females (red striped bars) lambs at 4 months of age * denotes a significant difference overall between male

and female relative brain weights (P<0.05).

*

Treatment group

CC CR HH HR

Rel

ativ

e br

ain

wei

ght (

g/kg

)

0

1

2

3

4

Treatment group

CC CR HH HR

Rel

ativ

e br

ain

wei

ght (

g/kg

)

0

1

2

3

4

*

100

Treatment group

CC CR HH HR

Abs

olut

e he

art w

eigh

t (g)

0

50

100

150

200

250

Treatment group

CC CR HH HR

Abs

olut

e he

art w

eigh

t (g)

0

50

100

150

200

250

Figure 3.3.10a Effect of periconceptional overnutrition and/or dietary restriction on absolute heart of 4 month old male

and female lambs

There was no effect of periconceptional overnutrition and/or dietary restriction on absolute heart weight at 4 months of age

when males and females were combined or in males (blue striped bars). There was a sex and treatment interaction effect of

the absolute heart weight of the lambs (P=0.02). Dietary restriction in the control ewes (CR) resulted in higher absolute heart

weights in the female offspring (red striped bars) when compared to HH and HR groups. Different superscripts (e.g. a,b)

denotes significant differences between mean values.

ab

b

a a

101

Treatment group

CC CR HH HR

Rel

ativ

e to

tal h

eart

wei

ght (

g/kg

)

0

2

4

6

8

Figure 3.3.10b Effect of periconceptional overnutrition and/or dietary restriction on heart weight relative to body

weight of 4 month old male and female lambs

There was no effect of periconceptional overnutrition and/or dietary restriction on the heart weight relative to body weight at 4

months of age when males and females were combined. There was a sex and treatment interaction effect of the relative heart

weight of the lambs (P=0.002). Dietary restriction in the control ewes (CR) resulted in higher relative heart weights in the female

offspring (red striped bars) when compared to HH and HR groups. Different superscripts (e.g. a,b) denotes significant

differences between mean values (P<0.05).

Treatment group

CC CR HH HR

Rel

ativ

e to

tal h

eart

wei

ght (

g/kg

)

0

2

4

6

8

ab

b

a a

102

Treatment group

CC CR HH HR

Rela

tive

tota

l adr

enal

wei

ght (

g/kg

)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

Treatment group

CC CR HH HR

Abs

olut

e ad

rena

l wei

ght (

g)

0.0

0.5

1.0

1.5

2.0

2.5


restriction on absolute adrenal weight and relative to body weight of 4

month old lambs

Periconceptional dietary restriction (CR and HR) resulted in higher adrenal and

relative adrenal weights at 4 months of age when compared with the CC and CC

and HH respectively. Different superscripts (e.g. a, b) denotes significant

differences between mean values, P<0.05

a ab

b b

a a

b b

103

Treatment group

CC CR HH HR

Tota

l fat

mas

s (g

)

0

1000

2000

3000

4000

.

Figure 3.3.12 Effect of periconceptional overnutrition and/or dietary restriction on total fat mass of male and female

lambs at 4 months of age

In female lambs (red striped bars) total fat mass was significantly higher in the HH group (n=5) than in the CC and CR groups.

Dietary restriction in the PCON ewes (HR, n=7) ablated the effect of PCON on total fat mass. Different superscripts (e.g. a,b)

denotes a significant difference between absolute total fat mass in female lambs at 4 months of age.

Treatment group

CC CR HH HR

Tota

l fat

mas

s (g

)

0

1000

2000

3000

4000

a a

b

ab

104

Donor weight at conception (kg)50 55 60 65 70

Tota

l fat

mas

s (g

)

1000

1500

2000

2500

3000

3500

4000

4500

Figure 3.3.13 Relationship between total fat mass in 4 month old female

offspring and the weight of the donor ewe at conception

There was a significant positive relationship between the total fat mass in female

offspring at 4 months of age and the weight of the donor ewe at conception

(Total fat mass= 63 (donor ewe weight ) – 1466.7, r2=0.31, P<0.05)

105

Treatment group

CC CR HH HR

Om

enta

l fat

mas

s (g

)

0

200

400

600

800

1000

Figure 3.3.14 Effect of periconceptional overnutrition and/or dietary restriction on omental adipose tissue depot weight

of 4 months old male and female lambs

There was no effect of periconceptional overnutrition or dietary restriction on the absolute omental fat mass. Female lambs

(red striped bars) had significantly more absolute omental fat mass than male lambs (blue striped bars) at 4 months of age.

* denotes a significant difference of fat mass between males and females, P<0.05.

Treatment group

CC CR HH HR

Om

enta

l fat

mas

s (g

)

0

200

400

600

800

1000

*

106

Treatment group

CC CR HH HR

Perir

enal

fat m

ass

(g)

0

200

400

600

800

1000

Figure 3.3.15 Effect of periconceptional overnutrition and/or dietary restriction on perirenal adipose tissue depot

weight of 4 months old male and female lambs

There was no effect of periconceptional overnutrition or dietary restriction on the absolute perirenal fat mass. However females

(red striped bars) had significantly more absolute perirenal fat mass than males (blue striped bars) at 4 months of age. *

denotes a significant difference of absolute fat mass between males and females, P<0.05.

Treatment group

CC CR HH HR

Per

iren

al fa

t mas

s (g

)

0

200

400

600

800

1000

*

107

Figure 3.3.16 Effect of periconceptional overnutrition and/or dietary restriction on subcutaneous adipose tissue depot

weight of 4 months old male and female lambs

There was no effect of periconceptional overnutrition or dietary restriction or gender on the absolute subcutaneous fat mass.

Male lambs are represented by the blue striped bars, female lambs by the red striped bars.

Treatment group

CC CR HH HR

Subc

utan

eous

fat m

ass

(g)

0

200

400

600

800

1000

1200

1400

Treatment group

CC CR HH HR

Subc

utan

eous

fat m

ass

(g)

0

200

400

600

800

1000

1200

1400

108

CC Female

Figure 3.3.17 The proportion of the total fat mass in the different adipose

depots in female lambs at 4 months of age

a a

b

a,b

2.4±0.2%

CR Female

38.0 ± 4.4%

6.5 ± 0.8%

3.1 ± 0.4% 5.8 ± 0.3%

26.3 ± 4.7%

20.1 ± 0.6% HH Female

34.5 ± 6.0%

6.7 ± 1.2%

2.3 ± 0.2% 4.4 ± 0.6%

25.0± 4.1%

27.0± 3.6%

HR Female

41.2 ± 3.1%

6.5 ± 0.6%

2.4 ± 0.2%

5.0 ± 0.4%

19.6 ± 1.1%

25.2 ± 4.1%

Subcutaneous fat Omental fat

Perirenal fat Axillary fat Pericardial fat Epididymal/Parametrial

37.6 ± 2.8%

9.1 ± 0.8%

3.0 ± 0.4%

6.6 ± 0.3% 21.7 ± 1.9%

21.9 ± 1.8%

109

Figure 3.3.18 The proportion of the total fat mass in the different adipose

depots in male lambs at 4 months of age

CC Male

46.5 ± 6.1%

10.2 ± 1.2%

4.9 ± 0.9% 6.5 ± 1.1%

15 ± 8.5%

16.6 ± 2.6%

Subcutaneous fat Omental fat

Perirenal fat Axillary fat Pericardial fat Epididymal/Parametrial

CR Male

39.0 ± 3.0%

9.3 ± 0.3%

3.6 ± 0.6% 6.8 ± 1.1%

17.4 ± 0.8%

23.8 ± 5.1%

HH Male

43.6 ± 2.0%

7.8 ± 1.8%

3.5 ± 0.3%

7.4 ± 0.8% 16.5 ± 0.6%

21.2 ± 1.7%

HR Male

41.6 ± 0.9%

10.0 ± 0.9%

4.2 ± 0.4%

7.0 ± 0.7%

17.6 ± 1.4% 1.4%

19.4 ± 0.5%

110

Table 3.3.2 Differences between the proportion of the total adipose tissue

in each fat depot between male and female lambs at 4 months of age

* Denotes a significant difference in the proportion of fat stored in the different

adipose tissue depots between male and female lambs

Adipose tissue depot

Proportion of total adipose tissue (%)

Male lambs Female lambs

Subcutaneous 41.81 ± 1.39 38.19 ± 1.89

Omental 18.38 ± 0.99 22.59 ± 1.35*

Perirenal 19.82 ± 1.88 23.80 ± 1.66

Axillary 7.02 ± 0.47* 5.45 ± 0.29

Pericardial 3.84 ± 0.26* 2.68 ± 0.14

Epididymal/ Parametrial 9.13 ± 0.64* 7.29 ± 0.48

Visceral (omental + perirenal)

38.20 ±1.74 46.39 ± 2.26*

111

3.4 DISCUSSION

The objective of this study was to investigate whether the plane of nutrition of the

ewe before conception and during early embryo development was an

independent determinant of the growth trajectory of the lamb at birth through to

the first 4 months of life. In particular, we wished to determine the impact of

periconceptional nutrition on the development of fat mass in the offspring.

We have demonstrated for the first time that there are important relationships

between the weight of the non-pregnant donor ewe during the periconceptional

period and the development of total fat mass in the female offspring at 4 months

of age, such that the ewe weight at conception is a predictor of adiposity in the

female offspring in later life. Furthermore it was observed that a short period of

moderate periconceptional nutrient restriction ameliorated the impact of

periconceptional overnutrition on the development of increased adiposity in

female lambs at 4 months of age.

3.4.1 LAMB GROWTH MEASUREMENTS

We previously showed in Chapter 2 that there was no effect of periconceptional

overnutrition and/or restriction on the weight, crown rump length, abdominal

circumference or shoulder height of lambs at birth. In this chapter we have found

that there was also no effect of periconceptional overnutrition and/or restriction

on the weights of the lambs at 4 months of age. Interestingly, male lambs were

born heavier and remained heavier than female lambs at 4 months of age.

112

3.4.2 LAMB PLASMA GLUCOSE, NON-ESTERIFIED FATTY ACID AND INSULIN

CONCENTRATIONS


the plasma glucose, NEFA and insulin concentrations between birth and 4

months of age. Periconceptional overnutrition and/or dietary restriction did not

affect the plasma glucose and insulin concentrations in response to a glucose

challenge at 3 months of age.

3.4.3 LAMB WEIGHTS

We demonstrated that periconceptional overnutrition decreased maternal insulin

sensitivity in early pregnancy in Chapter 2. Interestingly it has been shown that

reduced pre-gravid insulin sensitivity in women has the strongest correlation with

fat mass of the offspring at birth. Reduced insulin sensitivity in late gestation,

however, was strongly correlated with both birth weight and fat-free mass (lean

body mass) in the offspring (Catalano and Ehrenberg 2006). It has been

suggested that pre-gravid obesity, characterised by a decrease in maternal

insulin sensitivity and an increase in her pancreatic beta cell response, may alter

early placental functional development, resulting in an increased placental mass.

Therefore placental transport of nutrients in later pregnancy may be affected by

changes to placental development. In the current study, however, we found no

impact of periconceptional overnutrition on offspring birth weight (Chapter 2),

which suggests that in this study placental function was not enhanced by the

early exposure of the embryo to a high plane of nutrition during early

development.

113

3.4.4 LAMB ORGAN WEIGHTS

3.4.4.1 Lamb heart weights

It has been shown in experimental models of fetal nutrient restriction that critical

organs such as the heart, brain and adrenals may be “spared” from the effects of

nutrient restriction such that there is a redistribution of cardiac output to maintain

the appropriate growth of these essential organs at the expense of other organ

systems including the gut and liver (Haugen, Hanson et al. 2005).

A period of periconceptional nutrient restriction in the Control fed ewes resulted

in larger hearts relative to body weight in female lambs at 4 months of age. This

suggests that that heart growth in female lambs is particularly vulnerable to

periconceptional nutrient restriction. Nutrient restriction in the periconceptionally

overnourished ewes (HR) did not however, result in the development of

proportionately larger hearts in the female lambs. This suggests that heart

growth is more vulnerable when nutrition is restricted from a control plane of

nutrition compared to a high plane of nutrition in the periconceptional period.

Previous studies of the effects of poor maternal nutrition have shown that there

are long term consequences for the development and function of the

cardiovascular system in the offspring. The Dutch Hunger Winter Famine study

provided evidence that the timing of nutrient deprivation during pregnancy is

important for determining specific pathophysiological outcomes in the offspring

(Ravelli, van der Meulen et al. 1999; Roseboom, van der Meulen et al. 2000).

Exposure to the famine in the first trimester led to an increased risk of developing

114

coronary heart disease in the offspring. Furthermore in these studies,

hypertension in the offspring in later life was associated with any level of

maternal undernutrition which led to reduced fetal growth (Ravelli, van der

Meulen et al. 1999; Roseboom, van der Meulen et al. 2000). Similarly in an

experimental model of periconceptional undernutrition, in which the ewe was fed

a diet to provide 70% of metabolisable energy requirements for 60 days prior to

conception to 7 days post conception, it was found that there was an increase in

fetal arterial blood pressure, which was positively related to a rise in fetal ACTH

in twin fetuses (Edwards, Bryce et al. 2002; Edwards and McMillen 2002).

