optimisation of oocyte in vitro maturation using oocyte ... · optimisation of oocyte in vitro...

OPTIMISATION OF OOCYTE

IN VITRO MATURATION USING

OOCYTE SECRETED FACTORS

Jaqueline Sudiman, MD, MRepSc

Robinson Institute

School of Paediatrics and Reproductive Health

Research Centre for Reproductive Health

Discipline of Obstetrics and Gynaecology

University of Adelaide, Adelaide

Australia

Thesis submitted to the University of Adelaide in total fulfilment of the

requirements for admission to the degree Doctor of Philosophy

October 2014

This is dedicated to my beloved father who taught me that

women have the right and equal opportunity

to an education as men

i

Abstract

During follicular development, oocyte and somatic cells communicate by producing

signals such as proteins, growth factors, hormones that interact in complex harmony.

The oocyte plays an active role in this communication by secreting soluble oocyte

secreted factors (OSFs) including growth differentiation factor 9 (GDF9) and bone

morphogenetic factor 15 (BMP15). Those two growth factors have been implicated in

follicular development and are related to female fertility. In vitro matured oocytes

have aberrant gene expression and altered matrix protein profiles in cumulus cells

compared to their in vivo mature counterparts, which leads to a decrease in oocyte

quality and embryo development post fertilisation.

The first aim of this study was to determine the effect of exogenous native OSFs from

denuded oocytes (DOs) on mouse in vitro maturation (IVM). In a series of

experiments, native OSFs were used in various ways to observe the role of FSH,

cumulus cells, and temporal effects, on production of native OSFs. Overall, co-

culture of COCs with DOs improved mouse embryo development. The highest

improvement in embryo development and embryo quality post co-culture of COCs

with DOs was when COCs were matured in separate IVM media in the presence of

FSH, then denuded at 3h to produce DOs, which were used in co-culture with COCs

for another 14-15 h. Unfortunately, it is not practical to generate such large numbers

of DOs in a clinical scenario. Therefore the next step was to find the functional

bioactive forms of pure recombinant proteins that could improve IVM outcomes.

The above concept led to the main hypothesis of this thesis that the developmental

competence of IVM oocytes can be improved by novel variants of purified

recombinant GDF9 and BMP15. In these studies, GDF9 and BMP15 from various

sources and forms were tested in cattle and mouse IVM. Two different forms of

proteins: pro-mature complex and mature domain of GDF9 and BMP15, were used in

cattle and mouse IVM. In this study, pro-mature complex of human BMP15

(hBMP15) improved cattle blastocyst development, which may, in part, be due to an

increased in nicotinamide adenine diphosphate [NAD(P)H] and reduced glutathione

(GSH) levels. Mature region of BMP15 had a moderate, albeit non-significant, effect

on cattle embryo developmental outcomes, whilst mature region of GDF9 was

ii

ineffective. Similar results were observed using pro-mature hBMP15, which improved

the developmental competence of in vitro matured mouse oocytes, where a

combination of mouse GDF9 (mGDF9) and hBMP15 (both in pro-mature complex

form) produces the highest blastocyst rate.

The work presented in this thesis has provided evidence that exogenous native

OSFs, and recombinant hBMP15 in its pro-mature complex form, are important for

oocyte developmental programming and prove useful for improving mouse and cattle

IVM oocyte developmental competence. Moreover, the source, doses and form of

recombinant proteins play an important role in improving developmental competence

of IVM oocytes. These results may contribute and translate to improve the success

rate of in vitro matured human oocytes.

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Declaration

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

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

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

person, except where due reference has been made in the text.

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

being made available for loan and photocopying, subject to the provisions of the

Copyright Act 1968.

Jaqueline Sudiman

September 2014

iv

Acknowledgements

I would like to express my sincere gratitude to my supervisors: Robert Gilchrist,

Jeremy Thompson and David Mottershead. Thank you for the opportunity to learn,

for the advice and guidance during my PhD candidature. The lessons you have

taught me, which are to be more critical, cautious and enthusiastic in science have

been tremendous help during my candidature and for my future career.

I must also give an enormous thank you to Lesley Ritter for assisting me with my

experiments and Melanie Sutton-McDowall for guidance in oocyte metabolism. My

very special thanks go to Satoshi Sugimura for helping me with everything and

making me believes in myself during my candidature.

Thanks to all the members of Gilchrist, Thompson and Mottershead groups. Post-

doc, fellow students and research assistants over the past 3 years: Laura Frank,

Dulama Richani, Xiaoqian Wang, Ryan Rose, Hannah Brown, Deanne Feil, Marie

Anastasi, Melissa White and Jun Yan Shi. Thank for all your friendship and supports.

I would also like to thank to all members in Robinson Institute that I can’t mention one

by one for their friendship especially for Izza Tan, Noor Lokman, Kay Govin, Linda

Liu, Tod Fullston, Hong Liu, Nicole Palmer, Lauren Sandeman, Wan Xian Kang and

Gary Heinemann. Also thanks to the animal house staff and Don for taking care of

mice and transporting the ovaries. I would also like send my thanks to Michelle

Lorimer for statistical support.

I must thank two of my very special friends during my “insane period of time”: Pranay

Sharma and Riksfardini Ermawar. Thank you for your friendships and continuous

support during my hard times and the tears.

My sincere thanks to Kencana Dharmapatni, Tony and Nes Lian-Llyod, Jane Dunn

and Bert, and Sandra and Wayne Turner to show me different side of Australia (My

cubicle and lab station are only a small part of Australia )

My PhD was financially supported by the International postgraduate research

scholarship. I would also like to acknowledge the financial support of Faculty of

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Health Sciences and Robinson Institute for international and domestic travel

opportunities.

I would like to acknowledge my mum, brother, my very special sister, Josephine

Sudiman, for keeping me strong and supporting me with love and encouragement to

finish my PhD (no matter how hard it is!). Thanks to my grandma and dad for raising

me to believe that having education is a better way to grow up (I know you are

always here with me).

Finally, I would also like to say thanks to myself to be so strong and determine to

finish my PhD! Doing a PhD far away from your family is not an easy way but

Australia is the country where life seems easier and happier.

vi

Publications arising from this thesis

1. Sudiman, J, LJ Ritter, DK Feil, X Wang, K Chan, DG Mottershead, DM

Robertson, JG Thompson, and RB Gilchrist. (2014a) Effects of differing

oocyte-secreted factors during mouse in vitro maturation on subsequent

embryo and fetal development. J Assist Reprod Genet 31 295-306.

2. Sudiman, J, ML Sutton-McDowall, LJ Ritter, MA White, DG Mottershead, JG

Thompson, and RB Gilchrist. (2014b) Bone morphogenetic protein 15 in the

pro-mature complex form enhances bovine oocyte developmental

competence. PLoS One 9(7): e103563.

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Conference Proceedings

1. Sudiman J, Ritter LJ, Mottershead DG, Thompson JG and Gilchrist RB. The

Effect of Native and Recombinant Oocyte-Secreted Factors on In Vitro

Maturation. Poster was presented at Australian Society of Medical Research

in Adelaide (2011).

2. Sudiman J, Ritter LJ, Mottershead DG, Thompson JG and Gilchrist RB.

Oocyte Secreted Factors Improve the In Vitro Maturation of Mouse Oocytes

and Subsequent Embryo Development. Abstract was presented at Indonesian

Fertility and Endocrinology Society in Bali (2011).

3. Sudiman J, Ritter LJ, Feil DK, Wang X, Mottershead DG, Thompson JG and

Gilchrist RB. Effect of the Form and Temporal Addition of Oocyte-Secreted

Factors during In Vitro Maturation. Poster was presented at Robinson Institute

Research Symposium in Adelaide (2012), Ovarian Club in Prague (2012) and

Post graduate Research Conference in Adelaide (2013).

4. Sudiman J, Sutton-McDowall M, Ritter LJ, White MA, Mottershead DG,

Thompson JG and Gilchrist RB. Pro-mature Form of Bone Morphogenetic

Protein 15 (BMP15) in Oocyte Maturation Improves Subsequent Bovine

Embryo Development. Abstract was presented for Meat and Livestock finalist

award at Society for Reproductive Biology in Sydney (2013).

viii

Table of contents

Abstract………………………………………………………………………………………...i

Declaration……………………………………………………………………………………iii

Acknowledgements……………………………………………………………………….....iv

Publications arising from this thesis……………………………………………………….vi

Conference proceedings…………………………………………………………………...vii

Table of contents…………………………………………………………………………...viii

List of figures………………………………………………………………………………..xiii

List of tables………………………………………………………………………………....xv

Abbreviations……………………………………………………………………………….xvii

CHAPTER 1 LITERATURE REVIEW ......................................................................... 1

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

1.2 NATURAL FOLLICULAR AND OOCYTE DEVELOPMENT ..................... 3

1.2.1 Maintenance and triggering oocyte nuclear maturation………….. . 5

1.2.2 Cytoplasmic maturation……………………………………………… . 7

1.2.3 Development of oocytes after fertilisation………………………….. 8

1.3 OOCYTE-SOMATIC CELL INTERACTIONS ........................................... 9

1.3.1 Gap Junction Signalling………………………………………………. 9

1.3.2 Paracrine Signalling…………………………………………………. 10

1.3.2.1 Promotion of Follicular Growth ........................................................... 10

1.3.2.2 Regulation of Granulosa and Cumulus Cell Proliferation and Cumulus

Cell Expansion ................................................................................................ 11

1.3.2.3 Prevention of Cumulus Cell Apoptosis ............................................... 12

1.3.2.4 Regulation of Steroidogenesis (Luteinization) and Differentiation of

Granulosa and Cumulus Cells ......................................................................... 12

1.4 TGF-Β SUPERFAMILY .......................................................................... 13

1.4.1 GDF9………………………………………………………………….. 13

ix

1.4.2 BMP15………………………………………………………………… 14

1.4.3 Mutation of GDF9 and BMP15……………………………………… 14

1.4.4 TGF-β Superfamily Signalling………………………………………. 15

1.4.5 Structure and Production of In vitro Recombinant GDF9 and BMP15………………………………………………………………………... 18

1.5 FIBROBLAST GROWTH FACTORS (FGFS) ......................................... 22

1.6 OOCYTE METABOLISM........................................................................ 23

1.6.1 Glutathione…………………………………………………………… 25

1.6.2 Role of OSFs in Cumulus and Oocyte Metabolism………………. 27

1.7 IN VITRO OOCYTE MATURATION ....................................................... 27

1.7.1 Selection of Oocytes for In Vitro Maturation……………………… 28

1.7.2 In Vitro Maturation vs In Vivo Maturation…………………………. 29

1.7.3 Ooctye Secreted Factors in the In Vitro Maturation of Oocytes… 29

1.8 CONCLUSION ....................................................................................... 31

1.9 HYPOTHESIS AND AIMS ...................................................................... 32

1.9.1 Hypothesis…………………………………………………………….. 32

1.9.2 Aims……………………………………………………………………. 32

CHAPTER 2 FACTORS AFFECTING THE CAPACITY OF EXOGENOUS OOCYTE SECRETED FACTORS TO IMPROVE MOUSE EMBRYO AND FETAL DEVELOPMENT POST-IVM ..................................................................................... 33

2.1 ABSTRACT ............................................................................................ 34

2.2 INTRODUCTION .................................................................................... 35

2.3 MATERIALS AND METHODS ............................................................... 38

2.3.1 Isolation and maturation of COCs…………………………………. 38

2.3.2 Generation of denuded oocytes as a source of native OSFs……38

2.3.3 Oocyte nuclear maturation…………………………………………. 41

2.3.4 In vitro fertilization…………………………………………………… 41

2.3.5 In vitro culture of embryos………………………………………….. 41

x

2.3.6 Differential staining of blastocysts…………………………………. 41

2.3.7 Vitrification and warming of blastocysts…………………………… 42

2.3.8 Embryo transfer and assessment of fetal outcomes……………...42

2.3.9 Immunodetection of oocyte GDF9…………………………………. 43

2.4 STATISTICAL ANALYSES ..................................................................... 44

2.5 RESULTS ............................................................................................... 44

2.5.1 Effect of co-culture of intact mouse COCs with native OSFs during IVM……………………………………………………………………………. 44

2.5.2 Temporal effect of co-culture of intact mouse COCs with native OSFs during IVM……………………………………………………………. 46

2.5.3 Effect of exposure of COCs to native OSFs on nuclear maturation ………………………………………………………………………….49

2.5.4 Temporal effects on oocyte GDF9 levels………………………….. 50

2.5.5 The role of FSH and cumulus cells in the production of native OSFs…………………………………………………………………………. 52

2.5.6 Effect of exposure of COCs to native OSFs during IVM on cryo-survival of blastocysts………………………………………………………. 55

2.5.7 Effect of exposure of COCs to native OSFs on pregnancy rates and fetal outcomes………………………………………………………….. 56

2.6 DISCUSSION ......................................................................................... 59

CHAPTER 3 BONE MORPHOGENETIC PROTEIN 15 IN THE PRO-MATURE COMPLEX FORM ENHANCES BOVINE OOCYTE DEVELOPMENTAL COMPETENCE ......................................................................................................... 63

3.1 ABSTRACT ............................................................................................ 64

3.2 INTRODUCTION .................................................................................... 65

3.3 MATERIALS AND METHODS ................................................................. 67

3.3.1 Collection of cells…………………………………………………….. 67

3.3.2 Granulosa cell thymidine assay…………………………………….. 68

3.3.3 Sources of BMP15 and GDF9 and treatment of COCs…………. 68

3.3.4 In vitro fertilization (IVF) and embryo culture………………………69

3.3.5 Blastocyst differential staining……………………………………… 70

xi

3.3.6 Glucose and lactate measurements……………………………….. 70

3.3.7 Autofluorescence of NAD(P)H and FAD++………………………… 71

3.3.8 Reduced glutathione (GSH)………………………………………… 71

3.4 STATISTICAL ANALYSIS ...................................................................... 71

3.5 RESULTS ............................................................................................... 72

3.5.1 Bioactivity of different BMP15 and GDF9 forms………………….. 72

3.5.2 Dose-response of pro-mature BMP15 on oocyte developmental competence………………………………………………………………….. 73

3.5.3 Comparison of pro-mature BMP15, mature BMP15 and mature GDF9 during IVM on subsequent embryo development………………... 74

3.5.4 Effect of pro-mature BMP15, mature BMP15 and mature GDF9 on COC glucose consumption and lactate production……………………… 78

3.5.5 Effect of pro-mature BMP15, mature BMP15 and mature GDF9 on oocyte FAD++ and NAD(P)H autofluorescence………………………….. 80

3.5.6 Effect of pro-mature BMP15, mature BMP15 and mature GDF9 on oocyte GSH………………………………………………………………….. 82

3.6 DISCUSSION ......................................................................................... 83

CHAPTER 4 EFFECT OF INTERACTIONS BETWEEN DIFFERING FORMS OF RECOMBINANT OOCYTE SECRETED FACTORS ON MOUSE OOCYTE DEVELOPMENTAL COMPETENCE ........................................................................ 87

4.1 ABSTRACT ............................................................................................ 88

4.2 INTRODUCTION .................................................................................... 89

4.3 MATERIAL AND METHODS .................................................................. 91

4.3.1 Isolation and maturation of COCs…………………………………. 91

4.3.2 Sources of BMP15 and GDF9 and treatment of COCs………….. 92

4.3.3 In vitro fertilization……………………………………………………. 93

4.3.4 In vitro culture of embryos…………………………………………… 94

4.3.5 Differential staining of blastocysts…………………………………. 94

4.4 RESULTS ............................................................................................... 95

xii

4.4.1 The effect of pro-mature mGDF9 and hBMP15 alone or in combination during IVM on subsequent embryo development………… 95

4.4.2 Interactions between GDF9 and BMP15: effect of differing forms during IVM on subsequent embryo development……………………….. 97

4.5 DISCUSSION ....................................................................................... 100

CHAPTER 5 GENERAL DISCUSSION .................................................................. 104

BIBLIOGRAPHY ..................................................................................................... 115

APPENDICES ......................................................................................................... 143

xiii

List of figures

Figure 1.1 Follicular development stages .................................................................. ..5

Figure 1.2 Oocyte-somatic cell interactions that regulate in vivo matured oocytes......7

Figure 1.3 Schematic representations of GDF9 and BMP15 homodimers and putative

GDF9:BMP15 heterodimers signalling in regulating cumulus and granulosa cell

functions.. .................................................................................................................. 18

Figure 1.4 The forms of mouse GDF9 and BMP15……………………………………..20

Figure 1.5 The structure of GDF9 and BMP15-post purification (pro-mature

complex/processed protein) which consist of dimers of mature regions and

associated pro-regions……………………………………………………………………..22

Figure 1.6 Glucose utilization in cumulus cells and oocytes…………………………..24

Figure 2.1 Schematic illustration of methods of producing native OSFs based on

pretreatment of oocytes with and without cumulus cells and FSH ............................ 40

Figure 2.2 GDF9 Western blot comparison of denuded oocyte (DO) extracts at 0h

and 3h (3h+FSH+CC) of pretreatment ...................................................................... 50

Figure 2.3 Quantitative analysis of Western blot denuded oocyte extracts at 0 and 3h

(3h+FSH+CC) of pretreatment……………………………………………………………51

Figure 2.4 Effect of treatment with native OSFs during IVM on subsequent outcomes

.................................................................................................................................. 57

Figure 3.1 Bioactivity of different BMP15 and GDF9 forms ....................................... 72

Figure 3.2 Glucose consumption and lactate production .......................................... 79

Figure 3.3 Intra-oocyte NAD(P)H, FAD ++, REDOX ratio ........................................... 81

Figure 3.4 Intra-oocyte GSH level ............................................................................. 82

Figure 4.1 In vitro maturation of mouse COCs in low-binding tube…………………...92

xiv

Figure 5.1 Different forms of GDF9 and BMP15 proteins ........................................ 111

xv

List of tables

Table 2.1 Effect of native OSFs during IVM on subsequent embryo development ... 45

Table 2.2 Effect of native OSFs during IVM on inner cell mass (ICM), trophectoderm

(TE) and total cell numbers (TCN) of day 6 blastocysts ............................................ 45

Table 2.3 Temporal effect of co-culture of intact mouse COCs with native OSFs on

subsequent embryo development ............................................................................. 47


inner cell mass (ICM), trophectoderm (TE) and total cell numbers (TCN) of day 6

blastocysts ................................................................................................................ 48

Table 2.5 Effect of native OSFs added at 3 hours on oocyte nuclear maturation ..... 49

Table 2.6 Effects of cumulus cells and FSH on native OSFs efficacy at enhancing

post-IVM embryo development ................................................................................. 53

Table 2.7 Effects of cumulus cells and FSH on native OSFs on inner cell mass (ICM),

trophectoderm (TE) cells and total cell numbers (TCN) of day 6 blastocysts ............ 54

Table 2.8 Effect of native OSFs during IVM on the survival rate of post-warmed

blastocysts ................................................................................................................ 55

Table 2.9 Effect of native OSFs during IVM on subsequent fetal and placental weight

and crown-rump length ............................................................................................. 58

Table 3.1 Effect of graded doses of pro-mature BMP15 during IVM on oocyte

developmental competence ...................................................................................... 73

Table 3.2 Effect of supplementing COC cultures with different forms of BMP15

(mature BMP15 and pro-mature BMP15), mature GDF9 and FSH on oocyte

developmental competence ...................................................................................... 75

Table 3.3 Effect of supplementing COC cultures with different forms of BMP15

(mature BMP15 and pro-mature BMP15), mature GDF9 in the absence of FSH on

oocyte developmental competence ........................................................................... 76

xvi

Table 3.4 Number of total (TCN), inner cell mass (ICM) and trophectoderm (TE) cells

on day 8 expanded, hatching and hatched blastocysts following co-culture of COCs

with different forms of BMP15 (mature BMP15 and pro-mature BMP15), mature

GDF9 in the presence of FSH ................................................................................... 77

Table 3.5 Number of total (TCN), inner cell mass (ICM) and trophectoderm (TE) cells

on day 8 expanded, hatching and hatched blastocysts following co-culture of COCs

with different forms of BMP15 (mature BMP15 and pro-mature BMP15), mature

GDF9 in the absence of FSH .................................................................................... 77

Table 4.1 Effect of supplementing COC cultures with mGDF9, hBMP15 (pro-mature

complexes) and combination of both proteins on oocyte developmental

competence…………………………………………………………………………………96


complexes) and combination of both proteins on numbers of inner cell mass (ICM),

trophectoderm (TE) and total (TCN) cells in blastocysts ………….............................97

Table 4.3 Effect of supplementing COC cultures with a combination of hGDF9 and

hBMP15 (pro-mature complexes), a combination of mGDF9 and hBMP15 (isolated

mature region), or a hGDF9:hBMP15 heterodimer (isolated mature regions) on

oocyte developmental competence………………………………………………………98

Table 4.4 Effect of supplementing COC cultures with a combination of hGDF9 and

hBMP15 (pro-mature complexes), a combination of mGDF9 and hBMP15 (isolated

mature region), or a hGDF9:hBMP15 heterodimer (isolated mature regions) on

numbers of inner cell mass (ICM), trophectoderm (TE) and total (TCN) cells in

blastocysts ……………………………………………………………………………….....99

Table 5.1 Summary of developmental competency of mouse and cattle oocytes

undergoing IVM with the addition of native OSFs anddifferent forms and sources of

GDF9 and BMP15………………………………………………………………………...112

xvii

Abbreviations

ART assisted reproductive technology BSA bovine serum albumin BMP15 bone morphogenetic protein 15 hBMP15 human bone morphogenetic protein 15 BMPR2 bone morphogenetic protein receptor type II cAMP cyclic adenosine monophosphate COC cumulus oocyte complex DO denuded oocyte ECM extracellular matrix EGF epidermal growth factor FAD flavin adenine dinucleotide FAF fatty acid free FSH follicle stimulating hormone FGF fibroblast growth factor GC granulosa cell GDF9 growth differentiation factor 9 hGDF9 human growth differentiation factor 9 mGDF9 mouse growth differentiation factor 9 GSH reduced form of glutathione GV germinal vesicle GVBD germinal vesicle breakdown GJC gap junction communication HEK-293T human embryonic kidney cell line H-αMEM hepes-buffered minimum essential medium

xviii

H-TCM hepes-buffered tissue culture medium ICM inner cell mass IBMX 3-isobutyl-1-methylxanthine IVC in vitro culture IVF in vitro fertilization IVM in vitro maturation IVP in vitro production KL kit ligand LH luteinising hormone LHR luteinising hormone receptor MI metaphase I MII metaphase II NADPH nicotinamide adenine dinucleotide phosphate OOX oocytectomised complex OSF oocyte-secreted factor PMSG pregnant mare’s serum gondadotropin PPP pentose phosphate pathway TCM-199 tissue culture medium 199 TCN total cell number TE trophectoderm TGFβ transforming growth factor β TGFβR-II transforming growth factor β receptor type-II

Chapter 1: Literature Review

1

CHAPTER 1

LITERATURE REVIEW


2

1.1 INTRODUCTION

The demand for assisted reproductive technology (ART) has increased over the last

few decades due to the rising rate of infertility among couples. This technology has

been applied successfully, with some IVF clinics achieving pregnancy rates of 30-

40% per embryo transfer. However, this technology has a drawback with the use of

gonadotropin hormones to generate large numbers of mature oocytes. These

hormones are expensive, and in countries where the cost of using ART comes from

the patient’s pocket, it is a substantial financial burden for the patients. The use of

these hormones is also associated with patient inconvenience due to the

administration by subcutaneous or intramuscular injection and the risk of ovarian

hyperstimulation syndrome (OHSS). In vitro maturation (IVM) of oocytes is an

emerging technology that eliminates or substantially reduces the use of gonadotropin

hormones. It is especially useful for women with polycystic ovary syndrome (PCOS)

who are less able to tolerate the side effects of these hormones. IVM also

significantly decreases the cost of ART which could allow more infertile couples from

low economic levels to access this technology. However, there is a discrepancy in

the success rate of embryos derived from in vitro matured oocytes compared to in

vivo matured oocytes, with the pregnancy rate post-IVM less than half of the

pregnancy rate post-IVF (Gremeau, et al. 2012). To date, many ingredients have

been added to IVM medium to nurture the immature oocytes in the Petri dish (Albuz,

et al. 2010, Hussein, et al. 2006, Richani, et al. 2013, Yeo, et al. 2008). However,

further development is needed to achieve an acceptable pregnancy success rate for

IVM-derived oocytes. Therefore, there is an urgent need to extend and develop new

techniques/media to increase the quality of in vitro matured oocytes. This can be

achieved by understanding the mechanisms and components which are responsible

for oocyte developmental competence.

Oogenesis and folliculogenesis involve cellular and molecular communication

between the oocyte and its surrounding somatic cells. Endocrine, paracrine and

autocrine signalling regulate this process to provide the oocyte the capacity to

undergo meiotic resumption and support subsequent embryo and fetal development.

There are two major oocyte secreted factors (OSFs) that regulate granulosa/cumulus


3

cell function: bone morphogenetic protein 15 (BMP15), also known as GDF9b, and

growth differentiation factor 9 (GDF9) (Dube, et al. 1998, Nilsson and Skinner 2002).

