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Role of oocyte-secreted factors in prevention of cumulus cell apoptosis and enhancement of oocyte developmental competence
Tamer Hussein, MScMed
Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health, University of Adelaide, Australia.
A thesis submitted to the University of Adelaide in total fulf,rllment of the requirements for the degree of Doctor of Philosophy.
July 2006 Table of Contents
Abstract VI Declaration VIII Acknowledgements_...- IX XI Glossary/Abbreviations- -... ------Publications XIII XIV Conference Proceeding - Provisional Patent XV Visits to Overseas Laboratories & Seminars,.---.-.- XV Awards, Scholarship & Prizes..-. XVI
CHAPTER 1: LITERATURE REVIEW
1.1 Introduction 1.2 Fotlicle and Oocyte Development...-.-...- ---.-.
1.2.1 F ollicul ar development -.... -. - -. -. - - -. -. L2.2 Oocyte-follicular cell interactions -----.------. -
1.2.2.1 G ap -j unct i o nal communi cat ion. - 1.2.2.2 P aracrine s oluble factors--..-.-- 11 1.2.3 Granulosa cell regulation of oocyte growth ------15 1.3 Transforming Growth Factor -p Superfamily-.--.....------15 1 .3.1 TGF -13 sup erfamily signalling pathways .___..._.16 gr ow th -/3.. 1.3.2 Tran sþrmin g factor -. ------...... __.20 t.3.3 Activins and inhib int--....-..-..-.--. _....__..20
i at io n 9 1.3.4 Growth differ ent fac tor - -. - -. -. ------. .__.....22 1.3.5 Bone morphogenetic protein I5 ------...... 24 1.3.6 GDF-? & BMP-L5 deficient animal models...-...... _..26 1.3.7 OSFs regulation of COC function----- 28 t.4 Follicular Atresia, Apoptosis and Oocyte Quality-...... 28
1.4.1 F ollicul ar atresia. ------...... _...28 1.4.2 The cellular mechanism of apoptosis 29 1.4.3 Cumulus cell apoptosis and oocyte quality....-.-.---...... _...... 3 1 aa 1.5 Oocyte Maturation JJ I 33 1.5.1 O o cy t e nucl ear and cy top as mi c matur ati on ------. - -. - - 1.5.2 The effect of cumulus cells on oocyte developmental competence..-.--,35 L5.3 In vitro & in vivo oocyte maturation outcomes.--- -..--.--..--.37 1.6 Summary 38 1.7 Hypothesis and Aims for PhD Project..-.-...---...- 1.7.1 Hypothesis.------1.7.2 Aims
CHAPTER 2: OOCYTES PREVENT CUMULUS CELL APOPTOSIS BY MAINTAINING A MORPHOGENIC PARACRINE GRADIENT OF'BONE MORPHOGENETIC PROTEINS 4I
II 2.1 Abstract 42 )) Introduction 43 2.3 Materials and Methods 46 2.3.I Collection of bovine oocytes and culture conditions 46 47 2.3.2 Treatment of cumulus cells. - - - - -. - - - - - 2.3.2.1 Generation of oocytectomized complexes -- 47 2.3.2.2 Generation of denuded oocytes ------47 2.3.2.3 Growth factors and binding proteins --.---.------48 z.).J Determination of DNA damage by TUNEL (assessment of cumulus cell apoptosß) . . 48 2.3.4 Confocal microscopy and analysis 49
14/ t b I o s i,s 50 2.3.5 es ern t analy - -... -.. -.. -. ------. 51 2.3.6 Exp erimental D esign - - - - 2.3.6.I Experiment 1 ; Effect of oocytectomy on cumulus cell
apoptosis . 51 2.3.6.2 Experiment 2: Effect of oocyte-secretedfactors on cumulus cell apoptosls------51 2.3.6.3 Experiment 3: Pattern of apoptosis in relation to proximity to oocyte-secreted factor origin------.-. 52 2.3.6.4 Experiment 4: Dose response of GDF-9, BMP-6 & B MP- 1 5 on cumulus c ell apoptost.ç..--. ------. ---- 52 2.3.6.5 Experiment 5: Effect of oocytes, GDF-9 and BMP-15 on CC expression of Bcl-2 and Bax proteins ------53 2.3.6.6 Experiment 6: Effect of oocytes, BMP-6, and BMP-I5 on s t o 53 cumulu s c el I ap op t os is induc ed by aur sp orine. ------2.3.6.7 Experiment 7: Effect of BMP antagonists on cumulus cell 54 apoptosis ___ 2.3.6.8 Experiment B: Role of BMP-15 and BMP-6 in the anti- apoptotic actions of oocytes on cumulus cells ------54 2.3.6.9 Experiment 9: Effect of BMP-7 and its antagonist, gremlin, on cumulus cell apoptosis-..-.------55 55 2.3.7 Statis tic al analy sis. __ - -- 2.4 Results 56 2.4.l Experiment l: Effect of oocytectomy and FSH on cumulus cell apoptosls..-..---....-.... -...... 56 2.4.2 Experiment 2: Effect of oocyte-secretedfactors on cumulus cell apoptos,s.------..------.--.---56 2.4.3 Experiment 3: Pattern of apoptosis in relation to proximity to oocyte-secreted factor origin ------.-.--.------.59 2.4.4 Experiment 4: Dose response of GDF-9, BMP-6 and BMP-I5 on cumulus cell apoptosls--.-..-.-..--.-. --.---.--.--.61 2.4.5 Experiment 5: Effect of oocytes, GDF-9 and BMP-15 on CC expression of Bcl-2 and Bax proteins.------...... ------.61 2.4.6 Experiment 6: Protection of cumulus cells from staurosporine- induced apoplosis by oocytes, BMP-6 and BMP-15...... -.--..--...--.-..-..62 2.4.7 Experiment 7: Effect of BMP antagonists on cumulus cell
ilI 65 apoptosis --. 2.4.8 n*p"ri*"*t t, nàtà ;;; aui-o in the anti- apoptotic actions of "/aMP-îloocytes on cumulus cells 66 2.4.9 Experiment 9: Effect of BMP-7 and its antagonist,
gremlin, on cumulus cell apoptosls------..------... 67 2.5 Discussion 7l 2.6 Acknowledsements 80 2.7 References 81
CHAPTER 3: OOCYTE-SECRETED FACTORS ENHANCE OOCYTE DEVELOPMENTAL COMPETENCE 87
3.1 Abstract____ 3.2 Introduction.__._____._.._..______3.3 Materials and Methods 3.3.1 Collection of oocvtes and culture conditions
3.3.2 Treatment of cumulus-oocyte complexes --
3.3.2.1 Generation of denuded oocyte . ------3.3.2.2 Growth.factors & antagonists .---- 3.3.3 In vitro fertilization and embryo culture---.---- 94 3.3.4 Differential slainins 95 gn. 96 3.3.5 Exp erim en ta I desi - - - - 3.3.5.1 Experiment 1: Effect of co-culture of intact COCs with DOs
s c o mp et enc e 96 durin g IV M on ub s equ ent dev el op ment al - - - - -. - - - -. 3.3.5.2 Experiment 2: Effect of BMP-L5 and/or GDF-9 during IVM on oocyte developmental competence -.------97 3.3. s.3 Experiments 3 & 4: Effect of GDF-9 or BMP-I5 antagonisls on oocyte developmental competence 97 98 3.3.6 Statis tic al Analysis _ _... 3.4 Results 99 3.4.1 Experiment 1: Effect of co-culture of intact COCs with DOs during IVM on subsequent developmental competence ------99 3.4.2 Experiment 2: Effect of BMP-15 and/or GDF-9 during IVM c 99 on oocyte devel opment al omp etence ------. - - - - 3.4.3 Experiments 3 & 4: Effect of GDF-9 or BMP-I 5 antagonists o o o t el c et e 101 n cy e dev opment al omp enc . - -. ------3.5 Discussion. 105 3.6 Acknowledsements t12 3.7 References 113
CHAPTER 4: TEMPORAL EFFECTS OF OOCYTE-SECRETBD FACTOR(S) DURING IN VITRO MATURATION ON BOVINE OOCYTE DEVELOPMENTAL COMPETENCE I2O
ry 4.1 Abstract I2r 4.2 Introduction I 22 4.3 Materials and Methods 1 25 4.3.I Collection of oocytes and culture conditions 1 25 4.3.2 Treatment of cumulus-oocyte complexes -. 1 26 o o cytes 1 26 4.3.2.1 G eneration of denuded ------Gr ow th or s 1 26 4.3.2.2 fact ------. - - 4.3.3 In vitro fertilization and embryo culture------1 26 D ren tia I s taining. 1 28 4.3.4 ffi - -. -. 4.3.5 Eîperimentat design-' .-.....-.-----.--.-.....----.. .1 28 4.3.5.I Experiment I : Temporal effects of OSFs on oocyte developmental competence following co-culture of intact COCs with DOs at either 0 or t hour of IVM- t28 4.3.5.2 Experiment 2: Assessment of oocyte developmental competencefollowing treatment of COCs with GDF-9 or BMP-15 at either 0 or t hour of IVM.. ..-.--..-.-...... 130 4.3.6 Statistical analyses.-.-- ....------.130 4.4 Results------""r32 4.4.1 Experiment 1: Temporal fficts of OSFs on oocyte developmental competence following co-culture of intact COCs with DOs at either 0 or t hour of IVM- ....-..--...... 132 4.4.2 Experiment 2: Assessment of oocyte developmental competencefollowing treatment of COCs with GDF-9 or BMP-(5 at either 0 or t hour of IVM.. --...... 133 4.5 Discussion 136
4.6 Acknowled gements.__ ___. r40 4.7 References I4I
CHAPTER 5: FINAL DISCUSSION t47
Final Discussion 148 Future directions 153
REFERENCES 156
APPENDICES 181
Appendix I : Additional Experiments.....--- -....-..- 182 Appendix 2 : TUNEL Assay.....-..--- 187 Appendix 3: Culture Media 191 Appendix 4: Reagents 195 Appendix 5: Blastocyst Scoring System.....-. \97 Appendix 6: Published Version of Chapter 2 ----.----.---.---- 199 Appendix 7: Published Version of Chapter 3...... -.-...-.-.-. 200
V Abstract
Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence.
The aim of this thesis was to examine whether cumulus cells exhibit a low incidence of apoptosis due to their close association with oocytes and their exposure to OSFs, and to investigate if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM).
This thesis includes a series of studies designed to examine by various means the nature of the paracrine network of bone morphogenetic proteins (BMPs) and their binding proteins involved in the regulation of cumulus cell apoptosis. OSFs, in particular BMP-
15 and BMP-6, but not growth differentiation factor 9 (GDF-9), reduced apoptosis of cumulus cells by following a gradient from the site of the oocytes. Morever, follistatin
and a BMP6 ne:utralizing antibody, which antagonized the anti-apoptotic effects of
BMPl5 and BMP6, respectively, whether alone or combined, blocked -50% of the anti-
apoptotic actions of oocytes. These results demonstrated that OSFs, particularly BMP-15
and BMP-6, maintain the low incidence of apoptosis by establishing a localized gradienl
of bone morphogenetic proteins.
Results from the present thesis also demonstrated that OSFs enhance oocyte
developmental competence during IVM, whether in their native form as an
uncharacterized mix of growth factors secreted by the oocyte, throughout the oocyte
VI maturation period, or as exogenous BMP-15 and GDF-9, during the first t hour of IVM.
Also, OSFs improved embryo quality as evident by increased blastocyst total and trophectoderm cell numbers, These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor 3B-431542, which antagonize BMP-15 and GDF-9, respectively, neutralized the stimulatory effects of the exogenous growth factors, and impaired the developmental competence of control cumulus-oocyte complexes (COCs)'
The work presented in this thesis has provided multiple lines of evidence that OSF- regulation of the COC microenvironment is an important determinant of cumulus cell apoptosis and oocyte developmental programming.
VII Declaration
This thesis contains no material which has been accepted for the award of any other degree or diploma in any university or other ßfüary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference is made in the text. The experiments reported in this thesis were performed by myself and any assistance received from others is specifically pointed out and acknowledged.
I consent to this thesis being made available in the University Library if accepted for the award of the degree.
July 2006 u
Tamer Hussein
VIII Acknowledgements
I would like to express my most sincere gratitude to my two supervisors, Drs Robert
Gilchrist and Jeremy Thompson, for allowing me the opportunity to undertake a PhD in the field of research that I have grown to enjoy immensely. Many thanks for the continual support, advice and encouragement throughout my project. Your enthusiasm for the subject and never ending expertise in this area of research has been of tremendous help.
My very special thanks go to Dr. Karen Kind who has helped me in every aspect of my study and who has provided innumerous valuable comments. I must also give gargantuan thanks to Dave Froiland for his great support throughout my residency in Adelaide. I will miss our chats about almost everything and working with you in the lab. To me, you have been more than just a work colleague; you are a great friend.
An enormous thanks goes out to past and present staff and students of the Research
Centre for Reproductive Health. In particular, I would like to thank Lesley Ritter for all
her support over the years, and Samantha Schultz for assisting with experiments. I would
also like to thank Helen Holmes and Kara Cashman for too many things to name so I will
not even try. Exceptional thanks go to Michelle Lane, who has always been willing to
help me and who gave me the opportunity to train and work in clinical embryology.
Fellow students over the past 3 years please know that all your friendship and support
have been valuable to my sanity.
IX I must thank my incredible network of friends for all the love and support they have provided throughout the course of my PhD, especially at the end of it when I broke my ankle and stayed in the hospital. Raik, Sarah and Rach, I am so fortunate to have such wonderful, supportive friends and I appreciated everything that you have done to help me
Last but not least, my study and scientific work would not have been possible without the strong inspiration and support I receive from my family. I wish to express my deep gratitude to all members of my family; without their love and encouragement, life would have been intolerable. I would therefore like to dedicate this thesis to my family and to those whose names are not included, but who assisted me in one form or another. I sincerely thank them all.
These studies were financially supported by the National Health and Medical Research
Council Program Grant and Faculty of Health Science Postgraduate Award.
X Gloss arylAbbreviations
293H 293 human embryonic kidney cell line ALK activin receptor-like kinase AMH anti-Mullerian hormone ATP adenosine triphosphate BMP bone morphogenetic protein BMPR-II bone morphogenetic protein receptor type-Il BSA bovine serum albumin B-TCM bicarbonate-buffered tissue culture medium CAMP cyclic adenosine monophosphate
CC cumulus cell
CEEF cumulus expansion-enabling factor COC cumulus-oocyte complex cox2 cyclooxygenase-2 DO denuded oocyte ECM extra cellular matrix EGF epidermal growth factor FAF fatty acid-free
FCS fetal calf serum
FF follicular fluid FSH follicle stimulating hormone
GC granulosa cell GDF-9 growth differentiation factor 9 GV germinal vesicle GVBD germinal vesicle breakdown GJC gap junctional communication HA hyaluronan HAS2 hyaluronan synthase-2 HCG human chorionic gonadotrophin H-TCM hepes-buffered tissue culture medium ICM inner cell mass
XI IVF in vitro fertilisation IVM in vitro maturation IVP in vitro production (of embryos) c-kit kit-ligand receptor KL kit-ligand LH luteinising hormone LHR lteinising hormone receptor MAPK mito gen-activated protein kinase MGC mural granulosa cell MI metaphase I MII metaphase II oox oocytectomised complex OSF oocyte-secreted factor
PG prostaglandin PTX3 pentraxin-3 PVA polyvinyl alcohol PVP polyvinyl pyrolidone rhFSH recombinant human FSH sB-431542 ALK4I 5 17 kinase inhibitor SMAD mothers against decapentaplegic StAR steriodo genic acute regulator TCM-199 tissue culture medium 199
TGF B transforming growth factor B TGFBR-II transforming growth factor receptor type-Il
TSG6 tumour necrosis factor-cr,-stimulated gene-6 TUNEL terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling TZP transzonal cytoplasmic processes
UPA urokinase plasminogen activator
XII Publication and Conference Proceeding
Scientific Publications
1. Tamer S. Hussein, David Froiland, Jeremy G. Thompson and Robert B. Gilchrist. (2005). Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic
paracrine gradient of bone morphogenetic proteins . Journal of Cell Science. II8 (22):
5257-5268. flmpact Factor: 6.9]
2. Tamer S. Hussein, Jeremy G. Thompson and Robert B. Gilchrist. (2006). Oocyte paracrine factors enhance oocyte developmental competence. Developmental Biology. 296: 514-521. llmpact Factor: 5.51
3. Tamer S. Hussein, Robert B. Gilchrist and Jeremy G. Thompson. Temporal effect of oocyte-secreted factor(s) during in vitro maturation on bovine oocyte developmental
competence. Biology of Reproduction. (Manuscript to be submitted December 2006)
flmpact Factor: 3.6]
Other Journal Contributions 1. Tamer S. Hussein, David Froiland, Jeremy G. Thompson and Robert B. Gilchrist. (2004). Oocyte-secreted factor(s) regulate apoptosis of bovine cumulus cell
complexes. Reproduction, Fertility and Development; 16 (suppl); Ãbsftaclt252.
2. Tamer S. Hussein, David Froiland, Jeremy G. Thompson and Robert B. Gilchrist. (2005). Oocyte-secreted factors prevent bovine cumulus cell apoptosis: paracrine involvement of bone morphogenetic proteins. Biology of Reproduction; (Special
Issue); Abstract 79.
3. Tamer S. Hussein, David Froiland, Jeremy G. Thompson and Robert B. Gilchrist (2005). Oocyte prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins. Reproduction, Fertility and
Developmenr; I 7 (Suppl); AbstracT 232'
XIII 4. Tamer S. Hussein, Jeremy G. Thompson and Robert B. Gilchrist. (2006). Oocyte- secreted factors directly affect oocyte developmental competence during in vitro maturation of the bovine cumulus-oocyte complex. Reproduction, Fertility and
D evelopment; 18 (1,2); AbstracT 327 .
Conference Proceeding
1. International
1. Tamer Hussein, David Froiland, Jeremy G. Thompson and Robert B. Gilchrist. (2005). Oocyte-secreted factors prevent bovine cumulus cell apoptosis: paracrine involvement of bone morphogenetic proteins, Society for the Study of Reproduction
conference, Quebec, Canada. (Oral presentation)
2. Tamer Hussein, Jeremy G. Thompson and Robert B. Gilchris|. (2006). Oocyte- secreted factors directly affect oocyte developmental competence during in vitro maturation of the bovine cumulus-oocyte complex. International Embryo Transfer Society Conference, Orlando, USA. (Poster presentation)
3. Robert B, Gilchrist, Tamer Hussein, Jeremy G. Thompson (2006). Improved embryo development from in vitro matured oocytes by addition of native or recombinant
oocyte-secreted growth factors. 22"d annual Meeting of European Society of Human Reproduction and Embryology. Prague, czech Republic. (oral presentation)
2. National
1. Tamer Hussein, David Froiland, Jeremy G. Thompson and Robert B. Gilchrist, (2004), Oocyte-secreted factor(s) regulate apoptosis of bovine cumulus cell
XIV complexes. Proceedings of the 35th Annual Conference of the Society for Reproductive Biology, Sydney, Australia. (Oral presentation)
2. Tamer Hussein, David Froiland, Jeremy G. Thompson and Robert B. Gilchrist. (2004). Oocyte-secreted factor(s) regulate apoptosis of bovine cumulus cell complexes. North Western Adelaide Health Service Research Day, Adelaide; Abstract 14. (Oral presentation)
3. Tamer Hussein, David Froiland, Jeremy G. Thompson and Robert B. Gilchrist (2005). Oocyte prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins. Proceedings of the 36th Annual Conference of the Society for Reproductive Biology, Perth, Australia. (Oral presentation)
Provisional Patent Tamer Hussein, Jeremy G. Thompson, Robert B. Gilchrist and Rebecca Dragovic. "Modulation of Granulosa Cell Apoptosis" Australian Provisional Patent Application No. 2005903782. Filed l8l7 12005.
Visits to Overseas Laboratories & Seminars
2005 Prof Marc-Andre Sirard, Research Centre for the Study of
Repro ductio n, Lav al Univers ity, Queb ec, C anada.
2006 Prof Shunichi Shimasaki, Department of Reproductive Medicine, University of California, San Diego, USA.
2006 Robert B. Gilchrist and Tamer Hussein 'Oocyte regulation of cumulus- oocyte complex function' Department of Reproductive Medicine, University of California, San Diego, USA.