Periconceptional nutrient restriction did not alter the blood pressure response to

captopril, an angiotensin-converting enzyme inhibitor, suggesting that the

increase in blood pressure was not dependent on the activation of the renin-

angiotensin system (RAS) (Edwards and McMillen 2002). Gardner and

colleagues investigated the postnatal outcomes associated with peri-implantation

undernutrition in a cohort of lambs. It was shown that early nutrient restriction

increased pulse pressure and reduced rate pressure product in these offspring at

1 year of age which may contribute to long term cardiovascular dysfunction

(Gardner, Pearce et al. 2004). Maternal low protein diet imposed during only the

preimplantation period in the rat increased systolic blood pressure in male rats at

12 weeks of age however, there was no change in postnatal heart growth

(Kwong, Wild et al. 2000). In the present study blood pressure was not

measured.

Preimplantation maternal nutrient restriction reduced cell numbers in the ICM in

the early blastocyst and later in development reduced the cell numbers between

115

both the ICM and trophectoderm (Kwong, Wild et al. 2000). This restriction in

embryonic cellular proliferation may alter the potential to develop appropriately

sized stem-cell lineages which may impair the growth trajectory of the fetus

(Kwong, Wild et al. 2000). It has also been shown that angiotensin-converting

enzyme (ACE) activity is enhanced in mice in response to in vitro culture and

embryonic transfer procedures (Watkins, Platt et al. 2007). This suggests that

the developing embryo is sensitive to environmental factors either in vivo

(maternal diet) or in vitro (embryo culture) which may contribute to the onset of

cardiovascular dysfunction in later life (Watkins, Platt et al. 2007).

In the current study, an increase in heart size in the periconceptionally

undernourished lambs may potentially be attributed to a change in IGF

expression which is important for heart growth. It is well known that cardiac

development occurs by hyperplasia and hypertrophy of cardiac myocytes in fetal

life (Burrell, Boyn et al. 2003). It has also been documented that IGF-1 and 2

play a major role in the regulation of fetal organ system development throughout

gestation, exerting metabolic and mitogenic effects on fetal tissues including the

heart (Dong, Ford et al. 2005; Lumbers, Kim et al. 2009). Reiss and colleagues

(Reiss, Cheng et al. 1996) showed that an over-expression of IGF-1 in sheep

and mice leads to an increase in heart weight and number of cardiomyocytes.

IGF receptors are present in fetal and adult human cardiac myocytes. Their

action is related to cardiac growth and development of myocyte hypertrophy in

hypertrophic cardiomyopathy (Toyozaki, Hiroe et al. 1993). Early to mid

gestational nutrient restriction in the sheep leads to an enhanced expression of

116

IGF receptor and is associated with an increase in ventricular size in fetal sheep

(Dong, Ford et al. 2005). Furthermore Han and colleagues (Han, Austin et al.

2004) reported increased left ventricular hypertrophy in fetuses exposed to

nutrient deprivation. In the present study periconceptional nutrient restriction

altered the growth in the hearts of female lambs at 4 months of age. We suggest

that periconceptional nutrient restriction may lead to the asymmetric growth in

the hearts of female lambs at 4 months of age. Cardiomyocyte size and number

however, were not assessed in the current study. In contrast to the effect of

dietary restriction in the periconceptional period on heart growth in the female

lamb, heart growth in the male offspring was not altered in response to

periconceptional restriction at 4 months of age. In the current study blood

pressure was not measured, therefore we are unable to determine the impact of

blood pressure on heart weight.

The difference in response of the genders to periconceptional nutrient restriction

may be explained by the sexual dimorphism of plasma IGF-1 in lamb and adult

sheep. Plasma IGF-1 concentrations are higher in rams than non-pregnant ewes

(Van Vliet, Styne et al. 1983; Gatford, Fletcher et al. 1996). It may be possible

that IGF-1 may be up-regulated in female offspring in response to nutrient

restriction and/ or an increase in the abundance of IGF receptors in the cardiac

myocytes. Sexual dimorphism both in the size and nucleation of cardiac

myocytes and cardiomyocyte responses of the fetuses to IGF-1 were observed in

the study by Lumbers and colleagues (Lumbers, Kim et al. 2009). In this study

intra-fetal IGF-1 infusion at 121-125d gestation was associated with increased

myocyte volumes in male fetuses only, suggesting a promotion of cardiac growth

117

by hypertrophy. Female fetal sheep however, had larger myocytes and a greater

proportion of binucleated cardiac myocytes than males. This may suggest that

myocyte maturation may occur earlier in females than males. If programming of

cardiovascular dysfunction occurs during the periconceptional period in the

developing embryo then gender specific differences at this stage of development

may contribute to the pathogenesis of cardiovascular disease in later life.

3.4.4.2 Lamb brain weights

Previous studies have shown that chronic maternal feed restriction from 4 weeks

prior to conception to mid-gestation is associated with an increase in brain weight

relative to body weight at 60d gestation in fetal guinea pigs (term~69d)

accompanied by a decrease in body growth (Kind, Roberts et al. 2005). In the

current study, periconceptional nutrient restriction resulted in an increase in brain

weight in the CR group when compared to all other treatment groups in female

offspring at 4 months of age, with no change to body growth in these lambs. This

suggests that the effect of periconceptional dietary restriction in the CR lambs on

increasing brain growth is a specific effect on the brain and not whole body

growth and is indicative of brain sparring in these periconceptionally

undernourished lambs.

Female lambs at 4 months of age also had proportionately larger brains relative

to body weight. One possible factor which may account for the sexual

dimorphism of relative brain weight in the lambs may be the differential levels of

circulating estrogens. Previous studies have established a neuroprotective

function of natural and synthetic estrogens in the neurons of mammalian brains

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in vivo and in vitro (Beyer 1999). Estrogens are also involved in a wide array of

other functions including the regulation of growth factor supply (Cardona-Gómez,

Mendez et al. 2001), promotion of anti-apoptotic signalling, facilitation of

neuronal activity and synaptic performance and as a free radical scavenger in

the central nervous system, thereby reducing oxidative injury in females (Beyer

1999). Therefore the increased presence of natural estrogens in the female

compared with the male lambs may contribute to the increase in relative brain

weight in the female lambs at 4 months of age. In primates, however, it has been

shown that fetal and infant head circumference is larger in males with an

increase in brain weight, relative to females. (Beyer 1999). Therefore there may

be species differences in any interaction between the effects of periconceptional

nutrient restriction during critical stages of embryo development and offspring

gender on the development and growth trajectory of the brain in postnatal life.

3.4.4.3 Lamb adrenal weights

Lambs of ewes on a restricted diet during the periconceptional period

irrespective of the previous plane of nutrition (either Control or High) had

proportionately larger adrenals compared to lambs of ewes on a Control or High

diet during the periconceptional period, suggesting that nutritional changes

during the critical window of the periconceptional period may promote adrenal

growth. Interestingly it has been previously reported that PCUN from at least 60

days prior to conception until 7 days post conception had no effect on relative

adrenal weight at 140-147d gestation in single and twin fetal sheep (Edwards

and McMillen 2002).

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In the sheep model it has been well established that the prepartum activation of

the hypothalamic-pituitary-adrenal axis (HPA) axis is essential for the timing of

parturition and for the preparation for the transition to the extra uterine

environment (Whittle, Holloway et al. 2000; Whittle, Patel et al. 2001;

Kumarasamy, Mitchell et al. 2005; MacLaughlin, Walker et al. 2007). It has been

previously shown in sheep that a relatively severe undernutrition imposed in

ewes from before conception until 30 days after conception results in preterm

delivery in a number of ewes which is preceded by a prepartum fetal cortisol

surge. The rise in fetal cortisol concentration precedes the rise in fetal and

maternal prostaglandin concentrations, suggesting that accelerated maturation of

the fetal HPA axis results in a premature initiation of parturition (Bloomfield,

Oliver et al. 2003; Bloomfield, Oliver et al. 2004; Kumarasamy, Mitchell et al.

2005). Studies using a model of periconceptional undernutrition similar to the

present study (i.e. a more moderate nutrient restriction imposed before and for

one week after conception) have found that there was evidence for an enhanced

HPA activity in twin, but not singleton fetal sheep on gestation length (Edwards

and McMillen 2002). These studies highlight the possibility that nutrient

restriction in the periconceptional period may enhance the long term capacity of

the offspring to respond to stress. The key finding in the present study is that

periconceptional nutrient restriction resulted in an increased relative adrenal

weight in the postnatal lamb and that this occurred independently of whether the

periconceptional nutrient restriction was imposed in ewes on a Control or High

plane of nutrition. This suggests that the impact of periconceptional nutrient

restriction on the subsequent development of the HPA axis is independent of the

body weight and condition of the ewe at the time of conception.

120

3.4.5 ADIPOSE TISSUE WEIGHT

It has previously been reported that maternal overnutrition in late gestation

resulted in an increase in the size of the subcutaneous adipocytes and the

relative mass of the subcutaneous fat in lambs of well fed mothers (Muhlhausler,

Duffield et al. 2007). In the present study we have found that periconceptional

overnutrition resulted in a significant increase in total fat mass in female lambs at

4 months of age and that a short period of moderate dietary restriction in

previously overnourished ewes ablated this effect. This is the first study which

has demonstrated that a positive relationship exists between the donor ewe

weight at conception and the total fat mass in female offspring at 4 months of

age.

There is approximately a 900g increase in the total fat mass of female lambs

from the periconceptional overnourished group at 4 months of age compared to

the other treatment groups. This increased total mass is comprised

approximately of 27% perirenal fat, 35% subcutaneous fat and 25% of omental

fat. It has been suggested that altered maternal nutrition during early pregnancy

may program the susceptibility of the offspring to develop obesity by altering the

endocrine mechanisms that regulate body fat accumulation postnatally (Ravelli,

Stein et al. 1976). Interestingly, the perirenal adipose tissue depot is the first

depot to develop and is the major adipose tissue depot in fetal sheep

(Muhlhausler, Roberts et al. 2003; Symonds, Mostyn et al. 2003; Budge,

Edwards et al. 2004). As discussed in Chapter 4, we propose that

periconceptional overnutrition in the female lambs may alter the expression of

121

genes regulating adipogenesis and lipogenesis, altering the secretion of key

adipokines from adipose tissue and therefore, altering the endocrine

mechanisms that regulate body fat accumulation postnatally.

It may be possible that autocrine factors, paracrine factors, transcription factors

and key regulatory genes involved in the critical commitment of the

mesenchymal stem cells to favour the differentiation into the adipocyte cell

lineage, may be up-regulated by the exposure of the embryo to a high nutrient

environment during the periconceptional period. The high nutrient environment

may result in the induction of a response in which there is a favourable

differentiation of adipocytes derived from mesenchymal stem cells, in order to

prepare for a „predicted‟ nutrient rich environment in later life. The predictive

adaptive response may alter autocrine factors, paracrine factors, transcription

factors and key regulatory genes involved in adipogenesis and lipogenesis.

Our results showing that periconceptional overnutrition increases absolute total

fat mass in female offspring may suggest an altered embryonic environment

which favours the commitment of embryonic stem cells into the adipocyte

lineage, to deal with excess storage of nutrients in an energy rich postnatal life.

Interestingly our results which show that periconceptional nutrient restriction in

previously overnourished ewes ablates the increase in total fat mass in female

lambs at 4 months of age, suggests that the developing embryo is particularly

sensitive to an altered nutrient environment in early stages of pregnancy or it

may be able to increase adipogenic and lipogenic gene expression.

122

3.5 SUMMARY

The present study demonstrates that periconceptional overnutrition and

restriction alters the development of adiposity at 4 months of age. There was an

increase in absolute total fat mass in the female lambs at 4 months of age when

exposed to periconceptional overnutrition from at least 5 months prior to

conception until 7 days after conception. Furthermore this increase in total fat

mass in the female offspring was attributed to an increase in donor ewe weight

before conception. There was no effect of periconceptional nutrition on the

development of adiposity in the male offspring at 4 months of age. This study

may suggest that female offspring of periconceptionally overnourished ewes may

be more susceptible to the development of obesity in later life. Interestingly a

restriction of nutrient intake to 70% metabolisable energy requirements in

previously overnourished ewes resulted in a decrease in total fat mass to levels

which were similar to those in control lambs.

There was a greater effect of restricted periconceptional nutrition on the growth

of the heart in female offspring only and the adrenals in 4 month old lambs. This

suggests that the growth and development of the heart and adrenals may be

more vulnerable to the effects of periconceptional nutrient restriction. This has

implications for the development of any dietary intervention strategies in obese

women in the period immediately before conception.

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CHAPTER 4

124

CHAPTER 4: IMPACT OF PERICONCEPTIONAL

OVERNUTRITION AND/OR DIETARY RESTRICTION ON THE

EXPRESSION OF ADIPOGENIC AND LIPOGENIC GENES IN THE

POSTNATAL LAMB

4.1 INTRODUCTION

It has been proposed that there are critical windows of development in which

exposure to a high nutrient environment may result in changes in the biology of

the adipocyte which results in increased adiposity in later life (Plagemann,

Harder et al. 1997). Current studies however, have focused on determining

mechanisms by which a high nutrient environment during critical developmental

windows during gestation and postnatal life impact on the development of later

adiposity, and not on the impact of a high nutrient environment on the

developing embryo and how this may affect the programming of fat mass in the

offspring.