Both of these proteins are expressed exclusively in oocytes from an early stage of

follicular development and are important in regulating female fertility. This review will

focuses on bi-directional communication between the oocyte and somatic cells

(granulosa cells and cumulus cells), particularly the transfer of proteins and

molecules that sustain the development of the somatic cells and the oocyte itself.

Secondly, it will focus on the role of OSFs, notably GDF9 and BMP15 in regulating

the function of somatic cells and the interaction and signalling which occurs between

these proteins and somatic cells. The function of GDF9 and BMP15 in reproduction

and the production of GDF9 and BMP15 in vitro will then be explained, followed by

the role of OSFs in oocyte metabolism. Finally, how OSFs improve the IVM system

will be discussed.

1.2 NATURAL FOLLICULAR AND OOCYTE DEVELOPMENT

During fetal life the primordial germ cells (PGCs), which originate from epiblast cells

in the posterior region of the primitive streak, proliferate and migrate through the yolk

sac, hindgut endoderm, and the dorsal mesentery of the hindgut and finally rests in

the genital ridges at the ventral sides of the mesonephroi. The initiation signal for

epiblast cells to proliferate into PGSs is regulated in part by transforming growth

factor β (TGF-β) family members, bone morphogenetic protein 4 (BMP4), BMP8b

and BMP2 (Ying, et al. 2001). During the migration, the germ cells transform into an

irregular shape with protrusions and pseudopodia, with the formitself crucial for the

migration act (Lin, et al. 1982). In the mouse, c-kit receptor tyrosine kinase, kit-ligand

(KL) or stem-cell factor (SCF), TIAR 1 and leukemia inhibitory factor (LIF) are factors

that are required for proliferation and migration of germ cells (Cheng, et al. 1994,

Godin, et al. 1991, Matsui, et al. 1990). In the genital ridges, the PGCs differentiate

into either as oogonia or spermatogonia (Picton, et al. 1998). In the ovary, oogonia

undergo mitotic divisions and connect with each other by intercellular cytoplasmic

bridges which are known as germ cell clusters or cortical cords (van den Hurk and

Zhao 2005). In the human ovary, the population of oogonia is approximately 600,000

at 8 weeks of intrauterine life and can reach a peak of 6-7 million by 20 weeks of


4

gestation (Strauss III and Barbieri 2009). Those oogonia then undergo further DNA

replication and enter the first meiosis divisions: leptotene, zygotene and pachytene

stages of meiotic prophase I before arresting at the diplotene stage (van den Hurk

and Zhao 2005). At the same time the meiotic process begins, the oogonia, which

are now called primary oocytes, will invest themselves with one layer of squamous

somatic cells to form primordial follicles. Oogonia undergo apoptosis if those germ

cells do not enter meiosis by the seventh month of gestation (Strauss III and Barbieri

2009). At the end of gestation, only approximately 700,000 primordial follicles are

found in human ovaries (Strauss III and Barbieri 2009). This number further reduces

by approximately 300,000 in puberty and only 400-500 grow into mature antral

follicles during a woman’s reproductive life (Strauss III and Barbieri 2009).

The development of primordial follicles to primary follicles is marked by the changing

of squamous cells of granulosa cells into a single layer of cuboidal cells. The single

layer of cuboidal cells develops into several layers of cuboidal granulosa cells

surrounding primary oocytes to form secondary follicles, and those cells start to

develop follicle stimulating hormone (FSH), estrogen and androgen receptors

(Gosden 2002, Lintern-Moore, et al. 1974). Blood vessels develop to supply the

follicle, and the zona pellucida becomes more highly structured (Strauss III and

Barbieri 2009). During the proliferation of granulosa cells in secondary follicles,

oocytes grow in diameter and volume extensively (Braw-Tal 2002) followed by the

development of theca layers from interstitial stroma. Refer to Fig. 1.1. The theca has

two layers: the theca interna is adjacent to the basal lamina of the follicle and

consists of steroidogenic cells and is highly vascularised, whilst, the theca externa

consists of non-steroidogenic cells (Magoffin 2005). Both granulosa and theca cells

are required for the production of steroids, via what is known as the two cell-two

gonadotropin concept of follicle steroid biosynthesis. Luteinizing hormone (LH)

stimulates theca cells to express cholesterol side-chain cleavage cytochrome P450

(CYP11A), 3-hydroxysteroid dehydrogenase (3β-HSD), and 17α-hydroxylase/C17-20

lyase cytochrome P450 (CYP17) enzymes, which are used to synthesise

androstenedione from cholesterol. FSH stimulates the expression of aromatase

cytochrome P450 (CYP19) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in the


5

granulosa cells. Those enzymes metabolise androstenedione to estradiol (van den

Hurk and Zhao 2005). The small follicle has a low ratio of estrogen to androgen, but

this ratio is slowly reversed during follicular growth. The dominant follicle can be

distinguished from other follicles by an increased level of FSH in the antral fluid

(Sullivan, et al. 1999), high concentrations of estradiol (van Dessel, et al. 1996), low

levels of androgen (McNatty, et al. 1975), a high mitotic index of the granulosa cells

(Strauss III and Barbieri 2009) and a transfer of dependency from FSH to LH (van

den Hurk and Zhao 2005). Estradiol levels reach their peak in the ovulatory follicle,

and the pituitary ovulatory LH surge, leads to production of hyaluronic acid by the

cumulus cells and down regulation of CYP17 in theca cells to switch them from

androgen-producing to progesterone-producing cells (Magoffin 2005). Most follicles

do not develop to ovulatory follicle. In monoovular animals such as sheep, cattle and

humans, only one preovulatory follicle is ovulated each cycle to produce a mature

oocyte. On the other hand, polyovulatory animals (e.g. mouse) develop many

preovulatory follicles during each ovarian cycle.

Figure 1.1 Follicular development stages (taken from

https://www.med.unsw.edu.au)

1.2.1 Maintenance and triggering oocyte nuclear maturation

In the primary follicle, the oocyte remains arrested at prophase of the first meiotic

division (GV stage) by cyclic adenosine monophosphate (cAMP)-dependent protein

kinase A (PKA). cAMP is synthesized from G proteins (Gs) in the oocyte membrane

(Mehlmann, et al. 2002), as well as supplied by somatic cells, and enters the oocyte

through gap junctions (Anderson and Albertini 1976). Somatic cells also generate

cyclic guanosine monophosphate (cGMP) that also passes through gap junctions into

the oocyte, inhibiting the degradation of cAMP and thus blocking meiotic progression


6

(Norris, et al. 2009). In the event of an LH surge, the oocyte phosphodiesterase type

3 (PDE3) enzyme is activated degrading cAMP which induces the maturation

promoting factors (MPF), causing the oocyte to resume meiotic maturation (Tsafriri,

et al. 1996). This event also leads to disconnection of the gap junctions and loss of

receptivity of the oocyte receptors for cAMP and cGMP and lead to releasing the first

polar body of the oocyte (Norris, et al. 2008, Norris, et al. 2009, Sela-Abramovich, et

al. 2005, Sela-Abramovich, et al. 2006). Refer to Fig. 1.2.

The preovulatory LH and FSH surges also induce expression of the epidermal growth

factor (EGF)-like peptides: amphiregulin (AREG), epiregulin (EREG) and β-cellulin

(BTC) (Park, et al. 2004). Activation of the EGF receptor (EGFR) in granulosa and

cumulus cells through the ERK1/2 pathways is followed by the production of a

hyaluronan-rich matrix. Hyaluron synthase 2 (Has2), tumor necrosis factor (TNF)-α-

induced protein 6 (Tnfaip6), pentraxin (Ptx3), prostaglandin-endoperoxide synthase 2

(Ptgs2) (Park, et al. 2004, Russell and Robker 2007) are all involved in granulosa

and cumulus cell expansion. OSFs such as GDF9 and BMP15 are also known to be

involved in this process, through their receptors (bone morphogenetic protein

receptor type-II and activin receptor-like kinases) which activate the Sma and Mad

related (SMAD) intracellular signals to stimulate expansion. The signalling from OSF

receptors to granulosa and cumulus cells activates many functions of these cells,

such as proliferation, expansion, luteinisation, mucification and metabolism (Refer to

section 1.3, Fig. 1.2). Interestingly, during oocyte maturation, signals from both the

EGF and OSF receptors interact with each other in the cumulus cells, making a

complex harmony (Gilchrist 2011).


7

Figure 1.2 Oocyte-somatic cell interactions that regulate in vivo matured

oocytes. There are three processes involved in this pathway: interaction of

GDF9/BMP15 through SMAD2/3 signalling and EGFR; LH and FSH-induction of the

EGF-like peptides in granulosa and cumulus cells; and the role of cGMP, PDE and

cAMP regulating oocyte meiosis. Adapted and modified from (Gilchrist 2011). GJC=

gap junction communication.

1.2.2 Cytoplasmic maturation

Maturation of oocytes within selected follicles is accompanied by cytoplasmic

maturation. The process of cytoplasmic maturation is less obvious compared to

nuclear maturation. In mature human oocytes, actin cytoskeleton is moved and

organized in the cortex therefore oocytes become highly asymmetric to maintain

organelles, proteins and RNAs within the ooplasm (Coticchio, et al. 2014, Levi, et al.

2013). Moreover cortical granules, endoplasmic reticulum and mitochondria also

move toward the oocyte cortex (Strauss III and Barbieri 2009, Tian, et al. 2007).


8

Recent evidence shows Fyn kinase and thick filamentous microtubules in the human

ooplasm are important for successful maturation-competence of oocytes (Levi, et al.

2013).

Oocyte cytoplasmic maturation is important for the ability of the oocyte to be fertilised

and to develop into a zygote. It has also been shown that there is a strong correlation

between oocyte cytoplasmic maturation and the size of follicles. Mature oocytes

which are retrieved from large antral follicles have the capability to develop to the

blastocyst stage when compared to MII oocytes which are obtained from small antral

follicles (Leibfried and First 1979). Moreover, oocytes from pre-pubertal animals have

poorer cytoplasmic maturity, as evidenced by more abnormalities in their

ultrastructure, alterations in metabolic activity and lower embryo developmental

potential compared to those from adults (Leibfried and First 1979, O'Brien, et al.

1996, Succu, et al. 2007)

1.2.3 Development of oocytes after fertilisation

If there is a fertilisation event, the oocyte resumes the second meiotic division and

extrudes the second polar body. In cattle oocytes, male and female pronuclei

develop around 9-14 hours after insemination. The pronuclei enlarge and migrate

centrally, then fuse and undergo syngamy at around 17 hours after insemination. The

earliest cleavage in bovine can be seen at 24 hours after fertilisation and reaches its

maximum rate at 30 hours after fertilisation (Rizos, et al. 2002). Blastocyst formation

in bovine embryos starts at day 6 after insemination (Rizos, et al. 2002). In mouse,

these events occur faster. The formation and migration of the male and female

pronuclei are completed within 5 hours after insemination and the first cleavage of

mouse embryo takes place around 17-19 hours after insemination (Witkowska 1981).

The blastocyst formation in mouse starts at 4.5 days after insemination. This

embryonic structure consists of two different cell types: inner cell mass (ICM) and

trophectoderm (TE) cells (Thouas, et al. 2001). The inner cell mass (ICM) cell

number is a reasonable measure of blastocyst quality and probability of implantation

as the ICM is the portion of the blastocyst that derives the fetus. It has been shown

that there is a strong correlation between an increase in ICM cell numbers and


9

successful implantation and fetal development (Sudiman, et al. 2014a, Yeo, et al.

2008).

1.3 OOCYTE-SOMATIC CELL INTERACTIONS

There are important and multiple modes of communication between theca cells,

granulosa cells and the oocyte, required for follicular development. Communication

between the oocyte and somatic cells is mediated by gap junction and paracrine

signalling. Theca cells secrete factors such as transforming growth factor alpha

(TGF-α), and beta (TGF-β), keratinocyte-derived growth factor (KGF) and hepatocyte

growth factor (HGF) to regulate the development of granulosa cells (Nilsson and

Skinner 2001, Parrott, et al. 1994). The granulosa cells produce KL to promote theca

cell organization (Parrott and Skinner 1997). Oocytes actively regulate follicular

development by producing oocyte derived factors such as GDF9, BMP15, bone

morphogenetic protein 6 (BMP6) and fibroblast growth factors (FGFs) which are

transmitted to the granulosa cells via paracrine signalling. This bi-directional

communication is important to transfer molecules from the oocyte to somatic cells,

and vice versa to sustain granulosa and cumulus cell health and oocyte development

itself.

1.3.1 Gap Junction Signalling

Oocyte and cumulus cells are seperated from each other by the zona pellucida. To

allow small molecules, amino acids and cAMP to penetrate from the cumulus cells

into the oocyte and other cumulus cells, they develop trans-zonal cytoplasmic

projections that go through the zona pellucida and abut the oocyte membrane

(Albertini, et al. 2001). This type of channel is important in order to achieve the

development of follicles and oocytes. Cumulus cells perfuse small molecules such as

purines, linoleic acid, ions, cAMP and other low molecular weight peptides into the

oocyte to maintain meiotic arrest, induce meiotic resumption and support cytoplasmic

maturation of the oocyte (Tanghe, et al. 2002). This gap junctional communication

also occurs at all close cell apposition such as at the surface of oocyte microvilli,

between cumulus-cumulus cells and cumulus-granulosa cells (Kidder and Mhawi

2002).


10

Gap junction channels are composed of connexons, a cylindrical organelle that forms

an intercellar channel. Each connexon is made up from connexins, a hexamer of

protein subunits. Gap junctions allow inorganic ions, second messengers and small

metabolites (< 1 kDa) to pass from cell to cell (Kidder and Mhawi 2002). There are

many different kind of connexins, however, there are two well-known connexins

which are important in follicular and oocyte development: connexin-37 and connexin-

43 (Ackert, et al. 2001, Simon, et al. 1997). Homozygous mutant connexin-43 mice

show abnormalities in follicular development and retarded oocytes which failed to be

fertilized (Ackert, et al. 2001). Oocytes in mice deficient in connexin-37 arrest before

they reach meiotic completion (Simon, et al. 1997).

In oocytes, there is a strong correlation between the loss of gap junction

communication and the resumption of meiosis (Larsen, et al. 1986, Thomas, et al.

2004). It has been demonstrated that the gap junction communication between the

oocyte and cumulus cells is essential to maintaining meiotic arrest, especially in early

oocyte maturation (Thomas, et al. 2004). In bovine oocytes, gap junction

communication levels fall progressively from 0-9 hours of maturation (Thomas, et al.

2004).

1.3.2 Paracrine Signalling

Another type of communication between the oocyte and its somatic cells (granulosa

and cumulus cells) is paracrine signalling. Soluble growth factors secreted by the

oocyte to cumulus cells via paracrine signalling. Below the mechanism of OSFs

impacts on ovarian folliculogenesis and cumulus cells functions.

1.3.2.1 Promotion of Follicular Growth

OSFs such as GDF9 and BMP15 are important during follicular development. A

deficiency of GDF9 and/or BMP15 can reduce fertility due to follicular development

retardation (see section 1.4.3). In in vitro culture, addition of GDF9 or BMP15 alone

promotes growth of pre-antral follicles of the mouse only during the first 24 hours;

interestingly, after 24 hours in culture, BMP15 causes shrinkage and atresia of

follicles and increases apoptosis of the granulosa cells (Fenwick, et al. 2013).

However, a combination of both proteins induces the development of the follice


11

beyond 24 hours of culture (Fenwick, et al. 2013). The situation is different in bovine

in vitro cultured pre-antral follicles, where the addition of either BMP15 or a

combination of FSH and BMP15 enhances the growth of secondary follicles (Passos,

et al. 2013). These results confirm that GDF9 and BMP15 have different roles in

different species in term of promoting follicular development.

1.3.2.2 Regulation of Granulosa and Cumulus Cell Proliferation and Cumulus

Cell Expansion

An increase in granulosa and cumulus cell proliferation in response to OSFs can be

observed by [3H] thymidine uptake as an indicator of DNA synthesis in cells (Li, et al.

2000, Vanderhyden, et al. 1992). It has been shown that recombinant GDF9 or native

OSFs; which are produced by culturing denuded oocytes (DOs) with somatic cells,

play a significant role in creating extracellular matrix (ECM) for cumulus cell

expansion by inducing Has2, Tnfalp6 , Ptgs2 and Ptx3 mice gene expression

(Dragovic, et al. 2005, Dragovic, et al. 2007).

Mouse cumulus expansion depends on the presence of the oocyte (Buccione, et al.

1990, Salustri, et al. 1990). Oocytectomised complexes (OOXs) are cumulus oocyte

complexes (COCs) which have undergone microsurgical removal of the oocyte.

Mouse OOXs are unable to undergo cumulus expansion even in the presence of

FSH (Buccione, et al. 1990, Salustri, et al. 1990). FSH induced the synthesis of

hyaluronic acid, the protein that is required for cumulus expansion and stabilility of

the extracellular matrix, in the COC group but not in the OOX group (Buccione, et al.

1990). Interestingly, cumulus cell expansion of OOXs can be induced in the presence

of FSH by co-culturing OOXs with DOs or culturing the OOXs in the oocyte-

conditioned medium (medium produced by co-culturing the OOXs with DOs,

granulosa cells with DOs or COCs with DOs) (Buccione, et al. 1990, Gilchrist, et al.

2008, Salustri, et al. 1990). Therefore, the presence of FSH is important, but not

sufficient, to induce the cumulus expansion of mouse OOX (Salustri, et al. 1990).

Interestingly, in the pig and cow, expansion of the cumulus cells does not depend on

the presence of oocytes, even though they secrete OSFs (Gilchrist and Ritter 2011,


12

Prochazka, et al. 1991, Ralph, et al. 1995, Singh, et al. 1993, Vanderhyden 1993). It

has been shown that oocyte-conditioned medium from pig oocytes can induce the

cumulus expansion of mouse and rat OOX (Prochazka, et al. 1998, Vanderhyden

1993). Vanderhyden et al., (1993) also demonstrated that oocytes from different

species secrete these soluble factors at different times. In vitro matured mouse

oocytes are capable of secreting OSFs from the time they resume the first meiotic

divison until metaphase II of the second meiosis, whilst in pig, the oocyte secretes

OSFs only during the transition between GV and metaphase I (Nagyova, et al. 2000,

Vanderhyden 1993). In general, the capability of OSFs to regulate the functions of

cumulus and granulosa cells occurs mostly in fully grown oocytes in the antral phase,

declining after the LH surge with re-initiation of meiosis (Gilchrist, et al. 2008).

1.3.2.3 Prevention of Cumulus Cell Apoptosis

Hussein et al (2005) demonstrated for the first time how OSFs can prevent cumulus

cell apoptosis by co-culturing OOXs with denuded oocytes (Hussein, et al. 2005). In

this elegant experiment, the OOXs were exposed to DOs in a dose dependent

manner by incubating OOXs with increasing numbers of oocytes. The apoptosis level

reduced linearly with the increasing number of oocytes. The results also showed that

recombinant BMP15 and BMP6 are potent anti-apoptotic factors, but suprisingly,

recombinant GDF9 had little effect. Interestingly, the incidence of apoptosis in COCs

was higher in the outer layer of the cumulus cells whilst in OOXs+DOs, it was the

inner layer (Hussein, et al. 2005). This effect may, in part, be due to OSFs promoting

the expression of anti-apoptotic Bcl-2 and supressing pro-apoptotic Bax in cumulus

cells.

1.3.2.4 Regulation of Steroidogenesis (Luteinization) and Differentiation of

Granulosa and Cumulus Cells

There are two somatic cell populations in the ovarian follicle: those that attach

directly to the oocytes (cumulus cells) and the cells that line the follicle wall (mural

granulosa cells). Both populations are differentiated during the antral phase of

follicular development. LH receptors (LHR) and LHR mRNA are detected abundantly

in mural granulosa cells but only at low levels in cumulus cells; thus this is an

important marker that distinguishes granulosa and cumulus cells (Eppig, et al. 1997).


13

Since the production of progesterone triggers the luteinization of follicles, OSFs

suppress elevation of LHR mRNA in mural granulosa cells and cumulus cells.

However, if oocytes are microsurgically removed from COCs (OOX), the cumulus

cells express higher levels of LHR mRNA (Eppig, et al. 1997). Oocytes at GV stage

are more effective in suppressing expression of LHR mRNA in cumulus cells

compared to those at metaphase II (Eppig, et al. 1997). The same result was also

obtained by Li et al (2000) showing that OSFs inhibit the production of progesterone

in cumulus and granulosa cells. Moreover, recombinant GDF9 and BMP15 together

are able to inhibit FSH-stimulated progesterone production by granulosa cells

(McNatty, et al. 2005a). In contrast with progesterone production, mouse OSFs

induce the production of estradiol by granulosa cells (Vanderhyden, et al. 1993),

although this is not the case in bovine where OSFs suppress estradiol production

(Glister, et al. 2003).

1.4 TGF-β SUPERFAMILY

The TGF-β superfamily proteins are involved in the proliferation, differentiation and

death of cells. This superfamily is present in a diverse range of species, including

humans, and it consists of six sub-groups: TGF-β 1, 2, 3, Anti-Mȕllerian hormone

(AMH), inhibin A and B, activin A, B and AB, 20 different BMPs and 9 GDFs (Burt

and Law 1994, Ghiglieri, et al. 1995). GDF9 and BMP15 are members of the TGF-β

superfamily and important in female fertility.

1.4.1 GDF9

In 1993, McPherron and Lee discovered for the first time GDF9 in the mouse ovary

(McPherron and Lee 1993). In polyovulatory animals, the expression of GDF9 starts

from the primary follicle stage (Jaatinen, et al. 1999, Laitinen, et al. 1998), whilst in

monoovular animals such as cow, sheep and humans, the expression of GDF9 is

first observed in primordial follicles and is observed thereafter in the oocyte

(Aaltonen, et al. 1999, Bodensteiner, et al. 1999, Eckery, et al. 2002). GDF9 mRNA

expression has also been reported in non-ovarian tissue such as spermatocytes,

spermatids, sertoli cell cytoplasm, uterus and bone marrow (Fitzpatrick, et al. 1998).


14

Recombinant GDF9 can mimic the effect of OSFs such as a cumulus expansion

enabling factor in mouse oocytes (Dragovic, et al. 2005, Vanderhyden, et al. 2003).

Recombinant GDF9 stimulates Has2, cyclooxygenase 2 (Cox-2) and steroidogenic

acute regulator protein (StAR) mRNA sythesis, and suppresses urokinase

plasminogen activator (uPA) and LHR mRNA in granulosa and cumulus cells, thus

playing an important role in inducing cumulus cell expansion and differentiation

(Elvin, et al. 1999a). However, Dragovic et al (2005) showed that other proteins must

also be involved in mouse cumulus cell expansion, since a GDF9 antagonist is not

sufficient to completely neutralize oocyte-induced expansion.

1.4.2 BMP15

Dube and co-workers were the first group to discover BMP15, also referred to as

GDF9B, in mouse oocytes (Dube, et al. 1998). The first expression of BMP15

appears at a later developmental stage compared to GDF9. In mice the expression of

BMP15 is expressed in the oocyte beginning at the one-layer primary follicle stage

and continuing through ovulation (Dube, et al. 1998). Studies in human and sheep

oocytes have shown that the expression of BMP15 is at the late primary follicle stage

(Aaltonen, et al. 1999, Galloway, et al. 2000).

Similar to GDF9, BMP15 regulates rat cumulus and granulosa cell proliferation and

this effect is FSH-independent (Otsuka, et al. 2000). However, BMP15 is a selective

modulator of FSH action as it decreases FSH-induced progesterone production but

has no effect on estradiol production; therefore, BMP15 is a candidate for a potent

granulosa cell luteinization inhibitor (Otsuka, et al. 2000). BMP15 inhibits StAR, P450

aromatase, LHR and inhibin/activin subunit mRNA in rat granulosa cells (Otsuka, et

al. 2001b) but stimulates the expression of KL (Otsuka and Shimasaki 2002).

1.4.3 Mutation of GDF9 and BMP15

Unlike most other members of the TGF-β superfamily, the function and expression of

both GDF9 and BMP15 differ markedly among species. The ratio of GDF9 and

BMP15 in oocytes is species-specific. In the oocytes of polyovulatory animals such

as mouse and rat, the ratio of GDF9:BMP15 is around 5:1, with the exception of pig

oocytes, where it is much lower (0.5:1) (Crawford and McNatty 2012). In monoovular


15

animals such as cow and sheep, the level of BMP15 mRNA in oocytes is much

higher or at least equal to GDF9 (Crawford and McNatty 2012). Homozygous mutant

GDF9 mice are sterile due to a block in follicular development beyond the primary

follicle stage (Dong, et al. 1996), and display 2-3 fold increased in the levels of LH

and FSH (Erickson and Shimasaki 2000). The size of oocytes inside the primary

follicles of these mutant mice is much larger than control mice (Erickson and

Shimasaki 2000). Moreover, in the granulosa cells there is a lack of FSH receptors, a

decrease in mitosis, overexpression of KL and no apoptosis (Elvin, et al. 1999b).

However, BMP15 is not essential for mouse fertility. BMP15 null mice demonstrate

only a mild reduction in ovulation and fertilization rates (Yan, et al. 2001). Double

homozygote mice (BMP15 -/-, GDF9 -/-) have the same ovarian phenotype with

homozygous mutant GDF9 mice (Yan, et al. 2001).