XV Awards, Scholarship & Prizes
2003-2006 P o s t graduat e Aw ar d (Scholarship) Faculty of Health Sciences, University of Adelaide
2004 Meat & Livestock Australia New Scientist Award Society for Reproductive Biology Conference, Sydney
2004 New Investigator Award Finalist Society for Reproductive Biology Conference, Sydney
2004 Research Day Prize Finalist North Western Adelaide Health Service
2005 Faculty of Health Science Postgraduate Traveling Fellowship University of Adelaide
2005 Society for Reproductive Biology Travel Award Society for Reproductive Biology Conference, Perth
2005 Research Centre for Reproductive Health Travel Award University of Adelaide
2005 USA Department of Agriculture, Merit Travel Award Society for the Study of Reproduction, Quebec, Canada
2006 Network in Genes and Environment in Development
C o nferen c e P articip ati o n Aw ard International Embryo Transfer Society Conference, Orlando, USA
XVI Chapter One
Literature Review Chapter 1. Literature review
1.1 Introduction
Ovarian follicular development involves important morphological changes in follicular
cells including proliferation and cell death as well as cytoplasmic and nuclear maturation
of the oocyte. These processes require bidirectional communication between the oocyte
and the surrounding follicle cells (Albertini et a1., 2001). Cell-cell communication
between somatic cells and the oocyte is crucial for follicle development and oocyte
maturation (Carabatsos et al., 1998), Disruption of this communication induces apoptosis
of both cell types (Luciano eT a7., 2000). The two cell types are connected via gap junctions (Anderson and Albertini, 1976), which allow the transfer of nutrients,
metabolic precursors, and regulatory substances between cumulus cells (CCs) and the
oocyte (Eppig, 1991). Paracrine factors secreted by the oocyte are also crucial in
regulating many important aspects of follicle development (Gilchrist et at.,2004a).
The identification of these oocyte-secreted factors (OSFs) has been difficult. Howevet,
studies on the transforming growth factor-B GGF-B) superfamily, which consists of over
40 structurally related proteins, suggest a primary role for these growth factors through
their ability to mimic the actions of the oocyte in regulating granulosa cell (GC) activity
(Gilchrist et al., 2003). In particular, the closely related TGF-B superfamily members,
bone morphogenetic protein-6 (BMP-6), growth differentiation factor-9 (GDF-9) and
BMP-15, also called GDF-98, are all expressed in an oocyte-specific manner from an
early stage in the follicle and appear to play a key role in promoting follicle growth and
regulating female fertility (Aaltonen et al., 1999 ; McNatty et al., 2003).
2 Chapter 1. Literature revrew
Most follicles within the ovary do not ovulate; they instead degenerate and die by the process of atresia. The cellular mechanism of follicular atresia is generally caused by apoptosis (Jolly et al.,1994). Most of these cellular events are dependent on mRNA and protein synthesis, including expression of genes of the Bcl-2 family. During atresia in antral follicles, the CCs and the oocyte are the last compartments of the ovarian follicle to be affected by the atretic changes, which is primarily manifested as apoptosis in the mural granulosa cells (MCGs) and, at a later stage, in the theca cells (Yang and
Rajamahendran, 2000).
Oocyte maturation is the conclusion of an extended period of oocyte growth and
development within the growing follicle, and the short interval of meiotic maturation preceeds ovulation. The capacity of the oocyte to acquire developmental competence, i.e.
to gradually acquire the cellular machinery required to support early embryonic
development, is inherently linked to the process of folliculogenesis and to the health of
the developing follicle.
This review will firstly cover the bidirectional communication pathways between the
oocyte and granulosa-cumulus cells, particularly with respect to paracrine soluble factors
secreted by the oocyte. The review will then focus on putative OSF candidates within the
TGF-B superfamily, specifically GDF-9 and it's homologue BMP-15, and whether these
oocyte factors regulate apoptosis in the CCs, Finally, the numerous events that affect
oocyte maturation and the acquisition of developmental competence will be discussed
J Chapter l. Literature review with reference to the cumulus-oocyte communication and the importance of CCs to oocyte maturation in vitro (IVM).
1.2 Follicle and Oocyte Development
1. 2. I Follicular development
Cyclic ovarian follicular development is a complex process involving proliferation,
differentiation, and death of follicle cells (Luciano et al., 2000). Gonadotrophins
produced by the pituitary gland have a central role in the regulation ofthese processes. In
addition, a wide range of paracrine and autocrine signals produced within the ovary
regulate follicle growth and ovarian function,
The ovary has subsets of follicles at various stages of development. During fetal life, the
developing ovaries become populated with primordial germ cells (oogonia), which
continue to divide rapidly by mitosis until a few weeks before birth (Gosden, 2002).
From early in gestation, many fetal oogonia enter meiosis and progress to the first
prophase, where they arrest. At this stage these oogonia are called oocytes. The oocyte is
surrounded by a single layer of GCs forming a primordial follicle (Fig. 1). In response to
unknown signals, primordial follicles leave the resting pool and GCs initiate a phase of
growth, although proliferation is slow (Buccione et al., 1990a). The initiation of these
processes can be considered the first functional interaction between the oocyte and GCs
in the ovary. With the formation of the primary follicle, the GCs become cuboidal in
4 Chapter L Literature revle'w
shape and undergo cell division (Fig. 1). Subsequently, multiple layers of GCs surround the oocyte forming a secondary follicle (Gosden, 2002).
The preantral phase is characterised by growth of the oocyte and the formation of a
glycoprotein coat surrounding the oocyte, The zona pellucida. GCs maintain contact with
the oocyte through cytoplasmic projections that penetrate the zona pellucida and establish
specialised junctions with the oolemma (Mitchell and Burghardt, 1986). GCs become
proliferative, forming a stratified epithelium. At this stage, the GCs acquire high-affinify
receptors for follicle-stimulating hormone (FSH) (Hirshfield, 1986). Following preantral
development, the oocyte ceases growing and acquires competence to resume meiosis.
This initial phase of oocyte growth and preantral follicle development seems to occur
independently of gonadotrophic hormones (Tirone et al', 1993)'
During the antral phase of folliculogenesis, fluid accumulates between GCs and a
follicular antrum cavity is formed with the MGCs located at the periphery (Fig. 2). The
oocyte remains surounded by closely associated GCs, referred to as CCs (Fig 2),
forming the compact cumulus-oocyte complex (COC), At this stage of follicle
development, GCs located in different regions of the follicle acquire different
morphological and functional proprieties. Most notably, the CCs differ from MGCs in
distribution of luteinising hormone receptors (LHR) (Eppig et al., 1997b) and mRNA
coding for steriodeogenic enzyme such as cholesterol side-chain cleavage cytochrome
P450 (Zlotkin et al., 1986) and cytochrome P450 aromatase (Whitelaw et a1., 1992).
5 Chapter l. Literature revlew
Rr'sunrption of nrciosis
suct¡¡rda'v o0rùy¡0 primary oocyte &rrested itt prophase of Mei<¡sis I {rc$lc(l rl II @@o0 o0 35 ¿lr 120 qnr matuTntion
Nucleal rrtatul'ation
20 d f'tillicle \ diamctcr \ (nrm) r,5 nt 4" 20 mN
r0
5 o\ t ,r1j ' t ,o .'- I 2 -,5 lüu 0 5-g-c- - ñ 00,1 - 0l¡ììDr 02ilìnì tÌ2-0.4mnr 0,1)+ rll ,Ù, Initiation Sclection prirrrordial prinrary sccrlndtty crlr y antrut prcclvulittÕr-r' (car1v (Prcanlral) ¿urtrul grrrling)
Figure 1. A summary of the stages of follicle development and its relationship to oocyte growth and maturation. Adapted and modified from (Hardy et al., 2000)'
6 Chapter l. Literature revlew
Also, CCs possess the capacity to secrete hyaluronic acid and undergo expansion, which
MGCs do not (Salustri et a1., 1990b). In addition, CCs play an important role in the normal growth and development of the oocyte, while MGCs support the growth of the follicle (Li et al., 2000).
Eppig et aL., (1997a) demonstrated a role for oocytes in determining the differentiation of surounding GCs and in maintaining the organization of the antral follicle. They concluded that "without the influence of the oocyte, the default pathway of GC differentiation in antral follicles leads to the establishment of the MGC phenotype, and importantly, oocytes cancelled the pathway to promote the development of the CCs phenotype" (Eppig et al., I997a). Oocytes may do this in order to control their microenvironment through suppression of the MGC phenotype and by promoting differentiation of the CC phenotype (Gilchrist eT a1.,2004a). Furthermore, Eppig et a1.,
(I997b) examined the expression of LHR mRNA, as a marker of the phenotype of MGCs
in preovulatory follicles. They noticed down regulation of LHR expression in the CCs, but not in the MGCs. This f,rnding indicated that there is a difference between MGCs and
CCs in both function and fate and this difference is dependent to a large extent on soluble
OSFs. In the absence of the OSFs, CCs acquire some of the characteristics of the MGCs
phenotype. On the other hand, MGCs respond to OSFs by becoming more CC-like,
indicating that the factor(s) is able to at least attenuate, if not partially reverse, the
pathway of development to the MGC phenofype.
7 Chapter L Literature review
tlu:ca
orlmulus oûüyte gran*losa genninål vesiele I \ I zona pellucida
ântrum basement r*em.brane
Figure 2. Structure of the antral follicle. Adapted and modified from (Hardy et al.,
2000).
1. 2, 2 Oocyte-follicular cell interactions
Traditionally research has focused on just one direction of the communication axis
between the oocyte and the follicle cells, that is, on GC support of the developing oocyte.
However, recent studies have demonstrated the importance of a bi-directional
communication axis (Albertini et a1., 2001). Communication between oocytes and
companion follicle cells is mediated by both direct physical contacts (gap junctions) and
paracrine soluble factors (oocyte-secreted factors) (Fig. 3).
8 Chapter 1. Literature review
Cumulus cells to oocyte E.g. Kit-Ligand FF-MAS
Gap-junctional cAMP Metabolites Amino acids
Oocyte to cumulus cells Oocyte-secreted factors E.g. GDF-9 BMP-15 (GDF-gB) BMP-6
Figure 3. Bidirectional communication between oocyte and cumulus cells. Communication pathways occur via paracrine signaling (curved arrow) and gap junctions (straight arrow). Adapted and modified from (Sutton et a1.,2003).
1. 2. 2. 1 Gup-j unctíonal communication
The CCs penetrate the zona pellucida via trans-zonal cytoplasmic projections and connect with the oocyte membrane. Gap-junction transmembrane channels at the end of these projections (and between CCs) provide a communication axis for low molecular mass molecules (< 1000 Mr) (Kruip et al., 1983). During the growth of mammalian oocytes, there is continuous metabolic coupling with the surrounding follicle cells via gap junctions (Eppig, 1991). According to morphological evidence, this coupling begins as primordial follicles form and expands as folliculogenesis proceeds through primary,
9 Chapter 1. Literature revlew
secondary, and antral follicle stages (Mitchell and Burghardt, 1986). Gap junction channels are made up of two symmetrical units called connexons - each consisting of a hexamer of proteins from the connexin family (Haefliger et al., 1992). Many different connexin types and combinations have been localized in the ovarian follicle (Kidder and
Mhawi, 2002). Gap junctions allow the passage of nutrient and signaling molecules between cells, such as cyclic adenosine monophosphate (cAMP) (Byskov eÏ a1.,2002),
2002), amino acids (Eppig et al., 1996) and metabolites including glucose (Coskun and
Lin,1994) and nucleotides (Downs, 1997).
Connexin 43 (Cx43), the protein associated with gap junctions between GC (Ackert et a1.,200I), mRNA and protein is present in the bovine COC during IVM (Sutovsþ et al.,
1993). Connexin 43 has also been found in GCs of human follicles (Simon et a1.,1997).
Gap junctions may have an important role during bovine oocyte IVM as the blocking of gap junction signaling or the reduction of Cx43 protein resulted in impaired oocyte maturation (Yozzi et a1., 2001). Evidence from knockout mice with the connexin gene deleted, has strengthened the hypothesis that gap junctional communication is critical for folliculogenesis and oocyte maturation. In cultured neonatal ovaries from mice homozygous for a null mutation encoding connexin 43, folliculogenesis could not proceed beyond the primary stage, showing that this gap junction protein is essential for folliculogenesis (Juneja eT aL,1.999). Furthermore, mice deficient in connexin-37 (Cx37), the protein buitding block of oocyte-cumulus cell gap junctions, lack preowlatory
follicles and oocyte development is arrested before meiotic competence is achieved
(Simon er. al., 1997).
10 Chapter 1. Literature review
1.2.2.2 Paracrine soluble faclors
It is now widely accepted that the oocyte plays a very active role in promoting follicle growth. This is apparent in vivo in germ cell def,rcient mice and in women with Tumer's syndrome, where the germ cells degenerate and follicles fail to form. The impact of the oocyte on GC and CC functions and differentiations has been demonstrated by co- culturing oocytectomised complexes (OOX) (the surgical removal of the oocyte leaving a hollow ball of CCs) or GCs with fully-grown oocytes (Buccione et al., 1990b) (Fig. a).
Using this approach, it has been shown that the oocyte regulates GC proliferation of both preantral and antral follicles (Vanderhyden et al., 1992), steroidogenesis in mouse
(Vanderhyden et al., 1993) and porcine CCs (Coskun et al., 1995), deposition of extracellular matrix (Buccione et al., 1990a), and hyaluronic acid synthesis (Tirone et a1.,
1ee3)(Fig. s).
üunulus-ü0cyte cËll rnmFlelr
Intact rüflFlex tocytectomized ICOC} ffimÌtlex {üüxi
Figure 4. Microsurgical oocyte removal from the intact complex, retains the three- dimensional integrity and the cell-cell association in the oocyctectomised complex. Adapted and modihed from (Vanderhyden et al., 2003).
11 Chapter 1. Literature revlew
Recent findings highlight the ability of the oocyte to influence ovarian somatic cell function directly through paracrine factors. Soluble factors produced by the oocyte locally modulate the response of CCs and MGCs to gonadotrophin signals by maintaining the cells in a responsive state to these hormones. Moreover, mouse oocytes secrete paracrine signals that enable CCs to undergo expansion in response to FSH stimulation by down-regulating urokinase plasminogen activator (uPA) and up-regulating hyluronan synthase-2 (HAS-2) expression in CCs (Dragovic et al., 2005; Salustri, 2000;
Vanderhyden et al., 1990) (Fig. 5).
Conversely, follicle cells produce paracrine signals that affect the oocyte. Prochazka et al. (1998) observed that porcine CCs and GCs isolated from pre-antral to preovulatory- stage follicles were capable of producing cumulus expansion-enabling factor (CEEF) activity in vitro, involved in cumulus expansion. Kit-ligand (KL) mRNA expression has been demonstrated in GCs and acts in a paracrine fashion through an interaction with the kit-ligand receptor (c-kit) expressed at the oocyte surface, regulating the growth and function of the oocyte (Joyce et a1.,2000; Thomas and Vanderhyden, 2006). Moreover,
KL promotes recruitment of theca cells from the stroma surrounding the primordial follicle and has a direct effect on theca cell proliferation (Besmer et a1., 1993)'
A potential role for the oocyte in the regulation of follicular steroidogenesis and luteinization was first suggested by el-Fouly eT al., (1970), who demonstrated that removal of the oocyte from rabbit Graafian follicles in situ resulted in spontaneous
lutenization and increased progesterone production. Vanderhyden and co-workers
l2 Chapter 1. Literature revlew
(Vanderhyden et al., 1993; Vanderhyden and Tonary, 1995) demonstrated that factors released by the oocyte modulate gonadotrophin stimulated steroid hormone production by both mouse CCs and GCs. These factors modulate FSH-induced progesterone and oestradiol synthesis by CCs and MCGs and suppress FSH-induced LHR mRNA expression (Eppig et al., 1997b; Vanderþden and Macdonald, 1998) (Fig. 5).
Furthermore, Coskun et al. (1995) reported that porcine oocyte-secreted factors inhibit
CC and GC steroidogenesis.
Species variation seems to exist with regard to the role of the oocyte in the regulation of
GC function. Vanderhyden (1993) demonstrated that factor(s) secreted by mouse andral oocytes are necessary for their CCs to undergo expansion in response to FSH, epidermal growth factor (EGF) or cAMP. Regulation of cumulus expansion in ruminant complexes is different from that in rodents. While pig and bovine oocytes can secrete CEEF, expansionof pig(Singh etal.,1993) andbovine(Ralphetal., 1995)complexesdoesnot depend upon this factor.
Previous reports have shown that synthesis and secretion of paracrine factors by the oocyte is developmentally regulated and is dependent on the stage of oocyte growth and maturation (Eppig et a1.,1993; Gilchrist et a1.,2001; Vanderhyden et a1., 1990), Eppig et al. (1997b) reported that mature oocytes were less effective than fully grown immature germinal vesicle-stage oocytes in suppressing LHR mRNA expression in murine GCs.
Gilchrist et al., (2001) suggested that there was a relationship between the ability of the oocyte to promote mouse GC proliferation and its meiotic status, demonstrating that this
13 Chapter 1. Literature review
ability was increased with the acquisition of meiotic competence and declined during and after oocyte maturation.
These studies clearly illustrate that oocyte paracrine signalling to follicular GCs and CCs is crucial for regulating appropriate somatic cell growth and differentiation and hence for normal folliculogenesis and fertility. Members of the TGF-P superfamily are currently the prime candidate oocyte-secreted molecules that regulate such processes, due to their ability to mimic these in vitro (Gilchrist et al., 2003; Vanderhyden et al., 2003).
GDF.g GDF.gB 7?? a a
Growth ô DNA *ynlhcsir +\ Differentiation cnh¡nct IGF.I & FSll CC axoanslon ovulatlon stim uliltt-d prollfc rrlion 9l,tt nttr¡rtor t . ltÂS-2 -+ hynlumnic rcid f¡cllitatc and rogcn stin¡ ulatcd prcvcnt lutcinlsatlon - rcguletc ô f prollfrrrllon P4 rnd E2 prorluctlon ' * ul'À Tegulutc kit-ligrnd a I inhibin A nnrl ll synthcsis . ô (:ox-2
folliculogenes¡s, ovulation & ovulation rete
Figure 5. Oocyte-secreted soluble factors regulate a broad range of cumulus and granulosa functions including growth, differentiation, cumulus expansion and ovulation. The exact identities of these oocyte-secreted factors are still vague, but GDF-9 and its closely related homologue BMP-15 are impoftant oocyte-secreted factors that can mimic many of these processes in vitro. Adapted and modified from (Gilchrist et a1.,2004a).
I4 Chapter L Literature review
1.2.3 Grønulosa cell reguløtion of oocyte growth
GCs are the basic support system of the oocyte, affecting oocyte cycle events, nutrition, and maturation (de Loos et al., 1991). Mouse and cow studies have demonstrated that
GCs are metabolically coupled to oocytes via gap junctions, and nutritional and regulatory elements responsible for growth are able to pass through these gap junctions
(de Loos et a1., 1991; Richard and Sirard, 1996). In vivo, mammalian oocytes resume meiosis after the rnid-cycle surge of luteinizing hormone (LH). Since oocytes lack receptors for LH, the signal to resume meiosis must be mediated by the associated GGs
(Eppig et al., 1997b).
Co-culture of isolated oocytes with monolayers of GC or GC conditioned medium has similarly been shown to have an inhibitory effect on meiosis. The important role of GCs in the regulation of oocyte maturation was clearly demonstrated by Tasfiriri and
Changnning (I975) who reported that co-culture of porcine GCs with immature oocytes resulted in a density-dependent inhibition of spontaneous oocyte maturation. Later, Sirard and Bilodeau (Sirard and Bilodeau, 1990) showed that fresh GCs inhibited meiotic resumption of bovine oocytes. GCs can also affect the phosphorylation of oocyte proteins that are associated with the resumption of meiosis (Colonna et a1., 1989). GCs therefore, play a crucial role in supplying nutrient and regulatory elements during growth and initial stage of maturation.
1.3 Transforming Growth Factor -p Superfamily
15 Chapter l. Literature review
Although the identities of the OSFs involved in regulating oocyte/follicular development remains elusive, there is growing evidence that members of the TGF-B superfamily are involved in physiological events such as folliculogenesis, oocyte growth and maturation
(Knight and Glister, 2003). The TGF-B superfamily is a large family of growth factors comprised of more than 40 members, each similar in structure yet different in function.
Members of the superfamily include TGF-ß; which is present in three isoforms (TGF-P
I,2,3), anti-Mullerian hormone (AMH), two inhibins (inhibin A and B), three activins
(activin A, B and AB), 20 different bone morphogenetic proteins (BMPl to 20) and nine growth differentiation factors (GDF1 to 9) (Ghiglieri et a1., 1995). Various members of the TGF-B superfamily have been identified in follicular fluid, suggesting that they are probably released by follicle cell types within the ovary (Ackland eT al., 1992) (Fig. 6).
The action of many of the TGF-B superfamily members on GCs activity seems to be dependent on the context, particularly as it relates to stage of follicle development and the species (Vanderhyden et al., 2003). Three members of the TGF-B superfamily, growth differentiation factor-9 (GDF-9), bone morphogenetic protein-15 (BMP-15), and
BMP-6 are expressed by oocytes (Aaltonen et a1.,1999; Dube et al., 1998; Glister et al.,
2004; McGrath et a1.,1995).