It has previously been shown that pregnancies complicated by diabetes leads

to the development of fetal hyperglycaemia and hyperinsulinemia with

corresponding increases in both cord blood leptin and adiposity in infancy

(Cetin, Morpurgo et al. 2000; Tapanainen, Leinonen et al. 2001). Furthermore

maternal diabetes before and during pregnancy as well as mild glucose

intolerance during pregnancy are all risk factors for the development of obesity

and type 2 diabetes in the offspring with an increase in fat mass in early life

(Plagemann, Harder et al. 1997; Silverman, Rizzo et al. 1998; Catalano,

Thomas et al. 2007). Guo and Jen (Guo and Jen 1995) report increases in

125

body weight and fat composition in adult offspring of dams fed 40% fat during

pregnancy. Consistent with these findings, Khan and colleagues (Khan, Dekou

et al. 2004; Khan, Dekou et al. 2005) showed that offspring of dams fed a 24%

fat diet during pregnancy and suckling and reared on a normal diet were

significantly heavier and had more visceral fat deposits than offspring of control

fed dams. The mechanisms by which maternal overnutrition results in increased

fat mass in postnatal life in the offspring has not been clearly elucidated.

PPARγ is a pleiotropic transcriptional activator which co-ordinately induces the

expression of a suite of adipocyte specific genes resulting in growth arrest of

the preadipocyte and the terminal differentiation of adipocyte cells (Butterwith

1994). Furthermore PPARγ is also required for adipogenesis and insulin

sensitivity, regulating adipose tissue mass (Medina-Gomez, Gray et al. 2007).

Activation of PPARγ stimulates storage of fatty acids in mature adipocytes

through the activation of LPL and G3PDH. White adipose tissue also functions

as an endocrine tissue and secretes adipokines, leptin and the insulin

sensitizing hormone, adiponectin in response to PPARγ activation.

Studies on maternal overnutrition during late gestation in the sheep have shown

that increased exposure to maternal nutrition in late gestation results in an

increase in subcutaneous fat deposition in postnatal life and an increase in the

expression of key adipogenic, lipogenic and adipokine genes, PPARγ, LPL and

leptin mRNA expression within the major fetal adipose tissue depots in fetal life

(Muhlhausler, Duffield et al. 2007). This suggests that the key regulatory genes

involved in fat development and storage may be up-regulated in fetal life in

126

response to the nutrient rich environment. It has not been established as to

whether a nutrient rich environment before pregnancy or in the first week of

embryonic life may alter the programming of the offspring, predisposing a risk

for the development of adiposity later in life.

In Chapter 3 I showed that periconceptional overnutrition leads to an increase

in total fat mass in the female offspring at 4 months of age and that dietary

restriction imposed in overnourished ewes for more than one month prior to

conception ablates this increase in postnatal fat mass. No previous study has

explored the impact of maternal PCON on the programming of genes which

regulate the differentiation and the development of fat or the storage of lipids in

the postnatal offspring. We have therefore investigated the impact of maternal

overnutrition during the periconceptional period from 5 months prior to

conception until 7 days after conception on the expression of key adipogenic

and lipogenic genes of the major adipose tissue depots, omental, perirenal and

subcutaneous fat in the offspring at 4 months of age.


4.2.1 ANIMALS AND NUTRITIONAL FEEDING REGIME

As described in Chapter 2, donor ewes (n=23) were randomly assigned to one

of four treatment groups, either control-control (CC), control-restricted (CR),

high-high (HH) or high-restricted (HR) and fed according to the nutritional

protocol for at least 5 months prior to conception (see Chapter 2). The

127

reproductive cycles of all experimental ewes were synchronized and super

ovulation was induced. Fresh semen was collected from a ram of proven fertility

and the donor ewes were inseminated by laparoscopy. Following insemination

embryos were collected and single embryos were transferred to adult recipient

ewes maintained on a control diet (100% MER) at embryonic day 6-7 by

laparoscopy.

These recipient ewes were maintained on a diet calculated to provide 100%

energy requirements for the maintenance of a pregnant ewe bearing a singleton

fetus, as specified by the U.K Ministry of Agriculture, Fisheries and Food


Pregnancy was confirmed by ultrasonography at day 49 of gestation. The

pregnant ewes were allowed to give birth naturally in a lambing pen (term=150

± 3 days) (see Chapter 2).

4.2.2 POST MORTEM AND ADIPOSE TISSUE COLLECTION

At 4 months of age, lambs were killed with an overdose of sodium

pentobarbitone (Virbac Pty Ltd, Peakhurst, NSW, Australia). Samples of

omental, perirenal and subcutaneous adipose tissue were immediately

collected, weighed and snap frozen with liquid nitrogen and subsequently

stored at -80oC.

128

4.2.3 RNA EXTRACTION

RNA from the omental, perirenal and subcutaneous adipose tissue depots

(≈100mg) was extracted using Trizol reagent (Invitrogen Australia Pty Limited,

Mount Waverley, Australia) and chloroform. RNA was treated with 70%

ethanol and run through a purification process using the RNeasy Mini Kit

(QIAGEN Pty Ltd Australia, Doncaster, Australia). The quality and

concentration of the RNA was determined by measuring absorbance at 260

and 280 nm, and RNA integrity was confirmed by agarose gel electrophoresis.

cDNA was then synthesised using the purified RNA (≈5µg), Superscript 3

Reverse Transcriptase (Invitrogen Australia Pty Limited, Mount Waverley,

Australia) and random hexamers.

4.2.4 REAL TIME QUANTITATIVE REVERSE TRANSCRIPTION-PCR (QRT-PCR)

The relative expression of PPARγ, G3PDH, LPL, leptin and adiponectin mRNA

transcripts were measured by quantitative real time PCR (qRT-PCR) using the

Sybr Green system in an ABI prism 7900 Sequence Detection System (PE

Applied Biosystems, Foster City, CA) (Gibson et al. 1996, Heid et al. 1996). All

primers were designed with the aid of Primer Express (PE Applied Biosystems,

Foster City, CA) software and where possible one primer of each pair was

positioned over a splice site to prevent amplification of any residual genomic

DNA. For each transcript RT-PCR was performed using specific primers, all

primers for this study have been previously optimised (Muhlhausler, Duffield et

al. 2007; Duffield, Vuocolo et al. 2009) (Table 4.2.1). Each amplicon was

designed to be approximately 200 base pairs (bp) in length and was

129

sequenced to ensure the authenticity of the DNA product and qRT-PCR melt

curve analysis was performed to demonstrate amplicon homogeneity. Controls

containing no reverse transcriptase were also used. For the qRT-PCR

measurements, the primer concentrations were equivalent for all genes and

the amplification efficiencies were 0.987-0.999. A constant amount of cDNA

equating to 10ng of total RNA was used for each qRT-PCR measurement and

three technical replicates were performed for each gene.

Each qRT-PCR reaction (5µl total volume) contained: 2.5 µl 2x Sybr Green

master mix (Applied Biosystems); 0.25 µl of each primer giving a final

concentration of 450 µM, 1.0 µl of molecular grade H2O and 1.0 µl of a 1:10

dilution of the stock template. The cycling conditions consisted of 40 cycles of

950C for 15 seconds and 600C for 1 minute. At the end of each run, a

dissociation melt curve was obtained.

The abundance of each mRNA transcript was measured and its expression

relative to that of Ribosomal Protein Large Subunit P0 (RPLP0) was calculated

using Q-gene qRT-PCR analysis software. This provides a quantitative

measure of the relative abundance of a specific transcript normalised to a

reference gene in different tissues.

130

Table 4.2.1 Sequences of Real Time PCR primers for adipogenic,

lipogenic and reference genes

Gene name (GenBank

Accession no.) Forward (5’-3’) Reverse (5’-3’) Amplicon

size (bp)

PPARγ (AY179866)

5‟-atgtctcataatgccatcaggtt-3‟ 5‟-gataacaaacggtgatttgtctgtc-3‟ 225 bp

G3PDH (BT020681)

5‟-gctttggcgacaacacca-3‟ 5‟-agctgctcaatggactttcc-3‟ 208 bp

Leptin

(NM173928)

5‟-atctcacacacgcagtccgt-3‟ 5‟-ccagcaggtggagaaggtc-3‟ 202 bp

Adiponectin (NM174742)

5‟-atcaaactctggaacctcctatctac-3‟ 5‟-ttgcattgcaggctcaag-3‟ 232 bp

LPL (M16966)

5‟-taccctgcctgaagtttccac-3‟

5‟-cccagtttcagccagactttc-3‟

302 bp

RPLP0 (AF013214 )

5‟-caaccctgaagtgcttgacat-3‟

5‟-aggcagatgcatcagcca-3‟

220 bp

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=2653405




http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=14289336

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=14289336

131

4.2.5 STATISTICAL ANALYSIS

All data are presented as mean ± standard error of the mean (SEM). Data

were analysed using the Statistical Package for the Social Sciences version

17.0 (SPSS Inc., Illinois, USA) and STATA10: Data analysis and Statistical

Software for repeated measures (Stata Corp. LD., Texas, USA).

4.2.5.1 Adipogenic and lipogenic gene expression

The effects of periconceptional overnutrition and/or dietary restriction on the

expression of adipogenic and lipogenic genes were compared using

multifactorial ANOVA. Specified factors for the ANOVA included treatment

group (CC, CR, HH or HR) and gender. The depot specific expressions of

adipogenic and lipogenic genes were compared using multifactorial ANOVA

with repeated measures.

When a significant interaction between major factors was identified in ANOVA,

the data were split on the basis of the interacting factors and reanalyzed. The

Duncan‟s post hoc test was used post-ANOVA to identify significant

differences in between mean values and a probability level of 5% (P<0.05)

was taken as significant.

132

4.3 RESULTS

4.3.1 SUBCUTANEOUS ADIPOSE TISSUE

There was no effect of periconceptional overnutrition and/or dietary restriction

on the relative expression of PPARγ, G3PDH, LPL, leptin and adiponectin in

the subcutaneous fat depot (Figure 4.3.1). The relative expression of G3PDH

(P=0.04) and LPL (P=0.03) were higher and leptin mRNA expression tended to

be higher (P=0.07) in the subcutaneous fat of female lambs compared to male

lambs at 4 months of age.

There were also significant positive relationships between PPARγ mRNA

expression and adiponectin, LPL and G3PDH mRNA expression in the

subcutaneous adipose tissue of male and female lambs (Table 4.3.1).

4.3.2 PERIRENAL ADIPOSE TISSUE


on the relative expression of PPARγ, G3PDH, LPL and leptin in the perirenal

fat depot (Figure 4.3.2). The relative expression of G3PDH (P=0.04) and LPL

(P=0.01) mRNA in perirenal fat was higher in female lambs when compared to

male lambs at 4 months of age. There was an interaction between the effects

of nutritional treatment and gender (P=0.04) on the relative expression of

adiponectin in the perirenal fat depot. In male lambs adiponectin expression in

the perirenal fat was lower (P=0.05) in the HR group compared to the CC

group.

133

There were significant positive relationships between PPARγ mRNA

expression and adiponectin, G3PDH and LPL mRNA expression in the

perirenal adipose tissue in male and female lambs (Table 4.3.1).

There was also a significant positive relationship between donor ewe weight at

conception and the normalized gene expression of G3PDH in the perirenal

adipose tissue of female lambs at 4 months of age (Figure 4.3.4).

4.3.3 OMENTAL ADIPOSE TISSUE


on the relative expression of PPARγ, G3PDH, LPL, leptin and adiponectin in

the omental fat depot (Figure 4.3.3). The relative expression of G3PDH mRNA

(P=0.007) was higher, however, in the omental fat of female lambs compared

to male lambs at 4 months of age. There were also significant positive

relationships between PPARγ mRNA expression and LPL and leptin mRNA

expression in the omental adipose tissue in male and female lambs (Table

4.3.1).

4.3.4 COMPARISON OF ADIPOGENIC AND LIPOGENIC GENE EXPRESSION IN ALL FAT

DEPOTS

There was a significant difference between the expression of all adipogenic

and lipogenic genes between different adipose tissue depots, irrespective of

treatment group and gender of the lambs.

134

The expression of PPARγ, G3PDH, LPL, leptin and adiponectin mRNA in

omental adipose tissue was higher than in the subcutaneous or perirenal

adipose tissue depots in male and female lambs at 4 months of age, P<0.05

(Figures 4.3.6 to 4.3.9).

135

Subcutaneous adipose tissue depot

Treatment group

CC CR HH HR

Nor

mal

ised

PP

AR

mR

NA

exp

ress

ion

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Treatment group

CC CR HH HR

Nor

mal

ised

PPA

R

mR

NA

exp

ress

ion

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Treatment group

CC CR HH HR

Nor

mal

ised

G3P

DH

mR

NA

exp

ress

ion

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Treatment group

CC CR HH HR

Nor

mai

lsed

G3P

DH

mR

NA

exp

ress

ion

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Treatment group

CC CR HH HR

Nor

mal

ised

LP

L m

RN

A e

xpre

ssio

n

0

2

4

6

8

10

Treatment group

CC CR HH HR

Nor

mal

ised

LPL

mR

NA

exp

ress

ion

0

2

4

6

8

10

*

*

136

*

Figure 4.3.1 Normalised expression of adipogenic and lipogenic genes in

the subcutaneous adipose tissue depot of male and female lambs at 4

months of age

* denotes a significant difference of the normalised adipogenic and lipogenic

gene expression between male (blue striped bars) and female lambs (red

striped bars) at 4 months of age (P<0.05).