On the other hand, GDF9 and BMP15 deficient sheep (through passive immunization

or homozyogus for naturally occuring mutations) are sterile (Galloway, et al. 2000,

Hanrahan, et al. 2004). Interestingly, heterozygous GDF9 and BMP15 sheep have an

increased incidence of mutiple pregnancies and ovulation due to increased LH

sensitivity in secondary follicles that leads to a rise in the number of antral follicles

(Galloway, et al. 2000, Hanrahan, et al. 2004, Juengel, et al. 2004). In humans, rare

mutations and genetic variations in GDF9 may contribute to PCOS (Laissue, et al.

2006, Wang, et al. 2010) and dizygotic twinning (Montgomery, et al. 2004, Palmer, et

al. 2006), and low ovarian reserve (Dixit, et al. 2005, Wang, et al. 2013), whilst

mutations in BMP15 lead to premature ovarian failure (Di Pasquale, et al. 2004). In

contrast, BMP6 has shown no major effect on ovarian function and follicular

development. Homozygous null BMP6 mice are fertile with normal offspring

(Solloway, et al. 1998), but BMP6 plays a role in regulating steroidogenesis by

reducing the FSH-stimulated progesterone level without affecting FSH-induced

estradiol synthesis (Otsuka, et al. 2001a).

1.4.4 TGF-β Superfamily Signalling

The TGF-β superfamily proteins bind as homo or heterodimers to their type-I or type-

II receptors. These receptors have a serine/theronine protein kinase domain which

bind together and act as a dimeric assembly factor. Phosphorylated intracellular


16

receptor-regulated signal transducers, known as SMADs, transport the signals into

the nucleus of the cells (Massague 2000). SMADs are nuclear proteins and interact

with transcription factors. They control the expression of genes, either by activation

or repression of gene expression.

Both BMP15 and GDF9 activate the type-II receptor (BMPR2) before they trigger the

SMADs. BMP15 also activates the ALK 6 receptor whilst GDF9 activates the ALK 5

receptor (Kaivo-Oja, et al. 2005, Vitt, et al. 2002). The activation of BMPR2 and ALK6

by BMP15 phosphorylates SMAD 1/5/8 which assembles with co-SMAD 4 (Moore, et

al. 2003), whilst GDF9 triggers the activation of SMAD 2/3 which cooperates with co-

SMAD 4 (Kaivo-Oja, et al. 2005, Mazerbourg, et al. 2004), before they translocate to

the nucleus to regulate specific genes (Fig. 1.3). Moreover, the TGF-β family and

BMPs may also utilize the MAPK (mitogen-activated protein kinase) pathway (Moore,

et al. 2003). It has been reported that BMP2 activates p38 MAPK, c-Jun N-terminal

kinase (JNK) and triggers the activation of alkaline phosphatase activity (Nohe, et al.

2002). Both BMP2 and BMP4 also activate the nuclear factor-kB (NFKB) pathway

(Mohan, et al. 1998).

There is a strong synergistic effect between GDF9 and BMP15 on granulosa cell

proliferation, inhibition of FSH-stimulated progesterone production and stimulation of

inhibin production (McNatty, et al. 2005a). The combination of mature regions of

mouse GDF9 and human BMP15 at a low dose induces a synergistic interaction

which is mediated by SMAD 2/3 signalling (Mottershead, et al. 2012). This

synergisitic interaction also involves the EGF receptor-activated ERK1/2 (also known

as MAPK3/1) signalling pathway (Mottershead, et al. 2012). In sheep, the synergistic

effect of ovine GDF9 and BMP15 involves an initial interaction with BMPR2, and

BMPR1 is either recruited later or is already associated with BMPR2, to induce this

effect (Edwards, et al. 2008).

Interestingly, there are species differences in physiological functions and signalling of

GDF9 and BMP15. It has been shown that both ovine and murine GDF9+BMP15

have a synergistic effect on [3H] thymidine uptake on rat granulosa cells through

SMAD 2/3 (Reader, et al. 2011). However, there is a divergency in non-SMAD


17

signalling between these two species. Inhibitors of NFKB and p38-MAPK pathways

had no effect on murine GDF9+BMP15 but had a significant effect on ovine

GDF9+BMP15 stimulation of [3H] thymidine uptake (Reader, et al. 2011). Whilst,

addition of the JNK pathway inhibitor has no effect on ovine GDF9+BMP15 it has a

significant effect on murine GDF9+BMP15 on [3H] thymidine uptake (Reader, et al.

2011). In addition, murine GDF9+BMP15, but not ovine GDF9+BMP15, also utilise

the ERK-MAPK signalling pathway (Reader, et al. 2011).

Another combination of GDF9 and BMP15 is in the heterodimers form. Heterodimers

of GDF9:BMP15 use the BMPR2 in order to activate and signal through

phosphorylation of SMAD2/3 (Peng, et al. 2013), athough the existence and function

of a GDF9:BMP15 heterodimers is controversial (Mottershead, et al. 2013) (See

section 1.4.5 for GDF9 and BMP15 heterodimer structure). Heterodimers of

GDF9:BMP15 activate BMPR2 and ALK4/5/7 to trigger SMAD2/3 phosphorylation

with ALK6 co-receptor in mouse and human granulosa cells (Peng, et al. 2013).

Putative heterodimers of recombinant human or mouse GDF9:BMP15 enhance

granulosa cell activation ~3000 fold compared to BMP15 homodimers, and ~10-30

fold compared to GDF9 homodimers, as well as increase the expression of genes

such as Ptx3, Ptgs2 and Has2 in the ECM, compared to GDF9 and BMP15

homodimers alone or in combination (Peng, et al. 2013).


18

Figure 1.3 Schematic representations of GDF9 and BMP15 homodimers and

putative GDF9:BMP15 heterodimers signalling in regulating cumulus and

granulosa cell functions. Adapted and modified from (Peng, et al. 2013).

1.4.5 Structure and Production of In vitro Recombinant GDF9 and

BMP15

The most common method to date of treating COCs with OSFs is by co-culturing

them with DOs. However, it is impractical to use this procedure in human IVF clinics

due to the limited number of DOs available. Therefore, it is important to produce the

bioactive recombinant GDF9 and BMP15 proteins to be used practically.


19

GDF9 and BMP15 both consist of a pro-region and mature region (Fig. 1.4). The pro-

region is important for activities such as folding, formation of the disulfide bond and

regulation of protein bioactivity (Gray and Mason 1990, Harrison, et al. 2011).

To date, it is still unclear what forms of GDF9 and BMP15 are secreted by the oocyte

into the cumulus cells, and whether the form produced by oocytes matured in vitro is

similar to what may occur in vivo. In sheep follicular fluid, the majority of GDF9 and

BMP15 is detected as pro-protein (McNatty, et al. 2006), whereas sheep oocytes

secrete the mature domains of GDF9 and BMP15 during IVM (Lin, et al. 2012).

Mouse in vitro matured oocytes secrete GDF9 as a mixture of the unprocessed pro-

protein and mature domain (Gilchrist, et al. 2004b). Rat IVM oocytes secrete the

mature domains of GDF9 and BMP15 (Lin, et al. 2012).

Both GDF9 and BMP15 proteins lack a conserved cysteine residue found within the

mature region of other members of the TGF-β superfamily, and therefore the

dimerization of mature regions occurs non-covalently and lacks the intersubunit

disulphide bond (McPherron and Lee 1993). In theory, this may readily facilitate the

formation of GDF9:BMP15 heterodimers.


20

Figure 1.4 The forms of mouse GDF9 and BMP15. Un-processed protein/pro-

protein of GDF9 and BMP15 consist of two structures; the pro-region and mature

region. The molecular scale of un-processed GDF9 and BMP15 is ~65 KDa. During

processing, the pro and mature regions are separated at their protease processing

site. Processed mouse GDF9 from transfected 293H cells is generally secreted in the

media as a dimer of a pro-mature complex. The molecular size of the pro-region of

GDF9 is ~42 KDa and mature region ~17 KDa. Mouse BMP15 from transfected 293

cells is secreted as monomers of the pro-region (~40 kDa) or mature region (~28 kDa

for glycosylated mature region and ~24 KDa for un-glycosylated) or pro-protein (~65

kDa). Since the regions are attached only by electrostatic interactions (non-covalent

bonding), GDF9 and BMP15 may easily interact with each other, making

heterodimers. Adapted and modified from (McIntosh, et al. 2008).


21

In general, recombinant GDF9 and BMP15 are produced in vitro using a human

embryonic kidney cell line (HEK-293 T) as host cells. Either mouse, sheep or human

GDF9 and BMP15 full length cDNA is cloned into the pEFIRES-P expression vector

and transfected into the host cells using fugene 6 transfection reagent. The cells that

express high levels of the recombinant protein (assessed by increasing the

concentration of puromycin) are selected and the levels of GDF9 and BMP15 are

measured in comparison with a standard curve of Escherichia coli expressed oGDF9

or oBMP15 (Kaivo-Oja, et al. 2003, McNatty, et al. 2005a). This is the methodology

that was used over the previous decade, however, the GDF9 and BMP15 produced

by this method are not pure. Nevertheless, this partially purified GDF9 and BMP15

has been used in cattle and mouse IVM media, and they improved the quality of in-

vitro matured oocytes and thus subsequent embryo development and fetal survival

rate (Hussein, et al. 2011, Hussein, et al. 2006, Yeo, et al. 2008).

In the present study, novel purified recombinant human and mouse GDF9 and

recombinant human BMP15 was used (produced in-house by Drs D.Mottershead and

Assoc Prof R. Gilchrist) Briefly, BMP15 and GDF9 plasmids were transfected into

host cells (HEK-293T cell lines) which were cultured in Dulbecco’s modified eagles

medium (DMEM-Hams F12). An 8-histidine tag (His8-tag) and a strep II epitope tag

at the N terminus of the pro-region were used for purification of the pro-mature

complex (Fig. 1.5). The resultant BMP15 protein was secreted into the serum-free

media with a processing efficiency of the pro and mature regions of over 90%. The

BMP15 or GDF9-containing production media was then subjected to immobilized

Ni2+ based ion-affinity purification targeting the His-tag. This methodology generates

highly purified GDF9 and BMP15 preparation in the form of pro-mature complex (Fig.

1.5) (Pulkki, et al. 2011).


22

Figure 1.5 The structure of GDF9 and BMP15-post purification (pro-mature

complex/processed protein) which consist of dimers of mature regions and

associated pro-regions. SS: Strep-tag. (David Mottershead: personal

communication)

1.5 FIBROBLAST GROWTH FACTORS (FGFS)

Another family of proteins that are secreted by the oocyte are the fibroblast growth

factors (FGFs). FGFs consist of 22 members grouped into 11 subfamilies, and have

five known FGF receptor genes (Sleeman, et al. 2001). The expression of FGFs

differs among species. FGF2 is synthesized by immature oocytes in bovine

primordial follicles, and activates follicle growth via stimulation of granulosa cell

proliferation and follicular basement membrane synthesis (van Wezel, et al. 1995).

Whilst the expression of FGF2 starts in early gonad development in the rat (Koike

and Noumura 1994), FGF8 is expressed in mouse antral follicles but was not

detected in the fetal rat (Valve et al., 1997) or in adult human ovary (Valve et al.,

2000). However, the expression FGF8 is detected in bovine preantral follicles


23

(Buratini, et al. 2005a, Buratini, et al. 2005b). FGF17 is expressed in primordial,

primary and antral follicles and acts as a potential mediator of granulosa cell

differentiation by suppressing follicular steroidogenesis (Machado, et al. 2009).

Interestingly, FGF17 was also found abundantly in the granulosa and theca cells of

atretic follicles, indicating that it may be involved in the process of follicle atresia

(Machado, et al. 2009).

Recently, it has been demonstrated that FGF8 in combination with BMP15 enhances

the metabolism of glucose (glycolysis) in mouse cumulus cells (Sugiura, et al. 2007).

The products of glycolysis, such as pyruvate, are important in oocyte development

(Sugiura, et al. 2007). Another member of the FGF family, FGF10, improves the

blastocyst rate in cattle when added during the IVM phase (Zhang, et al. 2010). The

addition of exogenous FGF10 also increases BMP15 mRNA in cultured bovine

oocytes (Zhang, et al. 2010).

1.6 OOCYTE METABOLISM

The cumulus and granulosa cells surrounding the oocyte are essential to support

oocyte metabolism and its capacity to undergo maturation, fertilization and further

development. In the absence of cumulus cells, the immature oocyte demonstrates

undetected levels of glycolytic activity (Zuelke and Brackett 1992) due to being

incapable to utilize glucose (Biggers, et al. 1967). The oocye depends on somatic

cells to uptake amino acids and transport them through gap junctional channels to

the oocyte (Colonna and Mangia 1983). Only pyruvate and oxaloacetate are able to

be used by oocytes if no cumulus cells are present (Biggers, et al. 1967).

During maturation, glucose activity increases linearly with oocyte maturation. Mature

oocytes have significantly higher glycolytic activity compared to immature oocytes

(Spindler, et al. 2000). During fertilization (fusion and decondensation) the glycolytic

and pentose phosphate pathway (PPP) activities increase significantly (Urner and

Sakkas 1999).


24

Glucose

ROS

GSH GSSG

TCA

Cycle

ATP

Oxidative

phosphorylation

Pyruvate

(Sutton-McDowall et al 2010 Reprod)

PPP

NADPH

NADPH

PRPP

Meiotic

regulation

Glucose

PPP

Glucose

Oocyte

Cumulus CellGlycolysis

Pyruvate

Pyruvate

Lactate

Lactate

+ FSH

HBP ECM

expansion

O-linked

glycosylation

Post-translation

modifications

+ FSH

Figure 1.6 Glucose utilization in cumulus cells and oocytes. Glucose can be

utilized by cumulus cells via glycolysis, the pentose phosphate pathway (PPP), and

the hexosamine pathway (HBP). The end product of glucose utilization via glycolysis

is lactate or pyruvate. Pyruvate is converted to Acetyl CoA and further oxidised by

the TCA cycle in mitochondria, followed by oxidative phosphorylation in the

intermembrane of mitochondria to produce adenosine triphosphate (ATP) via

electron transfer. The final product of the HBP is hyaluronic acid for extracellular

matrix expansion of CCs. The PPP utilizes glucose to produce glyceraldehydehyde

3-P and fructose 6-P, which can be re-utilized in glycolysis. One of the most

important products of the PPP is phosphoribosylpyrophosphate (PRPP) which is

used for purine synthesis and is involved in oocyte nuclear maturation. Another

important product of the PPP is the reducing agent nicotinamideadenine dinucleotide

phosphate(NADPH), involved in glutathione metabolism for homeostasis in cells.

Adapted and modified from (Sutton-McDowall, et al. 2010). FAD= flavin adenine

dinucleotide; GSG= reduced glutathione; GSSG= oxidised glutathione.

NAD=Nicotinamide adenine dinucleotide.


25

Glycolytic activity contributes to ATP production since in this pathway, glucose is

converted into pyruvate as a source of Acetyl CoA for the TCA cycle where it is

further metabolised in the mitochondria by oxidative phosphorylation (Downs and

Utecht 1999, Dumollard, et al. 2007). In COCs, phosphofructokinase, as a key

enzyme for glycolytic activity, has the highest activity in glucose metabolism followed

by glucose-6-phosphate dehydrogenase (for PPP). It has been shown that there is a

correlation between low glycolytic activity and developmental competence of COCs.

The glycolytic activity is much lower in DOs compared to COCs (Downs and Utecht

1999). Oocytes derived from pre-pubertal cattle have lower blastocyst development

rates and also show a reduction and delay of glycolytic activity during maturation

compared to those derived from adults (Steeves and Gardner 1999). Moreover, in

vitro matured cat oocytes have significantly lower glycolytic activity and glucose

oxidation compared to in vivo counterparts (Spindler, et al. 2000). It has also been

shown that oocytes that utilize more glucose (≥ 2 pmol/oocyte/hr) during maturation

have a significantly higher blastocyst development rate compared to those which

utilize less glucose (0.1-2 pmol/oocyte/hr) (Spindler, et al. 2000).

The major product of the hexosamine pathway is hyaluronic acid which is important

to build ECM in cooperation with FSH-induced cumulus expansion (Sutton-McDowall,

et al. 2004). Supplementing IVM medium with glucosamine as an alternative hexose

substrate reduces significantly pig and cattle embryo development. However, the

effect of glucosamine induced reduction in developmental competence can be

reversed by using the inhibitor of o-linked glycosylation, as one of hexosamine

pathway substrates (Sutton-McDowall, et al. 2006). The pentose phosphate pathway

(PPP) is another pathway involved in glucose metabolism, and produces the

reducing agent NADPH which is a key regulator of glutathione metabolism (see

below). The PPP is also involved in meiotic maturation, as it synthesises

phosphoribosyl pyrophosphate (PRPP) (Downs, et al. 1996, Downs and Utecht

1999). Refer to Fig. 1.6.

1.6.1 Glutathione

Glutathione has an important role in the cell as a defence against oxidative stress.

Glutathione has two forms: the reduced form (GSH) and the oxidized form (GSSG).


26

The thiol groups of GSH consist of cysteine, proline and glutamine which can react

with hydrogen peroxide and be oxidised by glutathione oxidase into GSSG. In turn,

GSSG in the presence of NADPH can be converted by glutathione reductase back

into GSH (Meister and Anderson 1983). A direct correlation does not always appear

between GSH and GSSG concentrations. For example, a reduction in the

concentration of GSH is not always correlated with an increase in GSSG, because

the thiol groups can also bond with other molecules.

2GSH+H2O2 GSSG+ 2H2O Glutathione oxidase GSSG+NADPH+H+ 2GSH+NADP+ Glutathione reductase

GSH concentration reaches a peak in mature oocytes, with the concentration in MII

mouse oocytes approximately double the concentration of GV stage oocytes

(Perreault, et al. 1988, Yoshida, et al. 1993). The quality of oocytes is also related to

the concentration of GSH. It has been demonstrated that in vivo matured oocytes

contain more GSH compared to in vitro matured oocytes, which may be due to the

higher concentration of oxygen during IVM (Brad, et al. 2003, Luberda 2005).

Another function of GSH during oocyte maturation is to maintain meiotic spindle

structure to ensure the normal development of zygotes (Yamauchi and Nagai 1999,

Yoshida, et al. 1993). Exposure of matured oocytes during fertilization to diamide

(specific GSH oxidant) produces abnormal female pronuclei (Zuelke, et al. 1997).

Several low molecular weight thiol compounds such as cysteamine (2-

mercaptoethylamine, MEA) and N-acetylcysteine (NAC) promote cystine, which is

the only source of mammalian cells for GSH synthesis uptake. Thus these

compounds increase intracellular GSH biosynthesis (Issels, et al. 1988).

Supplementing IVM medium with cysteamine increases the intracellular

concentration of GSH in the oocyte and improves blastocyst development in cattle

(de Matos, et al. 1995), buffalo (Anand, et al. 2008, Gasparrini, et al. 2003), sheep

(de Matos, et al. 2002) and goat (Romaguera, et al. 2010).


27

1.6.2 Role of OSFs in Cumulus and Oocyte Metabolism

During maturation there are several genes involved in glycolysis such as Eno1,

Pkm1, Tpi, Aldoa, Ldh1 and Pfkp, which are upregulated predominantly in the

cumulus cells near the antrum (Sugiura, et al. 2005). Mouse OOXs display

decreased expression of cumulus cell genes encoding glycolytic enzymes; however,

if the OOXs are co-cultured with fully grown oocytes from antral follicles, the activity

is restored (Sugiura, et al. 2005). In contrast in the presence of FSH, there is no

difference in glycolytic activity in cumulus cells among intact bovine COCs, OOXs or

OOXs co-cultured with DOs (Sutton, et al. 2003b).

Co-culture of COCs with DOs increases GPX1 gene expression (related to the

production of glutathione peroxidise as an antioxidant in oocytes), and enhances the

expression of StAR, which contributes to nucleus and cytoplasmic maturation (Dey,

et al. 2012). Bovine oocytes matured in the presence of the mature form of BMP15

showed a significant increase in glucose uptake and in mRNA encoding GLUT1 and

GFPT1 at 22 hours, and GFPT2 at 12 hours and 22 hours culture, but interestingly

did not increase lactate production (Caixeta, et al. 2013). Whilst oocytes matured

with the pro-mature complex form of BMP15 showed that even though there is no

increase in glucose uptake, there is a significant increase in oocyte NAD(P)H and

FAD++ autofluorescence which is related to increased ATP production (Sutton-

McDowall, et al. 2012) structure and production of in vitro recombinant GDF9 and

BMP15.

1.7 IN VITRO OOCYTE MATURATION

In vitro maturation of cattle oocytes is used to reduce the costs of commercial

embryo production and transfer (Brackett, et al. 1982), decrease the generation

interval, increase the quality of offspring and further embryogenesis and assisted

reproduction research (Hoshi 2003). In 2005, the annual number of viable cattle

offspring derived from IVM was about 133 000 (Thibier 2006). In contrast, in humans,

the number of babies born from IVM is reported in the literature at only around 1300

births (Suikkari 2008).


28

Compared to conventional IVF, implantation, clinical pregnancy and live birth rates

are significantly lower using IVM (Gremeau, et al. 2012). In all studies reported so

far, babies born through IVM have normal body weight, height and head

circumference compared to IVF counterparts (Cha, et al. 2005) and the general

population (Soderstrom-Anttila, et al. 2006). However, some studies have also found

that 19% of IVM babies have minor neuropsychological developmental delays at the

age of 12 months, but by 2 years of age the development was within the normal

range compared to natural born children (Soderstrom-Anttila, et al. 2006). Looking at

obstetric complications, around 30-40% of pregnancies from IVM cycles ended with

Caesarean section (Soderstrom-Anttila, et al. 2006). However, it is difficult to

speculate whether these complications are due directly to IVM, since preterm

delivery, placenta praevia, gestational diabetes and pre-eclampsia also occur

significantly more often in IVF pregnancies compared to natural pregnancies

(Jackson, et al. 2004).

1.7.1 Selection of Oocytes for In Vitro Maturation

In animal embryo production, the diameter of the follicle and oocyte, the presence of

cumulus cells, the homogeneity of the cytoplasm and maternal age all affect the

maturation of oocytes (Leibfried and First 1979). Romaguera et al demonstrated that

oocytes which are generated from small follicles (<3mm) in prepubertal goats

produce significantly lower blastocyst rates following fertilization compared to oocytes

from follicles with a diameter greater than 3 mm (Romaguera, et al. 2010). In cattle

and sheep embryo production, it has been reported that oocytes aspirated from

prepubescent animals have poor cytoplasmic maturation, abnormalities in the

ultrastructure, alterations in metabolic activity and slower embyro development rates

compared to adult counterparts (Succu, et al. 2007). On the other hand,

prepubescent mice have a better response to gonadotrophin stimulation than adults

and these immature oocytes readily undergo IVM and develop blastocysts.

Developmental competence post-fertilization also has a positive correlation with the

size of oocytes (Otoi, et al. 1997). This study found that bovine oocytes at a diameter

of 115 microns have full meiotic competence but have not acquired full

developmental competence. They aquire this capibility to develop into blastocysts


29

when they reach a diameter of 120 microns. Leibfried and First (1979) reported that

there was a strong correlation between homogeneous cytoplasm and an intact

cumulus layer and a higher proportion of oocytes with homogeneous cytoplasm and

intact cumulus layers reach maturity (Leibfried and First 1979).

1.7.2 In Vitro Maturation vs In Vivo Maturation

There is clear evidence that oocytes aspirated from large antral follicles resume

spontaneous maturation in vitro (Pincus and Enzmann 1935). In in vivo matured

oocytes, the follicular environment is responsible for meiotic arrest and meiotic

resumption of the oocyte.

In vitro maturation of oocytes usually leads to the spontaneous maturation of the

nucleus, a lack of synchronization between nuclear and cytoplasmic maturation

(Succu, et al. 2007), abberant gene and protein expression in granulosa and cumulus

cells compared to in vivo matured oocytes (Dunning, et al. 2007, Kind, et al. 2013,

Richani, et al. 2013), and disregulation of oocyte-cumulus cell gap-junctional

communication (Thomas, et al. 2004). It has also been reported that mouse in vitro

matured oocytes have a different position, organization and distribution of the meiotic

spindle and cytoplasmic microtubules compared to in vivo counterparts (Sanfins, et

al. 2003). Using terminal deoxynucleotidyltransferase 3’nick end labelling (TUNEL), it

has been shown that in vitro derived porcine embryos express more TUNEL positive

cells compared to their in vivo counterparts, suggesting that the in vitro culture

system generates DNA fragmentation (Long, et al. 1998). All these factors lead to

reduced oocyte developmental competence of in vitro matured oocytes. Even though

the rate of nuclear maturation of in vitro matured oocytes is similar to that of in vivo

matured oocytes, the blastocyst rates of embryos derived from in vitro matured cattle

and sheep oocytes is less than half of in vivo matured oocytes (Leibfried-Rutledge, et

al. 1987, Thompson, et al. 1995).