1.3.1 TGF-fi superfamily signalling pathways
Members of the TGF-P superfamily signal through two types of membrane-bound serine theronine kinases, type-I and type-Il receptors. To date, seven type-I receptors (activin receptor-like kinase; ALK 1-7) and five type-Il receptors (ActR-II, ActR-IIB, BMPR-II,
TGFßR-II, AMHR-II) have been identified (Shi and Massague, 2003). Binding of the
16 Chapter l. Literature revlew
Primordiat O - Àhtlt B inlrhct flE È t E"É E inhihin c.e follirtulin s t rq¡ $'
ÉAÙ Early ann"øI
BlvlP4 t BlvfP-? I large $ntrül (preovulatory) \ t\ f\ I I Putative rotes: . GDF-gIBMP.Is E goa ard diffsrc¿tiðtion . TGN moduloteo granu{oea ood theca ccll pr,oLifenøtion and difftir€'trlíaüon¡ suuÞm¡sec tncst ardroesn øodr¡gion . BMF.+6, -7 detaye luæinizatiun/et¡eeia of l¡rge¡ntrul follisles (inerwees oË$tradiol c¡¡.d inhibi¡: deæaee* progesterone producaion) Actlvla *imulatse gruruloot oell pmliferation in preonraUenrly anral follicb ardrupr€grtsæp FSH rccogtoc and FSH-iuducEd arornatasê âÊtivþ; tlclayc lutpi"ni¿rdon/auwia of large mtr*l Íolliclss; eo$a¡c¿s o'ôryts mûümellcn . Follictrtin ûtænuatoø sbo$c acdonc of activin (and BMPI?); nroy promotc '' tutsiniuat¡mi¡akrgxit of large antal follicle¡ . Itrtrtbir e¡bû¡ccs LH-ind¡¡ced andqgen trÞdrction in Ìarge anual follicles . AMH
Figure 6. Expression of TGF-B superfamily members in the follicle and their putative roles as paracrinelautocrine signaling molecules, Superscripts (o, g, and t) refer to oocytes, granulosa cells and theca cells, respectively. Adapted and modified from (Knight and Glister, 2003)
t7 Chapter l. Literature revlew
TGF-P superfamily member to its receptor results in phosphorylation of the SMAD signaling molecules (Fig. 7). There are eight SMAD proteins in total (SMAD1-8).
Normally, SMAD 2 and3 are activated by TGF-Bs and activins, whereas, SMAD 1, 5, 8 are activated by members of the BMPs. Once phosphorylated, these SMADS combine with the co-SMAD (SMAD 4) and translocate to the nucleus where they interact with transcriptions factors involved in regulation of specific genes. SMAD 6 and 7 are inhibitory SMADs that prevent the activation of the SMAD signaling pathway (see review (Shimasaki et a1.,2004) (Fig. 7).
To date, SMADs are the only receptor substrates identified that mediate signals of the
TGF-P superfamily members. However, there is growing evidence thaT activation of
TGF-P receptors can also regulate other signaling pathways such as the mitogen- activated protein kinase (MAPK) pathways (Derynck and Zhang,2003). This indicates that that there is crosstalk between these two pathways. Several high-affinity binding proteins anlagonize BMP signaling, including follistatin, noggin, chordin/SOG, and members of the DAN family, including DAN, cerberus, and gremlin (Balemans and Van
Ht:J,2002; Moser et a1., 2003). Their primary mode of inhibition occurs by binding to the ligands which causes inhibition of the ligand bioactivity and prevents association with the receptor complex. Follistatin binds activin with high affinity and also inhibits the biological activities of BMP-i5 by forming an inactive complex (Glister et al., 2004;
Otsuka et al., 2001a).
18 Chapter 1. Literature review
BMP pathway TGFp/activin pathway
Rlt RI RI
Plasma membranc
SMAD 2
SM/\D 5 SMAD 3
SMATI B
F6., 11:'lnì-i*.",
Cytoplasm
SI\] ALì 8 SMAD 3
---É--- Nucleus \
SMAD B
Rcgulation of target genes
Figure 7. Simplif,red schematic of regulation of TGF-B superfamily signaling. The TGF- B ligand binds to it receptor complex, the type-I receptor and type-Il receptor, which phosphorylate receptor regulated SMAD. SMAD 2,3 is phosphorylated by activirV TGF- B, whereas SMADI, 5, 8 are phosphorylated by BMPs. These activated SMADs then form a complex with a common SMAD (SMAD 4) that is capable of entering the nucleus to interact with various transcription factors to regulate their target genes. Adapted and modified from (Piek et al.,1999).
t9 Chapter l. Literature review
1.3.2 Trønsþrming growth factor-fi
Expression and secretion of TGF-P by ovarian cell types suggests that TGF-p may function as an autocrine and paracrine regulator of ovarian function. Three isoforms of
TGF-Ê; TGF-81, TGF-P2 and TGF-83 have been identified in the mammalian ovary.
TGF-P enhance proliferation of less differentiated GCs from immature rat follicles
(Dorrington et a1., 1988) and pre-antral hamster follicles (Roy, 1993). The localization of
TGF-P1 immunoreactivity in the oocyte and production of TGFP-2 by GCs suggests a role for TGF-Bs in oocyte development (Shull and Doetschman, 1994). Vanderhyden et al, (2003) reported that TGF-BI promoted CC oestradiol production. Furtheffnore, studies by Salustri et al, (1990a) and Vanderhyden et al. (2003) demonstrated that TGF-
B1 mimiced the CEEF secretedby mouse oocytes invitro by enabling expansion of the
CCs. However, Gilchrist et al. (2003) did not detect the secretion of TGF-p1 or TGF-B2 by bovine oocytes and using TGF-P neutralising antibodies demonstrated that neither
TGF-Pl nor TGF-B2 could account for the actions of bovine oocytes on GC proliferation.
1.3.3 Activins and Inhibins
Activins and inhibins are disulphide-linked dimeric glycoproteins. Inhibins are heterodimers of a c¿ subunit linked to either a BA or BB subunit to generate inhibin A or inhibin B, respectively. Dimerization of B subunits alone gives rise to three isoforms of activin referred to as activin A (pApA), activinB (BBPB) and activinAB (BABB)
(Knight, 2001). To date, four types of ß-subunit genes (BA, PB, BC and BE) have been
20 Chapter L Literature revrew
identified, however only activin A, activin B and activin AB are biologically active and important to reproductive function (Drummond, 2005; Harison et al., 2005).
Activins and inhibins are produced by GCs and the pattern of expression of activin/inhibin subunits changes during folliculogenesis (Sidis et al., 1998). GCs express inhibin/activin BB subunit from the primary stage, inhibin cr subunit from the secondary stage and BA subunit from the early antral stage (McNatly el a1., 2000). Studies in vitro in a range of species support a role for activin in the regulation of GC steriodogenesis, proliferation and differentiation, although the nature of this role appears to vary with the stage of follicular development. Activin-induced proliferation has been observed with cultured rat GCs from both small and large follicles (Li et a1., 1995). Moreover, Findlay,
(1993) reported that activin promoted differentiation of GCs during the preantral and early antral stages of folliculogenesis and prevented premature luteinization in the later stage of antral follicle development.
In knockout mice lacking activin type IIB receptors, follicle development was arrested at an early antral stage, consistent with a key role for activin in GC proliferation and differentiation (Nishimori and Matzuk, 1996). Moreover, mice deficient in inhibin cr, exhibit an increase in serum FSH, hence supporting the role of inhibin in the regulation of FSH production (Matzuk eT al., 7992). Activin may play a role in the regulation of oocyte activity, as oocytes express activin receptors. For instance, activin accelerates in vitro meiotic maturation of oocytes in monkeys (Alak el al., 1996), rats (Sadatsuki et al.,
1993) and humans (Alak et al., i998). Gilchrist et al. (2006) recently illustrated that
2I Chapter l. Literature revrew activin does not account for the mouse oocyte-secreted mitogenic factor(s). Previous studies demonstrated the development dependent effect of activin on GCs steroidogenesis, such that aromatase activity is enhanced, p450 activity is inhibited, and progesterone production is suppressed as follicular maturation progresses (Miro and
Hillier, 1996).
Inhibin signals act in a paracrine and endocrine manner (Findlay, 1993). Inhibin suppresses FSH secretion from the anterior pituitary for release to the circulation
(Findlay, 1993). A paracrine action of inhibin is demonstrated through positively regulating LH-induced androgen production by theca cells (Knight and Glister, 2001).
Although evidence implicating inhibin as a potential modulator of oocyte maturation is less consistent, free inhibin cr, subunit but not inhibin A, was shown to reduce oocyte developmental competence in cumulus-enclosed bovine oocytes (Silva et at.,1999).
1,3.4 Growth diffirentiøtion factor 9
In 1993, Lee and co-workers discovered GDF-9 and showed that it was synthesised selectively in oocytes, The levels of GDF-9 increase dramatically at the time the oocyte undergoes its growth phase during preantral folliculogenesis, and remain high in oocytes through to ovulation (Aaltonen et a1,, 1999). After fertilization, GDF-9 mRNA decreases and the levels are low or undetectable in preimplantation embryos (Erickson and
Shimasaki, 2000). However, the expression pattern of GDF-9 is species dependent
(Nilsson and Skinner,2002). GDF-9 mRNA and protein are expressed in the oocytes of
22 Chapter L Literature revrew
primary, but not primordial, follicles in mice (McGrath et al., 1995), rats (Jaatinen et a1.,
1999), and humans (Aaltonen et a1., 1999). In contrast, GDF-9 mRNA is expressed in the primordial and subsequent stages of developing follicles in bovine, ovine and marsupial oocytes (Bodensteiner et al., 2000; Eckery et al., 2002).It was originally thought that
GDF-9 expression was found exclusively in the oocyte, however studies have shown that
GDF-9 mRNA is present in human CCs (Sidis et a1., 1998), as well as mRNA and protein can be found in the GCs in the rhesus monkey (Dufff, 2003).
GDF-9 promotes the formation and integrity of the COC by inducing an alternative pathway of GC differentiation from the MGC phenotype. GDF-9 can mimic many of the processes regulated by OSFs, specifically steroidogenesis, growth and differentiation
(Eppig, 2001; Gilchrist et al., 2004a; Vanderhyden et al., 2003). GDF-9 stimulates prostaglandin and progesterone synthesis and/or signaling pathways, in the absence of
FSH, in preowlatory cumulus granulosa cells (Vanderhyden et aI., 1993). In contrast,
GDF-9 suppresses uPA, KL mRNA expression and the luteinization of CCs by inhibiting
LH receptor expression (Elvin et al., 2000a; Joyce et al., 2000). Gilchrist et al. (2004b) showed that although GDF-9 promotes proliferation of MGCs, GDF-9 only accounts for approximately half of the total mitogenic activity of oocytes.
In vitro studies have demonstrated that GDF-9 can substitute for the oocyte in inducing cumulus expansion and promotes expression of several downstream target genes important for CC mucification. Recombinant GDF-9 regulates HAS2, cyclooxygenase-2
(COX2), prostaglandin E2 (PGE2), tumour necrosis factor-cr-stimulated gene-6 (TSG6),
23 Chapter L Literature review
steriodogenic acute regulator (SIAR) and pentraxin-3 (PTX3) mRNA expression in GCs and CCs (Elvin et al., 2000a; Elvin el al., 1999; Varani et a1.,2002). However, Dragovic et al. (2005) demonstrated that multiple members of the TGF-p superfamily, including
GDF-9, account for the critical OSFs regulating mouse cumulus expansion.
1.3.5 Bone morphogenetic protein 15
BMP15, also referred to as GDF9B, was first discovered in mice oocytes in 1998 (Dube et al., 1998). Although the exact function of BMP-15 is still emerging, Otsuka et al.
(2001c) have shown that BMP-15 stimulates proliferation of rat GCs and inhibits the biological action of FSH by suppression of FSH receptor gene expression, but not estradiol production in vitro (Otsuka et al., 2001c). An additional important function of
BMP15 is its ability to inhibit StAR, P450 aromatase, LHR and the inhbin/activin subunit mRNA expression in rat GCs (Otsuka et al., 2001c). Furthermore, BMPl5 stimulates the expression of Kt in rat GCs, unlike GDF9, which inhibits KL expression (Otsuka and
Shimasaki, 2002). BMP-6, unlike BMP-15, does not appear to influence follicular development. Based on knockout mouse models, BMP-6 does not play an essential role in ovarian function (Elvin et a1., 2000b). Likewise, Otsuka et al. (2001b) demonstrated that BMP-6 has no effect on rat GC proliferation but suppresses FSH-stimulated progesterone production without affecting FSH-induced estradiol synthesis.
At the DNA and protein sequence level, GDF-9 and BMP-15 are more homologous to each other than to any other TGF-P superfamily member and form a distinct subgroup
24 Chapter l. Literature revrew
(Dube et a1., 1998). Aaltonen et al. (1999) reported that both GDF-9 and BMP-15 genes are expressed predominantly in oocytes of primary follicles in human ovary and that the expression of the BMP-15 gene begins slightly later and at lower levels than that of
GDF-9 in the human oocyte during follicular development. The presence of transcripts for GDF-9 and BMP-15 at various stages during the course of folliculogenesis is compatible with a model in which GDF-9 and BMP-I5 may act aT all stages of follicle development, with changing roles at different stages (Erickson and Shimasaki, 2000;
Teixeira Filho et a1.,2002). The expression of both GDF-9 and BMP-15 in mice occurs in oocytes of primary follicles and continues throughout ovulation (Dube et al., 1998), while GDF-9 expression in bovine and ovine ovaries is detectable from the primordial stage of follicle development (Bodensteiner et al., 2000). This suggests a potential role for GDF-9 in the initiation of folliculogenesis in domestic ruminants. McNatty et al,
(2003) hypothesized that both follicular growth and ovulation rate are influenced by the dose of BMP-I5 and GDF-9 delivered to the somatic cells of the follicle in mammals with low ovulation rate phenotype (e.g. sheep, human, cattle), whereas in mammals with high ovulation rate phenotype (e.g. rat and mice), the follicular somatic cells are relatively insensitive to the dose of BMP-15 but have an absolute requirement for GDF-9.
Liao et al. (2003) demonstrated that BMP-15 and GDF-9 can form homo and heterodimers and that intercellular interaction of these molecules can affect the processing and secretion of the mature proteins. Moreover, in a recent study canied out by Hashimoto et al. (2005), it was illustrated that fully processed mouse BMPl5 cannot be secreted as an intact homodimer, This finding suggested that mouse oocytes may not
25 Chapter l. Literature revlew
secrete BMP15 as a homodimer, therefore implying that BMP15 in the mouse oocyte may be secreted as a GDFS/BMPI5 heterodimer. Recombinant GDF9 and BMP15 co- operate synergistically to stimulate GC proliferation and to regulate GC inhibin and progesterone production in vitro (McNatty et al., 2005a; McNatty et al., 2005b). The pathways by which BMP-I5 and GDF-9 interact to synergistically affect the development of CCs are currently unknown. A more complete inventory of genes differentially controlled by GDF-9 and the BMP-I5 will be necessary for understanding the mechanisms of BMP-15/GDF-9 signaling.
1.3.6 GDF-| & BMP-I5 deJi.cient animal models
Most of what is known about GDF-9 and BMP-15 is derived from studies conducted in
GDF-9 and BMP-15 deficient female mice and sheep. These studies have identified that both GDF-9 and BMP-I5 are essential factors for female fertility. Deletion of the GDF-9 gene in mice (GDF-9 knockout mice) leads to female infertility as a result of arrested follicle development at the primary follicle stage (Dong et al., 1996). GDF-9J- follicles have abnormal GCs and lack a theca layer, leading to a complete block in folliculogenesis, demonstrating GDF-9 is a critical factor in follicular function. However, male GDF-9-deficient mice had normal testes size and were fertile (Dong el a1.,1996).
Similarly, sheep with natural mutations in the GDF-9 gene or immunised against GDF-9, were also found to be infertile because of a block in folliculogenesis beyond the primary stage of development (Hanrahan et a1.,2004; Juengel eT a1.,2002). Interestingly, sheep heterozygous for GDF-9 exhibited increased ovulation and fertility rates (Hanrahan et al.,
26 Chapter 1. Literature review
2004), in contrast to GDF-9 heterozygous mice, which have normal fertility (Yan et al.,
2001).
In 2001, Yan et al. aimed to define the function of BMP-15 in mice by generating a
BMP-15 knockout mouse model. The BMP-15 null males were normal and had no fertility defects. In contrast, deletion of the BMP-15 gene in female mice had little effect on follicle development; however mice were subfertile, displaying reduced ovulation and fertilisation rates. Furthermore, BMP-15 heterozygous mice have no affect on fertility
(Yan et a1.,200I). Therefore, it appears that BMP-15 is a less critical factor for normal folliculogenesis than GDF-9 in mice.
In contrast to BMP-i5 deficient mice, observations in BMP-15 sheep homozygous show an arrest in follicular development at the primary stage of growth, leading to complete infertility (Galloway et al., 2000; Hanrahan eT al., 2004). In contrast, sheep which are heterozygous show an increase in ovulation rate which leads to multiple pregnancies
(Galloway et a1.,2000; Hanrahan eTa1.,2004). Therefore, both GDF-9 and BMP-15 have a dose-dependent effect on ovulation rate in sheep. Juengel el al. (2002) reported that immunoneuTralization of either GDF-9 or BMP-15 severely interferes with normal follicular development in sheep and affects ovulation rate and/or normal luteal function.
Together these studies in sheep have provided solid evidence for the involvement of
GDF-9 and BMP-15 in folliculogensis and infertility.
27 Chapter l. Literature review
1.3.7 OSFs regulation of COCfunction
During the antral phase and when the follicular antrum is formed, OSFs play a crucial role in regulating granulosa cell differentiation; separating them into two distinct sub- types, CCs and MGCs, which become phenotypically and functionally distinct from each other (Eppig et a1.,1997;Li et a1.,2000). It is now widely recognized that OSFs direct the function of their surrounding CCs, including promotion of cumulus cell growth, proliferation and expansion, and prevention of luteinization by regulating steroidogenesis and inhibin synthesis (Eppig,2001; Gilchrist et a1.,2004a). The above findings support the concept that, via secretion of OSFs, the oocyte actively promotes the CC phenotype, thereby maintaining a highly specialised microenvironment immediately surrounding itself. The capacity of oocytes to secrete these factors and hence regulate COC activity is a function of high quality oocytes.
1.4 Follicular Atresia, Apoptosis and Oocyte Quatity
1.4. 1 Follicular øtresiø
Once the pool of primordial follicles is established, the vast majority undergo atresia
(Tilly, 1996; Tilly et al., 1991). In fact, more than 99% of all ovarian follicles in the cow are destined never to ovulate, but undergo atresia at various stages of follicular development (Jolly et al.,1994).
Morphological characteristics of follicular atresia can be recognised in four different cell compartments (theca cells, GCs, CCs and oocyte) (Kruip and Dieleman,1982; Leibfried
28 Chapter 1. Literature revlew
and First, 1979). Apoptosis is first recognized by scattered pyknotic nuclei in the GC layers (Hirshfield, 1989), detachment of the GC layers from the basement membrane, fragmentation of the basal lamina (Tilly et al., 1991), and the presence of cell debris in the antrum of the follicle (Hirshfield, 1989). In addition, GCs of an atretic follicle have a reduced capacity to synthesise of DNA and protein (Hirshfield, 1989). In contrast to the
GCs, theca cells undergo hypertrophy during the first stage of follicular atresia (Banka and Erickson, 1985). The oocyte on the other hand undergoes meiosis-like changes including germinal vesicle breakdown (GVBD), followed by oocyte fragmentation, and disruption of oocyte-cumulus connections (Tsafriri and Braw, 1984). The morphological characteristics of the atretic follicle have been shown to be due to an apoptotic pathway,
since DNA of an atretic follicle is fragmented into multiple sizes of 185-200 bp (Tilly et
a1., 1991). In antral follicles, the earliest and most prominent features of atresia is death
of GCs, leading to total destruction of the GC layer lining the inner follicle wall (Jolly et
al., 1997). Jolly et al. (1994) demonstrated, using biochemical evidence, that GC death
during ovarian follicular atresia in bovine occurs by apoptosis. However, Van Wezel et
al. (1999) reported for the first time, that GCs within the middle layers of the membrana
granulosa die by apoptosis, in a different pathway from that of GCs close to the antrum,
which undergo death as a result of terminal differentiation.
1.4.2 The cellular mechanism of apoptosis
Apoptosis plays an important role in the loss of oogonia and oocytes throughout
follicular development (Tilly, 1996; Tilly et a1., 1991). At the cellular level, apoptosis is
charac1erized by cytoplasmic and nuclear fragmentation, chtomatin condensation, DNA
29 Chapter l. Literature revlew fragmentation, and phagocytosis. Nuclear changes typical for early stage apoptosis include DNA fragmentation, which can be detected using terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TLINEL) (Makarevich and Markkula,2002).
Apoptosis has been intensively studied in C.elegans. Three of the genes shown to be essential for developmental programmed cell death in C.elegans are ced-3, ced-4, and ced-9 (Hengartner et al., 1992; Yuan and Horvitz, 1990). Inactivation of ced-3 or ced-4 results in survival of cells otherwise destined to be deleted during development. Ced-9, on the other hand, is a survival factor, which inhibits apoptosis in cells that are not destined to die. These first studies in C.elegans have contributed to the finding of mammalian homologues. For example, the mammalian homologues of ced-9, ced-4 and ced-3 have been shown to be members of the Bcl-2 gene family and members of the caspase gene family, respectively.