† denotes a trend towards a significant difference of the normalised

adipogenic and lipogenic gene expression between male and female lambs at

4 months of age (P=0.07)

Treatment group

CC CR HH HR

Nor

mal

ised

Lep

tin m

RN

A e

xpre

ssio

n

0.00

0.05

0.10

0.15

0.20

0.25

Treatment group

CC CR HH HR

Nor

mai

lsed

Lep

tin m

RN

A e

xpre

ssio

n

0.00

0.05

0.10

0.15

0.20

0.25

Treatment group

CC CR HH HR

Nor

mal

ised

Adi

pone

ctin

mR

NA

exp

ress

ion

0

2

4

6

8

10

Treatment groupCC CR HH HR

Nor

mai

lsed

Adi

pone

ctin

mR

NA

exp

ress

ion

0

2

4

6

8

10

†

137

Perirenal adipose tissue depot

Treatment group

CC CR HH HR

Nor

mal

ised

PP

AR m

RN

A e

xpre

ssio

n

0.0

0.5

1.0

1.5

2.0

2.5

Treatment group

CC CR HH HR

Nor

mal

ised

PP

AR

mR

NA

exp

ress

ion

0.0

0.5

1.0

1.5

2.0

2.5

Treatment group

CC CR HH HR

No

rmal

ised

G3P

DH

mR

NA

exp

ress

ion

0.0

0.5

1.0

1.5

2.0

2.5

Treatment group

CC CR HH HR

Nor

mal

ised

G3P

DH

mR

NA

exp

ress

ion

0.0

0.5

1.0

1.5

2.0

2.5

*

Treatment group

CC CR HH HR

Nor

mai

lsed

LP

L m

RN

A e

xpre

ssio

n

0

5

10

15

20

25

30

Treatment group

CC CR HH HR

Nor

mai

lsed

LP

L m

RN

A e

xpre

ssio

n

0

5

10

15

20

25

30

*

138


the perirenal adipose tissue depot of male and female lambs at 4 months

of age



striped bars) at 4 months of age (P<0.05)

Different superscripts (e.g. a,b) denotes a significant difference between

adipogenic and lipogenic gene expression between nutritional treatment

groups

Treatment group

CC CR HH HR

Rel

ativ

e ex

pres

sion

of L

eptin

mR

NA

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Treatment group

CC CR HH HR

Nor

mal

ised

Lep

tin m

RN

A e

xpre

ssio

n

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Treatment group

CC CR HH HR

Nor

mal

ised

Adi

pone

ctin

mR

NA

exp

ress

ion

0

2

4

6

8

10

Treatment group

CC CR HH HR

Nor

mal

ised

Adi

pone

ctin

mR

NA

exp

ress

ion

0

2

4

6

8

10a

ab

ab

b

139

Omental adipose tissue depot

Treatment group

CC CR HH HR

Nor

mal

ised

LPL

mR

NA

exp

ress

ion

0

50

100

150

200

250

Treatment group

CC CR HH HR

Nor

mal

ised

PP

AR

mR

NA

exp

ress

ion

0

5

10

15

20

25

30

35

Treatment group

CC CR HH HR

Nor

mal

ised

PP

AR

mR

NA

exp

ress

ion

0

5

10

15

20

25

30

35

Treatment group

CC CR HH HR

No

rmal

ised

G3P

DH

mR

NA

exp

ress

ion

0

2

4

6

8

10

12

14

16

18

20

Treatment group

CC CR HH HR

Nor

mal

ised

G3P

DH

mR

NA

exp

ress

ion

0

2

4

6

8

10

12

14

16

18

20

*

Treatment group

CC CR HH HR

Nor

mal

ised

LP

L m

RN

A e

xpre

ssio

n

0

50

100

150

200

250

140


the omental adipose tissue depot of male and female lambs at 4 months

of age



striped bars) at 4 months of age (P<0.05)

Treatment group

CC CR HH HR

Nor

mal

ised

Lep

tin m

RN

A e

xpre

ssio

n

0

20

40

60

80

100

120

Treatment group

CC CR HH HR

Nor

mal

ised

Lep

tin m

RN

A e

xpre

ssio

n

0

20

40

60

80

100

120

Treatment group

CC CR HH HR

Nor

mal

ised

Adi

pone

ctin

mR

NA

exp

ress

ion

0

10

20

30

40

50

60

70

Treatment group

CC CR HH HR

Nor

mal

ised

Adi

pone

ctin

mR

NA

exp

ress

ion

0

10

20

30

40

50

60

70

141

Donor weight at conception (kg)

50 55 60 65 70 75

Perir

enal

G3P

DH

mR

NA

exp

ress

ion

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Figure 4.3.4 Correlation between donor ewe weight at conception and

G3PDH mRNA expression in the perirenal fat of female lambs at 4 months

of age

There was a significant positive relationship between the perirenal G3PDH

mRNA expression of 4 month old female lambs and donor ewe weight at

conception (perirenal G3PDH mRNA expression= 63 (donor weight at

conception) – 1466.7, r2= 0.24, P<0.05)

142

Subcutaneous G3PDH (y)

Subcutaneous LPL (y)

Subcutaneous Leptin (y)

Subcutaneous Adiponectin

(y)

Subcutaneous PPARγ (x)

NS y=1.86 + 6.34x

r2=0.18, P<0.05 NS

y=3.41 + 6.03x

r2=0.23, P<0.05

Perirenal

G3PDH (y) Perirenal LPL (y)

Perirenal Leptin (y)

Perirenal Adiponectin

(y)

Perirenal PPARγ (x)

y=0.17 + 0.41x

r2=0.17, P<0.05 y=2.06 + 6.50x

r2=0.21, P<0.05 NS

y=2.96 + 1.62x

r2=0.15, P<0.05

Omental

G3PDH (y) Omental LPL (y)

Omental Leptin (y)

Omental Adiponectin

(y)

Omental PPARγ (x)

NS y=91.56+ 3.56x

r2=0.21, P<0.05 y=34.32 +1.22x

r2=0.12, P<0.05 NS

Table 4.3.1 Correlations between the relative mRNA expression of PPARγ

and other adipogenic and lipogenic genes in the subcutaneous, perirenal

and omental adipose tissue depots of lambs at 4 months of age

143

Figure 4.3.5 Relative adipose tissue depot specific expression of PPARγ

mRNA of 4 month old lambs

There was a significant difference between the relative expression of PPARγ in

all adipose tissue depots, subcutaneous, omental and perirenal. Different

superscripts (e.g. a,b) denotes a significant difference between relative

expression of PPARγ mRNA in the subcutaneous, omental and perirenal

adipose depots at 4 months of age, P<0.05.


Subcutaneous Perirenal Omental

Nor

mal

ised

PPA

R

mR

NA

exp

ress

ion

0

2

4

6

8

10

12

14

16

a

b

c

144



Nor

mal

ised

G3P

DH

mR

NA

exp

ress

ion

0

2

4

6

8

10

Figure 4.3.6 Relative adipose tissue depot specific expression of G3PDH


There was a significant difference between the relative expression of G3PDH in

all adipose tissue depots, subcutaneous, omental and perirenal. Different


expression of G3PDH mRNA in the subcutaneous, omental and perirenal

adipose depots at 4 months of age, P<0.05.

b

c

a

145



Nor

mal

ised

LPL

mR

NA

exp

ress

ion

0

20

40

60

80

100

120

140

160

Figure 4.3.7 Relative adipose tissue depot specific expression of LPL


There was a significant difference between the relative expression of LPL in all

adipose tissue depots, subcutaneous, omental and perirenal. Different


expression of LPL mRNA in the subcutaneous, omental and perirenal adipose

depots at 4 months of age, P<0.05.

a b

c

146



Nor

mal

ised

Lep

tin m

RN

A e

xpre

ssio

n

0

10

20

30

40

50

60

Figure 4.3.8 Relative adipose tissue depot specific expression of leptin


There was a significant difference between the relative expression of leptin in all

adipose tissue depots, subcutaneous, omental and perirenal. Different

superscripts (e.g. a, b) denotes a significant difference between relative

expression of leptin mRNA in the subcutaneous, omental and perirenal adipose

depots at 4 months of age, P<0.05.

a

c

b 0.5

147

Adipose tissue depotSubcutaneous Perirenal Omental

Nor

mal

ised

Adi

pone

ctin

mR

NA

exp

ress

ion

0

10

20

30

40

50

60

Figure 4.3.9 Relative adipose tissue depot specific expression of

adiponectin mRNA of 4 month old lambs

There was a significant increase in the relative expression of adiponectin in the

omental adipose tissue depot compared with the subcutaneous and perirenal

depots. Different superscripts (e.g. a, b) denotes a significant difference

between relative expression of adiponectin mRNA in the adipose depots at 4

months of age, P<0.05.

a a

b

148

4.4 DISCUSSION

We have demonstrated that periconceptional overnutrition and/or dietary

restriction does not alter the expression of key adipogenic, lipogenic and

adipokine gene expression in the major adipose tissue depots of 4 month old

lambs. There was however a positive relationship between donor weight at

conception and fat mass (Chapter 3) and the expression of perirenal G3PDH in

female lambs. This may suggest that the increase in total fat mass in the female

lambs at 4 months of age may be attributed to an increase in triglyceride storage

in the fat, rather than hyperplasia of the fat cells. Further work however, is

required to investigate the impact of periconceptional nutrition on adipocyte cell

size and number.

Many studies have attempted to elucidate the importance of timing of nutritional

perturbations in order to determine critical windows during development which

are important in the programming of obesity in later life. Studies by Khan and

colleagues (Khan, Dekou et al. 2004; Khan, Dekou et al. 2005) have shown that

maternal consumption of high fat diets of lard for 10 days prior to conception

through to weaning lead to increased hypertension, hyperinsulinemia and

adiposity in the offspring at 6 months of age. It is difficult however, to elucidate

whether the conditions observed in the offspring are due to the early exposure of

a high fat diet during critical stages of embryonic development or whether they

are due to the exposure of a high fat diet throughout pregnancy through to

weaning or the cumulative effect of both. Dams fed the high fat diet, however,

gained significantly more weight during pregnancy than the control fed rats,

suggesting that this model may mimic the effects of maternal weight gain during

149

pregnancy rather than the specific effects of an increased body fat mass at

conception.

A study by Shankar and colleagues (Shankar, Harrell et al. 2008) determined the

impact of maternal obesity, by feeding female rats with 15% excess calories/day

for 3 weeks prior to mating and resulted in increased body weight, adiposity,

insulin, leptin and insulin resistance. Exposure to maternal obesity was limited to

the in utero environment by cross-fostering pups to lean dams with ad libitum

diets throughout lactation. They showed that there was no effect of maternal

obesity on body weight or size at birth; however offspring from obese dams

gained remarkably greater body weight and higher percentage body fat when fed

a high-fat diet. Their results suggest that maternal body composition at

conception is important in programming offspring adiposity in later life.

Furthermore in this study there was no difference in the gestational weight gain

in the obese or lean dams.

These studies highlight the importance of the nutritional environment in early

pregnancy particularly at critical stages of implantation (~16d gestation in the

sheep) and placentation (~24-30d gestation in the sheep) on the development of

postnatal adiposity in the offspring and their role in predicting adversity and

producing adaptive responses to the current environment. These studies suggest

that offspring adiposity may be determined by exposure to nutritional influences

and is susceptible during several critical windows during early development.

150

It has been suggested by previous studies that increased maternal nutrition late

gestation stimulates the transcriptional co activator PPARγ in the adipose tissue

before birth and subsequently activates a suite of adipogenic, lipogenic and

adipokine genes in the adipose tissue which may be involved in the

pathogenesis of obesity in later life (Muhlhausler, Duffield et al. 2007). The

current study however, shows that periconceptional overnutrition did not affect

the expression of PPARγ, G3PDH, LPL, leptin nor adiponectin, despite the

increase in fat mass in female lambs. We propose that periconceptional

overnutrition may have produced changes to the expression of key adipogenic

and lipogenic genes earlier in development however the postnatal development

of adipose tissues at 4 months of age in the lambs may no longer show

increased expression of adipogenic and lipogenic genes. This suggests that

other mechanisms may be involved in the development of postnatal obesity in

lambs exposed to periconceptional overnutrition, such as the increase in insulin

sensitivity in the female lambs which could alter the threshold of activation of

PPARγ, the transcriptional co activator of adipogenic, lipogenic and adipokine

genes. This change in activation may suggest that minute changes in gene

expression may produce an enhanced downstream response and hence

increased PPARγ activity may contribute to an increase in fat mass in the female

offspring. Further work is required to investigate the impact of periconceptional

overnutrition on the insulin signalling pathways in the major adipose tissue

depots.