1.7.3 Ooctye Secreted Factors in the In Vitro Maturation of Oocytes

It has been hypothesised that IVM oocytes may have decreased expression and

signalling of GDF9 and BMP15. Mester et al (2013) showed no expression of BMP15

protein in in vitro matured mouse oocytes. By contrast, in mouse oocytes undergoing


30

in vivo maturation, the mature form of BMP15 is visible from 6 hours post-hCG

administration and further up-regulated at 9 hours post-hCG, but this protein is not

detected in in vitro matured oocytes (Mester, et al. 2013). Another study showed that

the stimulating effect of BMP15 on cumulus expansion occurs only after the LH/hCG

surge (Yoshino, et al. 2006), whilst in vitro matured mouse oocytes post-PMSG are

unable to replicate this effect (Yoshino, et al. 2006).

To overcome the deficiency of GDF9 and BMP15 in in vitro matured oocytes,

previous studies have used DOs as a source of native exogenous OSFs, and co-

cultured these with immature COCs. As anticipated, DOs increased oocyte

developmental competence in animals such as cow (Dey, et al. 2012, Hussein, et al.

2011, Hussein, et al. 2006), goat (Romaguera, et al. 2010) and pig (Gomez, et al.

2012). The important part of the co-culture of COCs with DOs is the ratio and volume

of medium used. Hussein et al (2011) used a ratio of 1 COC: 5 DO with the volume

0.5 DO/µl medium in order to increase bovine blastocyst rate up to 20% higher

compared to controls. Gomez et al (2012) showed that the ratio of COCs to DOs in

pig should be at least 1:4, with 0.2 DO/µl to observe the beneficial effect of native

OSFs on pig oocytes. Moreover, DOs from small follicles also have a positive effect

on development competence of IVM oocytes as well as DOs from large follicles,

indicating that DOs from small follicles are also capable of producing OSFs

(Romaguera, et al. 2010). Other than follicle size, another factor that affects the

capacity of exogenous OSFs to improve developmental competence is the timing of

addition of the DOs (Hussein, et al. 2011).

Maturing COCs in the presence of exogenous recombinant BMP15 and GDF9

significantly increased bovine blastocyst development (Hussein, et al. 2011, Hussein,

et al. 2006). Even though mouse COCs treated with GDF9 did not show an

improvement in blastocyst rate, there is a significant increase in the inner cell mass

number which led to an improvement in the fetal survival rate (Yeo, et al. 2008). The

improved blastocyst rate can be reduced in the presence of SB431542 and follistatin

which are antagonists of GDF9 and BMP15, respectively (Hussein, et al. 2006,

Romaguera, et al. 2010).


31

1.8 CONCLUSION

It is becoming increasingly important to reduce the cost of ART, as well as to

eliminate the side effects of gonadotropin hormones. In vitro maturation of oocytes

has the potential to alleviate these problems, as it is a less invasive and cheaper

method compared to IVF. However, the success rate of in vitro matured oocytes as

measured by embryo development is much lower compared to in vivo matured

oocytes. Understanding the mechanisms that improve the quality of the in vitro

matured oocyte could improve the success rate post IVM.

It is now a well-known concept that there is an intimate communication between

oocyte and somatic cells. The oocyte secretes OSFs into the cumulus cells to

regulate the functions of cumulus cells, and in return the cumulus cells perfuse small

molecules and ions through gap junctions into the oocyte to maintain the functional

activity and developmental competence of the oocyte. Previous studies showed co-

culture of COCs with Dos, as a source of native OSFs, improved developmental

competence of in vitro matured oocyte, however, application of this method to

generate such a huge numbers of DOs is not practical in human IVF and animal

production. Therefore, the next step is to find the proteins/molecules in OSFs which

are functionally active to improve the quality of in vitro matured oocytes.

The preparations of GDF9 and BMP15 available commercially (R&D Systems)

contain different structure of proteins compared to native proteins, therefore their

roles in in vitro maturation are likely to be different. This thesis will examine the

influence of native OSFs on mouse in vitro matured oocytes and observe the

difference between commercially available GDF9 and BMP15 (R&D Systems), which

contain only the mature region of these proteins, to the pro-mature complex forms,

on in vitro matured cattle and mouse oocytes and subsequent embryo development.

This work will provide a new perspective on the importance of bi-directional

communication between oocytes and somatic cells and its implications for

improvement of in vitro matured oocytes and embryo production in domestic species

and human infertility.


32

1.9 HYPOTHESIS AND AIMS

1.9.1 Hypothesis

Based on the literature review above, the following hypothesis is proposed:

developmental competence of in vitro matured murine and bovine oocytes can be

improved by exposure to either native OSFs, added at differing time points during in

vitro maturation, or to novel purified recombinant GDF9 and BMP15 on their pro-

mature complex forms.

1.9.2 Aims

There are three main aims of this project.

1. To examine factors affecting the capacity of exogenous oocyte secreted

factors to improve mouse embryo and fetal development post-IVM.

2. To examine the effect of a novel variant of hBMP15 (pro-mature complex vs

mature domain) compared with the mature domain of mGDF9 on metabolic

activity in oocytes and development of embryos after IVM in cattle.

3. To examine the effect of a novel variant of GDF9 and BMP15 (both in pro-

mature complex forms) and a combination of different forms (pro-mature

complex vs mature domain) and sources (mouse and human) of GDF9 and

BMP15 on developmental competence of IVM mouse oocytes.

Chapter 2: Factors Affecting the Capacity of Exogenous Oocyte Secreted Factor

to Improve Mouse and Fetal Development Post-IVM

33

CHAPTER 2

FACTORS AFFECTING THE CAPACITY OF

EXOGENOUS OOCYTE SECRETED FACTORS TO

IMPROVE MOUSE EMBRYO AND FETAL

DEVELOPMENT POST-IVM

(Sudiman, et al. 2014a)



34

2.1 ABSTRACT

The aim of this study was to determine factors affecting the efficiency of exogenous

oocyte-secreted factors (OSFs) in improving the developmental competence of

mouse in vitro matured oocytes. We hypothesised that varying native OSF exposure

would have differential effects on post-in vitro maturation (IVM) embryo and fetal

development due to the different forms that are secreted by denuded oocytes (DOs)

under different conditions. Mouse cumulus oocyte complexes (COCs) were co-

cultured with DOs from 0h or 3h of IVM. DOs were matured for 3h as either intact

COCs+/-FSH before denuding, or as DOs+FSH. COCs were fertilised and blastocyst

development was assessed on days 5 and 6, and were then either differentially

stained to determine ICM numbers or vitrified/warmed embryos were transferred to

recipients. Embryos derived from COCs+DOs had significantly improved blastocyst

rates and ICM numbers compared to controls (P<0.05). The highest response was

obtained when DOs were first added to COCs at 3h of IVM, after being pre-treated

(0-3h) as COCs+FSH. Compared to control, co-culture with DOs from 3h did not

affect implantation rates but more than doubled fetal yield (21% vs 48%; P<0.05).

GDF9 Western blot analysis was unable to detect any differences in quantity or form

of GDF9 (17 and 65 kDa) in extracts of DO at 0h or 3h. This study provides new

knowledge on means to improve oocyte quality in vitro which has the potential to

significantly aid human infertility treatment and animal embryo production

technologies.



35

2.2 INTRODUCTION

In vitro maturation (IVM) of oocytes is an alternative system used in assisted

reproductive technology for generating embryos in vitro, which reduces or eliminates

the need to administer gonadotropins to patients. In addition, IVM is widely used in

the domestic animal sector to further advanced-breeding technologies. However,

there is a discrepancy in the success rate of conventional in vitro matured oocytes

compared to in vivo matured oocytes. In women, it is reported that the pregnancy

rate post-IVM is less than half of the pregnancy rate post-IVF (in vitro fertilisation)

(Gremeau, et al. 2012). To overcome this challenge, it is important to understand

factors that regulate the maturation and development of oocytes which can be

implemented in clinical and veterinary scenarios to improve the success rate of IVM

(Gilchrist 2011).

Oocytes acquire developmental competence in the ovarian follicle. There are

numerous molecules, proteins and cellular processes involved in the bi-directional

communication axis between the oocyte and follicular somatic cells (Gilchrist, et al.

2004a). The oocyte regulates this communication network to a significant degree.

The oocyte secretes soluble factors (OSFs) which are transmitted to cumulus cells

via paracrine signalling to regulate a multitude of important cumulus cell processes,

including; proliferation (Gilchrist, et al. 2006, Vanderhyden, et al. 1992), apoptosis

(Hussein, et al. 2005), differentiation (Li, et al. 2000), luteinisation (Salustri, et al.

1990), metabolism (Caixeta, et al. 2013, Su, et al. 2008, Sugiura, et al. 2007, Sutton-

McDowall, et al. 2012) and expansion (Dragovic, et al. 2005, Dragovic, et al. 2007).

Another type of communication between the oocyte and cumulus cells is via gap

junctional signalling, mediated by cumulus trans-zonal cytoplasmic projections that

abut the oocyte membrane, enabling transport of cAMP, purines/pyrimidines, amino

acids and other small regulatory molecules (Eppig and Downs 1984, Salustri and

Siracusa 1983, Salustri, et al. 1989, Thomas, et al. 2004). Cumulus cells provide

signals and molecules to the oocyte to regulate meiosis and promote developmental

competence (Dey, et al. 2012, Gilchrist, et al. 2008). Removal of cumulus cells during

maturation has a significant detrimental effect on nuclear maturation, normal

fertilization and developmental competence of oocytes (Wongsrikeao, et al. 2005,



36

Zhang, et al. 1995). Follicle-stimulating hormone (FSH) is typically added to IVM

medium to stimulate cumulus expansion (Salustri, et al. 1989) and increase normal

fertilization rates and fetal development of IVM oocytes (Ali and Sirard 2005,

Merriman, et al. 1998).

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15)

are two well-known soluble growth factors derived from oocytes that are important for

normal cumulus cell function and hence normal oocyte development (Gilchrist, et al.

2008). In mouse oocytes, the expression level of GDF9 mRNA is much higher

compared to BMP15 (Crawford and McNatty 2012), reflecting the relative importance

of their roles in folliculogenesis. Homozygous GDF9 mutant mice are sterile due to a

block in follicular development beyond the primary follicle stage (Dong, et al. 1996),

whereas BMP15 null mice demonstrate only a mild reduction in ovulation and

fertilization rates (Yan, et al. 2001). Homozygous GDF9 or BMP15 mutant sheep

derived either by natural mutation or active immunization are sterile, however,

heterozygous carriers of mutations in either GDF9 or BMP15 have an increased

ovulation rate and multiple pregnancies (Galloway, et al. 2000, Hanrahan, et al.

2004, Juengel, et al. 2004). GDF9 and BMP15 also play an important role in human

fertility, where several mutations of GDF9 may be potentially associated with

polycystic ovary syndrome (Wang, et al. 2010) and rare variants of GDF9 contribute

to dizygotic twinning (Palmer, et al. 2006). Moreover, rare mutations of GDF9 and

BMP15 are associated with premature ovarian failure (Chand, et al. 2006, Dixit, et al.

2005, Wang, et al. 2013).

GDF9 and BMP15 in the proteolytically processed form consist of two structural

parts: the pro-region and the mature region (McIntosh, et al. 2008). GDF9 and

BMP15 are processed and secreted as non-covalent complexes of the pro- and

mature regions, the mature region being the bioactive receptor binding region,

whereby the pro-region plays a vital and species-specific role in regulating bioactivity

(Al-Musawi, et al. 2013, Mottershead, et al. 2008, Simpson, et al. 2012). Immunizing

mice against the pro-regions of GDF9 or BMP15 caused ovary abnormalities and

smaller litter sizes (McIntosh, et al. 2012).



37

However, the exact forms of GDF9 and BMP15 secreted by oocytes in vivo and in

vitro are unclear. Mouse in vitro matured oocytes secrete GDF9 as a mixture of the

unprocessed pro-protein and mature domain (Gilchrist, et al. 2004b). Rat IVM

oocytes secrete the mature domains of GDF9 and BMP15 (Lin, et al. 2012). Sheep

follicular fluid contains GDF9 and BMP15 proteins in the unprocessed pro-protein

form only (McNatty, et al. 2006), whereas sheep oocytes secrete the mature domains

of GDF9 and BMP15 during IVM (Lin, et al. 2012). To our knowledge, the only

commercially available forms of GDF9 and BMP15 produced in a recombinant

mammalian cell expression system are from R&D Systems and these consist of

purified mature domains only.

IVM COCs have aberrant gene expression and altered matrix protein profiles in

cumulus cells compared to in vivo matured oocytes (Dunning, et al. 2007, Kind, et al.

2013, Richani, et al. 2013), and in vitro matured oocytes also have notably

decreased BMP15 protein compared to in vivo matured oocytes (Mester, et al. 2013).

Currently, studies show that addition of exogenous native OSFs during IVM

significantly improves subsequent goat (Romaguera, et al. 2010), cow (Dey, et al.

2012, Hussein, et al. 2011, Hussein, et al. 2006) and pig (Gomez, et al. 2012)

embryo development, and recombinant GDF9 and BMP15 in their pro-mature form

improve in vitro matured cattle (Hussein, et al. 2011, Hussein, et al. 2006, Sutton-

McDowall, et al. 2012) and mouse oocytes (Yeo, et al. 2008). In addition, differing

degrees of improved bovine oocyte competence were observed depending on the

temporal pattern of addition of OSFs during IVM (Hussein, et al. 2011). In the current

study we examine factors that affect the production of native OSFs by mouse

oocytes, such as the timing of addition of denuded oocytes (DOs), the presence of

cumulus cells and FSH before denuding COCs and addition to IVM, and whether

these factors affect the form of GDF9. Outcomes examined were embryo

development, cryotolerance of blastocysts and fetal measures following embryo

transfer.



38

2.3 MATERIALS AND METHODS

All chemicals and media were purchased from Sigma Chemical (St Louis, MO, USA)

unless indicated otherwise.

2.3.1 Isolation and maturation of COCs

Mice used in this study were maintained in the Animal House, Medical School,

University of Adelaide. This study was approved by the local animal ethics committee

and conducted in accordance with the Australian Code of Practice for the Care and

Use of Animals for Scientific Purposes.

Twenty-one to twenty-eight day-old SV129 mice were injected intraperitoneally with 5

IU equine chorionic gonadotropin (eCG; Folligon, Intervet, Castle Hill, Australia), and

ovaries were collected 46-48 hours (h) later. Ovaries were cleaned of any connective

tissues and placed in HEPES-buffered αMEM (handling medium, GIBCO, USA)

supplemented with 3 mg/ml fatty-acid free bovine serum albumin (FAF BSA, MP

Biomedicals, USA), 1 mg/ml fetuin and 50 µmol 3-isobutyl-1-methylxanthine (IBMX).

Antral follicles were punctured with 27-gauge needles and immature COCs collected

in handling medium, then washed twice with handling medium minus IBMX. Only

COCs with compact cumulus cells (CCs) were taken. The rationale behind the use of

IBMX was to maintain all COCs in the same nuclear stage before transfer into

maturation medium or being denuded. For the control group, 20 COCs were cultured

in 50 µl IVM drops [(αMEM supplemented with 3 mg/ml FAF BSA, 1 mg/ml fetuin and

50 mIU/ml FSH (Puregon, Organon, Oss, Netherlands)] overlaid with mineral oil in 60

mm Petri dishes (Falcon, Becton Dickinson, USA) for 17-18 h at 370C in a humidified

atmosphere of 5% CO2 in air. For treatment groups, 20 COCs were cultured with 50

DOs in each 50 µl IVM drop (1 DO/µl).

2.3.2 Generation of denuded oocytes as a source of native OSFs

DOs were generated by vortexing COCs for ~2 minutes in αMEM handling medium in

order to remove the CC.

The DOs were then subjected to three processing methods used to produce native

OSFs before co-culture with immature COCs (refer to Fig. 2.1):



39

1. COCs were denuded at 0 h before co-culture of 50 DOs with 20 COCs in IVM

medium containing FSH. For COCs+DOs, IBMX was not present in the

handling medium, and therefore these DOs were at the GVBD stage. This

group was utilized to assess the benefits of DOs as a source of native OSFs

and will be referred to as COCs+DOs. By contrast, for COCs+DOs (0h [GV]),

IBMX was present in the handling medium and therefore these DOs were at

the GV stage.

2. COCs were matured first for 3 h in separate IVM medium containing FSH.

After 3 h of IVM, COCs were denuded and 50 DOs were co-cultured with 20

COCs for another 14-15 h in IVM medium containing FSH (Fig. 2.1 A). This

group is based on the design by Hussein et al (2011), and was utilized to

examine temporal, cumulus cell and FSH effects on the production of native

OSFs. This group is referred to as COCs+DOs at 3h+FSH+CC.

3. Intact COCs were matured first for 3 h in separate IVM medium without FSH

(Fig. 2.1 B). After 3 h of IVM, COCs were denuded and 50 DOs were co-

cultured with 20 COCs for another 14-15 h in IVM medium containing FSH.

This group assessed the production of native OSFs in the absence of FSH

during the first 3 h of maturation and is referred to as COCs+DOs at 3h-

FSH+CC.

4. COCs were denuded at 0 h and then matured for 3 h as DOs in separate IVM

medium containing FSH, then 50 of these DOs were co-cultured with 20

COCs for another 14-15h in IVM medium containing FSH (Fig. 2.1 C). This

group is referred to as COCs+DOs at 3h+FSH-CC and was designed to

assess the effect of CCs on production of native OSFs.



40

Figure 2.1 Schematic illustration of methods of producing native OSFs based

on pretreatment of oocytes with and without cumulus cells and FSH. A. Intact

COCs cultured for 3 h in separate IVM medium containing FSH. After 3 h, COCs

were denuded of CCs and the resultant DOs were then co-cultured with a separate

cohort of COCs + FSH for 14-15 h, before the COCs underwent standard IVF and

embryo culture (3h+FSH+CC). B. COCs pre-cultured for 3 h in separate IVM medium

without FSH, before denuding and co-culture (3h-FSH+CC). C. COCs were denuded

at 0 h and then pre-cultured for 3 h in separate IVM medium containing FSH, then

these DOs were co-cultured with COCs for 14-15h (3h+FSH-CC).



41

2.3.3 Oocyte nuclear maturation

An experiment was performed to assess whether adding native OSFs affects the

timing of oocyte nuclear maturation. At 12 h and 15 h of IVM, COCs were denuded

and fixed in paraformaldehyde for 30 minutes, then stained with DAPI. The presence

of the first polar body was determined using an upright microscope (Nikon, TE 2000-

E) under UV light.

2.3.4 In vitro fertilization

Sperm from CC x Bagg, A strain (CBA) F1 male mice aged 6-24 weeks and of

proven fertility were incubated for 1 hour in IVF medium (COOK® IVF, Research

medium, Catalogue No: K-RVWA- 50, Australia) under 5% CO2 at 370C for

capacitation. Post-IVM, 5-10 COCs were transferred into a 90 µl equilibrated IVF

drop overlaid with mineral oil and then 10 µl of capacitated sperm were added into

the drops. Three hours post-fertilization under 5% CO2 at 370C, the presumptive

zygotes were denuded of sperm and cumulus cells. Insemination day was counted

as day 1 (0 h).

2.3.5 In vitro culture of embryos

Five to ten presumptive zygotes were cultured in 20 µl drops of equilibrated culture

media (COOK® IVC, Research medium, Catalogue No: K-RVCL-50, Australia)

overlaid with mineral oil under 6% CO2, 5% O2 and 89% N2. Five to ten embryos

were cultured for six days, with cleavage rates assessed on day 2 (24 h post-

fertilization) and blastocysts assessed on day 5 (96-100 h post-fertilization) and day 6

(120-24 h post-fertilization).

2.3.6 Differential staining of blastocysts

Differential staining was performed on day 6 blastocysts to assess inner cell mass

(ICM) and trophectoderm (TE) cells. Briefly, expanded, hatching and hatched

blastocysts were placed into pronase (5 mg/ml) at 370C until the zona dissolved. The

zona-free blastocysts were incubated in 10 mM trinitrobenzene sulfonic acid (TNBS)

in 0.4% polyvinyl alcohol (PVA) in phosphate-buffered saline (PBS) at 40C for 10

minutes then washed twice before being transferred into 0.1 mg/ml anti dinitrophenol-



42

BSA antibody at 370C for 10 minutes. Embryos were then incubated in 10 µg/ml

propidium iodide for 5-10 minutes at 370C, followed by 4 µg/ml Hoechst 33342 in

96% ethanol at 40C overnight. The blastocysts were transferred onto a glass

microscope side, covered by a cover slip and assessed immediately under UV

fluorescence using an upright microscope (Nikon, TE 2000-E, excitation, 340-380

nm; emission, 440-480 nm), where ICM cells appeared blue and TE cells appeared

pink.

2.3.7 Vitrification and warming of blastocysts

Hatching and expanded blastocysts on day 6 were vitrified and then within 1-2 weeks

those blastocysts were warmed and assessed for cryotolerance, prior to embryo

transfer. Vitrification and warming solutions were prepared in HEPES-buffered

αMEM. The vitrification solution consisted of 2.3 M dimethylsulfoxide (DMSO) + 3 M

ethylene glycol supplemented with 0.75 M sucrose. The corresponding equilibration

solution contained half the concentration of cryoprotectants of the vitrification solution

and no sucrose. Vitrification steps were performed at 370C. Blastocysts were placed

in equilibration solution for 3 minutes then transferred to vitrification solution, with

minimal transfer of equilibration solution (less than 3 µl), placed on a fibre-plugTM

(CVM kit, Cryologic, Victoria, Australia) and touched on a pre-cooled steel block in

liquid nitrogen, and then sealed in a pre-cooled straw (CVM kit, Cryologic) for storage

in liquid nitrogen. The time taken from leaving equilibration solution to vitrification on

the block was 30 - 40 sec. Vitrified blastocysts were warmed at 370C and sequentially

passed through 3 solutions of sucrose (0.3 M, 0.25 M and 0.15 M) in HEPES-

buffered αMEM for 5 minutes each. Blastocysts were held in culture medium for 2-3 h

and then blastocyst morphology was assessed before being transferred into foster

mothers. The criteria used for blastocyst cryosurvival were as follows; normal:

blastocysts expanded with no signs of lysis, collapse or degeneration; abnormal:

blastocysts with signs of lysis, collapse and degeneration (Gautam, et al. 2008).

2.3.8 Embryo transfer and assessment of fetal outcomes

Vitrified/warmed blastocysts assessed as normal were transferred into pseudo

pregnant recipients (Swiss female mice aged 12-20 weeks, mated with vasectomised



43

F1 CBA males). On day 3.5 of pseudo pregnancy, recipients were anaesthetized with

2% Avertin (0.015 ml/g body weight) prior to embryo transfer. For each recipient, six

normal expanded or hatching blastocysts post-vitrification/warming were transferred

to each uterine horn, with control embryos in one horn and treatment embryos in the

other. Eight replicates were performed with a total of 96 blastocysts transferred into 8

recipients. The number of implantation sites, fetuses, fetal and placental weights and

fetal crown to rump lengths were assessed on day 17 post-embryo transfer.

2.3.9 Immunodetection of oocyte GDF9

DOs and oocyte-conditioned media were collected for western blot analysis of the

form and quantity of GDF9 protein. Briefly, COCs were collected and denuded at 0 h

and suspended at a ratio of 8 DOs/µl in IVM medium in Eppendorf tubes. DOs were

matured for 17-18 h under 5% CO2 at 370C. Post-IVM, DOs were pelleted by

centrifugation and oocyte-conditioned medium was transferred to a separate tube

and snap frozen in PBS. Oocytes were washed once in PBS, after which the PBS

was removed and the cells snap frozen. Oocytes and oocyte-conditioned medium

were also collected from the 3h+FSH+CC group (Fig. 2.1 A). COCs were matured

first for 3 h in IVM medium, then denuded and matured for an extra 14-15 h in fresh

IVM medium. Post-IVM, oocytes and oocyte-conditioned medium were processed as

above. Samples (30-60 DOs or media equivalent) were boiled for 3 minutes in 1xLDS

reducing buffer [15ul, 1 x NovexNuPAGE LDS sample buffer (NP0009)] with β-

mercaptoethanol (NovexNuPAGE NP0007, Carlsbad, USA) and centrifuged. The

supernatant was fractionated by SDS-PAGE for 1h at RT on 4-12% Bis-Tris pre-cast

gel (BioRad, Hercules, Ca, USA, Cat#345-0125) in MES buffer (Novex, NuPAGE,

NP0002) followed by electrotransfer at 30V overnight at 40C onto a nitrocellulose

membrane (Hybond-C Extra, GE Healthcare Life Sciences, Bucks, UK). The

membrane was incubated with biotinylated GDF9- monoclonal antibody 53 (Oxford

Brookes University, Oxford, UK) at 1:2,000 for 1hr at RT then washed, followed by a

further incubation with horseradish peroxidase-conjugated anti-mouse IgG

(SNN2004, Lot No 892329B, Biosource), at 1:10,000 for 1h at RT. Immunoreactive

proteins were detected using Lumilightchemiluminesence reagents (Roche

Diagnostics, GmbH Mannheim, Germany). Membranes were scanned on the



44

BioRadChemiDoc MP system and the images analysed using Image J software

(Biorad). Recombinant mGDF9 (17k) was used as a reference protein. Biotinylated

molecular weight protein standards (Cat No. MW001) were obtained from R&D

Systems, (Minneapolis, USA). Parallel controls (blanks) were undertaken with oocyte

extracts in the absence of biotinylated antibody but in the presence of HRP-IgG

antibody. Five replicates were performed to detect the form and quantity of GDF9

protein.