The Bcl-2 family members have been shown to play a central role in determining whether a cell should be eliminated by apoptosis or not. The members of the Bcl-2 family can be divided into two groups, the death antagonists or antiapoptotic proteins (Bcl-2,
Bcl-xl, Bcl-w, Bfl-1, Brag-1, Mcl-1, A1) and the death agonists or proapoptotic proteins
(Bax, Bak, Bcl-Xs, Bad, Bik, Bid, Hrk, Boo and Egl-l). The majority of this family's members contains a transmembrane domain and is sited in the outer mitochondrial membrane. The Bcl-2 family inhibitors of apoptosis act by heterodimerization with proapoptotic proteins in the mitochondrial membrane (Marcelli et aL, 2000), The antagonists and agonists have been shown to be able to either heterodimerize or
30 Chapter l. Literature revlew
homodimerize with each other, and the net result of this dimerization can result in either apoptosis or the inhibition of the apoptotic stimulus (Decaudin eT al., 1,997). For instance,
Bcl-2 and Bcl-x heterodimerize with Bax. HomodimerizedBax has been shown to form pores in the mitochondrial membrane. Bcl-2 and Bcl-x are able to block this pore formation by inhibiting the homodimerization of Bax (Yang eT al., 1997).
In 1993 , Oltvai et al demonstrated that the ratio of the expression of Bcl-2 (antiapoptotic) to Bax (proapoptotic) is the critical determinant of either cell survival or death.
Expression of Bcl-2 has been detected in human GCs from antral follicles (Fukaya et al.,
1997). Ablation of Bcl-2 expression in transgenic mice is associated with reduced oocytes and primordial follicles (Ratts et al., 1995), whereas overexpression of Bcl-2 results in decreased follicle apoptosis and enhanced folliculogenesis (Yang and
Rajamahendran,2002). In contrast, Bax deficient mice show resistance to apoptosis and a delayed menopause (Knudson et al., 1995); whereas overexpression of Bax results in accelerated follicle apoptosis (Yang and Rajamahendran, 2002).
1.4.3 Cumulus cell apoptosis and oocyte quality
An essential component for establishing a precise balance between cell proliferation, differentiation, and apoptosis is intracellular communication (Kolle eT a1.,2003). It has been shown that disruption of cell to cell contact between oocytes and CCs is followed by reduction of cell proliferation and stimulation of apoptosis in CCs (Luciano et al.,
2000).
31 Chapter l. Literature revlew
Due to the intimate association between the oocyte and the CCs, it is likely that the apoptotic state of the CCs can influence the quality of the oocyte (Lee et al., 2001).
Conversely, the integrity of the CCs can be influenced by the oocyte (Perez and Tilly,
1997).In humans, it has been reported that apoptosis in CCs and GCs is signifrcantly correlated to maturation stage, specifically during the resumption of meiosis and to oocyte morphology (Driancourt and Thuel, 1998; Nakahara et al., 1997). Furthermore,
Host et al. (2002) and Saito et al. (2000) observed that oocytes surrounded by low apoptotic-index cumulus developed into good quality embryos, while failed fertilized oocytes or those that performed poorly had more apoptotic GCs.
The developmental potential of in vitro produced (IVP) embryos is obviously affected by oocyte quality (Makarevich and Markkula, 2002). Furthermore, the incidence of CC apoptosis has been proposed as a useful predictor of subsequent embryo developmental outcomes (Dell'Aquila et aL.,2003; Host et al.,2002; Ikeda et al, 2003; Lee et a1.,200I;
Saito et al., 2000). Paracrine factors secreted by oocytes act as mitogenic factors, affecting CC proliferation and attenuating differentiation. On the other hand, these oocyte factors may act in an antiapoptotic manner, by regulating cellular apoptosis within CCs.
Equally; these paracrine factors may then indirectly influence oocyte developmental competence. Importantly, Elvin et al., 1999 demonstrated that mouse GCs of GDF-9- deflrcient type 3b primary follicles fail to proliferate, but also fail to undergo cell death
(Elvin eT al., 1999). This suggests that GCs require GDF-9 for continued growth and also to become competent to undergo apoptosis. This suggests that a mechanism involved in the regulation of GCs apoptosis appears to involve oocyte paracrine communication.
JZ Chapter L Literature revlew
Currently, there are no studies investigating the influence of GDF-9/BMP-15 on the incidence of apoptosis in CCs.
1.5 Oocyte Maturation
The acquisition of oocyte meiotic and developmental competence is a gradual process that increases with follicular development. The quality of the oocyte is one of the main factors in determining developmental potential of the resulting embryo. The ability of an oocyte to resume meiosis and progress to metaphase II (MII) is not necessarily indicative of either its ability to be fertilized, or its developmental competence (Thibaút, 1972).
Nuclear and cytoplasmic maturation are distinct processes.
1.5.1 Oocyte nuclear and cyloplasmic møturation
Oocyte maturation is a complex process involving both nuclear maturation (the progression of the meiotic cycle) and cytoplasmic maturation, with the endpoint of both being the release of a mature (MII stage) oocyte from the follicle that is competent to support normal embryonic development. Proper cytoplasmic maturation of the developing oocyte increases the developmental potential of the oocyte after-fertilisation
(Bell et a1., 1997). Cytoplasmic maturation involves numerous biochemical and physiological events that have a significant effect on subsequent development (Eppig et a1.,1996; Hunter, 2000; Kastrop et al., l99la; Kastrop et al.,I99Ib). The acquisition of developmental competence is also related to the oragnisation of the cytoplasm at the germinal vesicle (GV) stage (Van Blerkom, 1996). Several distinct cellular phenotypes based on the organisation of the cytoplasm of GV stage, and matured, oocytes tnder in
JJ Chapter 1. Literature revtew
vitro conditi.ons have been determined in a study by Van Blerkom (1991), which predict the oocyte's competence and potential to develop following fertilization (Van Blerkom,
1ee1).
During the oocyte growth phase, the oocyte gradually and sequentially acquires the
capactty to resume meiosis, complete meiosis and undergo activation. During this phase
of oogenesis there is restructuring of the cytoskeleton, and reprogramming of protein
synthesis (Bell et aL, 1997) and post-translational modifications (Dominko and First,
lggi), while proteins, mRNA, and molecular precursors also accumulate in the
cytoplasm. These changes are required to occur prior to activation of the embryonic
genome for fertilisation and embryonic development to proceed normally (Prather et al.,
1937). Since meiotic maturation was first described by Pincus and Enzmann in 1935
(Pincus and Enzmann, 1935), many papers have been published which report that oocytes
aspirated from large antral follicles of many species spontaneously progress through to
the MII stage in vitro, The majority of which readily undergo fertilisation. In bovine
oocytes, COCs derived from follicles >1.8mm undergo spontaneous nuclear maturation,
requiring -24hrs to complete the first meiotic division. However, oocytes from follicles
<1.6mm have not completed their growth phase and consequently have an impaired
ability to complete the first meiotic division (Leibfried-Rutledge el al., 1987). Similarly,
it has been demonstrated that oocytes have a size-related ability to undergo meiotic
maturation, with rates of cleavage and development to blastocysts increasing with oocyte
diameter (Otoi et a1.,7997).
34 Chapter l Literature revtew
1.5.2 The effect of cumulus cells on oocyte developmental competence
CCs suround the mammalian oocyte throughout its development in the ovarian follicle
until ovulation, and in almost all species a certain proportion of these cells are still
present during fertilisation (Familiari et al., 1998). CCs play a very important role during
oocyte growth and maturation. They are known to supply nutrients (Laurincik et a1.,
1992) and messenger molecules which aid oocyte development (Buccione eT al.,l990a;
Thibault et al., 1987) and mediate the effects of hormones on the COC, all of which
contribute to cytoplasmic maturation of the oocyte (Zuelke and Brackett, 1990).
The role played by CCs in determining the fertilizability of mammalian oocytes has been
extensively studied by comparing the fertilization and embryonic development of oocyte
matured in vitro with or without CCs. Schroeder and Eppig (1984) demonstrated that
more cumulus-enclosed mouse oocytes fertilized than oocytes matured under cumulus
free conditions. Similar experiments using bovine oocytes matured and fertilized in vitro
indicated those denuded of CCs were unable to acquire developmental competence
compared with those associated with an intact CC complement (Zhang et al., 1995),
Zhang et al. (1995) also showed that co-culture of bovine zygotes with CC monolayers
enhanced development as compared with those matured in medium alone.
Nuclear maturation, the resumption of meiosis and completion of the first meiotic
division, occurs in vitro for all species of mammalian oocytes studied to date. However,
aberrations in cytoplasmic maturation are more apparent in in vitro matured oocytes than
in vivo matured oocytes, due to incomplete oocyte development (Moor and Seamark,
35 Chapter J. Literature revtew
1986; Trounson et al., 2001). It has been reported that CCs are involved in the cytoplasmic maturation of oocytes allowing for the acquisition of developmental competence (Chian and Sirard, 1995; Vanderhyden and Armstrong, 1989). The morphology of CCs prior to maturation and the extent of cumulus expansion following maturation are good indicators of oocyte maturation and developmental potential (de
Loos et al., 1989). A close correlation between the degree of cumulus expansion and oocyte fertilization rate has also been found in in vitro matured mouse COCs
(D'Alessandris et al,, 2001). One of the most commonly used selection criteria for IVM is the morphology of the COC, in particular the cumulus vestment. Factors such as increased cell layers and degree of compaction are related to improved developmental outcome compared to oocytes surrounded by compromised vestments and denuded oocytes (DOs) (Goud et al., 1998; Lonergan et a1.,1994; Madison et al., 1992), as well as there being a positive relationship between increased CC number in co-culture and developmental competence (Hashimoto et al., 1998).
The atretic status of the follicle has been proposed to have a considerable effect on the
acquisition of developmental competence by the oocyte, often assessed from the morphological appearance of the CCs, as well as the homogeneity of the oocyte
cytoplasm. Under normal conditions, oocytes acquire developmental competence late in
the follicular phase of the oestrous cycle (Blondin et al., 1995). It was commonly
assumed that oocytes derived from healthy, non-atretic follicles would give higher
developmental competence. However, the late onset of the atresiamay allow the oocytes
to retain their developmental competence for a period of time even if the follicular atresia
36 Chapter l. Literature revlew
has already started (Hazeleger et al., 1995). It is also possible that the positive effects of atresia on in vitro developmental potential might be the result of a longer growth period during which the oocyte is preparing itself for maturation, fertilization and development
(de Wit and Kruip, 2001). On the other hand, the developmental competence of the oocyte in vitro will be lower if follicular atresia is too advanced (Blondin and Sirard,
199s).
1.5.3 In vitro & in vivo oocyte møluration outcomes
In vitro, the quality of the large numbers of oocytes obtained from abattoir-derived ovaries is variable, which is reflected in lower developmental competence, as collection of these oocytes does not distinguish between oocytes retrieved from different follicle
sizes or between oocytes retrieved from partially atretic follicles. The proportion of oocytes competent to complete nuclear and cytoplasmic maturation increases during
follicular development, however some oocytes that are able to undergo nuclear maturation to MII are of low developmental potential and fail to develop to the blastocyst
stage. Khatir et al (1996) reported that 90o/o of bovine oocytes progress through meiosis to the MII stage under routine in vitro culture conditions, however most laboratories can
only produce blastocysts from 30-40% of these inseminated oocytes (Lonergan et al.,
1997). The nuclear maturity of an oocyte cannot be used as an indicator of its
developmental competence and the fact that in vivo mafured oocytes fertilised and
cultured in vitro yield twice as many blastocysts as those matured in vitro (Leibfried-
Rutledge et a1., 1987), suggests that suboptimal in vitro oocyte maturation conditions
lead to incomplete oocyte cytoplasmic maturation (Eppig eT al., 1994). The degree of
5t Chapter L Literature revrew
apoptosis in blastocysts derived in vitro has been found not to be dependent on the conditions used for oocyte maturation (Watson et al., 2000). It is therefore likely that the low rates of blastocyst production seen in many IVM/IVF programs are not only the result of poor oocyte quality at the start of IVM, but are also significantly influenced by the constituents of culture media.
1.6 Summary
It is becoming increasingly clear that the oocyte and CCs form a mutually interdependent unit (Hardy et a1., 2000). If intercellular communication between the two cell types is disrupted, each respective unit can no longer adequately function. The successful relationship between the oocyte and CCs is dependent on differentiation of both cell types. It is now widely accepted that oocyte-secreted factors play a crucial role in the control and regulation of granulosa-cumulus cell functions.
Due to the intimate association between the oocyte and the CCs, it is likely that the state of the CCs can influence the quality of the oocyte. Conversely, the integrity of the CCs can be influenced by the oocyte. CCs support oocyte growth and maturation, and are essential for oocyte developmental competence, as removal of CCs from the oocyte prior to maturation or fertilization significantly reduces the percentage of oocytes capable of undergoing maturation, fertilization and blastocyst development.
Apoptosis controls oocyte and follicle cell fates, depending on the expression of "cell death genes" and "cell survival genes". Cells proliferate when the cell survival genes are turned on and the cell death genes are turned off. Conversely, cells undergo apoptosis
38 Chapter L Literature revlew when cell death genes are turned on or overexpressed. Importantly, the incidence of apoptosis in CCs can be used to predict oocyte quality. Paracrine factors secreted by the oocyte maintain the distinctive CC phenotype by regulating a broad range of CC functions. These OSFs may also act in an anti-apoptotic manner, being responsible for the low incidence of cellular apoptosis within CCs. As we continue to discover paracrine factors that coordinate oocyte-cumulus cell development, it will be important to define their roles as determinants not only of ovarian functions, but also of oocyte and
embryonic development.
In vitro embryo production (IVP) involves the in vitro maturation and fertiliza|ion of
oocytes with subsequent in vitro ctlítre of embryos in the laboratory. IVP technology is
of potential significance to the livestock industries, as well as applications in human
assisted reproduction. Oocytes derived from IVM have a lower developmental
competence compared to in vivo matured oocytes, largely due to the poorly compatible
conditions compared To in vivo maturation. This thesis will examine the influence of
OSFs on bovine CC apoptosis and oocyte in vitro maturation. This work will provide a
major new perspective on the importance of oocyte-CC interactions, and has far-reaching
implications for improving the efficiency of oocyte IVM and embryo production in
domestic species and in human infertility treatment, and supports a role for measurement
of OSF production as a potential diagnostic for oocyte quality.
39 Chapter l. Literature revtew
1.7 Hypothesis and Aims for PhD Project
1.7.1 Hypothesis
Based on the literature reviewed above, the following hypothesis is proposed, the capacity of oocytes to secrete paracrine factors and attenuate the incidence of apoptosis in bovine CCs, and hence regulate COC activity, is a function of high quality oocytes and is a determinant of oocyte developmental competence. Therefore, oocytes compromised by IVM would benefit from exposure to additional OSFs, which will allow for the development of improved culture systems for the IVM of bovine oocytes.
1.7.2 Aims
Three main aims are proposed for the project. Ultimately, the goal is to examine the role of OSFs on bovine CC apoptosis and oocyte maturation in vitro.
1 To determine whether the incidence of apoptosis within bovine CCs is regulated by OSFs, and to examine the nature of the paracrine network of oocyte
growth factors and BMPs regulating this process.
2. To determine whether OSFs have a direct influence on oocyte developmental competence during in vitro maturation.
3 To investigate temporal effect of OSFs in the enhancement of oocyte developmental competence.
40 Chapt er 2
Oocytes Prevent Cumulus Cell Apoptosis by
Maintaining a Morphogenic Paracrine Gradient
of Bone Morphogenetic Proteins
Tamer S. Hussein*, David A. Froiland, Fred Amato, Jeremy G. Thompson and
Robert B. Gilchrist
Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology,
The (Jniversity of Adelaide, The Queen Elizabeth Hospital, Australia.
Journal ofCell Science 2005 118, 5257-5268
* Contributions to this work by each author listed in Appendix 6 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic
2.1 Abstract
Paracrine factors secreted by the oocyte regulate a broad range of cumulus cell functions. Characteristically, cumulus cells have a low incidence of apoptosis and we proposed that this is due to oocyte-secreted factors acting in an anti-apoptotic manner. Bovine cumulus-oocyte complexes (COC) were aspirated from abattoir-derived ovaries and oocytectomized (OOX) by microsurgical removal of the oocyte. OOX were treated with doses of either denuded oocytes (DO) or various growth factors for 24h (+/- rFSH; 0.1 IU/ml). Proportions of apoptotic cumulus cells were assessed using TUNEL and laser confocal scanning microscopy followed by image analysis. Quantification of Bcl-2 and Bax proteins in OOX was undertaken by Western analysis. Oocyte removal led to a significant increase in cumulus cell apoptosis compared to COC controls (35% vs. 9% TUNEL +ve, respectively; P<0.001). Levels of OOX apoptosis were significantly reversed (P<0.001) in a dose-dependent manner when co-cultured with oocytes, Furthermore, the anti-apoptotic effect of oocyte-secreted factors followed a gradient from the site of the oocyte(s). Growth differentiation factor 9 (GDF9) had no significant effect on cumulus cell apoptosis, In contrast, cumulus cell apoptosis was significantly (P<0.001) reduced by bone morphogenetic proteins (BMP) 15, 6 or 7. Accordingly, levels of anti-apoptoticBcl-2 were high in OOX + DO and OOX + BMP15 and low with OOX + GDF9 or OOX alone, whereas the reverse was observed for pro-apoptotic Bax. DO, BMP15 and BMP6 were also able to protect cumulus cells from undergoing apoptosis induced by staurosporine, FSH partially prevented apoptosis in all treatment groups (P<0.001). Follistatin and a BMP6 neutralizing antibody, which antagonized the anti-apoptotic effects of BMP15 and BMP6, respectively, whether alone or combined, blocked -50% of the anti-apoptotic actions of oocytes. These results are the first to demonstrate that oocyte-secreted factors, and particularly BMPl5 and BMP6, maintain the low incidence of cumulus cell apoptosis by establishing a localized gradient of bone morphogenetic proteins.
42 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins
2.2Introduction
Mammalian ovarian follicles are highly specialised structures that support the growth
and development of oocytes. Folliculogenesis results from a complex balance between
proliferation, differentiation and cell death, of both the somatic and germ cell
compartments of the follicle, Follicles leave the resting primordial follicle pool and
continue to grow, although only very small numbers of follicles ovulate. Instead, most
ovarian follicles degenerate and die by the process of follicular atresia. In fact, more than
99%o of all ovarian follicles in the cow are destined never to ovulate, but undergo atresia
at various stages of follicular development (Mariana et al., 1991).
In antral bovine follicles, the earliest and most prominent feature of atresia is death of the granulosa cells, eventually leading to total destruction of the granulosa cell layer lining the inner follicle wall (Irving-Rodgers et a1.,2001; Jolly et al, 7994). Jolly et al (1994) demonstrated, using biochemical methods, that granulosa cell death during follicular atresia occurs by the active process of programmed cell death or apoptosis (Jolly et al.,
1994). Van Wezel et al., (1999) reported that granulosa cells within the middle layers of the membrana granulosa undergo apoptosis, whereas granulosa cells closer to the follicular antrum die via an alternative pathway as a result of terminal differentiation. At the cellular level, apoptosis is characterized by cytoplasmic and nuclear fragmentation, chromatin condensation, DNA fragmentation, and phagocytosis (Hardy, 1999). Nuclear changes typical for early stage apoptosis include DNA fragmentation, which can be detected using terminal deoxynucleotidyl transferase biotin-dUTP nick-end labelling
(ruNEL).
43 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins
In the ovary, gonadotrophins and local growth factors have been shown to be regulators of granulosa cell apoptosis. FSH suppresses follicular cell apoptosis in murine pre- and early antral and pre-ovulatory follicles (Chun et a1., 1994; Chun et al., 1,996), and in cultured bovine granulosa cells (Yang and Rajamahendran, 2000). In ovarian antral follicles, there are two major phenotypes of granulosa cells that are anatomically and functionally distinct; mural granulosa cells, which line the wall of the follicle, and cumulus cells, which surround and are in intimate metabolic contact with the oocyte, forming the cumulus-oocyte complex (COC). Apoptosis can be initiated in at least four different cellular compartments of the antral follicle; theca cells, granulosa cells, cumulus cells and in the oocyte itself. The first signs of 'classical'or antral atresia of follicles are degeneration of the mural granulosa cells, which lose their aromatase activity and undergo apoptosis (Irving-Rodgers et al., 2001). Later, the theca cells undergo hypertrophy and their androsterone production decreases (Driancourt et al., 1998). The cumulus cells and the oocyte are only affected in the most advanced stages of follicular atresia (Kruip and Dieleman, 1982; Leibfried and First, 1979). As such, during atresia of antral follicles, it is common for mural granulosa cells to be undergoing apoptosis whilst cumulus cells remain healthy within the same follicle (Yang and Rajamahendran, 2000).
The mechanism by which oocytes and cumulus cells escape apoptosis is entirely unknown.
Cumulus cells surround and communicate with the oocyte via paracrine factors and through gap junctions (Albertini et al., 2001). Disruption of this communication axis reduces cumulus cell proliferation and induces apoptosis in both cell types (Luciano et
44 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morohosenetic Proteins al., 2000). The distinct phenotype of cumulus cells is maintained by oocytes via the secretion of paracrine growth factors that regulate a broad range of cumulus cell functions (Eppig eT al.,1997; Li et al., 2000). Oocyte-secreted factors promote cumulus cell growth, regulate inhibin synthesis while suppressing steroidogenesis and luteinizing hormone receptor expression (see review (Gilchrist eÍ a1.,2004a)). Currently there are no
data available as to whether these oocyte factors may also regulate and maintain the low incidence of cellular apoptosis within cumulus cells.