We demonstrated that PPAR mRNA expression was directly related to LPL

mRNA expression in subcutaneous, perirenal and omental adipose tissue and to

151

G3PDH in the perirenal adipose tissue which is consistent with studies in the

adult rodent in which the activation of PPAR promotes lipogenesis within mature

adipocytes by increasing the expression of genes, which regulate the

incorporation of circulating free fatty acids (LPL) or nutrient-derived factors

(G3PDH) into adipose cells (Semple 2006). Interestingly we found that maternal

donor weight at conception was positively associated with perirenal G3PDH

mRNA expression in the female lambs at 4 months of age which suggests that

the increase in fat mass observed in the female lambs may be attributed to an

increase in fat storage. Furthermore we propose that PPARγ activation may be

dependent on the timing of exposure to maternal overnutrition, and may occur as

a consequence of exposure to excess nutrients during gestation rather than

when exposure occurs during critical stages of embryo development.

In Chapter 2 I showed that maternal periconceptional overnutrition results in

increased insulin resistance in the ewes. It has been clearly demonstrated by

Catalano and colleagues that an increase in insulin resistance entering

pregnancy is associated with an increased risk of developing increased adiposity

in the offspring (Catalano 2003).

It has been shown that alterations in the nutrient environment of the developing

embryo in vivo and in vitro can alter the allocation of cells within the inner cell

mass and trophectoderm, therefore altering the growth and development of the

embryo (Kwong, Wild et al. 2000; Minge, Bennett et al. 2008). In a recent study

by Minge and colleagues (Minge, Bennett et al. 2008) murine oocytes exposed to

a high fat diet for 16 weeks prior to conception resulted in poor quality oocytes. It

152

was observed that there was a decline in the number of embryos undergoing

further differentiation into 4 to 8 cell blastocysts, as well as the abnormal

localization of blastomeres in the inner cell mass and an increase in the

proportion of cells in the trophectoderm. Further evidence from other animal

models has shown that maternal periconceptional undernutrition alters the

proportion of cells between the inner cell mass and trophectoderm (Erwich and

Robinson 1997; Kwong, Wild et al. 2000).

It has been previously shown that maternal nutrient restriction early in

development may alter the development of adiposity in the offspring. Nutrient

restriction in ewes from 8d post conception throughout gestation showed an

increase in fetal adiposity in twin fetuses when compared to periconceptional

nutrient restriction from 60d prior to conception to 7d post conception (Edwards,

McFarlane et al. 2005). Bispham and colleagues (Bispham, Gopalakrishnan et

al. 2003) also showed an increase in relative adiposity in fetuses of ewes which

underwent nutrient restriction between 28 to 80d gestation (at a time when

maximum growth of the placenta is occurring) and then fed to appetite (~150%

MER) until 140d gestation compared to fetuses exposed to an ad libitum diet

from 28 to 140d gestation.

We found that maternal periconceptional overnutrition increases maternal insulin

concentrations and it is possible maternal insulin resistance may affect the

developing blastocyst, consequently increasing adiposity in susceptible female

offspring in later life. Periconceptional overnutrition and/or dietary restriction may

alter the proportion of cells allocated to the inner cell mass or trophectoderm in

153

the developing blastocyst, as well as alter the embryonic environment affecting

the differentiation and developmental potential of adipocytes. These alterations

may be important mechanisms by which periconceptional overnutrition and/or

dietary restriction may alter the growth and development of the offspring. It is

clear that further work is required to investigate the impact of the nutrient

environment during the periconceptional period on the developing blastocyst.

4.5 SUMMARY

We have shown that periconceptional overnutrition does not affect the gene

expression of PPARγ, G3PDH, LPL, leptin and adiponectin of 4 month old

lambs. There was a greater expression of genes however, involved in the

storage of fatty acids in mature adipocytes across all major depots in female

offspring. Furthermore we found that there was a positive relationship between

maternal body weight at conception and the expression of perirenal G3PDH in

the female offspring. This suggests that the increase in total fat mass observed

in the female lambs compared to the male lambs may be due to an increase in

adipocyte hypertrophy postnatally. We proposed that the development of

maternal insulin resistance in the periconceptionally overnourished ewe

subsequently alters the embryonic environment resulting in the development of

insulin sensitivity in the offspring which could alter the threshold of activation of

PPARγ, the transcriptional co-activator of adipogenic, lipogenic and adipokine

genes.

154

CHAPTER 5

155

CHAPTER 5: SUMMARY AND CONCLUSIONS

Maternal obesity and/or glucose intolerance is a major determinant of the

relationship between birth weight and BMI in adulthood and it has been proposed

that an increase in fetal nutrient supply, as a consequence of these conditions, is

an important determinant of fetal body condition both in utero and in later life.

Interestingly a study by Catalano and colleagues (Catalano and Ehrenberg 2006)

showed that maternal weight and insulin resistance before pregnancy is strongly

correlated with an infant‟s fat mass, whereas the development of insulin

resistance before pregnancy is associated with infants fat free mass (e.g.

skeletal muscle). Few studies have attempted to distinguish between the

contribution of maternal obesity and the development of maternal glucose

intolerance at later stages of pregnancy. In this study I was able to determine the

contribution of a high maternal BMI and maternal insulin resistance entering

pregnancy on the development of the oocyte/embryo and on the development of

adiposity in the offspring in later life.

In the current study we have successfully developed a novel model of maternal

periconceptional overnutrition in the ewe. We have shown that periconceptional

overnutrition for at least 5 months prior to conception to one week post

conception resulted in a significant increase in weight gain and body condition

scores in the HH ewes. A short period of dietary restriction for one month prior to

conception to one week post conception in the periconceptionally overnourished

ewes resulted in lower weight gain than the HH group, although the HR ewes

remained significantly heavier and fatter than the control fed, CC and

156

periconceptionally nutrient restricted, CR ewes. Our data suggests that

overnourishing non-pregnant ewes for at least 5 months prior to conception, as

in the HH and HR ewes leads to an obese body condition accompanied by

increased plasma insulin concentrations and unaltered plasma glucose

concentrations, indicative of the pathogenesis of insulin resistance prior to

conception in these ewes.

There was no effect of periconceptional overnutrition or restriction on the growth

parameters at birth, including birth weight. Recent studies have debated whether

birth weight is in fact an appropriate end point for determining developmental

origins of adult disease. It has long been recognised that in the „developmental

origins of health and disease‟ (DOHAD) hypothesis was coined to explain the

associations between low and high birth weights and the development of a range

of diseases later in life, including obesity. Over time the hypothesis has

generated some controversy, since birth weight is a non-specific marker of a

number of conditions including premature birth, maternal health and fetal

genetics. Furthermore it is argued that postnatal growth may be a more

important risk factor for the subsequent pathogenesis of disease than growth in

utero (Cole 2004; Singhal and Lucas 2004). Therefore birth weight may be one

of many important markers for the increased risk of the development of diseases

later in life, but this may not always be the case.

It has been recently shown that periconceptional undernutrition from 45 days

prior to conception to 7 days post conception in the sheep led to an increase in

placental and fetal weights in twin fetuses (MacLaughlin, Walker et al. 2005).

157

This suggests that a compensatory response by the placenta occurs as a

consequence of the exposure of periconceptional undernutrition, therefore

altering the nutrient supply to the fetus. It has been suggested that PCUN alters

the allocation of cells within the blastocyst, which may restrict early embryonic

proliferation and differentiation of appropriately sized stem cell lineages,

therefore affecting the fetoplacental growth trajectory in early pregnancy. This

suggests PCUN alters the placental nutrient supply to the fetus which may

further impede the development of vital organ systems including the

cardiovascular system during gestation. In the CR group in the present study,

however there was no effect of periconceptional nutrient restriction on the birth

weight of the lambs which may suggest appropriate placental development or

compensatory growth of the placenta has occurred early in development.

At 4 months of age female lambs had significantly more body fat than male

lambs. Female lambs exposed to periconceptional overnutrition (HH) had higher

total fat mass than lambs from the CC and CR groups despite no change in

plasma insulin, glucose and non-esterified free fatty acid concentrations and this

was positively correlated with donor ewe weight at conception. Interestingly a

dietary intervention period of one month prior to conception to one week post

conception ablated this effect. In studies not reported in this thesis, we have also

shown that in the HH female lambs there was a decreased expression of hepatic

PEPCK mRNA, a key rate limiting enzyme involved in the gluconeogenic

pathway (personal communication Nichols, Rattanatray 2009). This suggests

that the liver of the HH lambs may be relatively more sensitive to the actions of

insulin.

158

I propose that exposure of the developing oocyte/embryo to maternal

overnutrition during the periconceptional period which results in an increase in

maternal weight and fat mass, may program adipose tissue and hepatic

metabolism to prepare for a future of predicted nutrient abundance. An increase

in total fat mass in the female offspring and a decrease in hepatic PEPCK mRNA

in the periconceptionally overnourished lambs suggests that the adipose tissue

and liver may be more sensitive to the actions of insulin in these lambs. Female

lambs are apparently more vulnerable to the effects of periconceptional

overnutrition and may also be more sensitive to the actions of insulin on the

adipose tissue (Figure 5.1). This enhanced insulin action in the adipose tissue in

the female lambs may explain the increase in fat mass in the female lambs,

whereas male lambs may be more sensitive to the actions of insulin in the

skeletal muscle. This would explain why the increase in fat mass in the postnatal

lambs exposed to periconceptional overnutrition was not associated with an

increase in PPARγ or other adipogenic gene expression.

Consistent with previous studies, male lambs were born heavier, with higher

plasma insulin levels than female lambs irrespective of treatment group (Duffield,

Vuocolo et al. 2009). Skeletal myogenesis occurs during early embryo

development involving the differentiation of mesodermal stem cells to myogenic

precursor cells. The majority of muscle fibre formation however, occurs mid-

gestation in the sheep. Studies have shown that the development of skeletal

muscle in the fetal sheep is susceptible to maternal nutrient availability during

pregnancy (Tong, Yan et al. 2009), the peri-implantation environment (Quigley,

159

Periconcep-tional

overnutrition/ insulin

resistance

↔Birth weight

↑ Susceptibility of obesity in

females in later life

↔ In utero environment

↑ insulin sensitivity in

adipose tissue

↑ Fat mass in female lambs at

4 months

Kleemann et al. 2005) and gender specific hormones (Galluzzo, Rastelli et al.

2009). I suggest that the skeletal muscle of male lambs is more insulin sensitive

than the skeletal muscle of female lambs and therefore is more inclined to take

up glucose, increasing the growth of muscle fibres in the male lambs before

birth. Further studies are required to determine the impact of periconceptional

overnutrition and gender on the insulin signalling pathways in key organs and

tissues regulating whole body glucose homeostasis, such as the liver, skeletal

muscle and adipose tissue.

Figure 5.1 Proposed mechanisms for the impact of periconceptional

overnutrition on the intergenerational cycle of obesity

160

In the current study I have shown that a dietary restriction period of one month

prior to conception to one week post conception in the periconceptionally

overnourished ewe, ameliorates the impact of periconceptional overnutrition on

the development of increased adiposity in the female lambs at 4 months of age.

This suggests that a dietary intervention period restores normal insulin action in

the adipose tissue in the postnatal lamb. The dietary restriction period however,

was also associated with changes which have implications for the development

of the hypothalamic pituitary adrenal (HPA) axis (increased adrenal weights in

the CR and HR groups) brain development (increase in brain weight in the CR

group) and the cardiovascular system (increase in heart weight in the CR group)

in these lambs postnatally.

In contrast to previous work which has investigated late gestational overnutrition

in the sheep (Muhlhausler 2002), periconceptional overnutrition did not alter the

expression of key adipogenic, lipogenic and adipokine genes. This suggests that

periconceptional overnutrition alone may act through an enhanced insulin action

in the adipose tissue and/or early programming of preferential differentiation of

mesenchymal stem cells into the adipocyte cell lineage, whereas late gestational

overnutrition increases the expression of the key adipogenic (PPARγ) and

lipogenic genes (Figure 5.2). The periconceptional and late gestational periods of

development may both independently increase fat mass in the offspring in later

life and the susceptibility to develop obesity. The cumulative effect of

periconceptional obesity and late gestational overnutrition may further amplify

the risk of developing obesity in later life by programming an enhanced insulin

signalling action in the adipose tissue early in development and an increase

161

Maternal late gestational

overnutrition

↑Birth weight ↑PPARγ mRNA expression in adipose tissue


later life

↑ Fetal nutrient

supply

↑ Fat mass ↑glucose ↑insulin

concentrations postnatally

expression of PPARγ mRNA later in development, increasing fat mass in the

offspring (Figure 5.3).

Figure 5.2 Proposed mechanisms for the impact of late gestational

overnutrition on the intergenerational cycle of obesity

162

Periconception and late

gestational overnutrition

↑Birth weight ↑insulin

sensitivity in adipose tissue ↑PPARγ mRNA


later life

Maternal insulin resistance

↑Fetal nutrient supply

↑ Fat mass ↑glucose ↑insulin

concentrations postnatally

Figure 5.3 Proposed mechanisms for the impact of periconceptional

overnutrition and late gestational overnutrition on the intergenerational

cycle of obesity

Therefore from the findings from this study, it would not be recommended to

acutely restrict dietary intake in pregnant women who are overweight or obese

immediately before conception. Further research is required to determine the

time period and level of dietary restriction which if imposed may ablate the

“intergenerational cycle of obesity”.