2.4 STATISTICAL ANALYSES

All proportional data for embryo development were arcsine transformed and analysed

using either an independent t-test or multivariate ANOVA with least significant

difference (LSD) post-hoc tests. Log transformation was performed for blastocyst cell

number when data were not normally distributed, and analysed using an independent

t-test or one way ANOVA with either LSD or Dunnet’s T3 post-hoc tests, depending

on whether the data did or did not pass the homogeneity of variance test,

respectively. Fetal measurements were analysed with independent t-tests with LSD

post-hoc tests. Oocyte nuclear maturation, post-warm survival rate of blastocysts,

and post-embryo transfer data for fetal survival and implantation rates were assessed

using a Chi-square test. All statistical analyses were performed using SPSS version

13 (SPSS Inc, Chicago, IL, USA). Differences were considered statistically significant

at P < 0.05. Data are expressed as means ± SEM.

2.5 RESULTS

2.5.1 Effect of co-culture of intact mouse COCs with native OSFs

during IVM

Exposure of intact COCs to exogenous native OSFs from DOs throughout IVM

significantly (P < 0.05) increased the cleavage rate compared to COCs cultured

alone. ICM cell numbers were also significantly increased in blastocysts derived from

COCs exposed to DOs, whereas TE and total cell numbers were not affected (Tables

2.1 and 2.2).



45

Table 2.1 Effect of native OSFs during IVM on subsequent embryo development

Values with different superscript within a column are statistically different (P < 0.05).

Data are presented as means ± SEM of 4 replicate experiments. Each replicate

experiment consisted of 53-60 oocytes.

1Percentage of cleaved embryos per total oocytes

2Percentage of blastocysts generated 96-100 h post-fertilization per cleaved embryo


4Percentage of hatching blastocysts generated 100-125 h post fertilization per

cleaved embryo

5 COCs were cultured without DOs

6GVBD-stage denuded oocytes (DO) added from t= 0h of IVM

Table 2.2 Effect of native OSFs during IVM on inner cell mass (ICM), trophectoderm

(TE) and total cell numbers (TCN) of day 6 blastocysts

Treatments ICM TE TCN

Control1

COC+DO2

18.5±0.8a

25.3±1.5b

55.8±2.1

55.5±2.5

74.3±2.6

80.8±3.5

Values with different superscripts within a column are statistically different (P < 0.05).

Data are presented as means ± SEM. Mean blastocyst cell numbers following

differential staining of 16-18 blastocysts.

1COCs were cultured without DOs

2GVBD-stage denuded oocytes (DO) added from t= 0h of IVM

Treatments No. of

Oocytes

Cleavage1

(%)

Blastocyst

on day 52

Blastoycst

on day 63

Hatching

Blastocyst

on day 64

Control5

COC+DO6

224

233

90.6±1.8a

96.1±0.8b

52.8±8.9

60.1±4.9

77.3±4.6

74.3±2.4

58.8±8.6

68.0±1.9



46

2.5.2 Temporal effect of co-culture of intact mouse COCs with

native OSFs during IVM

A previous study by Hussein, et al. 2011 demonstrated that there is a temporal effect

of native OSFs on bovine oocyte developmental competence. Based on this result,

we assessed mouse oocyte developmental competence following co-culture of COCs

with DOs in a temporal manner. COCs were co-cultured with DOs from 0 h (0h [GV]

stage) or with DOs precultured as COCs for 3h+FSH+CC (refer to Fig. 2.1 A).

Following co-culture of COCs with DOs from 0-17 h (0h [GV] stage), a significant

increase was observed in cleavage rate compared to controls (Table 2.3), however

there were no significant differences in blastocyst rates or ICM number (Table 2.4).

By contrast, treating COCs with DOs from 3h (3h+FSH+CC) significantly (P < 0.05)

increased blastocyst and hatching blastocyst rates measured at day 6 and

subsequent ICM cell numbers, compared to the control group (Tables 2.3 and 2.4).



47

Table 2.3 Temporal effect of co-culture of intact mouse COCs with native OSFs on subsequent embryo development


Data are presented as means ± SEM of 4 replicates experiments. Each replicate experiment consisted of 37-40 oocytes.

1Refer to Fig. 2.1 for explanation of experimental design

2 Percentage of cleaved embryos per total oocytes



5Percentage of hatching blastocysts generated 100-125 h post fertilization per cleaved embryo


Treatments Pre-treatment of

OSFs1

No. of

Oocytes

Cleavage2

(%)

Blastocyst

on day 53

Blastoycst

on day 64

Hatching

Blastocyst

on day 65

Control6

COC+DO

COC+DO

N/A

0h [GV]

3h+FSH+CCs

152

155

153

87.5±1.6a

95.5±2.2b

94.8±1.1a,b

52.2±7.2

57.5±2.6

65.5±1.7

67.4±6.0a

77.2±4.4a,b

83.4±2.6b

53.3±5.6a

66.9±2.8a,b

74.5±2.8b

Chapter 2: Factors affecting the capacity of exogenous oocyte secreted factor

to improve mouse

48


inner cell mass (ICM), trophectoderm (TE) and total cell numbers (TCN) of day 6

blastocysts







OSFs1

ICM TE TCN

Control2

COC+DO

COC+DO

N/A

0h [GV]

3h+FSH+CCs

18.7±1.9a

22.0±1.7a,b

24.8±1.3b

51.1±3.4

52.4±2.4

55.7±2.7

69.8±4.3

74.4±3.5

80.5±3.4


to improve mouse

49

2.5.3 Effect of exposure of COCs to native OSFs on nuclear

maturation

Native OSFs regulate CC cGMP levels (Wigglesworth, et al. 2013) which affects

meiosis and, according to Dey, et al. 2012, co-culture of COCs with DOs increased

nuclear maturation rate in bovine oocytes. Since the highest competence treatment

group from the previous experiment was COCs co-cultured with DOs from 3h

(3h+FSH+CC), we hypothesised that an effect on oocyte nuclear maturation could

account for this enhanced oocyte quality in mice. However, in the present

experiment, there was no significant difference in the maturation rates of control

oocytes or COCs exposed to DOs (3h+FSH+CC) at 12h or 15h of IVM (Table 2.5).

Table 2.5 Effect of native OSFs added at 3 hours on oocyte nuclear maturation


OSFs1

Maturation time

(h)

N MII oocytes

(%)

Control2

COC + DO

N/A

3h+FSH+CCs

12

15

12

15

111

74

113

78

92.8

91.9

93.8

93.6

Data are presented as means of 3 replicate experiments. Each replicate experiment

consisted of 33-39 oocytes.





50

2.5.4 Temporal effects on oocyte GDF9 levels

Given the beneficial effects of pre-culture of oocytes with their CCs for 3h with FSH

on OSF-stimulated oocyte developmental competence (Table 2.4), we attempted to

quantify the amount and form of GDF9 expressed within and secreted by oocytes.

The band for the un-processed pro-protein was observed at ~65 kDa and the mature

protein at ~17 kDa, with the mature protein less abundant in oocyte extracts

compared to that observed in the recombinant mGDF9 standard (Fig. 2.2). Three

hours of pre-culture (3h+FSH+CC) had no effect on the total quantity or the

proportion of pro-versus mature-GDF9 in oocytes (Fig. 2.3). No GDF9 bands were

observed in the DO conditioned media.

Figure 2.2 GDF9 Western blot comparison of denuded oocyte (DO) extracts at

0h and 3h (3h+FSH+CC) of pretreatment. mGDF9 refers to recombinant mGDF9

standard. Blank refers to DO sample in the absence of the GDF9 primary antibody.

The 65kDa band corresponds to the unprocessed GDF9 pro-protein while the 17kDa

band corresponds to the mature domain. The 90 kDa-180 kDa bands seen in all

samples are attributed to the non-specific presence of biotinylated proteins in the cell

extracts.



51

Figure 2.3 Quantitative analysis of Western blot denuded oocyte extracts at 0

and 3h (3h+FSH+CC) of pretreatment. Data from 5 separate experiments. The

Western blot image was quantified using BioRadChemiDoc MP system and analysed

using Image J software (Biorad)



52

2.5.5 The role of FSH and cumulus cells in the production of native

OSFs

We hypothesized that FSH and cumulus cells are two factors that affect the

production of native OSFs. In this experiment, DOs were generated using different

methods (refer to Fig. 2.1). Following co-culture of COCs with precultured DOs

(3h+FSH+CC; refer to Fig. 2.1 A), a significantly increased blastocyst rate on day 5

was observed compared to control, which contributed to an improvement in ICM

number compared to control (Tables 2.6 and 2.7). DOs that were precultured as

intact COCs but without FSH (3h-FSH+CC, refer to Fig. 2.1 B) did not stimulate

development competence in their co-cultured COCs; these exhibiting embryo

developmental parameters equivalent to the control and ICM numbers significantly

lower than those stimulated with FSH (3h+FSH+CC). Interestingly, there was a

significant improvement in blastocyst rate on day 6 from COCs co-cultured with DOs

that were without CCs from 0-3h (3h+FSH-CC, refer to Fig. 2.1 C) compared to the

control. Overall, all treatment groups co-cultured with DOs tended to show an

improvement in embryo development.



53

Table 2.6 Effects of cumulus cells and FSH on native OSFs efficacy at enhancing post-IVM embryo development

Values with different superscripts within a column are statistically different (P < 0.05)

Data are presented as means ± SEM of 5 replicate experiments. Each replicate experiment consisted of 36-39 oocytes.








OSFs1

No. of

Oocytes

Cleavage2

(%)

Blastocyst

on day 53

Blastoycst

on day 64

Hatching

Blastocyst

on day 65

Control7

COC+DO

COC+DO

COC+DO

N/A

3h+FSH+CCs

3h-FSH+CCs

3h+FSH-CCs

192

191

190

191

92.7±2.9

93.6±1.9

94.7±1.9

93.7±2.5

52.5±3.4a

65.2±5.6b

61.0±3.6a,b

63.8±3.5a,b

76.6±3.0a

83.2±2.0a,b

79.5±2.5a,b

86.2±2.9b

60.8±3.3

69.0±3.4

66.7±4.0

70.4±2.7


to improve mouse and fetal development post-IVM

54

Table 2.7 Effects of cumulus cells and FSH on native OSFs on inner cell mass (ICM),

trophectoderm (TE) cells and total cell number (TCN) of day 6 blastocysts







OSFs1

ICM TE TCN

Control2

COC+DO

COC+DO

COC+DO

N/A

3h+FSH+CCs

3h-FSH+CCs

3h+FSH-CCs

11.9±0.6a

13.8±0.5b

11.7±0.5a

12.2±0.7a,b

43.1±2.2

43.6±1.5

46.4±1.9

44.1±2.0

55.0±2.4

57.4±1.6

58.2±2.0

56.3±2.4



55

2.5.6 Effect of exposure of COCs to native OSFs during IVM on

cryo-survival of blastocysts

With respect to cryopreserved blastocysts, the blastocyst re-expansion rate post-

vitrification/warming (normal morphology of blastocysts) in blastocysts derived from

COCs exposed to native OSFs (COCs+DOs at 3h+FSH+CC) did not differ

significantly from controls (Table 2.8). Therefore exposure of COCs to native OSFs

did not measurably alter the cryotolerance of blastocysts.

Table 2.8 Effect of native OSFs during IVM on the survival rate of post-warmed

blastocysts

Data are presented as means of 6 replicate experiments. Each replicate experiment

consisted of 10-19 blastocysts.




OSFs1

No of

Blastocysts

Normal

(%)

Abnormal

(%)

Control2

COC+DO

N/A

3h+FSH+CCs

90

92

73 (81.1)

74 (80.4)

17 (18.9)

18 (19.6)



56

2.5.7 Effect of exposure of COCs to native OSFs on pregnancy rates

and fetal outcomes

After 2 h of warming, the morphologically normal blastocysts were transferred into

pseudo pregnant recipients. There was no difference in implantation rates between

the control and treatment group (COCs co-cultured with DOs [3h+FSH+CC]).

However, the proportion of fetuses that developed to day 17 per implantation site,

from COCs co-cultured with native OSFs, was more than double that of the control

(21% vs 48%, P < 0.05, Fig. 2.4). Moreover, based on gross morphological criteria,

those fetuses were normal, with no significant differences between treatments in fetal

or placental weight or fetal crown-to-rump length (Table 2.9).



57

Figure 2.4 Effect of treatment with native OSFs during IVM on subsequent

pregnancy outcomes. Blastocysts were derived from standard IVM (control) or from

COCs exposed to native OSFs during IVM (COC+DO; 3h+FSH+CC pretreatment).

Control and treatment blastocysts were vitrified on day 6 and then later warmed and

transferred to pseudo pregnant recipients. Six control and six treatment blastocysts

were transferred into each uterine horn of a recipient (n=8). Pregnancy outcomes

were analysed on day 17. Implantation rate; implantation sites/embryos transferred

(n = 48 embryos transferred/treatment). Fetal yield; day 17 fetuses/implantation sites



58

Table 2.9 Effect of native OSFs during IVM on subsequent fetal and placental weight

and crown-rump length

Data are presented as means ± SEM of 6 and 13 fetuses (control and treatment,

respectively) from 8 recipients.

1Refer to Figure 2.1 for explanation of experimental design


Treatments Pre-treatment

OSFs1

Placental

weight (g)

Fetal weight

(g)

Crown to

rump length

(mm)

Control2 N/A 0.2 ± 0.01 1.8 ± 0.1 24.5 ± 1.9

COC + DO 3h+FSH+CC 0.2 ± 0.01 1.7 ± 0.1 25.6 ± 0.8



59

2.6 DISCUSSION

Our results show that treatment of COCs during IVM with native OSFs, enhances

oocyte quality, as measured by subsequent embryo cleavage, blastocyst and fetal

survival rates. These results are supported by previous studies which have shown

that exogenous native OSFs (obtained by co-culture with DOs), when used as

supplements in oocyte IVM, significantly improve embryo development in cattle (Dey,

et al. 2012, Hussein, et al. 2011, Hussein, et al. 2006), pigs (Gomez, et al. 2012) and

goats (Romaguera, et al. 2010). In contrast, addition of recombinant mature

homodimers of GDF9 and/or BMP15 (R&D Systems) in IVM media did not improve

mouse embryo development (Sudiman, et al. 2014a). However, previous results from

our laboratory using a complex of pro- and mature-domains of GDF9 and BMP15

(pro-mature protein) showed a positive effect on bovine (Hussein, et al. 2011,

Hussein, et al. 2006, Sutton-McDowall, et al. 2012) as well as mouse (Yeo, et al.

2008) oocyte developmental competence.

From this study, we suggest that there are a number of factors which influence the

capacity of exogenous OSFs to improve mouse embryo and fetal development post-

IVM namely; the capacity of cumulus cells and FSH to regulate native OSF

production, and the temporally regulated potency of the native OSF pool.

Supplementation of IVM with OSFs has the potential to dramatically improve the

efficiency of IVM and therefore its applicability in human and veterinary clinics

(Gilchrist 2011), as we saw more than a doubling in fetal yield using this approach.

However, co-culture of COCs with denuded oocytes is not practical in a clinical

scenario. Even though the exact identity of growth factors secreted by denuded

oocytes in vitro remains poorly characterised, the principal OSFs are thought to be

GDF9, BMP15, fibroblast growth factor 8 (FGF8) and FGF10, with widely differing

roles for individual growth factors between species (Dong, et al. 1996, Galloway, et

al. 2000, Gilchrist, et al. 2004b, Hanrahan, et al. 2004, Juengel, et al. 2004, Lin, et al.

2012, Sugiura, et al. 2007, Zhang, et al. 2010). Notably, the only commercially

available recombinant preparations of GDF9 and BMP15 produced in mammalian

cells, had no effect on post-IVM embryo development, despite the extensive dose



60

range tested (Sudiman, et al. 2014a). By contrast, recombinant FGF10 added during

bovine IVM enhances subsequent embryo development (Zhang, et al. 2010).

When full length recombinant GDF9 and BMP15 are expressed in mammalian cells

such as human embryonic kidney 293H cells, the proteins are processed (pro and

mature domains are proteolytically cleaved) and secreted into the medium as dimers

or monomers of pro- and mature regions, that are non-covalently bound as pro-

mature complexes (McIntosh, et al. 2008). Our in-house produced GDF9 and BMP15

preparations (Gilchrist, et al. 2004b, Hickey, et al. 2005), which contain both the pro-

and mature domains, when added to IVM, significantly improve the quality of the

blastocysts generated (as measured by ICM cell number) and thus improve fetal

survival rates in mouse (Yeo, et al. 2008), as well as increase embryo development

in cattle (Hussein, et al. 2011, Hussein, et al. 2006, Sutton-McDowall, et al. 2012).

The current results demonstrated that blastocyst development and fetal survival rates

were further improved when COCs were co-cultured with DOs from 3 h (versus

control), if those DOs were matured as COCs for the first 3 hours in the presence of

FSH, then subsequently denuded and added to IVM. This result supports previous

findings in bovine IVM oocytes, which suggested a temporal effect on secretion of

OSFs (Hussein, et al. 2011). It has been demonstrated that even a short exposure of

COCs to FSH significantly increases subsequent bovine blastocyst development, via

protein kinase C activation (Ali and Sirard 2005). Hence, the FSH-treated CCs may in

some manner improve the quantity and/or quality of factors which are secreted by the

resultant DO. During the 3 h maturation, the oocyte and cumulus cells communicate

with each other through gap junctional communication and via paracrine factors,

which presumably provides enough time for the oocyte to obtain the beneficial effect

of FSH through the receptors in cumulus cells. This effect may be mediated more by

paracrine factors rather than gap junctional communication. After meiotic resumption,

gap junctional communication between the oocyte and cumulus cells is lost (Thomas,

et al. 2004). The action of OSFs can be observed even without direct contact

between cumulus cells and oocytes, suggesting that they are acting in a paracrine

manner (Hussein, et al. 2005). In cattle, the developmental competence of DOs co-



61

cultured with COCs is significantly improved compared to DOs cultured alone (Dey,

et al. 2012, Hussein, et al. 2006).

Based on the notable improvement in oocyte developmental competence from the

3h+FSH+CC group, we anticipated that COCs matured first for 3 h in medium

containing FSH would produce different quantities and/or forms of GDF9, compared

to oocytes denuded at 0 h. However, unfortunately we were unable to detect any

GDF9 in oocyte-conditioned medium, unlike the recent study by Lin et al (2012). This

may be due to our oocyte-conditioning medium procedures or the western blot being

insufficiently sensitive to detect actual secreted forms. Also, we could not observe

any effect of the 3 h pre-culture with FSH treated cumulus cells on the GDF9

produced within the oocyte. It showed that there was no difference in the amount or

the proportion of either the pro-protein or the mature form of GDF9 in DOs at 0 h or 3

h. While GDF9 is the principal OSF produced by mouse oocytes (Crawford and

McNatty 2012, Gilchrist, et al. 2004b), other OSFs such as BMP15 (Yoshino, et al.

2006), FGF10 (Zhang, et al. 2010) and FGF8 (Sugiura, et al. 2007) may be

processed and interact differently in DOs at 0 h and 3 h.

Overall, we have shown that there is an obvious advantage to treatment of COCs

with exogenous OSFs during IVM. Mouse exogenous OSFs did not affect oocyte

nuclear maturation, in contrast with bovine oocytes matured in vitro, where co-culture

with DOs improves nuclear maturation (Dey, et al. 2012). Clearly exogenous OSFs

have major effects on key aspects governing the developmental program of oocytes,

although it is not yet clear how and by which exact mechanisms this is achieved.

Other groups have shown that native OSFs play a significant role in; the activity of

hyaluronic acid synthase 2 (HAS2), which correlates with mucification and expansion

of CCs (Dragovic, et al. 2005), increased GPX1 gene expression (related to the

production of glutathione peroxidise as an antioxidant in oocytes), and enhanced

expression of steroidogenic acute regulatory protein (StAR), which contributes to

oocyte nuclear and cytoplasmic maturation (Dey, et al. 2012). Bovine oocytes

matured with the mature form of BMP15 showed a significant increase in glucose

uptake (Caixeta, et al. 2013), and those matured with pro-mature BMP15 exhibited



62

increased oxidative phosphorylation activity in the oocyte (Sutton-McDowall, et al.

2012).

The results of this current study confirm that OSFs notably improve embryo

development and fetal survival in mice, and extend this knowledge by demonstrating

for the first time that the effect of OSFs on mouse oocytes depends on timing,

presence of FSH and cumulus cells. The beneficial effect of native OSFs on oocyte

quality and subsequent embryo development can be applied in the veterinary clinic;

however, application of co-culture of COCs with DOs is not possible in human IVF

clinics since it is unethical and not feasible to generate supernumerary DOs simply

for OSF supplementation of media. Thus, the next step is to identify and purify the

proteins that are secreted by oocytes, such as GDF9 and BMP15, into a functional

form that can be added into IVM medium, to improve the quality of IVM oocytes and

subsequent embryo development and pregnancy rates.

Chapter 3: Bone Morphogenetic Protein 15 in the Pro-mature Complex Form

Enhances Bovine Oocyte Developmental Competence

63

CHAPTER 3

BONE MORPHOGENETIC PROTEIN 15 IN THE

PRO-MATURE COMPLEX FORM ENHANCES

BOVINE OOCYTE DEVELOPMENTAL

COMPETENCE

(Sudiman, et al. 2014b)



64

3.1 ABSTRACT

Developmental competence of in vitro matured (IVM) oocytes needs to be improved

and this can potentially be achieved by adding recombinant bone morphogenetic

protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM media. The aim of

this study was to determine the effect of a purified pro-mature complex form of

recombinant human BMP15 versus the commercially available bioactive forms of

BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo

development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were

matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15,

mature BMP15 or mature GDF9; each in the absence or presence of FSH. Metabolic

measures of glucose uptake and lactate production from COCs and autofluorescence

of NAD(P)H, FAD++ and GSH were measured in oocytes after IVM. Following in vitro

fertilisation and embryo culture, day 8 blastocysts were stained to determine the cell

numbers. COCs matured in medium +/- FSH containing pro-mature BMP15

displayed significantly improved blastocyst development (57.7±3.9%, 43.5 ± 4.2%)

compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH

(36.1±3.0%), respectively. The mature form of BMP15 produced intermediate levels

of blastocyst development; not significantly different to control or pro-mature BMP15

levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione

(GSH) levels were increased by both forms of BMP15 in the absence of FSH.

Exogenous BMP15 in its pro-mature form during IVM provides a functional source of

oocyte-secreted factors to improve bovine blastocyst development. This form of

BMP15 may prove useful for improving cattle and human artificial reproductive

technologies.



65

3.2 INTRODUCTION

The oocyte and the somatic cells surrounding the oocyte (cumulus cells in antral

follicles) communicate via a complex bi-directional signalling axis mediated via

paracrine and gap junctional means (Gilchrist, et al. 2008). This communication is

important for transporting molecules such as growth factors, cyclic nucleotides,

amino acids and other small regulatory molecules from cumulus cells into oocytes,

and vice versa, in order to sustain cumulus cell health and oocyte development

(Gilchrist, et al. 2008, Herlands and Schultz 1984, Ka, et al. 1997, Zhang, et al.

1995). Oocyte-secreted growth factors (OSFs) act on cumulus cells to regulate

multiple functions, including; differentiation of the cumulus cell lineage (Li, et al.

2000), the proliferation and expansion of cumulus cells (Dragovic, et al. 2005,

Dragovic, et al. 2007, Gilchrist, et al. 2006, Vanderhyden, et al. 1992), prevention of

luteinisation (Li, et al. 2000, Vanderhyden, et al. 1993) and apoptosis (Hussein, et al.

2005) and regulation of cumulus cell metabolism (Eppig 2005, Sugiura, et al. 2005,

Sutton-McDowall, et al. 2012). Healthy cumulus cells are vitally important for the

development of oocytes through the provision of substrates and regulatory

molecules, required for the oocyte to undergo appropriate developmental

programming in order to support early embryo development (Gilchrist 2011).

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15)

are two major and well-known OSFs, required for follicular development and

ovulation (Dong, et al. 1996, Galloway, et al. 2000, Hanrahan, et al. 2004, Juengel, et

al. 2002). The function and expression of both proteins differs markedly between

species (Crawford and McNatty 2012). In polyovular animals, oocyte expression of

GDF9 is notably higher than BMP15, suggesting a minor role for BMP15 in the

regulation of their reproduction. Consistent with this notion, homozygous mutant

GDF9 mice are sterile due to a block in follicular development beyond the primary

follicle stage (Dong, et al. 1996), whereas BMP15 homozygous mutant mice

demonstrate only a mild reduction in fertility (Yan, et al. 2001). In contrast, in

monoovulatory animals, the ratio of oocyte GDF9 to BMP15 is nearly equal

(Crawford and McNatty 2012), reflecting the importance of both of these growth



66

factors in follicular development in these species. Sheep homozyous for certain

naturally occuring mutations in either GDF9 or BMP15 are sterile (Galloway, et al.

2000, Hanrahan, et al. 2004). Interestingly, sheep heterozygous for these mutant

forms of GDF9 or BMP15 have an increased ovulation rate and incidence of mutiple

pregnancies due to increased LH sensitivity in secondary follicles that leads to a rise

in the number of antral follicles (Galloway, et al. 2000, Hanrahan, et al. 2004,

Juengel, et al. 2004). Furthermore, in humans, rare mutations and genetic variance

of GDF9 and BMP15 are associated with polycystic ovary syndrome (Wang, et al.