The mechanisms by which oocytes regulate cumulus cell functions, including the
identities of the oocyte-secreted factors, remain largely unknown. Members of the
transforming growth factor-B GGF-P) superfamily are candidate oocyte-secreted
molecules due to their ability to mimic the actions of an oocyte on cumulus cells and
granulosa cells in vitro (Gilchrist et al., 2004a); in particular growth differentiation
facTor-9 (GDF-9), bone morphogenetic protein-15 (BMP-15), also referred to as GDF-
98, and BMP-6, all three of which are expressed by the oocyte. BMPs have only
recently become recognised as autocrine/paracrine regulators of ovarian follicular
development, even though it is well known BMPs regulate growth and differentiation in a
broad range of other tissues (Shimasaki et a 2004L ). For example, it is known that BMP-
7 inhibits apoptosis in several tissues including eye and kidney, and plays a role in
skeletal patterning (Dudley eI a1.,1995; Luo et a1.,1995). Recent evidence suggests that
BMPs may have a role in regulating ovarian atresia, as a dramatic decrease in the
expression of BMP-4, -7, and -3b is a feature of atresia (Erickson and Shimasaki, 2003).
As oocytes express BMP-15 and BMP-6, these oocyte-secreted factors may play a role in
45 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morphogenetic Proteins preventing follicular atresia. Several binding proteins antagonize BMP actions, including follistatin, noggin and gremlin (Balemans and Van Htú,2002; Canalis et a1., 2003).
Although evidence is so far lacking, it seems plausible that one mode of regulation of the actions of these oocyte factors on cumulus cells may be by BMP antagonists expressed in the follicle such as gremlin and follistatin, as well as by interaction with related follicular
BMPs, such as BMP-4 and BMP-7, utilising common receptor and signalling pathways.
This study was conducted to investigate the nature of cellular interactions within the
ovarian follicle that are responsible for maintaining the distinctive COC microenvironment. We hypothesized that cumulus cells exhibit a low incidence of
apoptosis due to their close association with oocytes and their exposure to oocyte-
secreted factors. Experiments were designed to determine the effect of oocyte-secreted
factors on cumulus cell apoptosis in the presence and absence of FSH, and to examine the
nature of the paracrine network of BMP growth factors and their binding proteins
regulating cumulus cell apoptosis.
2.3 Materials and Methods
Unless otherwise specified, all chemicals and reagents were purchased from Sigma (St
Louis, MO).
2.3.1 Collection of bovine oocytes and culture conditions
46 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morphogenetic Proteins
Bovine ovaries were collected from local abattoirs and transported to the laboratory in warm saline (30-35 "C). COC were aspirated from antral follicles (2 to 8mm diameter) using an 18-gauge needle and a 10-ml syringe containiîg - 2 ml aspiration media
(Hepes-buffered Tissue Cultured Medium-l99; TCM-199, ICN Biochemicals, Irvine,
CA, USA) supplemented with 50 prg/ml kanamycin (Sigma-Aldrish, St.Louis, MO), and
4 mglml fatty acid-free bovine serum albumin (FAF-BSA; ICPbio Ltd, Auckland, NZ).
Intact COC with compact cumulus vestments greater than five cell layers and evenly pigmented cytoplasm were selected under a dissecting microscope and washed twice in
Hepes-buffered TCM-199 and once in corresponding culturing media. Complexes were
cultured with or without 0.1 IU/ml recombinant human FSH (Organon, Netherlands) in pre-equilibrated 50 ¡rl drops of culture media (bicarbonated-buffered TCM-199
supplemented with 0.23 mmol sodium pyruvate l-r and 0.3 mg/ml polyvinyl alcohol)
overlaid with mineral oil and incubated at 39"C with 5o/o COz in humidified air for 24
hours.
2.3.2 Treatment of cumulus cells
2. 3, 2, 1 G ener øtio n of o o cyte cto mized c o mp I exe s
The cytoplasm of each oocyte was microsurgically removed from the COC
(oocytectomy) using a micromanipulator as described in Buccione et al (Buccione et al.,
1990). The resulting oocytectomized complex (OOX) consists of a hollow zona pellucida
surrounded by several layers of intact cumulus cells.
2.3.2.2 Generation of denuded oocytes
47 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Monchosenetic Proteins
Denuded oocytes (DO) were generated by removing cumulus cells from COC by vortexing for - 4 minute in 2 ml H-TCM-199/BSA. Any remaining cumulus cells were removed by repeated passage of the oocytes through a fine-bore fire-polished glass pipette in H-TCM-1 99/BSA.
2,3,2.3 Growth factors and bind,ing proteins
Recombinant mouse GDF-9 and recombinant ovine BMP-15 were produced in-house as previously described (Kaivo-Oja et aL.,2003; McNatty et al., 2005) using transfected 293
human embryonic kidney cell lines (293H), generously donated by Dr Olli Ritvos
(Biomedicum, Helsinki, Finland). Recombinant proteins were partially purified using
hydrophobic interaction chromatography (HIC), as recently described (Hickey et al.,
2005), and their concentrations were then estimated by Western blot (Kaivo-Oja et al.,
2003; McNatty et al., 2005). Control conditioned medium (293H) was also produced
from untransfected 293H cells and purified by HIC. Recombinant human BMP-6,
recombinant human BMP-7, BMP-6 neutralizing antibody, and gremlin were obtained
from R&D systems (Minnaepolis, MN). Follistatin-288 was generously donated by Drs
S. Shimasaki (University of California, San Deigo, USA) and R. Rodgers (The
University of Adelaide, Adelaide, Australia).
2.3.3 Determination of DNA dømage by TUNEL (assessment of cumulus cell
apoptosis)
Cumulus cell apoptotic DNA was detected using TUNEL (Roche Diagnostic, Penzberg,
Germany) according to the manufacturer's instructions. Briefly, following culture COC
48 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morohosenetic Proteins and OOX complexes were washed twice in PBS (pH 7.\ containing 1% BSA, fixed in
4o/o paraformaldehyde in PBS (pH 7.\ overnight aI 4oC and washed twice with
PBS/BSA before placing on Cell-Tak-coated coverslips (Beckton Dickinson Biosciences,
Franklin Lakes, NJ). Complexes \Mere then permeabilized in 0.1% Triton X-100 in 0.lYo sodium citrate for t hour at room temperature and washed 3 times in PBS/BSA. The complexes were then incubated in fluorescein-conjugated dUTP and terminal deoxynucleotide transferase (TIINEL reagents, Roche) for t hour at 37"C in the dark,
Positive controls were incubated in DNAse 1 (0.005 U/¡rl), which cleaves all DNA, for
20 minutes at room temperature and washed fwice in PBS/BSA before TUNEL. Negative
controls were incubated in fluorescein-duTP in the absence of TdT. After TUNEL,
complexes were washed twice in PBS/BSA and counterstained with propidium iodide 0.5
Vglml (PI) plus RNase A (0.1 mg/ml) for t hour at room temperature in the dark to label
all nuclei. Complexes were then washed twice in PBS/BSA and mounted with slight
coverslip compression in VectaShield anti-bleaching solution (Vector Labs, Burlingame,
CA), and stored in the dark at 4oC for confocal analysis.
2.3.4 Confocal microscopy and ønalysis
Apoptosis in COC and OOX was visualised and quantified using confocal microscopy.
Dual fluorescence emission from cumulus cells was detected using a Nikon Cl Confocal
Scanning Head and a Nikon TE2000E microscope (Nikon, Toþo, Japan). Simultaneous
emission capture of the apoptotic signal (fluorescein, laser excitation 488nm, emission
510-53Onm) and the nuclear signal (propidium iodide, excitation laser 532, emission 590-
640nm) was performed.
49 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins
In order to generate an accurate representation of the overall apoptotic incidence for all complexes, the depth of each complex was measured through a Z series to divide the construct into three percentiles (optical Z plane sections) al25Yo, 50o/o and 75Yo. These optical section images were acquired and saved as independent colour channels (green, apoptotic cumulus fluorescence and red, nuclear cumulus fluorescence). The captured images were then processed in Scanalytics IPLab software Version 3.6. (Scanalytics,
Fairfax, VA). Quantification of cumulus cell number (for each colour channel) was independently measured using a macro script utilizing an auto segmentation filter for each optical section percentile (3 optical "z"-plane sections for each complex). A percentage of apoptotic nuclei were generated for each slice and the three percentile values were then averaged to achieve a representation of the total apoptotic nuclei percentage for the whole complex. These processes were repeated separately on each individual complex.
2,3.5 llestern blot ønalysis
Following culture treatments, OOX complexes were lysed in 25 pl RIPA lysis buffer (10 mM Tris lpIJT.41,150 mM NaCl, 1 mM EDTA, 1% Triton X-100) and stored at -80 "C.
Thawed lysates were mixed with 4X loading buffer containing 100 mM Dithiothreitol
(DTT) and subjected to SDS-PAGE (12% polyacrylamide gel). Proteins were subsequently electrotransferred to nitrocellulose membranes (Hybond-ECL, Amersham
Life Science, Ontario, Canada.) in 25 mM Tris - 192 mM glycine containing 20Yo methanol. Blots were blocked in 20 mM Tris (pH 7.6) containing 13.7 mM NaCl, 1%
50 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine of Bone Morohosenetic
Tween-2O, and2%o blocking agent (provided in ECL Advance Kit) for I hr at RT, then incubated overnight with Bcl-2 or Bax rabbit polyclonal antibodies (0.35 pglml; Santa
Cruz Biotechnology, CA, USA) al 4oC, followed by incubation with horseradish peroxidase-conjugated anti-rabbit antibody (1: 200 000; Silenus laboratories, Melbourne,
Australia) and detected using the sensitive Enhanced Chemiluminescence (ECL)
Advance system (Amersham Biosciences, Ontario, Canada). Images were then scanned using a flat bed scanner and the intensity of Bcl-2 and Bax bands in each sample was quantitated by the ImageJ Imaging System Software version 1.3 (lt{ational Institutes of
Health, USA).
2.3.6 Experimental Desígn
2.3.6.1 Experiment I: Effect of oocytectomy on cumulus cell apoptosis
The aim of this experiment was to determine whether intact COC have a different level
of apoptosis to OOX. Groups of 5 COC or OOX were cultured in 50 pl drops of culture
media for 24 h before apoptosis was assessed. Six replicate experiments were performed,
2.3,6.2 Experiment 2: Effect of oocyte-secreted factors on cumulus cell apoptosis
To determine whether oocyte-secreted factors are responsible for the low incidence of
apoptosis in cumulus cells of intact COC, OOX were cultured with increasing numbers of
denuded oocytes and compared to COC. 5,25 or 50 denuded oocytes were added to 50pl
culture drops containing 5 OOX. Three replicate experiments were performed.
5l Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine of Bone Morphogenetic Proteins
2.3.6,3 Experiment 3: Pqttern of apoptosis in relation to proximity to oocyte-secreted
factors origin
The aim of this experiment was to determine the distribution of apoptosis within the cumulus cell complex in relation to the complex's proximity to the oocyte. We quantified the apoptotic incidence in COCs, where the origin of the oocyte-secreted factors is central to the cumulus cell complex, and in contrast, in OOXs co-cultured with denuded oocytes, where the origin of the oocyte-secreted factors is on the outside of the cumulus cell complex. OOXs cultured alone were used as a control, and all complexes were cultured without FSH. Using the confocal microscope, the diameter of complexes was measured after the diameter of the oocyte region was subtracted using Scanalytics IPLab software Version 3.6. This was then divided into 3 equal layers; inner, middle and outer cumulus cell layers, forming 3 ring zones around the oocyte. Each layer was equivalent to aproportion of 33%o of the total radius. The incidence of apoptosis was then analysed independently in each layer.
2.3.6.4 Experiment 4: Dose response of GDF-9, BMP-6 & BMP-|5 on cumulus cell
apoptosis
In an attempt to examine which of the putative oocyte-secreted factors may be contributing to the low incidence of apoptosis observed in COC, OOX were cultured with increasing concentrations of either GDF-9 (0-175 nglml), BMP-6 (0-100 nglml) or BMP-
15 (0-20% v/v), either in the absence or presence of FSH. OOX were also treated with
l0% (vlv) 293H, which served as a parent cell line-conditioned media negative control
52 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morohosenetic Proteins for GDF-9 (equivalent To 132 ng/ml) andl}yo (v/v) BMP-I5. Three replicates of these experiments were performed using 10 OOX per treatment group per replicate experiment.
2.3,6,5 Experiment 5: Effect of oocytes, GDF-| and BMP-15 on CC expression of
Bcl-2 and Bax proteins
This experiment was conducted to confirm that treatment effects on cumulus cell apoptosis, as assessed by TUNEL, are concomitant with changes in expression of key proteins regulating cell death and survival. OOX were cultured for 24 h wtreated, or treated with 132 nglml GDF-9, 10% BMP-15, co-cultured with 35 denuded oocytes / well, or I0% 293H (control conditioned medium), and then subjected to Western blot for analysis of Bcl-2 and Bax expression.
2.3.6.6 Experiment 6: Effect of oocytes, BMP-6, and BMP-I5 on cumulus cell
apoptosis induced by staurosporine
A preliminary experiment was conducted to determine the apoptotic effect of staurosporine on bovine cumulus cells, which induced apoptosis in a dose-dependent manner (range; 0.1-100 ¡rM, data not shown), The aim of this experiment was to determine whether oocyte-secreted factors could prevent cumulus cells from undergoing apoptosis induced by staurosporine. OOX alone or co-cultured with 35 denuded oocytes,
10 nglml BMP-6 or l0%o BMP-15, were then exposed to either 0.1 pM or 1.0 pM staurosporine for the last 6 hours of the 24 hour incubation period. Three replicates of
53 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins this experiment were carried out using 10 OOX per treatment group per replicate experiment.
2.3.6.7 Experiment 7: Effict of BMP antagonists on cumulas cell apoptosis
Follistatin binds to both BMP-15 and activin with high affinity and antagonizes their bioactivify (Lin et a1., 2003; Otsuka et al., 2001). Gremlin is expressed in both mural granulosa cells and cumulus cells and selectively blocks BMP-4 and BMP-7 (Merino et
a1.,1999), and may anTagonize BMP-15. The aim of this experiment was to examine the effectiveness of these antagonists against BMP-6 and BMP-15-prevented cumulus cell apoptosis. OOX were cultured with 10% BMP-15 in the presence of increasing doses of follistatin (0-100 ¡rglml) or in the presence of increasing doses of gremlin (0-40 pglml).
In a separate experiment, OOX were treated with 10 nglml BMP-6 in the absence or presence of a high dose (20 ¡rglml) of a BMP-6 monoclonal neutralizing antibody (NAb).
Three replicates of each of these experiments were carried out using 10 OOX per treatment group per replicate experiment.
2.3.6.8 Experiment 8: Role of BMP-I5 and BMP-6 in the anti-apoptotic øctions of
oocytes on cumulus cells
In an attempt to neutralize the anti-apoptotic bioactivity of oocytes on cumulus cells,
OOX were co-cultured with 25 denuded oocytes, either in the absence or presence of 50
pglml follistatin, 20 pglml BMP-6 NAb, or in the presence of both antagonists. A
separate experiment was conducted to examine any additive effects of BMP-15 and
BMP-6, compared to OOX + oocytes. OOX were treated with denuded oocytes, or with
54 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins l0 ng/ml BMP-6 and/or 10% BMP-15. Three replicates of these experiments were carried out using 10 OOX per treatment group per replicate experiment.
2.3.6.9 Experiment 9: Effect of BMP-7 and its antagonist, gremlin, on cumulus cell
apoptosis
To examine the influence of BMP-7 and its antagonist, gremlin, on cumulus cell apoptosis, OOX were treated with 100 ng/ml BMP-7 and/or 10% BMP-15 in the presence or absence of 2 ¡t"glml gremlin. Three replicates of this experiment were carried out using 10 OOX per treatment group per replicate experiment.
2.3.7 Statistical analysis
Frequencies of cumulus cell apoptosis were analysed by ANOVA using SigmaStat software (SPSS Inc, Chicago, IL), and significant differences between means were determined using Tukey-Kramer post-hoc test for comparison of multiple means. All cell proportional data were arc-sine transformed prior to analysis. Differences were considered statistically significant at p< 0.05.
55 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins
2.4 Results
2.4.1 Experiment 7: Effict of oocytectomy ønd FSH on cumulus cell øpoptosis
TUNEL coupled with confocal scanning microscopy proved a highly effective means of visualising and quantiõring cumulus cell apoptosis. TLINEL positive and negative controls (Figs 1A and 1B; 99Yo and 0% apoptosis, respectively) demonstrated specificity,
COCs exhibited a low incidence of cumulus cell apoptosis (9%; Figure 1C), and removal of the oocyte led to a significant increase lo 35%o in OOXs (p<0.001; Fig lD)' As is expected with bovine cumulus cells, treatment with FSH induced cumulus expansion in both COCs and OOXs (Figs lC and 1D), and significantly decreased the incidence of apoptosis in OOXs (by 10%) and in COCs (by 6%) (p<0.001;Fig 2).
2.4.2 Experiment 2: Effict of oocyte-secretedfactors on cumulus cell øpoptosis
To determine if oocyte paracrine factors are responsible for low COC apoptosis, an attempt was made to reduce the incidence of apoptosis in OOX to COC levels, by co- culturing OOXs with increasing concentrations of denuded oocytes. Cumulus cell apoptosis was significantly reduced (p<0.001), in a dose-dependent manner, by incubating OOXs with increasing numbers of oocytes. Apoptotic levels in OOXs were completely restored to COC levels at the maximum number of oocytes (50 DO/well), whether in the presence or absence of FSH (Fig 2). These results indicate that oocyte- secreted factors prevent apoptosis within cumulus cells.
56 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morphogenetic Proteins
E F
a
Figure 1. Representative images of confocal laser scanning microscopy of DNA fragmentation in cumulus cells, as detected by TUNEL (green label). All cell nuclei are also stained with propidium iodide (red). Positive control DNAse 1-treated OOX showed very strong apoptotic staining (99%) (A), negative control did not reveal any apoptotic signals (0%) indicating specific labelling (B), expanded COC after culture with low apoptotic labelling (9%) (C), compared to OOX with higher apoptotic labelling (35%) (D). Light micrograph of an unexpanded intact COC before culture (E) and a denuded oocyte (F), generated by the mechanical removal of cumulus cells from a COC.
57 Chapter 2, Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morohosenetic Proteins
A
40 . FSH s r-r oox + OOX + DOs .9. U' r coc 9socl o CL b oit ZO to c ¿ =J c c E 10 J C)
0 0 52550 0 n denuded oocytes / 50pl well B + FSH s^40 .2 Ut 9go a o. o CL
ît zO o b tt, J =J E10 c ¿ o c c
0 0 52550 0 n denuded oocytes / 50pl well
Figure 2. A dose response of oocyte-secreted factors on cumulus cell apoptosis in the absence (A) or presence (B) of FSH. Oocytectomized complexes (OOX) were cultured with increasing numbers of denuded oocytes (DO) and at the maximum dose were effective at reducing apoptosis to the control COC levels. FSH also significantly reduced apoptosis in COCs and OOXs. Points represent average percentage of apoptotic cumulus u'b'" cells (mean + SEM). Values from points with different labels differs significantly (p<0.001).
58 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morohosenetic Proteins
2.4.3 Experiment 3: Pa,ttern of apoptosis in relation to proximity to oocyte-secreted
føctor origin
Qualitative observations of confocal images suggested that the apoptotic cells within
COCs were mostly distributed to the outer layer of complexes, whereas apoptosis was observed in the inner cumulus layers when OOXs were co-cultured with denuded oocytes. We therefore hypothesized that oocyte-secreted factors establish an anti- apoptotic morphogenic gradient through the cumulus cell layers. To test this hypothesis, we measured the diameter of COC and OOX complexes and then divided them into 3 layers; inner, middle and outer (Figure 3A). Within COCs, which contain an intact oocyte, the incidence of apoptosis increased significantly (P < 0.026) from the inner layer toward the outer layer (Figs 38 and 3C). Conversely, when OOXs were co-cultured with denuded oocytes, the incidence of apoptosis decreased from the inner layer toward the outer layer, which is closest to the source of oocyte-secreted factors (Figs 38 and 3D). To further illustrate this effect, the inner layer in COC, which is closest to the oocyte and has the lowest incidence of apoptosis, has a 4-fold and significantly (P<0.026) lower incidence of apoptosis, compared to its counterpart inner layer from the OOX+DO group, which has the highest incidence of apoptosis, being the furthest layer from the oocytes
(Fig 3B). To examine the effect of the oocytectomy procedure itself and the associated loss of oocyte-cumulus cell gap junctional communication, apoptosis was analysed in
OOX cultured alone for 24 h. Apoptosis was equally distributed in the 3 layers; inner, middle and outer (49.5,49.9 and45.5%o, respectively; P>0.05).
59 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Proteins
I riü{ER I mnou Il ourER
12 E * Ero "g gBU' gÈL :6 (to þ4 I É E (,t
0 coc oox{{lo
Figure 3. Pattern of apoptosis within cumulus complexes in relation to proximity to oocyte-secreted factor origin. Diameters of unexpanded COCs and OOXs were measured after culture without FSH, using confocal microscopy and divided into 3 layers; inner, middle and outer layers, each layer representing 33% of the radius (A). The incidence of apoptosis was lowest closest to the oocyte, regardless of whether the oocyte was inside the complex ([C], COC inner layer) or oocytes were on the outside of the complex ([D], OOX outer layer), and apoptosis increased with increasing distance from the oocyte (B). Both C and D images are exaggerated examples to pictorially illustrate the morphogenic gradient of apoptosis through the cumulus cell layers. +Treatment X Layers (2-way ANOVA;P:0.026).