163

In conclusion, the results of this thesis therefore highlight the complex interaction

between the early exposure to excess nutrition during early embryo development

and the subsequent development of the offspring. Maternal periconceptional

overnutrition has been found to alter the development of the lamb. Female lambs

were found to be particularly vulnerable to the development of obesity in later

life, which may be attributed to an increase in lipogenesis and/or insulin

sensitivity in the adipose tissue, when exposed to an excess nutrient

environment during critical stages of embryo development. Restricted

periconceptional nutrition from either a high plane or control plane of nutrition

resulted in increased adrenal growth in the postnatal lambs. This may suggest

that dietary restriction before conception and early stages of embryo

development may alter the development of the HPA axis. Periconceptional

dietary restriction also increased heart growth in female lambs which may

indicate an altered developmental trajectory of the cardiovascular system and

program a susceptibility to developing cardio vascular related diseases in later

life. Although dietary restriction in the overnourished ewes ablated the increase

in adiposity in female lambs postnatally, implications for the development of the

HPA axis and cardiovascular system suggest caution is required in providing

advice to obese/overweight mothers to lose weight rapidly in the weeks

immediately before conception.

164

BIBLIOGRAPHY

Adamiak, S. J., K. Mackie, et al. (2005). "Impact of Nutrition on Oocyte Quality: Cumulative Effects of Body Composition and Diet Leading to Hyperinsulinemia in Cattle." Biol Reprod 73(5): 918-926.

Ahima, R. S. (2006). "Adipose tissue as an endocrine organ." Obesity 5: 242S-249S.

Ahima, R. S. and J. S. Flier (2000). "Adipose Tissue as an Endocrine Organ." Trends in Endocrinology and Metabolism 11(8): 327-332.

Ahima, R. S. and J. S. Flier (2000). "Leptin." Annual Review of Physiology 62(1): 413-437.

Ahima, R. S., C. B. Saper, et al. (2000). "Leptin Regulation of Neuroendocrine Systems." Frontiers in Neuroendocrinology 21(3): 263-307.

Aihaud, G., P. Grimaldi, et al. (1992). "Cellular and molecular aspects of adipose tissue development." Annu. Rev. Nutr. 12: 207-233.

Alexander, G. (1978). "Quantitative development of adipose tissue in foetal sheep." Aust. J. Biol. Sci 31: 489-503.

Alexander, G. R. and A. W. Bell (1990). "Effect of prolonged glucose infusion into fetal sheep on body growth, fat deposition and gestation length." Journal of Developmental Physiology 13: 277–281.

Ashworth, C. J., I. Wilmut, et al. (1987). "Effect of an inhibitor of 3-hydroxysteroid dehydrogenase on progesterone concentrations and embryo survival in sheep." J Endocrinol 112: 205.

Auwerx, J. (1999). "PPARγ, the ultimate thrifty gene " Diabetologia 42(9): 1033-1049.

Barker, D., A. Dull, et al. (1990). "Fetal and placental size and risk of hypertension in adult life." BMJ 301: 259-262.

Barker, D. J. P. (2007). "The origins of the developmental origins theory." Journal of Internal Medicine 261(5): 412-417.

Barker, D. J. P., C. N. Hales, et al. (1993). "TYPE 2 (NON-INSULIN-DEPENDENT) DIABETES-MELLITUS, HYPERTENSION AND HYPERLIPEMIA (SYNDROME-X) - RELATION TO REDUCED FETAL GROWTH." Diabetologia 36(1): 62-67.

Barker, D. J. P. and C. Osmond (1986). "Infant mortality, childhood nutrition and ischaemic heart disease in Engalnd and Wales " The Lancet 327(8489): 1077-1081.

Bellver, J. and A. Pellicer (2004). "Impact of obesity on spontaneous abortion." American Journal of Obstetrics and Gynecology 190(1): 293-294.

Bellver, J., L. P. Rossal, et al. (2003). "Obesity and the risk of spontaneous abortion after oocyte donation." Fertility and Sterility 79(5): 1136-1140.

Beyer, C. (1999). "Estrogen and the developing mammalian brain." Anatomy and Embryology 199(5): 379-390.

Bispham, J., G. S. Gopalakrishnan, et al. (2003). "Maternal Endocrine Adaptation throughout Pregnancy to Nutritional Manipulation: Consequences for Maternal Plasma Leptin and Cortisol and the

165

Programming of Fetal Adipose Tissue Development." Endocrinology 144(8): 3575-3585.

Blache, D., R. L. Tellam, et al. (2000). "Level of nutrition affects leptin concentrations in plasma and cerebrospinal fluid in sheep." Journal of Endocrinology 165: 625–637.

Bloomfield, F. H., M. H. Oliver, et al. (2003). "Brief Undernutrition in Late-Gestation Sheep Programs the Hypothalamic-Pituitary-Adrenal Axis in Adult Offspring." Endocrinology 144(7): 2933-2940.

Bloomfield, F. H., M. H. Oliver, et al. (2004). "Periconceptional Undernutrition in Sheep Accelerates Maturation of the Fetal Hypothalamic-Pituitary-Adrenal Axis in Late Gestation." Endocrinology 145(9): 4278-4285.

Boland, M. P., P. Lonergan, et al. (2001). "Effect of nutrition on endocrine parameters, ovarian physiology, and oocyte and embryo development." Theriogenology 55(6): 1323-1340.

Boney, C. M., Verma, A.,Tucker, R. and Vohr, B. R. (2005). "Metabolic Syndrome in Childhood: Association With Birth Weight, Maternal Obesity, and Gestational Diabetes Mellitus." Pediatrics 115: 290-296.

Bray, G. A., D. A. York, et al. (1989). "Experimental obesity: a homeostatic failure due to defective nutrient stimulation of the sympathetic nervous system." Vitam Horm 45: 1-125.

Bruce, K. D. and C. D. Byrne (2009). "The metabolic syndrome: common origins of a multifactorial disorder." Postgrad Med J 85(1009): 614-621.

Budge, H., L. J. Edwards, et al. (2004). "Nutritional Manipulation of Fetal Adipose Tissue Deposition and Uncoupling Protein 1 Messenger RNA Abundance in the Sheep: Differential Effects of Timing and Duration." Biol Reprod 71(1): 359-365.

Burrell, J. H., A. M. Boyn, et al. (2003). "Growth and maturation of cardiac myocytes in fetal sheep in the second half of gestation." Anatomical Record Part A, Discoveries in Molecular, Cellular and Evolutionary Biology 274: 953-961.

Butterwith, S. C. (1994). "Molecular events in adipocyte development." Pharmacology & Therapeutics 61(3): 399-411.

Cardona-Gómez, G. P., P. Mendez, et al. (2001). "Interactions of estrogens and insulin-like growth factor-I in the brain: implications for neuroprotection." Brain Research Reviews 37(1-3): 320-334.

Catalano, P. M. and H. M. Ehrenberg (2006). "The short- and long-term implications of maternal obesity on the mother and her offspring." BJOG: An International Journal of Obstetrics & Gynaecology 113(10): 1126-1133.

Catalano, P. M., Kirwan, J. P., Haugel-de Mouzonz, S. and Kingzz, J. (2003). "Gestational Diabetes and Insulin Resistance: Role in Short- and Long-Term Implications for Mother and Fetus." J. Nutr. 133: 1674S–1683S.

Catalano, P. M., A. Thomas, et al. (2007). "Phenotype of Infants of Mothers with Gestational Diabetes." Diabetes Care 30(Supplement_2): S156-160.

Cetin, I., P. S. Morpurgo, et al. (2000). "Fetal plasma leptin concentrations: Relationships with different intrauterine growth patterns from 19 weeks to term." Pediatric Research 48: 646-651.

Chagas, L. M., J. J. Bass, et al. (2007). "Invited Review: New Perspectives on the Roles of Nutrition and Metabolic Priorities in the Subfertility of High-Producing Dairy Cows." J. Dairy Sci. 90(9): 4022-4032.

166

Chen, N. X., B. D. White, et al. (1995). "Glucocorticoid receptor binding in porcine preadipocytes during development." J. Anim Sci. 73(3): 722-727.

Clark, A., B. Thornley, et al. (1998). "Weight loss in obese infertile women results in improvement in reproductive outcome for all forms of fertility treatment." Hum. Reprod. 13(6): 1502-1505.

Cnattingius, S., Bergstrom, R., Lipworth, L. and Kramer, M. (1998). "Prepregnancy Weight and the Risk of Adverse Pregnancy Outcomes." New England Journal of Medicine 338: 147-152.

Cogswell, M. E., G. S. Perry, et al. (2001). "Obesity in women of childbearing age: risks, prevention, and treatment." Primary Care Update for OB/GYNS 8(3): 89-105.

Cole, T. J. (2004). "Children grow and horses race: is the adiposity rebound a critical period for later obesity?" BMC Pediatrics 4(1): 6.

Considine, R. V., M. K. Sinha, et al. (1996). "Serum Immunoreactive-Leptin Concentrations in Normal-Weight and Obese Humans." N Engl J Med 334(5): 292-295.

Creed, J., T. G. McEvoy, et al. (1994). "The effect of preovulatory nutrition on the subsequent development of superovulated sheep ova in an in vitro culture system." Animal Prod 58: 82.

Curhan, G. C., G. M. Chertow, et al. (1996). "Birth Weight and Adult Hypertension and Obesity in Women." Circulation 94(6): 1310-1315.

Dabelea, D., Hanson, R. L., Lindsay, R. S., Pettitt, D. J., Imperatore, G., Gabir, M. M., Roumain, J., Bennett, P. H and Knowler, W. C. (2000). "Intrauterine Exposure to Diabetes Conveys Risks

for Type 2 Diabetes and Obesity: A Study of Discordant Sibships." Diabetes 49: 2208–2211.

Dabelea, D., Snell-Bergeon, J. K., Hartsfield, C. L., Bischoff, K. J., Hamman, R. F. and McDuffie, R. S. (2005). "Increasing Prevalence of Gestational Diabetes Mellitus (GDM) Over Time and by Birth Cohort." Diabetes Care 28: 579–584.

Devaskar, S. U., R. Anthony, et al. (2002). "Ontogeny and insulin regulation of fetal ovine white adipose tissue leptin expression." American Journal of Physiology 282 R431–R438.

Dong, F., S. P. Ford, et al. (2005). "Maternal nutrient restriction during early to mid gestation up-regulates cardiac insulin-like growth factor (IGF) receptors associated with enlarged ventricular size in fetal sheep." Growth Hormone & IGF Research 15(4): 291-299.

Downing, J. A., J. Joss, et al. (1995). "Ovulation rate and the concentrations of gonadotrophins and metabolic hormones in ewes infused with glucose during the late luteal phase of the oestrous cycle." J Endocrinol 146(3): 403-410.

Duffield, J. A., T. Vuocolo, et al. (2009). "Intrauterine Growth Restriction and the Sex Specific Programming of Leptin and Peroxisome Proliferator-Activated Receptor [gamma] (PPAR[gamma]) mRNA Expression in Visceral Fat in the Lamb." Pediatric Research 66(1): 59-65.

Dunne, L. D., M. G. Diskin, et al. (1999). "The effect of pre- and post-insemination plane of nutrition on embryo survival in beef heifers." Animal Science 69(2): 411-417.

Edwards, L. J., A. E. Bryce, et al. (2002). "Maternal undernutrition throughout pregnancy increases adrenocorticotrophin receptor and steroidogenic

167

acute regulatory protein gene expression in the adrenal gland of twin fetal sheep during late gestation." Molecular and Cellular Endocrinology 196(1-2): 1-10.

Edwards, L. J., J. R. McFarlane, et al. (2005). "Impact of periconceptional nutrition on maternal and fetal leptin and fetal adiposity in singleton and twin pregnancies." Am J Physiol Regul Integr Comp Physiol 288(1): R39-45.

Edwards, L. J. and I. C. McMillen (2002). "Impact of Maternal Undernutrition During the Periconceptional Period, Fetal Number, and Fetal Sex on the Development of the Hypothalamo-Pituitary Adrenal Axis in Sheep During Late Gestation." Biol Reprod 66(5): 1562-1569.

Edwards, L. J. and I. C. McMillen (2002). "Periconceptional nutrition programs development of the cardiovascular system in the fetal sheep." Am J Physiol Regul Integr Comp Physiol 283(3): R669-679.

Ehrenberg, H. M., B. M. Mercer, et al. (2004). "The influence of obesity and diabetes on the prevalence of macrosomia." American Journal of Obstetrics and Gynecology 191(3): 964-968.

Erwich, J. J. and J. S. Robinson (1997). "Factors in early pregnancy and fetal growth and development." Contemp Rev Obstet Gynecol 5-10.

Fall, C. H. D., C. Osmond, et al. (1995). "Fetal and infant growth and cardiovascular risk factors in women." BMJ 310(6977): 428-432.

Fasshauer, M., R. Paschke, et al. (2004). "Adiponectin, obesity, and cardiovascular disease." Biochimie 86(11): 779-784.

Fraser, R. B., S. L. Waite, et al. (2007). "Impact of hyperglycemia on early embryo development and embryopathy: in vitro experiments using a mouse model." Hum. Reprod. 22(12): 3059-3068.

Frisancho, A. R. (2000). "Prenatal compared with parental origins of adolescent fatness." American Journal of Clinical Nutrition 72: 1186-1190.

Galluzzo, P., C. Rastelli, et al. (2009). "17{beta}-ESTRADIOL REGULATES THE FIRST STEPS OF SKELETAL MUSCLE CELL DIFFERENTIATION VIA ER{alpha}-MEDIATED SIGNALS." Am J Physiol Cell Physiol: 00188.02009.