2010), dizygotic twinning (Palmer, et al. 2006) and premature ovarian failure (Chand,

et al. 2006, Dixit, et al. 2005, Wang, et al. 2013).

Like other members of the TGF-β superfamily, the pro-proteins (un-processed

proteins) of GDF9 and BMP15 are composed of a pro-region and a mature region

(McMahon, et al. 2008). The pro-regions of these proteins can only be dissociated

from the corresponding mature regions after processing at their protease processing

site, after which they form a pro-mature complex held together by non-covalent

interactions (McIntosh, et al. 2008). There still remains considerable uncertainty

exactly which forms of GDF9 and BMP15 are secreted by the oocyte to act on the

surrounding cumulus cells, and whether the form of these proteins produced by

oocytes matured in vitro is similar to what may occur in vivo. For example, sheep

follicular fluid contains GDF9 and BMP15 proteins only in the unprocessed pro-

protein form (McNatty, et al. 2006), whereas sheep oocytes secrete the mature

domains of GDF9 and BMP15 during IVM (Lin, et al. 2012). In vitro matured mouse

oocytes secrete GDF9 as a mixture of the pro-protein and mature domain (Gilchrist,

et al. 2004a), whilst in vitro matured rat oocytes secrete mature domain GDF9 only

(Lin, et al. 2012). Commercially available GDF9 and BMP15 proteins from the one

supplier of these peptides (R&D Systems) contain only the mature regions of these

proteins; however, our novel BMP15 contains both pro and mature regions, as a

processed pro-mature complex. Consistent with the role of pro-regions of other TGF-

β superfamily members, it has now been demonstrated that the pro-regions of GDF9

(Watson, et al. 2012) and BMP15 interact with granulosa and cumulus cells (Mester,



67

et al. 2013). Hence, we hypothesised that the presence of the pro-region is important

to retain the full function of BMP15 or GDF9 and may affect oocyte developmental

competence.

Our group has previously shown that addition of partially purified GDF9 or BMP15,

which contains a mixture of proteins including the pro-mature complex form, to in

vitro maturation (IVM) medium improves mouse and cattle blastocyst development

(Hussein, et al. 2011, Hussein, et al. 2006, Yeo, et al. 2008). Moreover, addition of a

purified BMP15 pro-mature complex to IVM medium increases bovine embryo

development (Sutton-McDowall, et al. 2012). No studies to date have examined the

effect of the form of GDF9 or BMP15 added during IVM on embryo development. In

this study, we examined the effect of the mature (R&D Systems) and pro-mature (in-

house) forms of human BMP15, as well as the mature form of mouse GDF9 (R&D

Systems), on bovine oocyte developmental competence following IVM. We examined

the metabolic activity of COCs, including glucose uptake and lactate production, as

well as mitochondrial and antioxidant activity that occurred during the maturation of

bovine oocytes.

3.3 MATERIALS AND METHODS

Unless otherwise specified, all chemicals and reagents were purchased from Sigma

(St Louis, MO).

3.3.1 Collection of cells

Bovine ovaries were collected from a local abattoir and transported to the laboratory

in 30-350C saline. Cumulus-oocyte complexes (COCs) and mural granulosa cells

(GCs) were aspirated from 3-8 mm antral follicles using an 18-gauge needle and a

10 ml syringe. The follicular fluid containing COCs was transferred into a 10 ml

Falcon tube at 390C. The follicular aspirate was allowed to sediment and the cellular

pellet was washed with handling medium (HEPES-buffered tissue cultured medium-

199; TCM-199, MP Biomedicals, Solon, OH, USA) supplemented with 4 mg/ml fatty-

acid-free bovine serum albumin (FAF-BSA; ICPbio Ltd, Auckland, NZ). Intact COCs



68

with compact cumulus cells, >3 cell layers and with an evenly pigmented oocyte

cytoplasm, were selected under a dissecting microscope and washed twice in

handling medium.

3.3.2 Granulosa cell thymidine assay

To confirm that the forms of recombinant GDF9 and BMP15 used in this study were

bioactive on bovine ovarian cells, a standard granulosa cell tritiated thymidine

incorporation bioassay was performed, as previously described (Gilchrist, et al. 2003,

Gilchrist, et al. 2006). In brief, GCs were collected from 3-8 mm follicles. The GCs

were washed twice in protein-free culture medium (bicarbonate-buffered TCM-199).

Cells were added to the wells of 96-well plates (Falcon) at a final concentration of 50

x 104 cells/ml, and cultured at 370C, 96% with 5% CO2 in humidified air for 18 hours,

followed by a further 6 hour pulse of 15.4 kBq tritiated thymidine ([3H]-thymidine, MP

Biomedicals) under the same conditions. Following culture, GCs were harvested and

incorporated [3H]-thymidine was quantified using a scintillation counter as an

indicator of GC DNA synthesis. Each treatment was performed in duplicate and the

assay was repeated five times.

3.3.3 Sources of BMP15 and GDF9 and treatment of COCs

COCs were matured in base IVM medium [bicarbonate-buffered TCM 199 + 4 mg/ml

FAF BSA +/- 0.1 IU/ml FSH (Puregon, Organon, Oss, Netherlands)] in a 4-well Nunc

dish (Nunclon, Denmark) and cultured for 23 hours at 390C with 5% CO2 in humidified

air. Recombinant mouse mature region GDF9 (Cat No: 739-G9) and human mature

region BMP15 (Cat No: 5096-BM) were purchased from R&D Systems (Minnaepolis,

MN, USA).

The recombinant human pro-mature complex of BMP15 was produced from a stable

human embryonic kidney (HEK)-293T cell line generated in our laboratory. Briefly, an

expression cassette encoding the human BMP15 DNA sequence was synthesized

(Genscript USA, Inc., Piscataway, NJ) incorporating the rat serum albumin signal

sequence at the 5’ end followed by a His8 tag and a Strep II epitope tag at the N-



69

terminus of the BMP15 pro-region. This expression cassette was transferred to the

pEF-IRES expression vector, as previously described (Hobbs, et al. 1998) and stably

transfected HEK-293T cell lines producing human BMP15 were established via

puromycin selection. For production of the BMP15 protein, confluent monolayers

were transferred to serum-free media (DMEM/F12 + 0.1 mg/ml BSA + 125 IU/ml

heparin) for a 48 hour collection period. The resultant BMP15 protein was secreted

into the serum-free media with a processing efficiency of cleavage of the pro and

mature regions of over 90%. The BMP15 containing production media was subjected

to immobilized Ni2+ based ion-affinity purification targeting the His-tag as described

previously (Pulkki, et al. 2011). Samples were tested for the presence of pro-mature

BMP15 protein by silver staining. The dose of processed mature region was

quantified using Western blotting with the mab28 monoclonal antibody (Pulkki, et al.

2011) and the commercially available mature region of hBMP15 (R&D Systems) as a

standard.

The effects of graded doses of the purified pro-mature BMP15 were tested at 10, 100

and 200 ng/ml. Four replicate experiments were performed with 27-55 COCs per

dose per replicate. The concentration of pro-mature BMP15 was chosen based on

this dose-response assessment of embryo development. For all further experiments

the treatment media contained pro-mature BMP15, mature BMP15 or mature GDF9

at 100 ng/ml +/- 0.1 IU/ml FSH. Six replicate experiments were performed with 20-46

COCs per treatment per replicate.

3.3.4 In vitro fertilization (IVF) and embryo culture

In vitro production of embryos was undertaken using defined, serum-free media

(Bovine Vitro series of media, IVF Vet Solutions, Adelaide, Australia), as previously

described (Hussein, et al. 2006). Frozen semen from a single bull of proven fertility

was used in all experiments. Briefly, thawed semen was layered over a discontinuous

(45%: 90%) Percoll gradient (GE Health Care, Australia) in a 12 ml Falcon tube

(Becton Dickinson, Franklin Lakes, NJ, USA) and centrifuged for 20-25 minutes at

271xg. The supernatant was removed and the pellet resuspended in wash medium



70

[VitroWash (IVF Vet Solutions) + 4 mg/ml FAF BSA)] and centrifuged for 5 minutes at

56 xg. The supernatant was removed and the pellet re-suspended in IVF medium

[VitroFert (IVF Vet Solutions) + 4 mg/ml FAF BSA + 10 IU/ml heparin] with a final

concentration of 1x106 spermatozoa/ml. COCs were inseminated in pre-equilibrated

drops of IVF medium for 23 h at 390C in 6% CO2 in humidified air. Post-insemination,

cumulus cells were removed by manual pipetting with a 1000 µl Gilson pipette. Five

presumptive zygotes were cultured in 20 µl drops of pre-equilibrated cleavage

medium [VitroCleave (IVF Vet Solutions) + 4mg/ml BSA + 1 mM L-carnitin] overlaid

with mineral oil at 390C in 5% O2, 6% CO2 and 89% N2, for 5 days. On day 5,

embryos were transferred into pre-equilibrated blastocyst medium [VitroBlast (IVF

Vet Solutions) + 4mg/ml BSA + 1 mM L-carnitin] and cultured for another 3 days.

Blastocysts were assessed on day 7 and day 8.

3.3.5 Blastocyst differential staining

Differential staining was performed using a modified method as previously described

by Thouas, et al. 2001. Expanded, hatching and hatched blastocysts were placed

into 1% (v/v) Triton X-100 containing 1 mg/ml propium iodide for 20-30 seconds.

Blastocysts were washed in absolute ethanol before incubation in 2.5 mg/ml Hoechst

33342 solution for 2 minutes, then mounted in a drop of glycerol in PBS on

microscope slides and covered with a cover slip. Embryos were examined under a

fluorescence microscope (Nikon, TE 2000-E) at 200x equipped with an ultraviolet

filter (excitation, 340-380 nm; emission, 440-480 nm). The inner cell mass (ICM)

appeared blue and trophectoderm (TE) cells nuclei stained pink.

3.3.6 Glucose and lactate measurements

Groups of 10 COCs were matured in 100 µl of IVM medium. After 23 h maturation,

spent media were analysed for glucose and lactate levels using a Hitachi 912

chemical analyser (F. Hoffmann-La Roche Ltd; Basel, Switzerland). Glucose uptake

and lactate production are expressed as pmol/COC/h. Twelve replicate experiments

were performed with 10 COCs per treatment, per replicate.



71

3.3.7 Autofluorescence of NAD(P)H and FAD++

Ten COCs were matured in 100 µl of IVM medium. At 23 h maturation, COCs were

denuded and oocytes were transferred into 10 µl drops of handling medium overlaid

with mineral oil in glass-bottomed confocal dishes (Cell E&G; Houston, TX, USA).

The autofluorescent intensity of FAD++ was observed using a blue laser (excitation,

473 nm; emission, 490-520 nm), and NAD(P)H using a violet laser (excitation; 405

nm, emission: 420-520 nm) with a confocal microscope (Olympus; Fluoview FV10i).

The fluorescence from the oocytes was normalized using a fluorescence standard

(Inspeck; Molecular Probes). Laser power, gain and pin-hole settings were kept

consistent. Four replicates were performed with 10 oocytes per replicate per

treatment group.

3.3.8 Reduced glutathione (GSH)

At 23 h maturation, COCs were denuded and washed in handling medium. Denuded

oocytes were incubated for 30 minutes in 12.5 µM monochlorobimane (MCB, Catt

No: 69899) and transferred into 10 µl drops of handling medium overlaid with mineral

oil in glass-bottomed confocal dishes (Cell E&G; Houston, TX, USA). The fluorescent

intensity was observed using a DAPI filter (excitation, 358 nm; emission, 461 nm)

with a confocal microscope (Olympus; Fluoview FV10i). Ten COCs were measured

per treatment group per replicate and each experiment was replicated 3 times.

3.4 STATISTICAL ANALYSIS

All proportional data for embryo development were arcsine transformed prior to

analysis using multivariate analysis of variance (ANOVA). Log transformation was

performed if the data were not normally distributed, and analysed using one-way

ANOVA for blastoycst cell numbers, glucose production and lactate consumption.

Autofluorescence of NADP(H), FAD++ and GSH were analysed by two-way ANOVA.

In all cases there was no main effect of FSH (P > 0.05), hence subsequently the form

of GDF9/BMP15 was tested within +/- FSH treatment by one-way-ANOVA followed

by a least-significant difference (LSD) post hoc test. All analysis was performed with



72

SPSS version 13 (SPSS Inc, Chicago, IL, USA). Differences were considered

significant at P < 0.05. Data are expressed as means ± SEM.

3.5 RESULTS

3.5.1 Bioactivity of different BMP15 and GDF9 forms

All forms of the three recombinant proteins (pro-mature BMP15, mature BMP15 and

mature GDF9) were bioactive on bovine GC, as evidenced by significant stimulation

of [3H]-thymidine incorporation compared to the control (Fig. 3.1). The mature region

of GDF9 protein tended to cause the highest level of GC [3H]-thymidine incorporation

and pro-mature BMP15 the least.

Figure 3.1 Bioactivity of different BMP15 and GDF9 forms. Granulosa cell tritiated

thymidine incorporation following exposure to mature GDF9, mature BMP15 or pro-

mature BMP15 at 100 ng/ml. Bars represent means ± SEM and different lowercase



73

letters indicate a statistically significant difference (P<0.05). Data were derived from 5

independent replicates.

3.5.2 Dose-response of pro-mature BMP15 on oocyte

developmental competence

Supplementation of IVM media with pro-mature BMP15 had no effect on subsequent

embryo cleavages rates; however, significantly increased blastocyst development on

day 8 at each dose examined, compared to control and buffer groups (P < 0.05;

Table 3.1). There was no observable dose-response to BMP15 ranging from the

lowest dose (10 ng/ml) to the highest dose (200 ng/ml); however, 200 ng/ml

produced a high blastocyst rate of 60%; a 1.5-fold higher embryo yield than under

control IVM conditions. No adverse effect of elution buffer (PBS/500 mM imidazole

buffer) during IVM on either cleavage or blastocyst development was observed

(Table 3.1).

Table 3.1 Effect of graded doses of pro-mature BMP15 during IVM on oocyte

developmental competence




Treatments No. of oocytes Cleavage

(%)

Blastocyst

day 8 per cleaved

(%)

Control

Buffer (Imidazole) 500 mM

BMP15 10 ng/ml

BMP15 100 ng/ml

BMP15 200 ng/ml

151

159

151

150

156

76.5±3.3

82.2±6.3

87.4±5.1

83.6±4.9

85.8±1.3

39.9±2.3a

40.9±4.6a

54.0±3.3b

55.5±6.0b

59.7±4.4b



74

3.5.3 Comparison of pro-mature BMP15, mature BMP15 and mature

GDF9 during IVM on subsequent embryo development

In the presence of FSH, addition of pro-mature BMP15 to IVM medium significantly

increased blastocyst development on day 7 compared to control (Table 3.2).

Irrespective of treatment, there were no significant differences in cleavage rates, day

8 blastocyst rates, inner cell mass, trophectoderm or total cell numbers (Tables 3.2

and 3.4). There was a trend towards an improvement in blastocyst development on

day 8 from pro-mature BMP15 compared to control, but this was not significant (p =

0.07). The mature form of BMP15 produced intermediate levels of blastocyst

development which were not significantly different to pro-mature BMP15 or controls.

The mature region of GDF9 did not improve any embryo parameter and blastocyst

development on day 7 was significantly lower than both forms of BMP15 (Table 3.2).

As expected, in the absence of FSH, all embryo development and embryo quality

measures were generally lower than in the presence of FSH (Tables 3.3 and 3.5). In

the absence of FSH, blastocyst development with pro-mature BMP15 was also

significantly higher than the control on day 7 (P < 0.05). The trend for improved

blastocyst development with mature BMP15 in the presence of FSH was not evident

in the absence of FSH. Mature GDF9 without FSH did not improve embryo

development or quality.



75

Table 3.2 Effect of supplementing COC cultures with different forms of BMP15 (mature BMP15 and pro-mature BMP15),

mature GDF9 and FSH on oocyte developmental competence



Treatments No. of

Oocytes

Cleavage

(%)

Blastocyst

day 7

per cleaved

(%)

Blastocyst

Day 8

per cleaved

(%)

Control

GDF9 mature 100 ng/ml

BMP15 mature 100 ng/ml

BMP15 pro-mature 100 ng/ml

214

195

207

210

94.2±1.4

88.8±3.1

92.1±1.8

89.1±1.6

43.3±2.4a

36.1±3.0a

50.1±2.6b

57.7±3.9b

52.8±3.9

52.0±4.1

60.0±3.8

61.7±3.5



76

Table 3.3 Effect of supplementing COC cultures with different forms of BMP15 (mature BMP15 and pro-mature BMP15),

mature GDF9 in the absence of FSH on oocyte developmental competence

Values with different superscript within a column are statistically different (P < 0.05). Data are presented as means ± SEM of

6 replicate experiments. Each replicate experiment consisted of 27-40 oocytes.

Treatments No. of

Oocytes

Cleavage

(%)

Blastocyst

day 7

per cleaved

(%)

Blastocyst

Day 8

per cleaved

(%)

Control




191

189

195

188

76.4±4.8

85.5±3.1

84.9±7.3

81.6±4.3

28.9±3.7a

33.1±9.5a,b

32.3±4.5a,b

43.5±4.2b

39.3±3.2

38.4±4.1

38.5±3.7

48.7±3.8



77

Table 3.4 Number of total (TCN), inner cell mass (ICM), trophectoderm (TE) cells in

day 8 expanded, hatching and hatched blastocysts following co-culture of COCs with

different forms of BMP15 (mature BMP15 and pro-mature BMP15), mature GDF9 in

the presence of FSH


Control




30.5±1.6

28.7±1.9

31.8±3.0

34.6±1.8

59.7±3.5

69.7±5.0

68.4±4.4

63.4±2.6

90.2±4.7

98.4±6.0

100.2±6.2

97.9±3.6



Table 3.5 Number of total (TCN), inner cell mass (ICM), trophectoderm (TE) cells in

day 8 expanded, hatching and hatched blastocysts following co-culture of COCs with

different forms of BMP15 (mature BMP15 and pro-mature BMP15), mature GDF9 in

the absence of FSH




Control




24.5±1.9

27.4±2.0

26.5±1.8

29.1±1.8

77.3±5.8

73.8±4.6

72.1±6.7

78.6±6.3

101.8±6.3

101.3±5.2

98.6±7.9

107.7±7.1



78

3.5.4 Effect of pro-mature BMP15, mature BMP15 and mature GDF9

on COC glucose consumption and lactate production

There was an increase in COC glucose uptake and lactate production (almost 2-fold)

in response to FSH, across all BMP15/GDF9 treatment groups and controls (Fig.

3.2). Similar results have been reported that FSH significantly increases glucose

uptake and lactate production (Roberts, et al. 2004). None of the BMP15 or GDF9

proteins tested altered COC glucose consumption or lactate production, regardless of

FSH treatment.



79

Figure 3.2 Glucose consumption and lactate production. Post 23 h maturation of

COCs after treatment with different forms of BMP15 (mature BMP15 and pro-mature

BMP15) or mature GDF9 at 100 ng/ml, in the absence or presence of FSH, spent

IVM medium were analysed for glucose and lactate levels. A. Glucose uptake. B.

Lactate production. Bars represent the means ± SEM. Data were derived from 12

independent replicates for each treatment.



80


on oocyte FAD++ and NAD(P)H autofluorescence

Across all treatments, FSH had no effect on intra-oocyte NAD(P)H, FAD++, or the

REDOX ratio, as assessed by confocal microscopy and analysed by 2-way ANOVA

(main effect FSH; P > 0.05). In the absence of FSH, there was significantly higher

autofluorescence of NAD(P)H in the oocytes treated with pro-mature BMP15

compared to controls (P < 0.05, Fig. 3.3 A). However, in the presence of FSH, the

NAD(P)H level in controls increased to the same level as pro-mature BMP15. There

was a significantly lower level of NAD(P)H autofluorescence in oocytes matured in

the presence of GDF9 compared to control and pro-mature BMP15 groups in the

presence of FSH. No increase in autofluorescence of FAD++ (Fig. 3.3 B) or change in

REDOX ratio (Fig. 3.3 C) in any treatment groups was observed compared to

controls.



81

Figure 3.3. Intra-oocyte NAD(P)H, FAD++, REDOX ratio. Effect of treatment of

intact COCs with different forms of BMP15 (mature BMP15 and pro-mature BMP15)

or mature GDF9 at 100 ng/ml, +/- FSH on intra-oocyte metabolic activity. A.

Autofluorescence of NAD(P)H. B. Autofluorescence of FAD++. C. REDOX ratio

(FAD++/NAD(P)H). Bars represent the means ± SEM. Data were derived from 4

independent replicates for autofluorescence on intra-oocyte NAD(P)H, FAD++, and

REDOX ratio. Columns with different superscripts are significantly different (P <

0.05); a,bminus FSH, x-yplus FSH.



82


on oocyte GSH

Since a significant increase in NAD(P)H levels in oocytes treated with pro-mature

BMP15 (-FSH) was detected, we then examined if this was associated with an

increase in oocyte GSH, on the basis that if NADPH increased, then the capacity to

regenerate reduced GSH was a likely consequence. In the absence of FSH, both

forms of BMP15 significantly increased oocyte GSH levels compared to the controls

(Fig. 3.4). Mature GDF9 produced an intermediate level of GSH. However, in the

presence of FSH, the GSH level of controls was comparable with all other

treatments. There was no overall effect of FSH on oocyte GSH levels (main effect

FSH; P > 0.05).

Figure 3.4 Intra-oocyte GSH level. Effect of treatment of intact COCs with different

forms of BMP15 (mature BMP15 and pro-mature BMP15) or mature GDF9 at 100

ng/ml, +/- FSH on intra-oocyte metabolic activity. Bars represent the means ± SEM.

Data were derived from 3 independent replicates. Columns with different superscripts

are significantly different (P < 0.05)



83

3.6 DISCUSSION

In this present study, we compared the effects of different forms of BMP15 (pro-

mature and mature) and the mature form of GDF9 during IVM on bovine embryo

development. The pro-mature form of BMP15 was the only form of OSF that

significantly increased subsequent embryo development relative to the control.

Mature domain GDF9 was ineffective and mature domain BMP15 lead to a

moderate, albeit non-significant, improvement in embryo yield. We have also

produced pro-mature human GDF9; however, the utilization of this protein is

problematic in culture, as we have found that it has a very high binding affinity for

plastic culture-ware (Mottershead DG, unpublished data) and we have not yet

determined a suitable culture environment.

Oocyte-secreted growth factors have been studied extensively either in their native

form (i.e. as secreted by oocytes) or as recombinant growth factors (Gilchrist, et al.

2008). It is now a well-known concept that co-culture of denuded oocytes (as a

source of native exogenous OSFs) with COCs improves developmental competence

of pig (Gomez, et al. 2012), cattle (Dey, et al. 2012, Hussein, et al. 2011, Hussein, et

al. 2006), goat (Romaguera, et al. 2010) and mouse (Sudiman, et al. 2014a) IVM

oocytes. However, a full characterisation of the forms and indeed the complete

identities of native OSFs have not been undertaken. Previous results showed that

addition of either non-purified BMP15 or GDF9 (containing pro-mature complexes)

improved cattle blastocyst development, and pro-mature mouse GDF9 improved

mouse fetal yield (Hussein, et al. 2011, Hussein, et al. 2006, Yeo, et al. 2008).

However, so far, there is no published report of any significant improvements after

addition of the only commercially available mammalian cell expressed mature

regions of GDF9 or BMP15. Our previous findings (Sudiman, et al. 2014a) indicated

that addition of the mature regions of GDF9 or BMP15 (R&D Systems) did not

improve the developmental competence of mouse IVM oocytes.

Although the form of GDF9 and BMP15 which are produced by the oocyte and

released into the somatic cell compartment of the follicle remains elusive, it is known

that both the pro-region and mature region play important roles in the function of



84

these proteins. The mature domain of TGF-β superfamily proteins is the bioactive

receptor binding region, and while the mature domain is complexed with the pro-

region after processing to form the pro-mature complex, this form can either be latent

or active depending on the particular superfamily member (Brown, et al. 2005, Gray

and Mason 1990, Wakefield, et al. 1988). In the case of human GDF9, the

association of the pro-region with its mature region confers latency (Mottershead, et

al. 2008, Simpson, et al. 2012). Further, the pro-region has important functions in

protein folding, formation of the disulfide bonds and regulation of bioactivity (Gray

and Mason 1990, Harrison, et al. 2011). It seems plausible that the pro-domain of

BMP15 may interact with the extracellular matrix of cumulus cells during maturation

and facilitate presentation of the mature domain to cumulus cell receptors. In support

of this concept, it has been shown that the GDF9 pro-mature complex binds strongly

to heparin sepharose, suggesting that heparan sulfate proteoglycans on cumulus

cells may act as co-receptors mediating oocyte secreted factor signalling (Watson, et

al. 2012). Moreover, the pro-region of GDF9 and BMP15 interact with mural

granulosa cells (Mester, et al. 2013). Collectively, these results indicate that the pro-

domains of GDF9 and BMP15 exhibit important interactions with ovarian somatic

cells, and may explain why the isolated mature regions of GDF9 and BMP15 appear

to have little effect on oocyte developmental competence.