60 Chapter 2, Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins
2.4.4 Experiment 4: Dose response of GDF-9, BMP-6 and BMP-15 on cumulus cell
apoptosis
These experiments were conducted to determine the effect of these putative oocyte- secreted factors on the regulation of cumulus cell apoptosis. OOX complexes were treated with increasing doses of GDF-9, BMP-6 and BMP-15. GDF-9 had no significant effect on the incidence of cumulus cell apoptosis in the presence or absence of FSH, as at the highest dose (175 nglml) of GDF-9, apoptosis was not significantly different to the
293H control conditioned medium group (Figs 44, 4B). With an increasing dose of
BMP-6, cumulus cell apoptosis significantly decreased (P<0.001), whether in the presence or absence of FSH (Figs 4C, D). Cumulus cell apoptosis was significantly reduced (p<0.001) in a dose-dependent manner, by treating OOX with an increasing dose of BMP-I5, having maximal effect at 20%BMP-15 (Fig 4E). A similar response was observed when BMP-15 was used in combination with FSH (Fig aÐ. Consistent with experiment 2, FSH had an additive effect in preventing cumulus cell apoptosis, independently decreasing the incidence regardless of growth factor treatment or complex type.
2,4,5 Experiment 5: Effect of oocytes, GDF-9 and BMP-I5 on CC expression of
Bcl-2 qnd Bax proteins
This experiment was conducted to study the pattern of Bcl-2 and Bax expression tn cumulus cells of OOX complexes. Expression of Bcl-2 protein was significantly
(P<0.001) higher in cumulus cells when OOX were co-cultured with denuded oocytes
6I Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morohosenetic Proteins and BMP-15 compared to when untreated or treated with GDF-9 (Fig 5). In contrast, expression of Bax protein was found to be significantly (P<0.001) higher in cumulus cells when OOX were untreated or co-cultured with GDF-9 compared to OOX co- cultured with denuded oocytes or BMP-15, where Bax levels were barely detectable (Fig
5). These results support our TUNEL results; namely that oocytes and BMP-I5, but not
GDF-9, are associated with the prevention of cumulus cell apoptosis, and that oocytes and BMP-15 alter the ratio of Bcl-2 ToBax in favour of cell survival (Oltvai et al., 1993), whereas GDF-9 has no effect on the Bcl-2 to Bax ratio.
2.4.6 Experiment 6: Protection of cumulus cells from staarosporine-induced
apoptosis by oocytes, BMP-6 and BMP-15
The aim of this experiment was to determine whether oocytes are capable of protecting cumulus cells from an apoptosis-inducing event and whether such an effect can by mimicked by BMP-15 and BMP-6. Staurosporine significantly increased (P<0.001) the incidence of cumulus cell apoptosis from 4I%o to 5lo/o and74o/owhen treated with 0.1 and 1.0 pM, respectively (Figure 6). The apoptosis-inducing effects of both doses of staurosporine were completely negated when staurosporine-treated OOXs were co- cultured with denuded oocytes, with apoptosis reduced to COC levels. Also, OOX treated with l0% BMP-15 or l0 nglml BMP-6, exposed to the same 2 doses of staurosporine, showed significantly decreased apoptosis; I7%o and 2l% (0.1 ¡t}ú); 25% and 31% (I
¡rM), respectively (P< 0.001). These results indicate that the anti-apoptotic actions of oocyte-secreted factors can protect cumulus cells from an apoptotic insult and that both
BMP-15 and BMP-6 can mimic this effect.
62 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins
H do A -FS @ B + FSH Lr lrh E:gJH
tr;. ,sO t 50 : oox + OOX+CDF-9 a .- I COC ¿'l(r 10 '¿ hl) = -10 _10 E¿o .]U ? !to t0
o 0 109;2s3lJ 0 ..lJ s8 l::\:l l?.s coc t09f,2cìH 0 -l-{ ,$8 l3':, 175 L-oC Ct)F-q (ng/nìl) CDF-9 (ugtntl)
({l C] tn D _FS H + FSH
5D ,: .1) ¿ : oox + oox+Bt\tP-ó '1, 4J 4tJ e r c--c)c _l _ì, E a) U =:ú Ð = u lfl l0
0 0 0,I I t0 100 coc u 0.l I l0 100 c0c (¡Ulml) ßþtP-6 BÀlP-6 (n/nnlr
fiJ u _FSH 11) [i :2q.ìH + FSH Ì=- oox itu 5{) + oc¡x+llMP-t,5 .t r coc 'r llt {) 7B '' ì0 õ x) U \zo E= ol0= t()
() 0 l0f¡ 29-¡i{ $ 2.5q" -5â, l0r¡i. 2ú',ry" C O C UY¡2t)Íl O 25So ,sri¡ lDry4 2.]Ç¿ CO C BNIP-l -\ lq.vtu) BIüP- L5 t*¡vlrì Figure. 4. Dose response of the putative oocyte-secreted factors; GDF-9, BMP-6, BMP- 15 on cumulus cell apoptosis, OOX were cultured with increasing concentrations of GDF-9 (0-175 nglml), BMP-6 (0-100 ng/ml), and BMP-I5 (0-20% v/v), either in the absence (4, C, E) or presence (8, D, F) of FSH. Cumulus cell apoptosis was unaffected by GDF-9 and attenuated in a dose-dependent manner by BMP-6, but more notably by BMP-15. FSH independently attenuated apoptosis regardless of treatment or complex type. Points represent average percentage of apoptotic cumulus cells (mean + SEM). Vãlues from points with different labelsu'b'" differ significantly (A-B; p<0.001). Asterisks represent significant difference (p<0.001) relative to the control (OOX) for that factor (C- F).
OJ Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Momhosenetic Proteins 1?.t45 fl?,-kÐa
Bax 19-lqËa 4 b
'dl gt g+ t\tl #0 v @ .E {J a¡ .E L b uI Þr 'wk tr t& "3 *lg a Ð !t t L t ?Ð3H rtx GBF-g B[üP-{5 DO
Figure. 5 Effect of denuded oocytes (DO), GDF-9 and BMP-15 on OOX expression of Bcl-2 and Bax proteins as examined by Western blot analysis. Groups of 35 OOX were loaded in each lane after the following treatments: lane I, I1yo vlv 293H (control conditioned medium); lane 2, control (OOX alone); lane 3, I32 nglml GDF-9; lane 4, I0o/o vlv BMP-15; lane 5,0.7 DO/¡rl. Band intensities were quantified by densitometry and are expressed relative to the 293H control, from three replicate experiments (mean * b v-'Bax) SEM). Bars with different superscripts within a group 1u Bcl-2, are significantly different (P<0.001).
64 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine of Bone Mornhosenetic Proteins
coc oox + 80 OOX STS * --+-- OOX+BMP.6+STS o oox+BMP-15+STS s -a- OOX+DO+STS .2 60 U' * o CL o f40CL o * rJ * ,,x Ø :I \ .Y-:-: E20 '..*'-" o t'
0 coG 0 0.1 1 STS (pM)
Figure. 6 Protection of cumulus cells from staurosporine-induced apoptosis by denuded oocytes (DO), BMP-6 and BMP-15. OOX alone or co-cultured with 35 DO, 10 nglml BMP-6, or I0%o v/v BMP-15, were exposed to either 0.1 ¡rM or 1.0 ¡rM staurosporine (STS) in the last 6 hours of incubation, Oocytes, BMP-6 and BMP-15 all prevented staurosporine-induced curnulus cell apoptosis. Asterisks represent OOX means significantly different (p<0.001) relative to the OOX control.
2,4.7 Experiment 7: Effecl of BMP antagonists on cumulus cell øpoptosis
These experiments were conducted to examine whether BMP-15 and BMP-6 antagonists
could neutralize the anti-apoptotic effects of BMP-i5 and BMP-6 on cumulus cell
apoptosis. There was a significant (P<0.001), dose-dependent increase in apoptosis when
BMP-15-treated OOXs were cultured with increasing concentrations of follistatin (Fig
65 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morphogenetic Proteins
7A). OOX complexes cultured with 10 nglml BMP-6 were treated with a high dose (20 pglml) of a BMP-6 neutralizing antibody. Fig 78 illustrates that the BMP-6 antagonist signif,rcantly (P<0.001) neutralized the anti-apoptotic effect of 10 ng/ml BMP-6.
2.4,8 Experiment 8: Rote of BMP-L5 und BMP-6 in the anti-apoptotic actions of
oocytes on cumulus cells
To further determine if the anti-apoptotic effects of oocytes on cumulus cells can be attributed to either BMP-15 and/or BMP-6, an attempt was made to neutralize oocyte- secreted factors using follistatin with and without a BMP-6 NAb. Fig 8A illustrates that
OOX co-cultured with oocytes have a reduced incidence of cumulus cell apoptosis compared to OOX alone, which was comparable to the COC control. Either follistatin alone or the BMP-6 NAb alone significantly anhagonized - 50% of the anti-apoptotic effects of oocytes on cumulus cells (P<0.001). The effects of follistatin and the BMP-6
NAb were not additive as their combined presence did not further restore apoptosis levels.
The results from Figure 8A suggested that oocyte-secreted BMP-15 and BMP-6 act redundantly to prevent cumulus cell apoptosis, and as such, should not act in an additive fashion. An experiment was conducted to test this proposal. Co-culturing OOX with denuded oocytes or treatment with BMP-15 alone or BMP-6 alone significantly
(P<0.001) decreased cumulus cell apoptosis (Fig 8B). Combined treatment of OOXs with BMP-6 and BMP- 15 did not further decrease apoptosis levels beyond that of BMP-
15 alone (P>0.05), suggesting no additive effect of these two BMPs.
66 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhogenetic Proteins
2,4.9 Experiment 9: Elfect of BMP-7 ønd ìts øntagonist, gremlin, on cumulus cell
apoptosís
This experiment was conducted to determine the effect of adding BMP-7 and its antagonist, gremlin, in the presence of BMP-15, on the regulation of cumulus cell apoptosis. BMP-7 and BMP-15 significantly reduced cumulus cell apoptosis (P<0.001;
Fig 9). The anti-apoptotic effects of BMP-15 were unaffected by gremlin, including at high doses (Fig 9A). Conversely,2 p"glml $emlin significantþ (P<0.001) reversed the inhibitory effect of 100 nglml BMP-7, but not when BMP-15 was present (Fig 9B).
67 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins
r coc oox -¡: OOX + BMP-15 A + oox + BMP-15 + Follistatin 40 b ø OOX+ Follistatin s b at, b 'õ so o b CL o <20CL ab oo a Eto.n a E o 0 GOC OOX 0 1 10 100 00x+FS Follistatin (Fg / ml) B 50 I . BMP.6 NAb : + BMP.6 NAb c 8oo c c a 6 o &go CL b o20o o ¿ = Ê a o=10
0 coc oox+BMP-6 00x
Figure. 7 Effect of BMP antagonists on cumulus cell apoptosis. OOX were cultured with l0%ovlv BMP-15 in the presence of increasing doses of follistatin (0-100 pglml) (A), and OOX were cultured with l0 ng/ml BMP-6 in the absence or presence of a high neutralizing dose of 20 p,glml of a BMP-6 neutralizing antibody (NAb) (B). Suppression of cumulus cell apoptosis by BMP-15 was antagonized by follistatin. The NAb effectively antagonized the anti-apoptotic effects of BMP-6. Points and bars represent average percentage of apoptotic cumulus cells (mean + SEM). Values from points with u'o'' different labels differs significantly (p<0.001).
68 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Mon¡hosenetic Proteins
A 50 coc - OOX + DO Øø OOX+DO+FS c s 40 N oox + Do + BMP-6 Nab .9 + + + tt, I oox Do Fs BMP-6 Nab o q oox b 30 o -¡ b CL b o o 20 an a =¿ E 10 a o
0 coc oox+Do oox
B I coc 50 OOX + DO @ OOX + BMP-15 + BMP-6 c \o N oox e40 I ooX+BMP-15+BMP-6 .9 U' : oox o tso ct b
o
0 coc OOX+DO oox
Figure. I Role of BMP-15 and BMP-6 in the anti-apoptotic actions of oocytes on cumulus cells. OOX co-cultured with denuded oocytes (25 DOs) were treated with 50 ¡rg/ml follistatin, 20 p,glml BMP-6 NAb, or a combination of the two (A). Both follistatin and the BMP-6 NAb were effective at partially anlagonizing the anti-apoptotic effects of oocytes, however neither completely restored apoptosis to OOX levels, either alone or combined. Co-culturing OOX with DO or treatment with BMP-15 alone or BMP-6 alone decreased cumulus cell apoptosis (B). Combined treatment of OOXs with BMP-6 and BMP-15 did not further decrease apoptosis levels beyond that of BMP-15 alone, suggesting no additive effect of these two BMPs. Bars represent average percentage of apoptotic cumulus cells (mean + SEM). Values from bars with different u'b'" labels differs significantly (p<0.001).
69 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morphogenetic Proteins
r coc : oox : OOX + BMP-15 40 A + oox + BMP-15 + Gremlin b 8777 oox + Gremlin s b .9 30 to o CL o o- f20 c õ c c o tn J a Elo a of
0 COC OOX 0 0.4 4 40 OOX+GR Gremlin (pg/ml) : oox t- oox + BMP-7 50 B S OOX + BMP.15 Ø oox + BMP-7 + Gremlin a æ oox + BMP-7 + BMP-15 + Gremlin 8oo .9 to o d âgo o. o20o b c, J =¿ o=10
0 oox
Figure. 9 Effect of BMP-7 and its antagonist gremlin on cumulus cell apoptosis. OOX were cultured with I0%o vlv BMP- 15 in the presence of increasing doses of gremlin (0-40 pglml) (A). OOX were also co-cultured with 100 nglml BMP-7 and/or 10 % BMP-I5 in the presence or absence of 2 ¡.t"glml gremlin (B). Gremlin did not antagonize the suppressive effect of BMP-15 on cumulus cell apoptosis, whereas it did that of BMP-7. Bars and points represent average percentage of apoptotic cumulus cells (mean + SEM), a' b' c Values from bars with different labels ¿ ffers significantly (p<0.001).
70 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins
2.5 Discussion
The current study was undertaken to test the hypothesis that ovarian cumulus cells exhibit a low incidence of apoptosis due to their close association with oocytes and their exposure to oocyte paracrine factors. This hypothesis was formulated based on the observation that during atresia in antral follicles, the COC is the last compartment of the ovarian follicle to undergo atresia, which is primarily manifested as apoptosis in the mural granulosa cell layer and, at later stages, in the theca cells (Tajima eI al., 2002;
Yang and Rajamahendran, 2000). The present study demonstrates that removal of the oocyte from the COC by oocytectomy leads to a substantial increase in cumulus cell apoptosis. However, low apoptosis levels can be restored by co-culturing OOX with oocytes, which reduces the incidence of apoptosis in a dose dependent manner, and which is completely restored to COC levels at the maximum concentration of 50 oocytes/well. These findings demonstrate that the low level of CC apoptosis is largely dependent on the presence of the oocyte. Furthermore, the characteristically low incidence of cumulus cell apoptosis can be specifically attributed to soluble paracrine signals from the oocyte, rather than oocyte gap junctional signalling to cumulus cells, since; l) the paracrine effects of oocytes were observed in a co-culture environment devoid of direct oocyte-cumulus cell contact, and 2) disrupting oocyte-cumulus cell gap junctional communication by oocytectomy led to a homogenous increase in apoptosis in all cumulus cell layers, not just in the inner layer, which has the highest junctional contact with the oocyte.
7I Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine t of Bone Momhosenetrc
The study demonstrates by various means that oocytes actively prevent cumulus cell apoptosis by establishing a morphogenic gradient of oocyte-secreted factors. Firstly, the reduction in cumulus cell apoptosis was assessed by two different methods; we used
TUNEL together with quantitative confocal microscopy, and also examined the expression of key proteins regulating apoptosis by Western blot. Exposure of OOX to oocytes dramatically induced anti-apoptotic Bcl-2 expression. Conversely, pro-apoptotic
Bax expression was high in OOX alone and was notably reduced by oocyte-secreted factors. These results suggest that oocytes prevent apoptosis within cumulus cells by altering the ratio of Bax to Bc1-2 in favour of cell survival. Secondly, the anti-apoptotic actions of oocytes followed a gradient from the site of the oocyte(s). In intact COCs, the incidence of apoptosis was lowest in the inner most layer of cumulus cells and increased with increasing distance from the oocyte. Conversely, in OOX f denuded oocytes, where the oocytes are outside the complex and the OOX is hollow, the outer layer of cumulus cells closest to the oocytes had the lowest level of apoptosis. This is the first direct evidence of a very localised morphogenic gradient of oocyte-secreted factors in the COC, which was proposed some time ago (Eppig 2001). This gradient of oocyte-secreted factors is likely to play a significant role in the spatial organisation and functional characteristics of cumulus cells within the COC. Thirdly, oocyte-secreted factors were able to protect cumulus cells from an apoptotic insult. Staurosporine induces apoptosis via a cellular signal cascade (to date uncharacterized) as opposed to causing indiscriminate DNA damage (Weil et al., 1996; Yuan et al., 2004). Oocyte-secreted factors prevented apoptosis induced by staurosporine demonstrating that oocytes are able to protect cumulus cells from an apoptosis-inducing event. Together these results
72 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morohosenetic Proteins demonstrate that oocytes secrete a potent anti-apoptotic factor(s) that acts in a very localised manner.
Supplementation of media with FSH also decreased the incidence of cumulus cell apoptosis in both COCs and OOXs. This is consistent with other studies (Chun et al.,
1996;Yang and Rajamahendran, 2000) and with the notion that FSH is an indispensable hormone driving follicular growth and that the primary cause of follicular atresia is inadequate exposure to FSH. It is also noteworthy that the anti-apoptotic effects of FSH were additive, whether in the presence of oocytes, BMP-15 or BMP-6.
Evidence exists, now from many different groups, of the many roles that oocytes play in regulating ovarian follicle development. Oocyte-secreted factors regulate folliculogenesis by modulating a broad range of functions associated with growth and differentiation of granulosa/cumulus cells (reviewed; (Eppig,200l; Gilchrist et a1.,2004a)). In general, oocyte-secreted factors promote granulosa/cumulus cell proliferation, particularly in synergy with local mitogenic agents such as IGF-I and androgens (Hickey eta1,,2005;Li et al., 2000), and simultaneously prevent gonadotrophin-induced differentiation and luteinisation. Adding to this developing model, this study provides the first direct evidence that oocyte-secreted factors also prevent cumulus cell apoptosis. Hence it seems oocytes help stimulate cumulus cell growth by actively preventing differentiation, whilst simultaneously providing a protective mechanism against cell death and promoting cellular DNA synthesis and proliferation.
73 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morpho senetic Proteins
Despite the dramatic and critical effects of oocyte-secreted factors on cumulus cell functions, the exact identities of these key oocyte molecules remains largely unknown.
To date, the best candidate molecules are members of the TGFP superfamily, in particular GDF-9 and BMP-15, which can mimic many of the actions of oocytes on granulosa/cumulus cells in vitro (Gilchrist et al, 2004a). It is interesting to note that
GDF-9 had no significant effect on the incidence of cumulus cell apoptosis in the present study, despite the fact that GDF-9 is an exceptionally potent granulosa cell mitogen
(Gilchrist et al., 2004b; Hayashi et a1., 1999; Yitt et al., 2002). Instead, cumulus cell apoptosis was markedly reduced by BMP-I5, BMP-6 and BMP-7, which in general are weak mitogens. This study provides multiple lines of evidence that BMP signalling, and not GDF-9 signalling, prevents cumulus cell apoptosis; 1) all three BMPs tested reduced the incidence of cumulus cell apoptosis in a dose-dependent manner, 2) both BMP-6 and
BMP-15 protected cumulus cells from apoptosis induced by staurosporine, 3) expression of cumulus cell anti-apoptotic Bcl-2 was stimulated by BMP-I5 but not by GDF-9, and in contrast, 4) pro-apoptotic Bax expression was inhibited by BMP-15 but not by GDF-9.
These findings support the concept proposed by Oltvai et al. ( 1 993) that the ratio of Bcl-2 to Bax determines whether a cell lives or dies (Oltvai et al., 1993), and that BMP-15 and
BMP-6 can regulate that ratio.
There are two divergent signalling pathways activated by the TGF-B superfamily; the
BMP pathway and the TGFB/activin pathway. GDF-9 was recently shown to signal through the ALK5 (Mazerbourg et al., 2004) and BMPRII (Vitt et a1.,2002) receptors, activating SMAD 213 molecules and hence eliciting a TGF-p-like intracellular response
74 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morohosenetic Proteins
(Kaivo-oja eta1.,2003; Roh et a1.,2003). on the other hand, BMP-15, BMP-6 and BMP-
7 havebeen shown to signal through ALK6 and BMPRII receptors, thereby activating the alternate SMAD 1/5/8 pathway (reviewed by (Shimasaki et a1.,2004)). Hence it seems likely that bovine oocyte-secreted factors stimulate both signalling pathways simultaneously in cumulus cells; activation of the SMAD 1/5/8 pathway by BMP-15 and
BMP-6 transmitting the anti-apoptotic actions of the oocyte, and activation of the alternate SMAD 213 pathway by GDF-9 conveying the oocyte's mitogenic signal.