Garbaciack, J. A., Richter, M., Miller, S., Barton, J.J. (1985). "Maternal weight and pregnancy complications." Am J Obstet Gynecol 152(2): 238-245.

Garbaciak, J. A., Richter, M.D., Miller, M.D. and Barton, J.J (1985). "Maternal weight and pregnancy complications." Am J Obstet Gynecol 152: 238-245.

Gardner, D. S., S. Pearce, et al. (2004). "Peri-Implantation Undernutrition Programs Blunted Angiotensin II Evoked Baroreflex Responses in Young Adult Sheep." Hypertension 43(6): 1290-1296.

Gatford, K. L., T. P. Fletcher, et al. (1996). "Sexual dimorphism of circulating somatotropin, insulin-like growth factor I and II, insulin-like growth factor binding proteins, and insulin: relationships to growth rate and carcass characteristics in growing lambs." J. Anim Sci. 74(6): 1314-1325.

Gemmell, R. and G. Alexander (1978). "Ultrastructural development of adipose tissue in foetal sheep." Australian Journal of Biological Science 31: 505-515.

Gillman, M. W., Rifas-Shiman, S., Berkey, C. S., Field, A. E. and Colditz, G. A. (2003). "Maternal Gestational Diabetes, Birth Weight, and Adolescent Obesity." Pediatrics 111: 221-226.

168

Gluckman, P. and M. A. Hanson (2004). "The developmental origins of the metabolic syndrome." Trends in Endocrinology & Metabolism 15: 183-187.

Gregoire, F. M. (2001). "Adipocyte Differentiation: From Fibroblast to Endocrine Cell." Experimental Biology and Medicine 226(11): 997-1002.

Gregoire, F. M., C. Genart, et al. (1991). "Glucocorticoids induce a drastic inhibition of proliferation and stimulate differentiation of adult rat fat cell precursors." Exp. Cell Res. 196(2): 270-278.

Gregoire, F. M., Smas, C. M. and Sul, H.S. (1998). "Understanding Adipocyte Differentiation." Physiol. Rev. 78(3): 783-809.

Guo, F. and K. L. C. Jen (1995). "High-fat feeding during pregnancy and lactation affects offspring metabolism in rats." Physiology & Behavior 57(4): 681-686.

Hales, C. and D. Barker (1992). "Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis." Diabetologia 35: 595-601.

Hales, C. N. and D. J. P. Barker (2001). "The thrifty phenotype hypothesis: Type 2 diabetes." Br Med Bull 60(1): 5-20.

Han, H.-C., K. J. Austin, et al. (2004). "Maternal nutrient restriction alters gene expression in the ovine fetal heart." The Journal of Physiology 558(1): 111-121.

Haugen, G., M. Hanson, et al. (2005). "Fetal Liver-Sparing Cardiovascular Adaptations Linked to Mother's Slimness and Diet." Circ Res 96(1): 12-14.

Hausman, G. J. (1992). "Responsiveness to adipogenic agents in stromal-vascular cultures derived from lean and preobese pig

fetuses: An ontogeny study." J. Anim . Sci. 70: 106. Hausman, G. J., T. R. Kasser, et al. (1982). "The Effect of Maternal Diabetes

and Fasting on Fetal Adipose Tissue Histochemistry in the Pig." J. Anim Sci. 55(6): 1343-1350.

Hausman, G. J., J. T. Wright, et al. (1993). "Cellular and molecular aspects of the regulation of adipogenesis." J. Anim. Sci. 71(Suppl 2): 33.

Havel, P. J., S. Kasim-Karakas, et al. (1996). "Relationship of plasma leptin to plasma insulin and adiposity in normal weight and overweight women: effects of dietary fat content and sustained weight loss." J Clin Endocrinol Metab 81(12): 4406-4413.

Hu, E., P. Tontonoz, et al. (1995). "Transdifferentiation of Myoblasts by the Adipogenic Transcription Factors PPAR{gamma} and C/EBP{alpha}." PNAS 92(21): 9856-9860.

Hube, F., U. Lietz, et al. (1996). "Difference in leptin mRNA levels between omental and subcutaneous abdominal adipose tissue from obese humans." Horm. Metab. Res. 28(12): 690-693.

Huxley, R., A. Neil, et al. (2002). "Unravelling the fetal origins hypothesis: is there really an inverse association between birthweight and subsequent blood pressure?" The Lancet 360(9334): 659-665.

Hwang, C.-S., T. M. Loftus, et al. (1997). "Adipocyte differentiation and leptin expression " Annual Review of Cell and Developmental Biology 13(1): 231-259.

Iwaki, M., M. Matsuda, et al. (2003). "Induction of Adiponectin, a Fat-Derived Antidiabetic and Antiatherogenic Factor, by Nuclear Receptors." Diabetes 52(7): 1655-1663.

Jensen, D. M., Damm, P., Sørensen, B., Mølsted-Pedersen, L., Westergaard, J. G., Ovesen, P. and Beck-Nielsen, H. (2003). "Pregnancy outcome and

169

prepregnancy body mass index in 2459 glucose-tolerant Danish women." American Journal of Obstetrics and Gynecology 189: 239-244.

Jolly, M. C., Sebireb, N. J., Harrisc, J. P., Regand, L. and Robinsona, S. (2003). "Risk factors for macrosomia and its clinical consequences:

a study of 350,311 pregnancies." European Journal of Obstetrics & Gynecology and

Reproductive Biology 111: 9-14. Kadowaki, T. and T. Yamauchi (2005). "Adiponectin and Adiponectin Receptors."

Endocr Rev 26(3): 439-451. Kajantie, E., T. Hytinantti, et al. (2004). "Cord Plasma Adiponectin: A 20-Fold

Rise between 24 Weeks Gestation and Term." J Clin Endocrinol Metab 89(8): 4031-4036.

Kakar, M. A., S. Maddocks, et al. (2005). "The effect of peri-conception nutrition on embryo quality in the superovulated ewe." Theriogenology 64(5): 1090-1103.

Kasser, T. R., J. H. Gahagan, et al. (1982). "Fetal Hormones and Neonatal Survival in Response to Altered Maternal Serum Glucose and Free Fatty Acid Concentrations in Pigs." J. Anim Sci. 55(6): 1351-1359.

Kendrick, K. W., T. L. Bailey, et al. (1999). "Effects of Energy Balance on Hormones, Ovarian Activity, and Recovered Oocytes in Lactating Holstein Cows Using Transvaginal Follicular Aspiration." J. Dairy Sci. 82(8): 1731-1740.

Kersten, S. (2001). "Mechanisms of nutritional and hormonal regulation of lipogenesis." EMBO Reports 2(4): 282-286.

Khan, I., V. Dekou, et al. (2004). "Predictive Adaptive Responses to Maternal High-Fat Diet Prevent Endothelial Dysfunction but Not Hypertension in Adult Rat Offspring." Circulation 110(9): 1097-1102.

Khan, I. Y., V. Dekou, et al. (2005). "A high-fat diet during rat pregnancy or suckling induces cardiovascular dysfunction in adult offspring." Am J Physiol Regul Integr Comp Physiol 288(1): R127-133.

Kind, K. L., C. T. Roberts, et al. (2005). "Chronic maternal feed restriction impairs growth but increases adiposity of the fetal guinea pig." Am J Physiol Regul Integr Comp Physiol 288(1): R119-126.

Klaus, R. (1997). "Functional differentiation of white and brown adipocytes." Bioessays. 19(3): 215-223.

Kleemann, D. O., S. K. Walker, et al. (1994). "Enhanced fetal growth in sheep administered progesterone during the first three days of pregnancy." J Reprod Fertil 102: 411-417.

Kral, J. G. (2004). "Preventing and Treating Obesity in Girls and Young Women to Curb the Epidemic." Obes Res 12(10): 1539-1546.

Kristensen, J., Vestergaard, M., Wisborg, K., Kesmodel, U. and Secherc, N. J. (2005). "Pre-pregnancy weight and the risk of stillbirth

and neonatal death." International Journal of Obstetrics and Gynaecology 112: 403-408.

Kumarasamy, V., M. D. Mitchell, et al. (2005). "Effects of periconceptional undernutrition on the initiation of parturition in sheep." Am J Physiol Regul Integr Comp Physiol 288(1): R67-72.

Kwong, W. Y., A. E. Wild, et al. (2000). "Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities

170

and programming of postnatal hypertension." Development 127(19): 4195-4202.

LaCoursiere, D. Y., L. Bloebaum, et al. (2005). "Population-based trends and correlates of maternal overweight and obesity, Utah 1991-2001." American Journal of Obstetrics and Gynecology 192(3): 832-839.

Laitinen, J., Power, C. and Järvelin, M-R. (2001). "Family social class, maternal body mass index, childhood body mass index, and age at menarche as predictors of adult obesity." American Journal of Clinical Nutrition 74: 287-294.

Lazar, M. A. (2004). "PPARg, 10 years later." Biochimie 87: 9-13. Lehmann, J. r. M., L. B. Moore, et al. (1995). "An Antidiabetic Thiazolidinedione

Is a High Affinity Ligand for Peroxisome Proliferator-activated Receptor (PPAR)." Journal of Biological Chemistry 270(22): 12953-12956.

Leroy, J., A. Van Soom, et al. (2007). "Reduced Fertility in High-yielding Dairy Cows: Are the Oocyte and Embryo in Danger? Part II Mechanisms Linking Nutrition and Reduced Oocyte and Embryo Quality in High-yielding Dairy Cows*." Reproduction in Domestic Animals 43(5): 623-632.

Lozano, J. M., P. Lonergan, et al. (2003). "Influence of nutrition on the effectiveness of superovulation programmes in ewes: effect on oocyte quality and post-fertilization development." Reproduction 125(4): 543-553.

Lucas, A. (1998). "Programming by Early Nutrition: An Experimental Approach." J. Nutr. 128(2): 401S-.

Lumbers, E. R., M. Y. Kim, et al. (2009). "Effects of intrafetal IGF-1 on growth of cardiac myocytes in the late gestation fetal sheep." Am J Physiol Endocrinol Metab In Press.

MacLaughlin, S. M., S. K. Walker, et al. (2007). "Impact of Periconceptional Undernutrition on Adrenal Growth and Adrenal Insulin-Like Growth Factor and Steroidogenic Enzyme Expression in the Sheep Fetus during Early Pregnancy." Endocrinology 148(4): 1911-1920.

MacLaughlin, S. M., S. K. Walker, et al. (2005). "Periconceptional nutrition and the relationship between maternal body weight changes in the periconceptional period and feto-placental growth in the sheep." The Journal of Physiology 565(1): 111-124.

Maeda, N., M. Takahashi, et al. (2001). "PPARg Ligands Increase Expression and Plasma Concentrations of Adiponectin, an Adipose-Derived

Protein." Diabetes 50(9): 2094-2099. Martin, J. L., K. A. Vonnahme, et al. (2007). "Effects of dam nutrition on growth

and reproductive performance of heifer calves." J. Anim Sci. 85(3): 841-847.

McEvoy, J. D., C. S. Mayne, et al. (1995). "Production of twin calves with in vitro fertilised embryos: effects on the reproductive performance of dairy cows." Vet Rec 136(25): 627-632.

McEvoy, T. G., J. J. Robinson, et al. (1995). "Dietary-induced suppression of pre-ovulatory progesterone concentrations in superovulated ewes impairs the subsequent in vivo and in vitro development of their ova." Anim. Reprod. Sci 39: 89-107.

McMillen, I. C., L. Rattanatray, et al. (2009). The Early Origins of Later Obesity: Pathways and Mechanisms. Early Nutrition Programming and Health Outcomes in Later Life: 71-81.

171

Mcmillen, I. C. and J. S. Robinson (2005). "Developmental origin of the metabolic syndrome: prediction, plasticity and programming." Physiol Rev 85: 571-633.

Medina-Gomez, G., S. Gray, et al. (2007). "Adipogenesis and lipotoxicity: role of peroxisome proliferator-activated receptor ? (PPAR?) and PPAR?coactivator-1 (PGC1)." Public Health Nutrition 10(10A): 1132-1137.

Merklin, R. (1973). "Growth and distribution of human fetal brown fat." Anta Rec 178: 673.

Mertens, I. L. and L. F. Van Gaal (2000). "Overweight, Obesity, and Blood Pressure: The Effects of Modest Weight Reduction." Obes Res 8(3): 270-278.

Minge, C. E., B. D. Bennett, et al. (2008). "Peroxisome Proliferator-Activated Receptor-{gamma} Agonist Rosiglitazone Reverses the Adverse Effects of Diet-Induced Obesity on Oocyte Quality." Endocrinology 149(5): 2646-2656.

Ministry of Agriculture, F. a. F. (1984). Energy Allowances and Feeding Systems for Ruminants. London, England, Her Majesty's Stationery Office.

Montague, C. T., J. B. Prins, et al. (1997). "Depot- and sex-specific differences in human leptin mRNA expression: implications for the control of regional fat distribution." Diabetes 46(3): 342-347.

Muhlhausler, B. S., C. L. Adam, et al. (2005). "Impact of glucose infusion on the structural and functional characteristics of adipose tissue and on hypothalamic gene expression for appetite regulatory neuropeptides in the sheep fetus during late gestation." J Physiol (Lond) 565(1): 185-195.