In vitro matured COCs have aberrant expression of genes and proteins compared to

COCs matured in vivo (Dunning, et al. 2007, Kind, et al. 2013, Richani, et al. 2013).

Notably, it has been shown that mouse COCs express processed BMP15 protein

during in vivo oocyte maturation, but not during IVM (Mester, et al. 2013). From our

data, it is clear that BMP15 improved bovine embryo development compared to

GDF9. However, GDF9 may have the capability to improve oocyte quality during IVM

if it were in a different form; e.g. as a pro-mature complex or if bovine or human

GDF9 were used instead of mouse GDF9. Different species origins of recombinant

OSFs have different effects on granulosa cell sterol biosynthesis and bioactivity

(McNatty, et al. 2005a, b). Interestingly, mouse GDF9 in its mature form is

significantly more bioactive on bovine granulosa cells compared to BMP15 (both



85

forms; current study). Hence, the lack of effect of mouse GDF9 on oocyte

competence was not due to lack of bioactivity of this preparation on bovine cells.

The improvement of embryo development after addition of pro-mature BMP15 was

accompanied by an increase in oocyte autofluorescence of NAD(P)H. However,

there was no increase in oocyte FAD++ or change in REDOX status. These results

differ slightly from our previous published data which showed that the addition of pro-

mature BMP15 increased not only NAD(P)H but also increased FAD++ levels (Sutton-

McDowall, et al. 2012). Unfortunately, we cannot distinguish between

autofluorescence from NADPH derived from the pentose phosphate pathway (PPP)

or isocitrate dehydrogenase activity, and autofluorescence from NADH, which is a

product of several metabolic pathways including glycolysis, and the tricarboxylic acid

(TCA cycle). If the increasing level of NAD(P)H in the oocyte after treatment with pro-

mature BMP15 is in part due to increased NADPH, then this may also relate to the

higher levels of GSH in oocytes, as NADPH is required for reduction of oxidised

glutathione via the enzyme glutathione reductase. Glutathione is an antioxidant

molecule in the cell and acts as a defence mechanism in oxidative stress (Luberda

2005, Meister and Anderson 1983). Reactive oxygen species (ROS), such as

superoxide anion, react with the thiol of GSH and oxidise glutathione (GSSG)

(Luberda 2005). As a reducing agent, NADPH aids in the conversion of GSSG into

GSH. Raising the level of GSH during IVM is well documented to improve oocyte

quality during bovine oocyte maturation (de Matos and Furnus 2000, de Matos, et al.

1997). Interestingly, even though there was no increase in NAD(P)H levels in COCs

matured in the presence of mature BMP15, the level of GSH was significantly

increased compared to control and GDF9 mature treatments. This is perhaps due to

lower levels of ROS production in this group, something that could be confirmed in

future experiments.

The aim of testing the medium in the absence of FSH was to observe the function of

exogenous growth factors on IVM oocytes without any influence of metabolic

activators. Cumulus cell function during IVM is profoundly altered by FSH treatment,

including cumulus cell energy metabolism (Sutton, et al. 2003b). The present study

shows that in the absence or presence of FSH, the addition of pro-mature BMP15 to



86

IVM increases significantly embryo development on day 7 and yielded more

blastocysts on day 8 compared to any other group. However, in the presence of FSH,

the influence of recombinant OSFs on NAD(P)H and GSH levels were diminished.

These results are similar with the previous reports that showed FSH can mask the

influence of OSFs on cumulus cell metabolism (Sutton-McDowall, et al. 2012, Sutton,

et al. 2003a). Moreover, it has been shown that in the absence of alteration of

metabolism by FSH, the influence of OSFs on glycolytic activity leads to the

activation of oxidative phosphorylation in mitochondria to produce ATP, rather than

lactate production (Sugiura, et al. 2005, Sutton-McDowall, et al. 2012). Our

collaborators have shown that addition of mature BMP15 increases glucose uptake

but has no effect on lactate production by Nellore (Bosindicus) bovine COCs, under

slightly different culture conditions (Caixeta, et al. 2013). We did not observe any

increases in glucose uptake in any OSF treatment group, including with mature

BMP15, in the presence or absence of FSH. This result is supported by our previous

study (Sutton-McDowall, et al. 2012).

In conclusion, we found that the form of the oocyte-secreted growth factors GDF9

and BMP15 is an important determinant of their function during oocyte IVM, and that

it affects subsequent embryo development. Only the pro-mature form of BMP15

added in IVM medium significantly improved oocyte developmental competence, and

importantly from a practical perspective, there was no evidence that the only

commercially available mammalian forms of GDF9 and BMP15 could increase cattle

embryo production. Improvement of bovine IVM oocyte competence, may be, in part,

due to an increase in oocyte NAD(P)H and GSH.

Chapter 4: Effect of Interactions Between Differing Forms of Recombinant Oocyte

Secreted Factors on Mouse Oocyte Developmental Competence

87

CHAPTER 4

EFFECT OF INTERACTIONS BETWEEN DIFFERING

FORMS OF RECOMBINANT OOCYTE SECRETED

FACTORS ON MOUSE OOCYTE DEVELOPMENTAL

COMPETENCE



88

4.1 ABSTRACT

The aim of this study was to determine the effect of various sources and forms of

recombinant growth differentiation factor 9 (GDF9) and bone morphogenetic protein

15 (BMP15) during in vitro maturation on embryo development, including

investigation of any additive effects of the two proteins. Cumulus oocyte complexes

(COCs) were collected from mice 24-25 h post-eCG (low developmental competence

model) and were matured in vitro in basic in vitro maturation medium (αMEM+FSH)

or treated with pro-mature human BMP15 (hBMP15), pro-mature mouse GDF9

(mGDF9) and combinations of different forms (pro-mature complex vs mature

domain) and sources (mouse and human) of GDF9 and BMP15. Following in vitro

fertilisation and embryo culture, day 6 blastocysts were stained to determine the cell

numbers. COCs matured in medium containing pro-mature hBMP15 or a

combination of hBMP15 and mGDF9 at 50 ng/ml each (both in pro-mature form)

displayed significantly improved blastocyst development compared to controls and

pro-mature mGDF9 (58.1±3.0%, 68.4±4.5% vs 37.6±4.9% and 40.4±6.8%

respectively, P < 0.05). In contrast, no improvement in developmental competence

was observed after addition of a combination of the mature domains of hBMP15 and

mGDF9, or heterodimers of human GDF9 (hGDF9) and hBMP15 (consisting of

mature domains only). These results show that hBMP15 in pro-mature complex form

improves mouse blastocyst developmental competence. Mature domain proteins did

not affect development suggesting that GDF9 and BMP15 pro-domains play an

important role in oocyte developmental competence.



89

4.2 INTRODUCTION

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15)

are two major proteins that are secreted from oocytes. These proteins are important

in ovarian folliculogenesis and required for fertility in a species-specific manner

(Dong et al., 1996, Dube et al., 1998, Galloway et al., 2000). Unlike most of the TGF-

β superfamily members, the expression and bioactivity of GDF9 and BMP15 differ

markedly among species (Crawford and McNatty, 2012). In polyovular animals such

as mouse and rat, the expression of GDF9 is higher compared to BMP15, and

mouse GDF9 (mGDF9) is readily bioactive, hence GDF9 is more important than

BMP15 for fertility in these species (Dong et al., 1996, Yan et al., 2001). In

monoovulatory animals such as cattle and sheep, the ratio of GDF9 to BMP15

expression is closer to 1:1 (Crawford and McNatty, 2012), therefore BMP15 plays a

more prominent role, although both factors are needed.

GDF9 and BMP15 are part of the TGF-β superfamily, and consist of two regions: the

pro-region, and the mature region, which together are known as a pro-protein (un-

processed protein). After cleavage at their protease processing site, the pro-region is

held together with the mature region by non-covalent interactions, creating the pro-

mature complex form (McIntosh et al., 2008, Mottershead et al., 2008). The wild type

human GDF9 (hGDF9) is secreted in a latent complex as pro and mature regions,

whilst mouse GDF9 is secreted in an active form (Mottershead et al., 2008).

Sequence-wise, 64% of the amino acids in the pro-region of hGDF9 are identical to

those of mGDF9, and the mature domains of mouse and human GDF9 share 90%

amino acid homology (Simpson et al., 2012). Separation of the mature region of

hGDF9 from its pro-region activates the protein, allowing it to stimulate the SMAD3

signalling pathway, indicating that the pro-domain contributes to the latency of the

hGDF9 pro-mature complex (Mottershead et al., 2008). In contrast to hGDF9, human

BMP15 (hBMP15) is a potent stimulator of rat (Otsuka et al., 2000) and human

granulosa cell proliferation (Di Pasquale et al., 2004). Ovine pro-mature GDF9

(oGDF9), ovine BMP15 (oBMP15) and mouse BMP15 (mBMP15) are also probably

secreted in latent forms (McNatty et al., 2005). However, a combination of oBMP15



90

with either mGDF9 or oGDF9 increases granulosa cell bioactivity by 3-fold compared

to addition of each growth factor alone (McNatty et al., 2005). Hence, GDF9 and

BMP15 presumably interact with each other, which in some cases lead to the

activation of otherwise latent proteins.

After processing, the mature region of these proteins is non-covalently associated

with the pro-region, therefore the mature or pro-regions of GDF9 can interact with

mature or pro-regions of BMP15 and create heterodimers (McIntosh, et al. 2008).

Recently, Peng et al (2013) claimed to have produced a recombinant GDF9:BMP15

heterodimer, and showed that it is a potent activator of mouse and human granulosa

cell proliferation, and also increases expression of cumulus cell expansion genes,

such as Ptx3, Has2, and Ptgs2. However, the molecular composition of this

heterodimer is controversial (Mottershead et al., 2013). While it is not always the

case that GDF9 or BMP15-stimulated granulosa cell proliferation translates into an

improvement in embryo development following in vitro maturation (IVM), some

studies have demonstrated that high expression of cumulus expansion-related

genes, which are downstream of GDF9, is correlated with improvement in oocyte

quality and embryo development (Anderson et al., 2009, McKenzie et al., 2004).

Together, this information shows that compared to most members of the TGFβ

superfamily, a particularly complex level of species-specific control of protein

bioactivity exists in the case of GDF9 and BMP15. This means it is not necessarily

obvious which form of these oocyte-secreted factors (OSF) should be added to in

vitro maturation (IVM) medium. Furthermore, to date, little is known about the form

these proteins are secreted in, and how they interact with receptors. The only form of

GDF9 and BMP15 that can be detected in sheep follicular fluid is the unprocessed

pro-protein (McNatty et al., 2006), whereas in vitro sheep oocytes secrete the mature

domains of GDF9 and BMP15 (Lin et al., 2012). Mouse IVM oocytes secrete GDF9

as a mixture of the unprocessed pro-protein and mature domain (Gilchrist et al.,

2004, Lin et al., 2012), whilst rat IVM oocytes secrete the mature domains of GDF9

and BMP15 (Lin et al., 2012).



91

Supplementation of cattle IVM oocytes with recombinant pro-mature oBMP15 or

hBMP15 is more effective at increasing subsequent developmental competence than

the mature region of hBMP15 or the mature of region mGDF9 (Hussein et al., 2006,

Hussein et al., 2011, Sudiman et al., 2014b, Chapter 3). The only commercially

available forms of GDF9 and BMP15 from R&D Systems consist of only the mature

region of these proteins, and although very bioactive, these proteins are ineffective in

mouse IVM (Sudiman et al., 2014a). Therefore the objective of this study was to

examine the effects of different forms (pro-mature complex and mature), different

sources (mouse and human), and putative interactions between GDF9 and BMP15

(heterodimers GDF9:BMP15), during mouse IVM on subsequent embryo

development.

4.3 MATERIAL AND METHODS

All chemicals and media were purchased from Sigma Chemical (St Louis, MO, USA)

unless indicated otherwise.

4.3.1 Isolation and maturation of COCs

Mice used in this study were maintained in the Animal House, Medical School,

Adelaide University. The study was approved by local animal ethics committees and

conducted in accordance with the Australian Code of Practice for the Care and Use

of Animals for Scientific Purposes.

Twenty-one to twenty-eight day-old SV129 mice were injected intraperitoneally with 5

IU equine chorionic gonadotrophin (eCG; Folligon, Intervet, Castle Hill, Australia),

and ovaries were collected 24 h later. This in vivo priming protocol was chosen as it

produces oocytes that are inherently less developmentally competent than after 48 h

of eCG (Nogueira et al., 2003). Ovaries were cleaned of any connective tissue and

placed in HEPES-buffered αMEM (handling medium, Life Technologies)

supplemented with 3 mg/ml fatty-acid free bovine serum albumin (FAF BSA, MP

Biomedicals, LLC) and 1 mg/ml fetuin. Antral follicles were punctured with 27-gauge

needles and immature COCs collected in handling medium. Only COCs with



92

compact cumulus cells were taken. Twenty COCs were cultured in 200 µl IVM

medium [αMEM supplemented with 3 mg/ml FAF BSA, 1 mg/ml fetuin and 50 mIU/ml

FSH (Puregon, Organon, Oss, Netherlands)], in a low-binding Eppendorf tube

(LoBind Tube 0.5 ml, Eppendorf AG) overlaid with 100 µl mineral oil (Fig. 4.1). The

aim to use the Eppenderf tube was to minimize interactions between proteins of

interest and the plastic. Ten small holes were made in the lid to ensure gas

equilibration of IVM medium. The COCs were matured for 17-18 h at 370C in a

humidified atmosphere of 5% CO2 in air.

Figure 4.1 In vitro maturation of mouse COCs in low-binding tube

4.3.2 Sources of BMP15 and GDF9 and treatment of COCs

Recombinant mouse mature region GDF9 (catalogue No: 739-G9) and human

mature region BMP15 (catalogue No: 5096-BM) were purchased from R&D Systems

(Minnaepolis, MN, USA). Recombinant human pro-mature complexes of GDF9 and

BMP15 were produced in-house as described in Chapter 3 using a stable human

embryonic kidney (HEK)-293T cell line. The GDF9:BMP15 covalent heterodimer was

produced by David Mottershead (University of Adelaide) by co-expressing mutated

human BMP15 and GDF9 DNA sequences in HEK-293T cells. Both of the respective

COCs



93

DNA sequences had a Ser within the mature region mutated to a Cys residue, to

produce proteins with the intact “Cys-knot” structure characteristic of members of the

TGF-β superfamily. This also enabled production of mature regions of GDF9 and

BMP15 capable of forming covalent dimers. When these mutated DNA sequences

are co-expressed, the cells produce a mixture of covalent homodimers and

heterodimers. Conditioned medium containing the secreted GDF9 (Ser-Cys) and

BMP15 (Ser-Cys) mutant proteins was purified using nickel immobilised metal affinity

chromatography (IMAC) targeting the His-tag on the N-terminus of the pro-regions,

as described previously (Pulkki et al., 2011). After IMAC, the mature regions were

separated from the pro-regions by using rpHPLC (reverse phase high performance

liquid chromatography). This purification was performed by Adelaide Proteomics

Centre using Jupiter 5u C4 300A HPLC columns with a Widepore C4 4x3.0 mm

SecurityGuard Cartridge (Phenomenex, Lane Cove, Australia). One column was

dedicated for heterodimers of GDF9:BMP15 and eluted fractions were collected from

this column. The GDF9:BMP15 heterodimer preparation consists of only the mature

regions of hGDF9 and hBMP15 linked together by a disulphide bridge. The dose of

processed mature region was quantified using Western blotting with the mab28

monoclonal antibody (Pulkki, et al. 2011) and standardised with commercially

available mature region of GDF9 and BMP15 (R&D Systems).

For the first experiment, pro-mature mGDF9 or pro-mature hBMP15, each at 100

ng/ml, or a combination of these proteins at 50 ng/ml each, were added to IVM media

to observe mouse embryo development and quality. For the second experiment, the

effect of the combinations of pro-mature hGDF9 and pro-mature hBMP15 at 100

ng/ml each, mature mGDF9 and mature hBMP15 (R&D Systems) at 100 ng/ml each,

or heterodimers of mature region hGDF9:hBMP15 at 100 ng/ml were tested. Three

replicate experiments were performed with ~30 COCs per treatment per replicate.

4.3.3 In vitro fertilization

Sperm from CBA F1 male mice aged 6-24 weeks and of proven fertility were

incubated for 1 hour in IVF medium (COOK® IVF, Research medium, Catt No: K-

RVWA- 50) under 5% CO2 at 370C for capacitation. Post-maturation, 5-10 COCs



94

were underwent IVF into a 90 µl equilibrated IVF drop overlaid with mineral oil, and

10 µl capacitated sperm was added to each drop. After three hours of incubation

under 5% CO2 at 370C, the presumptive zygotes were denuded of sperm and

cumulus cells and placed into culture drops. Insemination day was counted as day 1

(0 h).

4.3.4 In vitro culture of embryos

Five to ten presumptive zygotes were cultured per 20 µl equilibrated culture drop

(COOK® IVC, Research medium, Cat. No: K-RVCL-50) overlaid mineral oilat 370C in

5% O2, 6% CO2 and 89% N2. Five to ten embryos were cultured for six days, with

cleavage rates assessed on day 2 (24 h post-fertilization) and blastocysts assessed

on day 5 (96-100 h post-fertilization) and day 6 (120-24 h post-fertilization).

4.3.5 Differential staining of blastocysts

Differential staining was performed on day 6 blastocysts to assess inner cell mass

(ICM) and trophectoderm (TE) cells counts. Briefly, expanded, hatching and hatched

blastocysts were placed into pronase (5 mg/ml) at 370C until the zona dissolved. The

zona-free blastocysts were incubated in 10 mM trinitrobenzene sulfonic acid (TNBS)

in 0.4% polyvinyl alchol (PVA) in phosphate-buffered saline (PBS) at 40C for 10

minutes, then washed twice in PBS before being transferred into 0.1 mg/ml anti

dinitrophenol-BSA antibody at 370C for 10 minutes. Embryos were then incubated in

10 µg/ml propidium iodide for 5-10 minutes at 370C, followed by 4 µg/ml Hoechst

33342 in 96% ethanol at 40C overnight. The blastocysts were then transferred onto a

glass microscope side, covered by a cover slip and assessed immediately under UV

fluorescence using an upright microscope (Nikon, TE 2000-E, excitation, 340-380

nm; emission, 440-480 nm), where ICM cells appeared blue and TE cells appeared

pink.



95

4.4 RESULTS

4.4.1 The effect of pro-mature mGDF9 and hBMP15 alone or in

combination during IVM on subsequent embryo development

Addition of pro-mature hBMP15 alone or in combination with pro-mature mGDF9 to

IVM medium, led to a substantial, almost 2-3-fold, increase in blastocyst

development on days 5 (P < 0.01) and 6 (P < 0.05), compared to controls and pro-

mature mGDF9 alone (Table 4.1). There were no significant differences in cleavage

rates or inner cell mass, trophectoderm cells or total cell numbers across all

treatment groups and controls (Tables 4.1, 4.2). The highest blastocyst rate was

derived from COCs exposed to a combination of these two proteins, however it did

not significantly differ from those exposed to hBMP15 alone.



96

Table 4.1 Effect of supplementing COC cultures with mGDF9, hBMP15 (pro-mature complexes) and combination of both

proteins on oocyte developmental competence

Treatments No. of

Oocytes

Cleavage

(%)1

Blastocyst

on day 52

Blastocyst

on day 63

Hatching

Blastocyst

on day 64

Control

mGDF9 pro-mature 100 ng/ml

hBMP15 pro-mature 100 ng/ml

mGDF9 pro-mature 50 ng/ml + hBMP15 pro-mature 50 ng/ml

94

86

92

92

63.1±6.2

60.7±3.0

73.2±6.5

58.6±12.8

17.2±1.0a

19.4±5.4a

41.7±7.0b*

45.4±4.3b*

37.5±5.0a

40.4±6.8a

58.1±3.0b

68.4±4.5b*

20.3±4.1

27.0±7.2

46.2±6.9

42.2±8.1

Values with different superscripts within a column are statistically different (P < 0.05), * = P < 0.01 compared to control and

mGDF9



2Percentage of blastocysts generated 96-100h post-fertilization per cleaved embryo





97


complexes) and combination of both proteins on numbers of inner cell mass (ICM),

trophectoderm (TE) and total (TCN) cells in blastocysts



4.4.2 Interactions between GDF9 and BMP15: effect of differing

forms during IVM on subsequent embryo development

While there was a slight improvement in blastocyst development with a combination

of pro-mature hGDF9 and hBMP15, this was not significantly different compared to

controls. No significant differences in embryo quality were found among treatment

groups compared to controls (Tables 4.3, 4.4).


Control

mGDF9 pro-mature 100 ng/ml


mGDF9 pro-mature 50 ng/ml +


13.7±1.3

12.0±1.0

14.8±1.5

15.3±1.7

30.0±3.1

28.5±2.9

41.9±4.2

34.0±4.6

45.0±3.3

40.5±3.4

56.7±5.2

49.3±5.0



98

Table 4.3 Effect of supplementing COC cultures with a combination of hGDF9 and hBMP15 (pro-mature complexes), a

combination of mGDF9 and hBMP15 (isolated mature regions), or a hGDF9:hBMP15 heterodimer (isolated mature regions) on

oocyte developmental competence.

Treatments Form No. of

Oocytes

Cleavage

(%)1

Blastocyst

on day 52

Blastocyst

on day 63

Hatching

Blastocyst

on day 64

Control

hGDF9 100 ng/ml + hBMP15 100 ng/ml

mGDF9 100 ng/ml + hBMP15 100 ng/ml

hGDF9:hBMP15 heterodimer 100 ng/ml

N/A

Pro-mature

Mature

Mature

94

86

92

92

64.2±9.9

71.8±1.6

67.4±5.0

72.6±5.8

26.1±10.0

32.6±6.2

17.8±7.8

20.0±9.1

49.5±10.7

55.5±0.5

46.0±4.5

44.4±6.6

29.8±7.7

33.2±8.5

25.5±11.3

21.8±5.1








99

Table 4.4 Effect of supplementing COC cultures with a combination of hGDF9 and hBMP15 (pro-mature complexes), a

combination of mGDF9 and hBMP15 (isolated mature regions), or a hGDF9:hBMP15 heterodimer (isolated mature regions) on

numbers of inner cell mass (ICM), trophectoderm (TE) and total (TCN) cells in blastocysts

Data are presented as means ± SEM. Mean blastocyst cell numbers following differential staining of 8-10 blastocysts.

Treatments Form ICM TE TCN

Control

hGDF9 100 ng/ml + hBMP15 100 ng/ml

mGDF9 100 ng/ml + hBMP15 100 ng/ml

hGDF9:hBMP15 heterodimer 100 ng/ml

N/A

Pro-mature

Mature

Mature

14.2±1.8

17.5±1.6

15.7±2.4

13.7±1.9

40.9±4.1

36.9±4.7

39.4±5.7

28.2±3.5

55.1±3.9

54.5±5.7

55.1±7.3

41.8±5.2



100

4.5 DISCUSSION

In this study, mouse oocytes were matured in vitro in low-binding Eppendorf tubes in

order to minimize interactions between the proteins of interest and the plastic of Petri

dishes. In previous studies, it was found that the pro-mature hGDF9 protein has a

very high binding affinity for plastic culture-ware (Mottershead DG, unpublished

data). However, by using low-binding Eppendorf tubes, we minimized the interaction

between pro-mature hGDF9 and the tube. Preliminary experiments showed that

there was no adverse effect on subsequent embryo development after maturation in

low-binding Eppendorf tubes compared to Petri dishes (see Appendix 3).

Based on the previous work by Nogueira et al (2003), we also used immature COCs

collected 24-25 h post-PMSG injection, instead of 46-48 h post-injection, in order to

create a COC IVM system which does not have unnaturally elevated oocyte

developmental competence. This proved effective as control blastocysts rates were

approximately 30-40% lower than in Chapter 2, and hence enabled us to detect any

beneficial effects on embryo development from treatments.

In the present study, pro-mature hBMP15 alone and the combination of mGDF9 and

hBMP15, in their pro-mature forms, substantially improved embryo development

compared to the control group and mGDF9 alone. The improvement in embryo

development observed was enhanced further with the combination of both proteins.

This supports similar results from Chapter 3, which showed that pro-mature hBMP15

significantly increased bovine embryo development at 100 ng/ml. Recently, it has

been demonstrated that in vitro matured oocytes are deficient in BMP15 protein

expression, compared to their in vivo counterparts (Mester, et al. 2013), thus addition

of exogenous pro-mature hBMP15 in IVM medium improves in vitro matured oocyte

developmental competence. Interestingly, even though the expression of GDF9 is

notably higher than BMP15 in polyovular animals (Crawford and McNatty 2012), and

pro-mature mGDF9 is a potent activator in granulosa cell proliferation assays

(Mottershead, et al. 2008), it did not improve embryo development post-IVM in the

current study. Likewise, mature region mGDF9 did not improve the development of



101

mouse IVM oocytes (Sudiman, et al. 2014a). In contrast, addition of partially purified

pro-mature mGDF9 to in vitro maturing bovine COCs increased blastocyst

development, and in mouse improved fetal survival rates (Hussein, et al. 2005,

Hussein, et al. 2011, Yeo, et al. 2008). The differences in results are likely to be due

to the different purification methods used to produce proteins. Previous studies have

used unpurified or partially-purified recombinant mGDF9 preparations, which contain

other proteins that are produced by the host cells (Kaivo-Oja, et al. 2003, McNatty, et

al. 2005a). GDF9 clearly benefits from the presence of BMP15, and it may also

benefit from interactions with other proteins, which may have been present in the

preparations used in previous studies.