A further objective of the current study was to attempt to at least partially identi$r the native oocyte-secreted factors preventing cumulus cell apoptosis. This is most easily achieved through experimental netúralization of the effects of oocytes on cumulus cells, as actual purification of oocyte-secreted factors has so far proved unfeasible. Several high-affrnity binding proteins anlagonize BMP actions, including follistatin and gremlin.
Follistatin, which is highly expressed by granulosa cells in developing follicles, inhibits the biological activities of activins and BMP-15 by forming inactive complexes (Otsuka et a1.,2001; Shimasaki et a1.,2004). In the current study, follistatin and a BMP-6 neutralizing antibody were able to antagonize the anti-apoptotic effects of BMP-15 and
BMP-6, respectively. Whereas gremlin, which is expressed in granulosa cells and cumulus cells and is a known BMP-2, BMP-4 and BMP-7 antagonist (Sudo eT a1.,2004), did not anlagonize the anti-apoptotic actions of BMP-15'
We next went on to examine the capacity of the BMP antagonists to neutralise the anti-
apoptotic effects of the oocyte. Follistatin or the BMP-6 neutralizing antibody alone was
75 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morphogenetic Proteins able to partially antagonize the anti-apoptotic actions of the oocyte, suggesting that this action by bovine oocytes can be attributed in part to BMP-15 and/or BMP-6. These findings describe an entirely novel function for these oocyte-secreted molecules. BMP-15 and BMP-6 appear to act redundantly to prevent cumulus cell apoptosis. The recombinant proteins did not have an additive effect on apoptosis when added together, and simultaneous neutralization of native oocyte BMP-15 and BMP-6 did not increase the effect of neutralizing either alone. These data provide the first direct evidence that endogenous BMP-15 and BMP-6 oocyte proteins are important anti-apoptotic oocyte- secreted factors, but also that these molecules account for only part of the total anti- apoptotic activity of the oocyte (-50%), the remaining portion of which is yet to be determined, Candidate molecules may include other members of the TGFp superfamily, however it seems unlikely to include the subfamily of TGFps and activins, as stimulation of their SMAD 213 signalling pathway, as exemplified here by GDF-9, has no effect on cumulus cell apoptosis. A putative oocyte-secreted GDF-9/BMP-15 heterodimer may play some role in regulating cumulus cell apoptosis, however at this stage, little is known of this molecule or of its signalling pathway (Juengel and McNatty, 2005).
BMPR-II is an indispensible receptor for the transmission of the paracrine actions of oocytes to cumulus/granulosa cells, as it is the sole type-Il receptor for BMP-15 and
GDF-9 and an important receptor for BMP-6 (Shimasaki et al., 2004). Efficacy of oocyte-secreted factors on cumulus cells would be reduced if alternate non- oocyte- secreted factors ligands expressed in the follicle that bind BMPR-II, such as BMP-7,
BMP-4 or BMP-2, competed for BMPR-II binding thereby reducing its availability. In
76 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morohosenetic Proteins the present study, we show that BMP-7 can mimic the action of oocyte-secreted BMP-15 or BMP-6 in preventing cumulus cell apoptosis, even though it is not an oocyte-secreted factor and is only expressed by theca in the follicle (Lee et al., 2001). Gremlin is a
BMP-binding protein expressed by granulosa cells in response to GDF-9 and BMP-4 which selectively inhibits certain BMPs without affecting GDF9 (Pangas et a1.,2004).
Gremlin, which is known to be highly effective at antagonizing BMP-2 and BMP-4 actions (Sudo et al., 2004), neutralized the anti-apoptotic effect of BMP-7 but was ineffective against BMP-15 (present study). As such, the anti-apoptotic actions on cumulus cells of the endogenous oocyte-product, BMP-15, were unaffected by the combined presence of BMP-7 and gremlin. Such interactions suggest an additional level of complexity of mechanisms regulating communication between the oocyte and its surrounding cumulus cells.
These results support and extend the model proposed by Pangas et al (2004) whereby gremlin modulates GDF9 and BMP interactions (Pangas eÏ a1.,2004), which \Me propose are crucial for maintenance of the highly specialized COC microenvironment (see model;
Figure 10). This extended model hypothesizes that oocyte-GDF9 induces cumulus cell gremlin expression, which in tum blocks theca and granulosa cell-derived BMPs from competing with BMP-15 and GDF-9 for BMPR-II binding. In addition, follistatin produced by mural granulosa cells and present in follicular fluid, may limit the anti- apoptotic effects of BMP-15 specifically to the COC. Apoptosis and subsequent atresia of the mural granulosa cell layer may be able to proceed despite the apparent presence of
BMP-15 in follicular fluid (McNatty et a1.,2004), due to follistatin neutralisation of
77 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins
BMP-15 at this site. This model illustrates our hypothesis that oocytes actively prevent death of cells in their immediate microenvironment only, by establishing a morphogenic gradient of BMP-15 and BMP-6 emanating from the oocyte. This gradient of BMPs is either sufficiently diluted andlor adequately neutralized outside the COC microenvironment, such that mural granulosa cell apoptosis and follicular atresia can proceed whilst the COC remains relatively healthy. We hypothesize that this is the mechanism whereby, in large mammals, the cumulus cells and oocyte are the last cell types to die during advanced follicular atresia.
Collectively, the evidence presented in this paper demonstrates for the first time that oocytes, in particular the oocyte-secreted factors BMP-6 and BMP-15, are responsible for the low incidence of apoptosis within cumulus cells, through the establishment of a paracrine network of BMP growth factors and their binding proteins, Thus prevention of cumulus cell apoptosis can be added to the growing compendium of follicular cell functions regulated by oocytes. This adds further support to the emerging doctrine that oocytes secrete these paracrine factors to establish and maintain an immediate microenvironment which is distinct from that of the rest of the follicle (Eppig, 2001;
Gilchrist ef. a1.,2004a). Specific knowledge of GDF-9 and BMP interactions and about the spatial and temporal regulation of their native antagonists is required, as regulation of this oocyte-cumulus cell communication network will have physiological implications for oocyte growth and development, oocyte maturation, ovulation, and developmental competence of the ensuing embryo.
78 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morphogenetic Proteins
Theca
ranulosa Cumulus
{
Follicular Fluid
Figure. 10 Proposed model by which the paracrine network of BMP/GDF-9 growth factors and their binding proteins interact to regulate apoptosis in the COC microenvironment. Oocyte-secreted BMP-15 and BMP-6 signal through the cumulus cell receptor BMPR-II to actively prevent cumulus cell death. Oocyte-secreted GDF-9, also acting through BMPR-II but using a different type-I receptor to the BMPs, does not prevent cumulus cell apoptosis but induces cumulus cell gremlin expression. GDF-9- stimulated gremlin expression may in turn block theca and granulosa cell-derived BMPs from competing with BMP-15, BMP-6 and GDF-9 for BMPR-II binding. In addition, follistatin produced by mural granulosa cells and present in follicular fluid, may limit the anti-apoptotic effects of oocyte BMP-15 specifically to the COC microenvironment.
79 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Momhosenetic Proteins
2.6 Acknowledgements
TS Hussein is supported in part by the Faculty of Health Sciences, University of
Adelaide. RB Gilchrist is the recipient of the University of Adelaide's FTT Fricker
Medical Research Associateship. This project was supported by a National Health and
Medical Research Council (NHMRC) Program Grant (250306) and the Research Centre for Reproductive Health, University of Adelaide. Follistatin was generously donated by
Drs S, Shimasaki and R. Rodgers. The authors would like to thank Dr. Olli Ritvos
(University of Helsinki) for generously donating the GDF-9 and BMP-15-expressing cell lines. The authors would like to thank Samantha Schulz, Chris Kraft, Karen Kind, Lesley
Ritter and Alexandra Harvey for helpful technical and editorial suggestions
80 Chapter 2. Oocytes Prevent Cumulus Cell Apoptosis by Maintaining a Morphogenic Paracrine Gradient of Bone Morphogenetic Proteins
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86 Chapter 3
Oocyte-secreted Factors Enhance Oocyte
Developmental C omp etence
Tamer S. Hussein, Jeremy G. Thompson and Robert B. Gilchrist
Research Centrefor Reproductive Health, Department of Obstetrics and Gynaecology,
The (Jniversity of Adelaide, The Queen Elizabeth Hospital, Australia.
Developmental Biolo gy 29 6 (2006), 514-521
* Contributions to this work by each author listed in Appendix 7 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
3.1 Abstract
The capacity of fully-grown oocytes to regulate their own microenvironment by paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) may in turn contribute to oocyte developmental competence. Here we investigated if OSFs have a direct influence on oocyte developmental competence during in vitro maturation (I\lM). Bovine cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries and matured in serum-free medium. COCs were either co-cultured with denuded oocytes (DOs) or treated with specific OSFs: recombinant bone morphogenetic protein-15 (BMP- 15) and/or growth differentiation factor-9 (GDF-9). Following maturation, oocytes were fertilized and embryos cultured in vitro and blastocyst development and cell number were assessed on Day 8. Co-culturing intact COCs with DOs did not affect cleavage rate, but increased (P<0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination frorrr 39%o f.o 5lo/o. OSFs also altered blastocyst cell allocation, as co- culture of COCs with DOs significantly increased total and trophectoderm cell numbers, compared to control COCs. BMP-15 alone, GDF-9 alone or the two combined, all (P<0.05) increased the proportion of oocytes that reached the blastocyst stage post insemination from 4IYo (conTrols) to 58yo, 50yo and 55%o, respectively. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which anlagonize BMP-I5 and GDF-9, respectively, neutralized the stimulatory effects of the exogenous growth factors, and impaired the developmental competence of control COCs. These results demonstrate that OSFs, and particularly BMP-15 and GDF-9, enhance oocyte developmental competence, and provide evidence that OSF-regulation of the COC microenvironment is an imporlant determinant of oocyte developmental programming.
88 Chapter 3. Oocyte- Secreted Factors Enhance Oocyte Developmental Competence
3.2 Introduction
The capacity of a mature oocyte to support the very earliest stages of life; fertilization,
preimplantation embryo development and implantation, is termed oocyte developmental
competence and is a measure of intrinsic oocyte quality. Oocytes gradually and
sequentially acquire developmental competence during the course of folliculogenesis, as
the oocyte grows and its companion somatic cells differentiate (Eppi g et al., 1994). Many
factors during follicular development affect oocyte competence, for example; (1) the
origin of the oocyte, where oocytes derived from large follicles are more competent than
those derived from small follicles (Lonergan et al., 1994), (2) follicle health, where
follicle dominance and atresia are associated with developmental competence (Blondin
and Sirard, 1995; Hagemann, 1999), (3) hormonal stimulation of follicle development
clearly improves oocyte competence (Sirard et al., 2006), and (4) communication
between the oocyte and its surrounding cumulus cells (CCs) is necessary for the
development of a competent oocyte (Krisher, 2004). Importantly, oocytes matured in
vitro have a lower developmental competence compared to in vivo matured oocytes, in
part due to inadequacies of in vitro environments to support complete oocyte maturation
(Greve et a|.,1987; Rizos e/ a|.,2002; Sutton et a|.,2003)
Oocytes and their companion somatic CCs maintain a close association from the earliest
stages of follicular development, and this coupling is necessary to maintain oocyte health
and CC development (Buccione et al., 1990a). Furthermore, the oocyte is dependent on
CCs to provide nutrients and regulatory signals which are necessary to promote oocyte
nuclear and cytoplasmic maturation and hence the acquisition of developmental
B9 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
competence (Chian and Sirard, 1995; Ka et al., 1997} Removal of CCs at the beginning of oocyte in vitro maturation (IVM) or shortly before in vitro fertilization is detrimental to oocyte maturation, fertilization and subsequent development (Fatehi et a|.,2002; Fukui and Sakuma, 1980; Zhang et al., 1995).
As follicles grow and the antrum is formed, the oocyte and its secreted paracrine growth factors play a crucial role in regulating granulosa cell differentiation; separating them into two distinct sub-types, CCs and mural granulosa cells, which become phenotypically and functionally distinct from each other (Eppig et al., 1997; Li et a1,,2000).It is now widely recognized that oocyte-secreted factors (OSFs) direct the functions of their surounding CCs, including promotion of cell growth and prevention of death, and prevention of luteinization by regulating steriodogenesis and inhibin synthesis and suppressing lutenizing hormone receptor expression (Eppig, 2001; Gilchrtst et a|,,2004a;
Hussein et a|.,2005).
The identities of the OSFs that regulate granulosa and CC functions are still emerging, however so far is seems clear that growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-ls (BMP-15, also referred to as GDF-9B) are crucial oocyte- derived growth factors, as evidenced by the reproductive defects in animals with mutations in these genes [reviewed (McNatly et al., 2004)] and by the ability of recombinant forms of these growth factors to mimic the actions of the oocyte on granulosa cells and CCs in vitro [reviewed (Gilchrisl et al.,200aa)]. GDF-9 and BMP-
15 belong to the transforming growth factor-p (TGF-P) superfamily, which has two
90 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence divergent intracellular signaling pathways; the BMP pathway utilizing SMAD 1/5/8 mossengers and the TGFp/activin pathway using SMADs 2 and 3. GDF-9 utilizes an unusual combination of receptors and elicits a TGF-B-like intracellular response by activating SMAD 2/3 molecules whereas BMP-15 activates the alternate SMAD 1/5/8 pathway freview by (Shimasaki et a1.,2004)].
Oocytes induce expression of genes in CCs that have been associated with oocyte maturation and subsequent embryo developmental potential. For example, GDF-9 upregulates CC gene expression of hyaluronic acid synthase 2 (HAS2), cyclooxygenase 2
(COX2; PTGS2) and gremlin (GREMI) (Elvin et a1.,1999; Pangas et a1.,2004). OSFs play crucial roles in processes of cumulus expansion and in mice GDF-9 as well as other
OSFs are involved (Buccione et al., 1990b; Dragovic et al., 2005). Oocytes are responsible for maintaining CCs in a non-luteinized state, promoting growth and limiting steroid production (Li et a|.,2000). We have recently shown that OSFs (especially BMP-
15) prevent CC apoptosis by maintaining alocalized gradient of anti-apoptotic factors within the COC (Hussein et a1.,2005). Furthermore, OSFs including GDF-9 regulate CC amino acid and energy substrate uptake and transport to the oocyte (Sugiura and Eppig,
200s)..
The above findings support the concept that, via the secretion of OSFs, the oocyte actively promotes the CC phenotype, thereby maintaining a highly specialized microenvironment immediately surrounding itself. We hypothesized that the capacity of oocytes to secrete these factors and hence regulate COC activity is a function of high
9l Chapter 3. Oocyte- Secreted Factors Enhance Oocyte Developmental Competence
quality oocytes and is a determinant of oocyte developmental competence, and as such, oocytes comprornised by IVM would benefit from exposure to additional OSFs. The aim of this study was to determine if OSFs enhance oocyte developmental competence during
IVM.
3.3 Materials and Methods
Unless otherwise specified, all chemicals and reagents were purchased from Sigma (St
Louis, MO).
3.3.1 Collection of oocytes and culture conditions
Bovine ovaries were collected from local abattoirs and transported to the laboratory in warïn saline (30-35 'C). COCs were aspirated from antral follicles (3 to 8mm diameter) using an l8-gauge needle and a 10-ml syringe containing - 2 ml aspiration medium
(Hepes-buffered Tissue Cultured Medium-l99; TCM-199, ICN Biochemicals, Irvine,
CA, USA) supplemented with 50 pglml kanamycin, 0.5 mM sodium pyruvate, 50 pglml heparin and 4 mglml fatty acid-free bovine serum albumin (FAF-BSA; ICPbio Ltd,
Aukland, NZ). Intact COCs with compact cumulus vestments > -5 cell layers and evenly pigmented cytoplasm were selected under a dissecting microscope and washed twice in
Hepes-buffered TCM-199 and once in Hepes-buffered TCM-199 supplemented with 10% fetal calf serum (FCS) (Invitrogen, Carlsbad, CA). The basic medium for oocyte maturation was Bovine VitroMat (Cook Australia, Eight Mile Plains, Qld, Australia), a medium based on the ionic composition of bovine follicular fluid (Sutton-McDowall e¡ al., 2005). All IVM treatments were supplemented with 0.1 IU/ml FSH (Puregon,
92 Chapter 3. Oocyte- Secreted Factors Enhance Oocyte Developmental Competence
Organon, Oss, Netherlands). Complexes were cultured in pre-equilibrated 300 pl drops overlaid with mineral oil and incubated at 39"C with 5%o COz in humidified air for 24 hour,
3.3.2 Treatment of cumulus-oocyte complexes
3.3.2.1 Generation of denuded oocytes
Denuded oocytes (DO) were generated by removing CCs from COCs by vortexing for -
4 minutes in 2 ml Hepes-buffered TCM-199. Any remaining CCs were removed by repeated passage of the oocytes through a fine-bore fire-polished glass pipette in Hepes- buffered TCM-199.
3.3.2.2 Growth føctors & antagonists
Recombinant mouse GDF-9 and recombinant ovine BMP-15 were produced and partially purified in-house as previously described (Gilchrist et a1.,2004b; Hickey et al., 2005;
Hussein et al.,2005) using transfected2g3 human embryonic kidney cell lines (293H) originally donated by O. Ritvos (University of Helsinki). Control conditioned medium
(hereafter designated '293H') was produced from untransfected293H cells and subjected to the same chromatography procedures as GDFS and BMP15 conditioned media, as described (Hickey et a|,,2005).
SB-43I542, generously donated by GlaxoSmithKline (Stevenage, UK), acts as competitive ATP binding site kinase inhibitor, specifically anïagonizing the activities of
93 Chapter 3. Oocyte- Secreted Factors Enhance Oocyte Developmental Competence
activin receptor-like kinases (ALKs) 4,5 and 7, without affecting the activities of ALKs
I,2,3 or 6 or other cellular kinases (Inman et a1.,2002). Consequently, 3B-431542 potently antagonizes the ALK 4/5 ligands; TGF-p1, the activins and GDF9, without affecting BMP signaling (Inman et a1.,2002; Gilchrist et a1.,2006). We have recently demonstrated that SB-431542 completely antagonizes the growth-promoting actions of native OSFs and GDF-9 on granulosa cells (Gilchrist et a1.,2006). Follistatin-288 was generously donated by S. Shimasaki (University of California San Diego, USA) and we have previously shown that this binding protein antagonizes the bioactivities of native
OSFs and recombinant BMP15 in CCs (Hussein et a1.,2005).
3.3.3 In vitro fertilizstion and embryo culture
In vitro production of embryos was undertaken using defined, serum-free media (Bovine
Vitro series of media, Cook Australia). Frozen semen from a single bull of proven fertility was used in all experiments. Briefly, thawed semen was layered over a discontinuous (45%: 90%) Percoll gradient (Amersham Bioscience) and centrifuged (RT) for 20-25 mins at 700 g. The supematant was removed and the sperm pellet was washed with 500 pl Bovine VitroWash (Cook Australia) and centrifuged for a further 5 minutes aT 200 g. Spermatozoa were resuspended with IVF medium (Bovine VitroFert, Cook
Australia), then added to the fertilization media drops (Bovine VitroFert, supplemented with 0.01 mM heparin, 0.2 mM penicillamine and 0.1 mM hypotaurine) at a final concentration of 1 x 106 spermatozoalml. COCs were inseminated at a density of 10 ¡rl of
IVF medium per COC for 24h, at 39"C in 60/o COz in humidified air.
94 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
CCs were removed by gentle pipetting 23-24 h post insemination and five presumptive zygotes were transferred into 20pl drops of pre-equilibrated Cook Bovine VitroCleave medium (Cook Australia) and cultured under mineral oil at 38.5'C in 7% Oz, 6Yo COz, balance Nz, for five days (day I to day 5)
On Day 5, embryos were transferred in groups of 5-6 to 20 pl drops of pre-equilibrated
Bovine VitroBlast (Cook Australia) at 38.5oC overlaid with mineral oil and cultured to
Day 8. Embryos were assessed for quality atDay 8 according to the definitions presented in the Manual of the International Embryo Transfer Society (Stringfellow, 1998) and were performed independently and blinded by an experienced bovine embryologist.
3. 3. 4 Dilferential staining
Cell counts were perforrned using a modified version of the technique described by
(Fouladi-Nashta et al., 2005). Briefly, expanded/hatched blastocysts were placed into t acid Tyrode's solution to remove lhe zona, followed by a brief wash in 4 mg mL poly- vinyl alcohol (PVA) in phosphate-buffered saline (PBS/PVA). Zona-free embryos were then incubated in 10 mM trinitrobenzene sulfonic acid (TNBS) in PBS/PVA at 4"C for 10 min. Following this, embryos were subsequently incubated with 0,1 mg mL-1 anti- dinitrophenol-BSA antibody (Molecular Probes, Eugene, OR, USA) at 37oC for l0 min.