Muhlhausler, B. S., J. A. Duffield, et al. (2007). "Increased Maternal Nutrition Increases Leptin Expression in Perirenal and Subcutaneous Adipose Tissue in the Postnatal Lamb." Endocrinology 148(12): 6157-6163.

Muhlhausler, B. S., J. A. Duffield, et al. (2007). "Increased Maternal Nutrition Stimulates Peroxisome Proliferator Activated Receptor-{gamma}, Adiponectin, and Leptin Messenger Ribonucleic Acid Expression in Adipose Tissue before Birth." Endocrinology 148(2): 878-885.

Muhlhausler, B. S., C. T. Roberts, et al. (2003). "Determinants of Fetal Leptin Synthesis, Fat Mass, and Circulating Leptin Concentrations in Well-Nourished Ewes in Late Pregnancy." Endocrinology 144(11): 4947-4954.

Muhlhausler, B. S., Roberts, C. T., McFarlane, J. R., Kauter, K. G. and McMillen, I. C. (2002). "Fetal Leptin Is a Signal of Fat Mass Independent of Maternal Nutrition in Ewes Fed at or above Maintenance Energy Requirements1." Biology of Reproduction 67: 493-499.

Norman, R. J., M. Noakes, et al. (2004). "Improving reproductive performance in overweight/obese women with effective weight management." Hum Reprod Update 10(3): 267-280.

Ntambi, J. M. and K. Young-Cheul (2000). "Adipocyte Differentiation and Gene Expression." J. Nutr. 130(12): 3122S-3126.

O'Callaghan, D. and M. P. Boland (1999). "Nutritional effects on ovulation, embryo development and the establishment of pregnancy in ruminants." Animal Science 68: 299-314.

O‟Callaghan, D., H. Yaakub, et al. (2000). "Effect of nutrition and superovulation on oocyte morphology, follicular fluid composition and systemic hormone

172

concentrations in ewes." Journal of Reproduction and Fertility 118: 303-313.

Ogden, C. L., K. M. Flegal, et al. (2002). "Prevalence and Trends in Overweight Among US Children and Adolescents, 1999-2000." JAMA 288(14): 1728-1732.

Oken, E. and M. W. Gillman (2003). "Fetal Origins of Obesity." Obesity 11(4): 496-506.

Pantaleon, M. and P. L. Kaye (1998). "Glucose transporters in preimplantation development." Reviews of Reproduction 3: 77-81.

Parr, R. A. (1992). "Nutrition-progesterone interactions during early pregnancy in sheep." Reproduction, Fertility and Development 4(3): 297-300.

Parr, R. A., I. F. Davis, et al. (1987). "Overfeeding during early pregnancy reduces peripheral progesterone concentration and pregnancy rate in sheep." J Reprod Fertil 80(1): 317-320.

Parsons, T. J., C. Power, et al. (2001). "Fetal and early life growth and body mass index from birth to early adulthood in 1958 British cohort: longitudinal study." BMJ 323(7325): 1331-1335.

Pedersen, J. (1954). "Glucose content of the amniotic fluid in diabetic pregnancies correlations with the maternal blood sugar." Acta Endrocinologica 15: 342-350.

Pedersen, J. (1954). "Weight and length at birth of infants of diabetic mothers." Acta Endrocinologica 16: 330-342.

Pedersen, J., Bojsen-Moller, B. and Poulsen, H (1954). "Blood sugar in the new born infants of diabetic mothers." Acta Endrocinologica 15: 33-52.

Phillips, D., D. Barker, et al. (1994). "Thinness at birth and insulin resistance in adult life." Diabetologia 37(2): 150-154.

Picard, F. d. r. and J. Auwerx (2002). "PPARÎ³ AND GLUCOSE HOMEOSTASIS." Annual Review of Nutrition 22(1): 167-197.

Pietiläinen, K. H., Kaprio, J., Räsänen, M., Winter, T., Rissanen, A. and Rose, R. J. (2001). "Tracking of Body Size from Birth to Late Adolescence: Contributions of Birth Length, Birth Weight, Duration of Gestation, Parents‟ Body Size, and Twinship." American Journal of Epidemiology 154(1): 21-29.

Plagemann, A., T. Harder, et al. (1997). "Glucose tolerance and insulin secretion in children of mothers with pregestational IDDM or gestational diabetes." Diabetologia(40(9)): 1094-1100.

Quigley, S. P., D. O. Kleemann, et al. (2005). "Myogenesis in sheep is altered by maternal feed intake during the peri-conception period." Animal Reproduction Science 87(3-4): 241-251.

Rasmussen, F. and M. Johansson (1998). "The relation of weight, length and ponderal index at birth to body mass index and overweight among 18-year-old males in Sweden " European Journal of Epidemiology 14: 373-380.

Ravelli, A. C. J., J. H. P. van der Meulen, et al. (1999). "Obesity at the age of 50 y in men and women exposed to famine prenatally." Am J Clin Nutr 70(5): 811-816.

Ravelli, G. P., Z. A. Stein, et al. (1976). "Obesity in young men after famine exposure in utero and early infancy." N Engl J Med 295(7): 349-353.

Reiss, K., W. Cheng, et al. (1996). "Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice."

173

Proceedings of the National Academy of Sciences of the United States of America 93(16): 8630-8635.

Rhind, S. M. (2004). "Effects of maternal nutrition on fetal and neonatal reproductive development and function." Animal Reproduction Science 82-83: 169-181.

Rhind, S. M., M. T. Rae, et al. (2001). "Effects of nutrition and environmental factors on the fetal programming of the reproductive axis." Reproduction 122(2): 205-214.

Rhodes, F. M., L. A. Fitzpatrick, et al. (1995). "Sequential changes in ovarian follicular dynamics in Bos indicus heifers before and after nutritional anoestrus." J Reprod Fertil 104(1): 41-49.

Roseboom, T. J., J. H. P. van der Meulen, et al. (2000). "Coronary heart disease after prenatal exposure to the Dutch famine, 1944-45." Heart 84(6): 595-598.

Roseboom, T. J., J. H. P. van der Meulen, et al. (2001). "Effects of prenatal exposure to the Dutch famine on adult disease in later life: an overview." Molecular and Cellular Endocrinology 185(1-2): 93-98.

Rosen, E. D., C.-H. Hsu, et al. (2002). "C/EBPalpha induces adipogenesis through PPARgamma : a unified pathway." Genes Dev. 16(1): 22-26.

Rosen, E. D., Sarraf, P., Troy, A.E., Bradwin, G., Moore, K., Milestone, D.S., Spiegelman, B.M. nad Mortensen, R.M. (1999). "PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro." Molecular cell 4(4): 611-617.

Rosen, E. D. and B. M. Spiegelman (2000). "Molecular regulation of adipogenesis." Annual Review of Cell and Developmental Biology 16(1): 145-171.

Russel, A. J. F., J. M. Doney, et al. (1969). "Subjective assessment of body fat in live sheep." J Agric Sci (Camb) 72: 451-454.

Shankar, K., A. Harrell, et al. (2008). "Maternal obesity at conception programs obesity in the offspring." Am J Physiol Regul Integr Comp Physiol 294(2): R528-538.

Sherwood, L. (2004). Human physiology from cells to systems. CA, U.S.A, Thomson Learning- Brooks/Cole.

Silverman, B., T. Rizzo, et al. (1998). "Long-term effects of the intrauterine environment. The Northwestern University Diabetes in Pregnancy Center." Diabetes Care 21: B142-149.

Silverman, B., T. Rizzo, et al. (1991). "Long-term prospective evaluation of offspring of diabetic mothers." Diabetes 40(Suppl 2): 121-125.

Singhal, A. and A. Lucas (2004). "Early origins of cardiovascular disease: is there a unifying hypothesis?" The Lancet 363(9421): 1642-1645.

Sloboda, D. M., T. J. M. Moss, et al. (2005). "Hepatic glucose regulation and metabolism in adult sheep: effects of prenatal betamethasone." Am J Physiol Endocrinol Metab 289(4): E721-728.

Spiegelman, B. S. and H. Green (1980). "Control of Specific Protein Biosynthesis during the Adipose Conversion of 3T3 Cells." The Journal of Biol Chem 255(18): 8811-8881.

Stanner, S. A., K. Bulmer, et al. (1997). "Does malnutrition in utero determine diabetes and coronary heart disease in adulthood? Results from the Leningrad siege study, a cross sectional study." BMJ 315(7119): 1342-1348.

174

Stevens, D., G. Alexander, et al. (1990). "Effect of prolonged glucose infusion into fetal sheep on body growth, fat deposition and gestation length." J Dev Physiol 13(5): 277-281.

Symonds, M. E., A. Mostyn, et al. (2003). "Endocrine and nutritional regulation of fetal adipose tissue development." J Endocrinol 179(3): 293-299.

Tapanainen, P., E. Leinonen, et al. (2001). "Leptin concentrations are elevated in newborn infants of diabetic mothers." Horm Res 55: 185-190.

Tavakkol, A., F. A. Simmen, et al. (1988). "Porcine Insulin-Like Growth Factor-I (pIGF-I): Complementary Deoxyribonucleic Acid Cloning and Uterine Expression of Messenger Ribonucleic Acid Encoding Evolutionary Conserved IGF-I Peptides." Mol Endocrinol 2(8): 674-681.

Tong, J. F., X. Yan, et al. (2009). "Maternal obesity downregulates myogenesis and {beta}-catenin signaling in fetal skeletal muscle." Am J Physiol Endocrinol Metab 296(4): E917-924.

Tontonoz, P., Hu, E. and Spiegelman, B.M. (1994). "Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor." Cell 79(7): 1147-1156.

Toyozaki, T., M. Hiroe, et al. (1993). "Insulin-like growth factor-1 receptors in human cardiac myocytes and their relation to myocardial hypertrophy." Jpn. Circ. J 57(1120-27).

Trayhurn, P. and J. H. Beattie (2001). "Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ." Proceedings of the Nutrition Society 60(03): 329-339.

Trayhurn, P., C. Bing, et al. (2006). "Adipose Tissue and Adipokines--Energy Regulation from the Human Perspective." J. Nutr. 136(7): 1935S-1939.

Trayhurn, P., M. Hoggard, et al. (1999). "International journal of obesity.". Van Vliet, G., D. M. Styne, et al. (1983). "Growth hormone treatment for short

stature." N Engl J Med 309: 1016–1022. Villafuerte, B. C., J. B. Fine, et al. (2000). "Expressions of Leptin and Insulin-Like

Growth Factor-I Are Highly Correlated and Region-Specific in Adipose Tissue of Growing Rats." Obesity 8(9): 646-655.

Voet, D. a. V., J.G, Ed. (2004). Biochemistry. Hoboken, NY, J. Wiley & Sons. von Eynatten, M., J. G. Schneider, et al. (2004). "Decreased Plasma Lipoprotein

Lipase in Hypoadiponectinemia: An association independent of systemic inflammation and insulin resistance " Diabetes Care 27(12): 2925-2929.

Wabitsch, M., H. Hauner, et al. (1995). "The role of growth hormone/insulin-like growth factors in adipocyte differentiation." Metabolism 44(10 Suppl 4): 45-49.

Wallace, J. M., D. A. Bourke, et al. (1999). "Switching Maternal Dietary Intake at the End of the First Trimester Has Profound Effects on Placental Development and Fetal Growth in Adolescent Ewes Carrying Singleton Fetuses." Biol Reprod 61(1): 101-110.

Watkins, A. J., D. Platt, et al. (2007). "Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure." Proceedings of the National Academy of Sciences 104(13): 5449-5454.

Whitaker, R. C., Pepe, M. S., Seidel, K. D., Wright, J. A. and Knopp, R. H. (1998). "Gestational Diabetes and the Risk of Offspring Obesity." Pediatrics 101(2): e9.

175

Whittle, W. L., A. C. Holloway, et al. (2000). "Prostaglandin production at the onset of ovine parturition is regulated by both estrogen-independent and estrogen-dependent pathways." Endocrinology 141: 3783-3791.

Whittle, W. L., F. A. Patel, et al. (2001). "Glucocorticoid Regulation of Human and Ovine Parturition: The Relationship Between Fetal Hypothalamic-Pituitary-Adrenal Axis Activation and Intrauterine Prostaglandin Production." Biology of Reproduction 64(4): 1019-1032.

Yaakub, H., S. A. Williams, et al. (1997). "Effect of dietary intake and glucose infusion on ovulation rate and embryo quality in superovulated ewes." J Reprod Fertil 19: 57-58.

Yajnik, C. S. (2002). "The lifecycle effects of nutrition and body size on adult adiposity, diabetes and cardiovascular disease." Obesity Reviews 3(3): 217-224.

Yang, W.-S., W.-J. Lee, et al. (2001). "Weight Reduction Increases Plasma Levels of an Adipose-Derived Anti-Inflammatory Protein, Adiponectin." J Clin Endocrinol Metab 86(8): 3815-3819.

Yu-Poth, S., G. Zhao, et al. (1999). "Effects of the National Cholesterol Education Program's Step I and Step II dietary intervention programs on cardiovascular disease risk factors: a meta-analysis." Am J Clin Nutr 69(4): 632-646.

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