The second experiment further examined interactions between GDF9 and BMP15

proteins and their effects on oocyte development in vitro. Even though there was a

slight improvement in blastocyst rate with the combination of pro-mature hGDF9 and

pro-mature hBMP15, it did not significantly differ compared to the control group. Pro-

mature hGDF9 alone is latent and is unable to induce granulosa cell proliferation

(Mottershead, et al. 2008); however, the combination of hGDF9 and hBMP15 is

capable of inducing granulosa cell proliferation (Mottershead, unpublished data).

Hence, the lack of effect of hGDF9 + hBMP15 on competence is interesting, in the

context of the positive effect of mGDF9 + hBMP15. This suggests tightly regulated

species-specific OSF control of oocyte competence. Consistent with this, pro-mature

hBMP15 interacts differently with pro-mature GDF9 sourced from mouse versus

human (McNatty, et al. 2005a).

It has been shown that the maximum synergistic effect of GDF9 and BMP15 in

stimulating granulosa cell proliferation is obtained at a dose of 12.5 ng/ml each

(Mottershead, et al. 2012). In general terms, COCs are less sensitive than isolated

granulosa cells to GDF9 and BMP15, and usually higher doses are used in IVM

(Dragovic, et al. 2005). Nonetheless, it is possible that using the higher doses of

these proteins induced an inhibitory, rather than stimulatory effect, on cumulus

expansion and competence. Unfortunately, we are unable to score the cumulus cell

expansion due to the COCs forming clumps during maturation in the low-binding



102

Eppendorf tubes. No positive effect on embryo development could be observed from

a combination of mature region mGDF9 and hBMP15 (R&D Systems), a result which

supports previous data from our lab showing that in combination, doses from 50

ng/ml up to 200 ng/ml did not improve mouse oocyte developmental competence

(Sudiman, et al. 2014a).

The first records of production of a recombinant GDF9:BMP15 heterodimer were in

2013, and this is the first attempt at using these preparations in oocyte IVM in any

species. The construct used in this study has a mutated cysteine in the mature region

and was purified by IMAC followed by HPLC chromatography, to generate an

isolated covalent heterodimer of the mature regions of hGDF9 and hBMP15

(Mottershead, unpublished data). This preparation is exceptionally potent on isolated

mouse granulosa cells (Mottershead, unpublished data). Nonetheless, the

heterodimer had no effect on mouse oocyte developmental competence (current

study). Since the heterodimer consists only of the mature regions of hGDF9 and

hBMP15, we hypothesize that the pro-regions may also be important for heterodimer-

mediated effects, as they are for homodimer-mediated effects, on developmental

competence of oocytes. Since heterodimers of GDF9:BMP15 use both receptors

from GDF9 and BMP15 (BMPR2 + ALK 4/5/7:ALK6) before triggering

phosphorylation of SMAD 2/3 in cumulus cells (Kaivo-Oja, et al. 2005, Peng, et al.

2013), we believe that this form has important implications for female fertility.

However, further studies are required to examine this hypothesis and other effects of

a heterodimer on oocyte function and the physiological dose that is required to

improve oocyte developmental competence.

The present results clearly show that hBMP15 in pro-mature complex form improves

mouse oocyte developmental competence. However, no effect could be observed of

the mature domain of these proteins, alone or in combination, on mouse IVM

oocytes. This suggests the pro-mature complex is the functional form required to

improve the developmental competence of IVM oocytes. Moreover, since there was a

trend toward further improvement with a combination of the pro-mature forms of

mGDF9 and hBMP15, it suggests that these proteins interact with each other.



103

Nevertheless, since different species have different requirements for GDF9 and

BMP15, and the source of these recombinant proteins (mouse, human, sheep)

affects their interaction with granulosa and cumulus cells, further studies are required

to observe the species-specific dose and form requirements of these proteins for

optimal embryo development.

Chapter 5: General Discussion

104

CHAPTER 5

GENERAL DISCUSSION


105

In vitro maturation (IVM) of oocytes has been successfully used in laboratory/small

animals and in the livestock industry for the production of embryos in vitro (Thibier

2006). In contrast, since the IVM procedure was introduced into human assisted

reproductive technology (ART), only 1300 live births have been produced by this

method (Suikkari 2008). Even though IVM in human ART has so far yielded limited

success, this emerging technology has the potential to substitute for standard human

in vitro fertilisation (IVF) protocols, or become an adjuvant to IVF for many reasons.

This method does not require the use of gonadotropin hormones (used in standard

IVF), therefore the side effects associated with gonadotropin use, such as ovarian

hyperstimulation syndrome (OHSS), and patient inconvenience, can be avoided. It

also reduces the cost of ART which makes this technology available for patients with

lower economic status. To date, several studies have demonstrated improvements to

the IVM process via different methods, such as administration of lower doses of

gonadotropin hormone (Jurema and Nogueira 2006), modification of the maturation

medium by adding exogenous ATP (Xue, et al. 2004), EGF-like peptides (Richani, et

al. 2013), or cAMP modulators (Albuz, et al. 2010), or prolonging gap junction

communication between the oocyte and cumulus cells (Albuz, et al. 2010, Thomas,

et al. 2004). Recent studies have shown that co-culture of denuded oocytes (DOs)

with cumulus oocyte complexes (COCs) improves embryo development derived from

in vitro matured oocytes in the cow (Dey, et al. 2012, Hussein, et al. 2011, Hussein,

et al. 2006), goat (Romaguera, et al. 2010) and pig (Gomez, et al. 2012). It has been

demonstrated that the oocyte actively secretes soluble paracrine factors (oocyte

secreted factors; OSFs) which act on the surrounding somatic cells and allow the

oocyte to control its own microenvironment (Gilchrist et al., 2008). Lower embryo

development post-IVM is at least in part due to aberrant gene and protein expression

(including BMP15), in in vitro matured oocytes compared to in vivo counterparts

(Dunning, et al. 2007, Mester, et al. 2014). Therefore the addition of native

exogenous OSFs derived from denuded oocytes, or exogenous recombinant proteins

such as GDF9 and BMP15, can be used to improve the developmental competence

of in vitro matured oocytes.


106

This study focused on OSFs and recombinant proteins (GDF9 and BMP15) and their

capacity to improve IVM systems. Results from Chapter 2 showed that exogenous

native OSFs improved the developmental competence of mouse IVM oocytes, and

led to a doubling in fetal yield. Moreover, this study gives an insight into factors

affecting the capacity of these native OSFs to improve mouse IVM, such as the

presence of cumulus cells and FSH, and the temporal regulation of the native OSF

pool. However, even though co-culture of COCs with DOs can be used effectively in

cattle and animal reproduction, it is not possible to collect the necessary numbers of

DOs for use in clinical human ART. Therefore, the next step was to examine the

effect of recombinant proteins that can be used to mimic the beneficial effects of

exogenous native OSFs.

GDF9 and BMP15 are two major growth factors secreted by oocytes, and are critical

for female fertility. Homozygous mutations in genes encoding these proteins

contribute to reduced fertility in mouse and sheep (Dong, et al. 1996, Galloway, et al.

2000, Hanrahan, et al. 2004, Juengel, et al. 2002). In humans, genetic variance and

rare mutations of GDF9 and BMP15 contribute to PCOS, dizygotic twinning and

premature ovarian failure (Chand, et al. 2006, Dixit, et al. 2005, Palmer, et al. 2006,

Wang, et al. 2013). Like all members of the TGF-β family, GDF9 and BMP15 consist

of pro and mature regions (together called the pro-protein). After processing, the pro

and mature regions of these proteins are held together by non-covalent bonding. To

date, it is still unclear which forms of GDF9 and BMP15 are secreted by in vivo and in

vitro matured oocytes, and there appears to be important species differences

(Gilchrist, et al. 2006, Lin, et al. 2012, McNatty, et al. 2006). The only commercially

available mammalian cell derived GDF9 and BMP15 is sold by R&D Systems, and

consist only of the mature domains of these proteins (refer to Figs. 5.1 C and D).

Previous studies from Hussein et al (2006, 2011) and Yeo (2008) using partially-

purified recombinant mouse GDF9 and sheep BMP15, which contain pro and mature

regions of these proteins, in IVM showed an improvement in mouse and cattle

blastocyst development and fetal yield. So far, there are no other studies showing

improvement in embryo development after addition of the mature domains of GDF9

and BMP15.


107

We suspected that the pro-region of these proteins plays an important role in

determining oocyte developmental competence. Therefore, the main aim of chapter 4

was to observe the effect of different forms of GDF9 and BMP15 (pro-mature and

mature domain only) from difference sources (mouse and human). We used purified

recombinant proteins from different sources that were purified using a His-tag

purification method (Pulkki, et al. 2011). Refer to Figs. 5.1 A and B. Even though

Western blot results from Chapter 2 were unable to detect any differences in the

quantity and form of GDF9 (17 and 65 kDa) from extracts of DO at 0h or 3h, we

found that human BMP15 (hBMP15) in its pro-mature complex form was the most

effective at increasing developmental competence of in vitro matured cattle and

mouse oocytes (Chapters 3 and 4, Table 5.1). We also demonstrated that pro-mature

hBMP15 increases oocyte metabolism during maturation (Chapter 3). However, the

mechanisms by which pro-mature complex proteins interact with the cumulus cell

matrix and the receptors, and what the exact function of the pro-region is during

oocyte maturation, are still unclear. In future experiments, different forms and

sources of recombinant proteins in IVM medium could be used and observed on

measures such as the activation of the Smad signalling in cumulus cells and any

differences in the production of diffusible factors such as cAMP and cGMP by the

cumulus cells. It would be also interesting to examine differences in meiotic activity

and the developmental programming of oocytes in response to these different protein

forms and sources.

In vivo matured oocytes from polyovular animals express GDF9 as the major

component of OSFs (Crawford and McNatty 2012). However in this present study,

mouse GDF9 (mGDF9) in pro-mature complex form did not improve mouse embryo

development (Chapter 4, Table 5.1). Similar to results from our previous study, no

improvement was observed after addition of mGDF9 and hBMP15 (both mature

domains only) or a combination of both proteins at a range of doses (without the

presence of the pro-domain, Sudiman et al. 2014a, Chapter 4, Table 5.1).

Nevertheless, when pro-mature mGDF9 was combined with pro-mature hBMP15 at a

dose of 50 ng/ml each, there was a significant improvement in mouse embryo

development (Chapter 4, Table 5.1). Moreover, results from our previous study

showed that the mature region of BMP15 combined with fibroblast growth factor 17


108

(FGF17) increased the number of inner cell mass cells in cattle blastocysts

(Appendix 2). Therefore, it remains to be investigated whether BMP15 or any other

OSFs such as BMP6 (Solloway, et al. 1998), FGF10 (Zhang, et al. 2010), FGF11

(Sugiura, et al. 2007) are required for GDF9 in order to have the optimal impact on

developmental competence of IVM oocytes. The challenge is to find the optimal dose

of these recombinant proteins which is effective in improving in vitro matured oocyte

developmental competence. Results from Chapter 4 showed that a combination of

pro-mature hGDF9 and hBMP15 at 100 ng/ml each had a small positive impact on

mouse embryo development (refer to Table 5.1). Therefore the effect of recombinant

proteins depends on the dose, and the source, of proteins and this suggests tightly

regulated species-specific OSF control of oocyte competence.

Even though this study provides evidence that the pro-mature form of hBMP15, and

a combination of pro-mature mGDF9 and hBMP15, are effective, it may be

problematic to apply these results to human ART. GDF9 and BMP15 are biologically

active substrates, and it could be argued that the only source which should be used

for human ART should be the human recombinant proteins. Unfortunately, wild type

hGDF9, which consists of pro and mature regions, is secreted in a latent complex

(Mottershead, et al. 2008). In the future, it will be interesting to observe the effect of

bioactive mutants of pro-mature hGDF9, produced by substituting the Gly residue

with aArg residue at position 391, on human IVM oocytes (Simpson, et al. 2012).

Moreover, in monovular animals such as cattle, the ratio of GDF9 and BMP15 is

nearly equal (Crawford and McNatty 2012) and a combination of pro-mature hGDF9

and pro-mature hBMP15 is bioactive (Mottershead, unpublished data). Therefore, we

propose that a combination of hGDF9 and hBMP15 in their pro-mature complex

forms is also a promising area to investigate for the purpose of improving human IVM

success.

The choice of animal experimental model is an important issue for future research in

this area. In Chapter 4 we used the approach of different timing for the collection of

COCs (24-25 h post-PMSG), since the conventional model using immature COCs

collected 46-48 h post-PMSG from pre-pubertal mice, produces a very high level of

blastocyst development (Chapter 2). It can be argued that this model has


109

questionable physiological relevance to the types of oocytes used in human or

veterinary IVM. The low-developmental competence model allows us to better detect

an actual improvement resulting from different treatments. Animal models such as

pig (Coy and Romar 2002), buffalo (Gautam, et al. 2008) or goat (Romaguera, et al.

2010), which have lower developmental competence, perhaps are more useful and a

better model for human applications, since IVM may be of particular importance for

polycystic ovary syndrome (PCOS) patients whose oocytes have compromised

developmental competence. For future experiments, it will also be interesting to

observe the effect of native OSFs or pro-mature GDF9 and BMP15 on oocytes

derived from small follicles and pre-pubertal animals. Commonly, researchers use

oocytes from large follicles (Leibfried and First 1979) and derived from adult animals

(Damiani, et al. 1996), due to better embryo development. Previous studies have

shown that co-culture of COCs derived from small follicles with DOs from either large

or small follicles increased goat blastocyst development (Romaguera, et al. 2010).

However, whether recombinant pro-mature GDF9 and BMP15 also have the same

positive effect on COCs from small follicles is still unknown.

Future directions from this study may also include design of functional heterodimers

of OSFs; notably of GDF9 and BMP15. Even though the concept of heterodimers is

well described (McIntosh, et al. 2008), the production of the putative protein was only

first described in 2013 (Peng, et al. 2013). However, the validity of a heterodimer of

GDF9:BMP15 in this study is still controversial since there is a lack of evidence to

show the molecular composition of this preparation, which may contain a mix of

dimers of homodimers of GDF9 and BMP15 (Mottershead, et al. 2013). This current

study is the first to use a heterodimer of GDF9:BMP15 which has a mutated cysteine

in the mature region and forms covalent heterodimers of the mature regions

(Mottershead, unpublished data; see Chapter 4 for production of this protein). It has

been shown that GDF9 pro-mature complex binds strongly to heparin sepharose

(Watson, et al. 2012), and the pro-regions of GDF9 and BMP15 interact with isolated

mural granulosa cells (Mester, et al. 2014). We suggested that the pro-region of

BMP15 may interact with the extracellular matrix of cumulus cells during maturation

and facilitate presentation of the mature domain to cumulus cell receptors. Therefore,

by creating heterodimers with a covalent bond, we hypothesised that the dimers of


110

the mature regions of GDF9:BMP15 interact with the receptors in cumulus cells

instead of extracellular matrix of cumulus cells (refer to Fig. 5.1 E). However, results

from Chapter 4 showed that heterodimers of GDF9:BMP15 mature regions do not

improve oocyte developmental competence (refer to Table 5.1). It seems probable

that the pro-mature complex form of this protein is also required. Therefore for future

experiments, the effect of heterodimers of GDF9:BMP15 which contain both the pro

and mature regions, on embryo development should be observed (refer to Fig. 5.1

F). Another direction would be to investigate the signalling pathway of heterodimers,

specifically in cumulus cells.


111

Figure 5.1 Different forms of GDF9 and BMP15 proteins. A and B. The pro-mature

complex forms of GDF9 and BMP15 consist of homodimers of mature regions and

their associated pro-regions. C and D. The commercially available GDF9 and BMP15

consist of homodimers of mature regions only. E. Heterodimers of GDF9:BMP15

consist of dimers of GDF9 and BMP15 mature regions with a covalent bond, as used

in Chapter 4. F. Our proposed heterodimers of GDF9:BMP15 which consist of dimers

of pro and mature regions of GDF9 and BMP15 for future experiments.


112

Table 5.1 Summary of developmental competency of mouse and cattle oocytes undergoing IVM with the addition of native

OSFs and different forms and sources of GDF9 and BMP15

Treatments Form Embryo development

Mouse IVM oocytes Cattle IVM oocytes

Native OSFs Possibly mix of pro-mature and mature domains of GDF9 and BMP15

↑↑ (Chapter 2, Sudiman et al. 2014a)

↑↑ (Dey, et al. 2012, Hussein, et al. 2011, Hussein, et al. 2006)

mGDF9 Partially purified (Pro-mature) ↑↑ (Yeo, et al. 2008)

↑ (Hussein, et al. 2011, Hussein, et al. 2006)

oBMP15 Partially purified (Pro-mature) N/A ↑↑ (Hussein, et al. 2011, Hussein, et al. 2006)

mGDF9 + oBMP15 Partially purified (Pro-mature) N/A ↑↑ (Hussein, et al. 2006)

mGDF9 Pro-mature - (Chapter 4)

N/A

hBMP15 Pro-mature ↑↑ (Chapter 4)

↑↑ (Chapter 3, Sudiman et al. 2014b)


113

↑↑ = large increase, ↑ = increase, - = no change, N/A= not available

Treatments Form Embryo development

Mouse IVM oocytes Cattle IVM oocytes

mGDF9 Mature - (Sudiman et al., 2014a)

- (Chapter 3, Sudiman et al., 2014b)

hBMP15 Mature - (Sudiman et al., 2014a)

↑ (Chapter 3, Sudiman et al., 2014b)

mGDF9 + hBMP15 Mature - (Sudiman et al., 2014a)

N/A

mGDF9 + hBMP15 Pro-mature ↑↑ (Chapter 4)

N/A

hGDF9 + hBMP15 Pro-mature ↑ (Chapter 4)

N/A

hGDF9:hBMP15 Heterodimers - (Chapter 4)

N/A


114

To date, there are no definitive and non-invasive technologies available to measure

developmental competence of immature oocytes. This study measures various

metabolic activities of oocytes matured in the presence of different recombinant

proteins. However, the common use of FSH, a metabolic activator, in IVM medium

(including human) masks these measurements (Chapter 3, Sudiman et al. 2014b).

Therefore, in future experiments, we need to investigate measurements which are

not affected by metabolic activators, such as; analysis of gene arrays in cumulus

cells, substrates released or energy production by the oocyte during maturation,

meiotic competence and so on.

There is still a long way to go to determine the optimal IVM conditions for human

oocytes, to improve the success rate to a level comparable to that of traditional IVF.

However, implementing IVM systems in IVF practice is important since this

technology is cheaper and safer in some patients. This study contributed to IVM

system development by showing that native oocyte secreted factors improve in vitro

matured oocyte developmental competence, and investigated factors that affect

production of OSFs. Furthermore, it demonstrated that the beneficial effect of native

OSFs can be mimicked by adding recombinant proteins (GDF9 and BMP15) in pro-

mature complex form. However, testing the effect of various forms, combinations,

sources and doses of BMP15 and GDF9 during IVM, on a wide range of outcome

measures for different species, including human, are important future steps in this

process.

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115

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Appendices

143

APPENDICES

Appendices

144

Appendix 1: The effect of native pig OSFs on mouse

IVM oocyte developmental competence

This experiment was performed to observe the effect of pig OSFs on mouse in vitro

matured oocytes. Mouse COCs were cultured in 50 µl drops of base medium (α MEM

supplemented with 3 mg/ml fatty-acid free bovine serum, 1 mg/ml fetuin and 50

mIU/ml FSH). Mouse COCs were distributed into 4 treatment groups: (1) 20 mouse

COCs were cultured in 50 µl IVM drop (control), (2) 20 mouse COCs were co-

cultured with 25 pig COCs, (3) 20 mouse COCs were co-cultured with 50 pig COCs,

(4) 20 mouse COCs were co-cultured with 25 pig DOs, (5) 20 mouse COCs were co-

cultured with 50 pig DOs. After 17 h in maturation medium, mouse COCs were

inseminated and blastocyst development was assessed on days 5 and 6.

Result:

As shown in Table 1, there was no difference in blastocyst development after

addition of pig denuded oocytes or cumulus oocyte complexes on mouse embryo

development. Co-culture of mouse COCs with 50 pig DOs significantly decreased

cleavage rate compared to control group.

Appendices

145

Table 1 Effect of native pig OSFs on mouse IVM oocytes on subsequent embryo

development







4Percentage of hatching blastocysts generated 100-125 h post fertilization per

cleaved embryo

Treatments No. of

Oocytes

Cleavage2

(%)

Blastocyst

on day 53

Blastocyst

on day 64

Hatching

Blastocyst

on day 65

Control

COC + 25 DO

COC + 50 DO

COC + 25 COC

COC + 50 COC

156

132

133

140

124

91.1±3.8a

82.1±3.2a,b

79.8±5.2b

84.1±4.1a,b

88.4±2.5a,b

37.5±5.5

48.8±7.8

49.0±10.8

54.2±5.5

42.2±6.3

62.3±6.1

68.2±4.0

74.0±8.9

76.1±2.9

61.8±2.9

43.1±3.9

51.7±8.6

59.5±8.7

61.1±6.8

48.3±3.9

Appendices

146

Appendix 2: The effect of FGF17 and BMP15 on

bovine in vitro matured oocytes and subsequent

embryo development

The aim of this study was to determine interactive effects between FGF17 and

BMP15 on bovine in vitro matured oocytes and subsequent embryo development.

COCs were cultured in base medium (bicarbonate-buffered TCM 199 + 4 mg/ml FAF

BSA + 0.1 IU/ml FSH), with the following additional supplements: (1) none (control),

(2) 100 ng/ml FGF 17 (R&D System), 100 ng/ml BMP15 (R&D System) and (4) 100

ng/ml FGF17 + 100 ng/ml BMP15 (both R&D System).

Results:

There was no significant difference on bovine embryo development between control

and treatment groups, however, there was a significant improvement on inner cell

mass number in the combined of FGF17 and BMP15 group compared to the control

group which suggests an interaction of these combined proteins on embryo quality.

Appendices

147

Table 2 Effect of supplementing COC cultures with BMP15, FGF17 and combination

both proteins at 100 ng/ml dose on bovine oocyte developmental competence

Data are presented as mean ± SEM of 4 replicate experiments. Each replicate


Table 3 Effect of supplementing COC cultures with BMP15, FGF17 and combination

both proteins at 100 ng/ml on numbers of inner cell mass (ICM), trophectoderm (TE)

cells and total cell numbers (TCN) of blastocyst on day 6


Control

FGF17

BMP15

FGF17 + BMP15

25.3±1.6a

27.7±1.7a,b

26.9±1.4a,b

31.4±2.0b

80.9±4.2

76.6±3.9

81.4±3.2

83.2±5.2

105.9±4.2

104.3±4.4

108.3±3.3

114.7±5.9


Data are presented as mean ± SEM of 4 replicates. Mean blastocyst cell numbers

following differential staining of 29-40 blastocysts.

Treatments No. of

Oocytes

Cleavage

(%)

Blastocyst

on day 7

Blastocyst

on day 8

Hatching

Blastocyst

on day 8

Control

FGF17

BMP15

FGF17 + BMP15

156

157

153

154

84.6±3.8

91.7±1.9

88.9±4.4

88.3±2.4

49.2±5.7

44.4±4.6

49.3±1.1

50.7±3.1

61.4±5.4

55.6±6.7

59.6±2.7

61.8±3.8

50.6±5.6

40.0±10.4

46.9±5.5

44.0±1.8

Appendices

148

Appendix 3: The effect of Petri Dish and Eppendorf

Tube on Mouse IVM oocytes

This prelimenary experiment for chapter 4 was performed to observe the effect of

Eppendorf tube compared to Petri dish as a conventional compartment to culture IVM

oocytes.

COCs were collected from 3-4 wks and were injected intraperitoneally with 5 IU

equine chorionic gonadotrophin (eCG; Folligon, Intervet, Castle Hill, Australia), and

ovaries were collected 24-25 h later. COCs then were matured in vitro in basic in

vitro maturation medium (α MEM supplemented with 3 mg/ml fatty-acid free bovine

serum, 1 mg/ml fetuin and 50 mIU/ml FSH) in Petri dish or in Eppendorf tube (Figure

4.1). Twenty COCs were matured in either a 50 µl IVM drop overlaid with mineral oil

on Petri dish or in a 200 µl drop overlaid with mineral oil in an Eppendorf tube.

As shown in Table 4, there was no obvious difference in embryo development after

cultured in Petri dish or Eppendor tube.

Table 4 Effect of maturation of mouse COCs in Petri dish or Eppendorf tube on

mouse embryo development

Data are presented as means ± SEM of 1 replicate experiment.

Treatments No. of

Oocytes

Cleavage

(%)

Blastocyst

on day 5

Blastocyst

on day 6

Hatching

Blastocyst

on day 6

Petri dish

Eppendorf tube

20

23

14 (70%)

20 (87%)

4 (29%)

6 (30%)

8 (57.1%)

13 (65%)

5 (62.5%)

10 (76.9%)

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