Following complement-mediated lysis using guinea-pig complement, embryos were washed and incubated in 10 pg ml,-t propidium iodide for 20 min at 37"C (to stain the trophectoderm), followed by 4 pg ml.-r bisbenzimide (Hoechst 33342; Sigma-Aldrich) in
95 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence rc}% ethanol at 4"C overnight (to stain both the inner cell mass (ICM) and trophectoderm). Embryos were then whole mounted in a drop of 80% glycerol in PBS on microscope slides and coverslips were sealed with nail polish. Embryos were then examined under a fluorescence microscope (Olympus, Tokyo, Japan) at 400x equipped with an ultraviolet filter and a digital camera attached to determine total and compartment cell counts where inner cell mass (ICM) nuclei appeared blue and trophectoderm (TE) nuclei stained pink.
3. 3. 5 Experimental design
3.3.5.1 Experiment l: Effect of co-culture of intact COCs with DOs during IVM on
s ub s e q uent dev elop menta I c omp et ence
To determine the effect of native OSFs on oocyte developmental competence, COCs were randomly allocated into 2 lreatmenl groups during IVM; treatment (1), 30 COCs were cultured in a 300 ¡rl drop for 24h,treatment (2),30 COCs were co-cultured from 0 to 24 h with 150 DOs in a 300 pl drop, after which the 30 complexes were removed and fertilized (Fig 1). Treatment 2 yields a ratio of 1 COC to 5 DOs in a 10 pl drop, giving a concentration of 0.5 DO/pl which is within the range required to examine the influence of OSFs (Gilchrist et a1.,2006; Husseinet a1.,2005). After IVM, all complexes were fertilized and the number and quality of blastocyst formation was assessed on day 8. Six replicate experiments were performed.
96 Chapter 3. Oocyte- Secreted Factors Enhance Oocyte Developmental Competence
3.3.5.2 Experiment 2: Effict of BMP-15 ønd/or GDF-9 during IVM on oocyte
developmental comp etence
This experiment was conducted to determine if addition of exogenous recombinant OSFs,
GDF-Ç and/or BMP-15, during IVM improves subsequent oocyte developmental competence. COCs were cultured for 24 h in the base IVM medium described above, with the following additional treatments 1) none (control), 2) I75 ng/ml GDF-9, 3) l0% v/v BMP-15,4) I0% v/v BMP-15 and 175 nglml GDF- 9, and 5) 10% vlv 293H. Aftet
IVM, all complexes were fertilized and blastocyst formation was assessed on day 8. Four replicates of these experiments were performed using 50 COCs per treatment group per replicate experiment.
3.3.5.3 Experiments 3 & 4: Effect of GDF-9 or BMP-|5 antagonists on oocyte
developmental competence
The aim of this experiment was two-fold; (1) to examine the effect of inhibiting the
GDF-9 or BMP-15 secreted by the oocyte within an intact COC on subsequent development, and (2) to specifically neutralize the effects of the recombinant OSFs on
COCs, as these preparations are not pure. COCs were either cultured alone, with 175 nglml GDF-9 or I\Yovlv 293H, either in the presence or absence of 4 pM SB-431542
(GDF-9 antagonist). In a separate experiment, COCs were cultured alone, or with 10% v/v BMP-15 or l0%o vlv 293H, either in the presence or absence of 10 pglml of follistatin-288 during IVM. After IVM, all complexes were fertilized and blastocyst formation was assessed on day 8. Three replicates of these experiments were performed using 60 COC per treatment group per replicate experiment.
97 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
3.3.6 Statistical Analyses
All replicated proportional development data were arc-sine transformed prior to analysis.
Statistical analyses were carried out by ANOVA using SigmaStat software (SPSS Inc,
Chicago, IL), and significant differences between means were determined using Tukey-
Kramer post-hoc test for comparison of multiple means. Differences were considered statistically significant at p< 0.05.
Treatmeil l: frsütmenl â coc ûOG + Dß
ft I 0h
Intd mmplex loocl
2¡ft t{ñ - I fvF tvF
Figure l. Diagrammatic illustration of the experimental design to expose COCs to oocyte-secreted factors (OSFs) during IVM. COCs were cultured either alone or co- cultured with denuded oocytes (COC + DOs) at a concentration of 0.5 DO/pl for the duration of IVM. Oocytes were subsequently fertilized and embryo development was used to assess oocyte developmental competence.
98 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
3.4 Results
3.4.1 Experiment 1: Effect of co-culture of intøct cocs with Dos during IVM on
subseq uent developmental competence
Exposure of intact COCs to native OSFs from DOs significantly increased (P<0.001) the proportion of oocytes that reached the blastocyst stage post-insemination (51%), compared with COCs cultured alone (39%o) (Table 1A). FurtheÍnore, the cell numbers of the ensuing blastocysts was significantly (P<0.05) increased, with more total and trophectoderm cell numbers, compared to control COCs (Table 1B). However, cleavage of oocytes was not significantly affected by exposure to OSFs during IVM (P>0.05;
Table 1A).
3.4.2 Experiment 2: Effect of BMP-15 ønd/or GDF-| during IVM on oocyte
deve lopmental comp etence
Addition of BMP-I5 to maturing COCs dramatically enhanced (P<0.05) their development to the blastocyst stage, by 160/o compared to control COCs ot 30%o compared to 293H-treated COCs (Table 2). Conditioned medium from the parent 293H cell line adversely affected oocyte developmental potential, lowering blastocyst rates by
l4o/o compared to control COCs (P<0.001). GDF-9 also increased (P<0.05) blastocyst yield compared to 293H-treated COCs, but not compared to COCs cultured alone. There was no additive effect on blastocyst yield of GDF-9 above that of BMP-15 alone,
Cleavage of oocytes was not significantly affected by the treatment groups, although rates were notably lower in those exposed to the 293H control conditioned medium.
99 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
Table I
A. Effect of co-culture of intact COCs with DOs during IVM on subsequent oocyte developmental competence.
Number "/o Cleavage %o Blastocysts Treatment of rate from oocytes cleaved coc r82 83.0 + 0.9 38.8 + 0.9" COC + DOs 176 82.5 + 0.7 51.0 + 0.9b u'b Values with different superscripts within the same column represent a statistically significant difference (P<0.001). Values are expressed as (mean + SEM).
B. Number of total, inner cell mass and trophectodetm cells (mean + SEM) of Day 8
expanded and hatched blastocysts following co-culture of COCs with DOs.
Treatment Blastocyst Total cells ICM TE (n) Mean cells Proportion Mean cells Proportion coc 40 148.3 + 1.2^ 50.0 + 1.3" 33.6 + 0.6" gg.2 + 0.8' 66.4 +0.6'
COC+DOs 51 156.1 + 1.3b 49.4 + lll^ 31.5 + 0.5b 105.7 + 1.3b 68.510.5" u'o Values with different superscripts within the same column represent a statistically significant difference (P<0.05).
100 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
Table 2. EffecT of GDF-9 and/or BMP-15 during IVM on oocyte developmental competence.
Number o/o Cleavage 7o Blastocysts Treatment of rate from oocytes cleaved Control 205 86.5 +3.2 41.0 + 0.9u"
GDF-9 191 88.1 + 2.0 49.5 + 3.gub
BMP-15 189 88.7 + 4.2 57.5 +2.4b GDF-9 + BMP-15 184 88.9 +2.9 55.1 + 4,5b 293IJ 187 80.4 +2.2 27.l +3.r" u-' Values with no common superscripts within the same column are significantly different (P<0.05). Values are expressed as (mean + SEM).
3.4.3 Experiments 3 & 4: Effect of GDF-9 or BMP-I5 antagonists on oocyte
developmental co mpelence
The adverse effect of adding2g3H during IVM on cleavage rate observed in the previous experiment 2 was also observed in both of these experiments; the difference from control groups was now significant (P<0.05; Figs 2A and 3A). The GDF-9 antagonist, SB-
431542, which is an ALK 41517 inhlbitor, had no influence on the cleavage rate of the embryo (2-way ANOVA, p>0.05; Fig 2A). In contrast, treatment of COCs with follistatin, regardless of BMP-15 treatment, significantly decreased cleavage rate of embryos (follistatin, 71.8 + 1.3; control , 76.2 + 1.3; p:0,007; Fig 3A). Consistent with experiment 2, treaTment of COCs with GDF-9 did not significantly alter cleavage rates compared to the control, nor did treatment with BMP-15, independent of any effects of follistatin (Figs 2A and 3A).
101 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
Treatment of control COCs with SB-431542 or follistatin significantly (P<0.05) decreased blastocyst development, compared to untreated COCs, suggesting that these
antagonists were able to at least partially neutralize the effect of endogenous GDF-9 or
BMP-15, respectively, that was secreted by the oocyte (Fig. 2B & 3B). Consistent with
experiment 2, exogenous GDF-9 and BMP-15 both increased blastocyst yields (P<0.05)
in these experiments (Figs 28 and 3B), and these increases were ablated by the addition
of their respective antagonists. Addition of SB-431542 or follistatin not only reduced
blastocyst development to levels similar to untreated control COCs, but further depressed
blastocyst rates to the levels of the control COCs treated with antagonists (Figs. 2B &,
3B). This suggests that SB-431542 and follistatin are neutralizing The effects of both
exogenous and endogenous GDF-9 or BMP-15 on developmental competence of the
oocyte. Blastocyst development rates from COCs matured with293H were substantially
reduced (P<0.05), regardless of whether 3B-431542 or follistatin were added (Fig. 28 &
3B).
t02 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
r - sB-431542 100 A r----r + SB-431542 a a
b 80 s *60 É. o 940Et oG o 20
0 Control GDF.9 293H 60 B c
50 r - 58431542 s a b ;40 b G É. d ö30 d o o 820 õ
10
0 Gontrol GDF-9 293H
Figure 2. Effect of treatment of intact COCs with GDF-9 in the presence or absence of the GDF-9 antagonist, 58-431542, d:uring IVM on subsequent cleavage (A) and developmental competence (B). Following IVM, all complexes were fertilized and the cleavage rate was assessed on day 2 and blastocyst formation on day 8.293H is control- conditioned medium from untransfected 293H cells. Bars represent percentages (mean t u-d SEM) and bars or grouped bars within a graphwith different labels differ significantly (p<0.05). Cleavage rate was not affected by SB-431542buf was by 293H.
103 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
A r - Follistatin 100 + Follistatin a a
b I 80 *60 É. o ctt 940 G' (JI 20
0 Control BMP.15 293H
B 60 c r - Follistatin
50 s ;40 fit É. b bd ü'30 d d o o Ë20 m
10
0 Control BMP.15 293H
Figure 3. Effect of treatment of intact COCs with BMP-15 in the presence or absence of follistatin during IVM on subsequent cleavage (A) and developmental competence (B). After IVM, all complexes were fertilized; cleavage rate was assessed on day 2 and blastocyst formation on day 8. 293H is control-conditioned medium from untransfected 293H cells, Bars represent percentages (mean + SEM) and bars within a graph with different labels u-d differ significantly (p<0.05).
104 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
3.5 Discussion
For at least three decades it has been known that the oocyte is dependent on granulosa cells and CCs to provide nutrients and regulatory signals through gap junctions
(Anderson and Albertini,1976; Buccione et a1.,1990a). More recently, a growing body of evidence has shown that paracrine factors secreted from the oocyte potently regulate follicular somatic cell functions, including proliferation, steroidogenesis, differentiation, apoptosis and expansion (Eppig, 2001; Gilchrist et a|.,2004a; Hussein et a\.,2005). We now provide evidence that oocyte-secreted factors enhance oocyte developmental competence during in vitro maturation, whether in their native form as aÍt uncharacterized mix of growth factors secreted by the oocyte, or as recombinant exogenous BMP-I5 and GDF-9 (Figure 4). Our data demonstrates that not only do OSFs regulate development of their neighbouring somatic cells, but also in doing so, the oocyte, through the secretion of paracrine signals within the COC, contributes to the very processes of oocyte maturation and the acquisition of its own developmental competence during in vitro maturation.
The capacity of an oocyte to support fertilization and preimplantation embryo development, termed oocyte developmental competence, is acquired during folliculogenesis. While oocyte meiotic competence is acquired during early folliculogenesis, developmental competence is acquired first during the antral phase, as the oocyte approaches ovulation (Eppig et al., 1994). During this latter phase of
development, the oocyte maintains intimate contact with, and is reliant on, its CCs, and is
relatively isolated from the rest of the follicle. The COC provides a highly specialized
105 Chapter 3. Oocyte-S ecreted Factors Enhance Oocyte Developmental Competence microenvironment and the CCs are functionally distinct from the remaining granulosa cells in the follicle - features that are dependent on paracrine signals from the oocyte
(Eppig et a\.,1997;Li et al.,2000). We have previously hypothesized that the process of maintenance of the specialized COC microenvironment by OSFs may be required for appropriate programming of the oocyte cytoplasm to support early development
(Gilchrist et a1.,2004a). The findings from the current study provide strong support for this hypothesis, especially during in vitro maturation, where the culture conditions are most likely to be sub-optimal for CC function in comparison to the follicular environment. Hence, the capability of individual oocytes to secrete paracrine factors, presumably to appropriately direct CC functions, appears to be a determinant of oocyte developmental competence.
It is not yet clear which particular functions of CCs regulated by OSFs impart developmental competence on the oocyte, even though it is now widely accepted that the characteristic phenotype of CCs is under OSF control (Eppig, 2001; GilchrisT et al.,
2004a). Diffusible paracrine factors produced by oocytes promote the expression of an array of CC genes and regulate a broad range of CC functions. For example, CC gene expression of hyaluronic acid synthase 2 (HAS2), cyclooxygenase 2 (COX2; PTGS2) and gremlin (GREMl), which are regulated by GDF-9 (Elvin et a1.,1999;Pangas et al',
2004), have been correlated to developmental competence of human oocytes (McKenzie et al., 2004). Mucification and expansion of CCs are essential for ovulation, sperm capacitation and ferlllization (Tanghe et al., 2003), and this feature of CCs that distinguishes them from mural granulosa cells is under OSF control (Buccione et al.,
106 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
1990b; Dragovic et a1.,2005). Furthermore, the incidence of CC apoptosis has been proposed as a useful predictor of subsequent embryo developmental outcomes (Host et
al., 2002; Ikeda et al., 2003; Lee et al., 200I). These CC processes are regulated by
oocyte-derived paracrine factors (Dragovic et a1.,2005; Elvin et a1.,1999; Eppig, 2001;
Hussein et al., 2005; Pangas et al., 2004). Recently, it has been demonstrated that
glycolysis and amino acid uptake in CCs and transfer of these substrates to the oocyte are
regulated by OSFs (Eppig et a1.,2005; Sugiura and Eppig, 2005). Thus, the oocyte is
dependent on CCs for the supply of many small regulatory molecules, such as cAMP,
and energy substrates, in particular pyruvate, such that CCs nurture the oocyte towards
developmental competence (Sutton et a1.,2003). This may be an important mechanism
by which OSFs improve oocyte developmental competence.
The present study demonstrates that treating intact COCs with OSFs during IVM
substantially increases the proportion of oocytes reaching the blastocyst stage of
development. In a bovine serum-free model, the blastocyst rate from IVM of a mixed
pool of oocytes collected from 3mm to 8mm follicles is generally limited to about 30-
40% (Rizos et al., 2002). In the current study, a blastocyst rate of -60% was achieved
using serum-free defined medium, simply by the addition of naturally occuring OSFs,
which is a substantial increase to levels similar to that achieved using in vivo matured
oocytes (Rizos et a|.,2002). Nevertheless, we have yet to determine if oocytes from any
particular sub class of follicles are particularly affected by the addition of OSFs.
107 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
In addition to increasing blastocyst yield, exposure of COCs to OSFs also altered the characteristics of resulting blastocysts, by increasing the total cell numbers. Increased total cell numbers in mouse embryos is associated with improved embryo quality and post-implantation developmental potential (Lane and Gardner, 1997). In the current study, the observed increase in total cell numbers was attributed to an increase in trophectoderm cell numbers, with no change to the ICM. Although improved levels of post-implantation development is associated with increased ICM cell numbers rather than trophectoderm cell numbers in the mouse (Lane and Gardner, 1997),little is known of
'When the impact of such alterations in bovine embryos. Koo et al. (2002) compared the total, trophectoderm and ICM cell numbers of in vivo to in vitro derived blastocysts, and to blastocysts derived from somatic cell nuclear transfer, the ICM cell number did not vary significantly (approximately 42-49 cells for each type of blastocyst). In contrast, trophectoderm cell numbers varied from a mean of 80.5 for in vivo blastocysts (highest developmental potential), To 62.4 cells for in vitro derived blastocysts, and 49.5 cells for nuclear transfer blastocysts (lowest developmental potential). Hence, while the increased total and trophectoderm cell numbers of blastocysts derived from COCs exposured to
OSFs could reflect improved embryo quality, embryo transfer experiments are required to verify this.
Since the beneficial effects of denuded oocytes on oocyte developmental competence were observed in a co-culture system that does not require physical contact between the cells, the active substances are most likely soluble paracrine signals secreted by the oocyte acting on CCs. Furthermore, the improvement was achieved with OSFs either in a
108 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
native form as an uncharacterized mix of growth factors secreted from the oocyte or as recombinant GDF-9 or BMP-15. Recent important studies have demonstrated the critical involvement of GDF-9 and BMP-15 in the regulation of fertility through their roles in regulating early follicle growth and ovulation rate freviewed; (McNatty et al., 2004)).
Moreover, the current study shows that GDF-9 and BMP-15 play an important role in regulating oocyte developmental programming and sheds new light on the importance of oocyte-CC interactions during the final stages of oocyte development (Figure 4).
It is interesting to note that both BMP-15 and GDF-9 increased oocyte developmental competence independently, despite the fact that they elicit different intracellular responses (Shimasaki et a1.,2004). This suggests that both the SMAD ll5l8 paThway
(activated by BMP-15) and the SMAD2/3 pathway (activated by GDF-9) in CCs are in someway involved in regulating developmental competence of the oocyte. We recently demonstrated that OSFs prevent CC apoptosis and that this is mediated by BMP, not
GDF-9, signaling (Hussein et a1.,2005). As both GDF-9 and BMP-I5 are required for fertility in sheep (McNatty et a1.,2004), bovine oocytes probably activate both TGF-B superfamily signaling pathways in CCs, in contrast to mice, where murine oocytes predominately activate SMAD2/3 (Gilchrist et al., 2006), consistent with BMP-15 playing a less critical role in this species (Yan et a|.,2001). BMP-15 and GDF-9 are also known to interact synergistically to regulate some granulosa cell functions (McNatty el al., 2005a; McNatty et a1.,2005b), although in the current study, combined treatment with BMP-15 and GDF-9 did not further increase developmental competence of oocytes beyond that of BMP-15 alone. However, this may be due to a doubling of the intrinsic
109 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
inhibitory factors produced by the parent cell line present in the 293H conditioned media.
These growth factors are prepared in a partially purified form (Hickey et a|.,2005) and it is noteworthy that conditioned medium from untransfected 293H cells had a marked inhibitory effect on oocyte developmental competence, suggesting that purified GDF-9 and/or BMP- 15 may even further enhance oocyte quality.
Due to the impurity of GDF-9 and BMP-15 preparations used in this study, a further objective was to confirm that the improvement in oocyte developmental competence was due specifically to GDF-9 or BMP-15. This was achieved in GDF-9 and BMP-15 neutralization experiments. Follistatin is an activin and BMP-15 binding protein and neutralizes their bioactivities (Hussein et a|.,2005; Otsuka et al., 200I). SB-431542 is a kinase inhibitor that antagonizes the biological activities of TGF-p1, activin and GDF-9, but has no effect on the BMPs (Gilchrist et a1.,2006; Inman et a1.,2002). 3B-431542 and follistatin ablated the positive effects of exogenous GDF-9 and BMP-15, respectively, on oocyte developmental competence. Interestingly, when added to untreated COCs, these antagonists also decreased oocyte developmental potential, possibly by antagonizing the positive effects of endogenous GDF-9 and BMP-15 in the intact COC. This is consistent with a previous study where addition of follistatin had an adverse effect on blastocyst yield (Silva and Knight, 1998), and provides further evidence that oocyte secretion of
BMP-15 enhances the developmental program of the oocyte.
Collectively, the evidence presented in this study demonstrates, for the first time, that oocytes, in particular the oocyte-secreted factors BMP-15 and GDF-9, enhance oocyte
110 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
developmental competence during in vitro maturation by their known effects on CC function. These results have signif,rcant implications for improving the efficiency of oocyte maturation technologies, and support the role of OSF production by oocytes as a diagnostic marker for developmental competence.
coc fi 40% developmental competence
æ
COC + OSFs
native OSFs
OR 60% developmental exogenous competence GDF9 BMP15
Figure 4. Diagrammatic illustration of the hypothetical model derived from this study. Exposure of COCs during oocyte maturation to oocyte-secreted factors (OSFs), whether in their native form as an uncharacterized mix of growth factors secreted by the oocyte or as exogenous recombinant BMP-15 or GDF-9, substantially improves subsequent oocyte developmental competence (from -40% to -60%). OSFs are known to regulate a multitude of cumulus cell functions and this model proposes that these may include positive regulatory factors that pass back to the oocyte (bold arrows), improving subsequent development.
111 Chapter 3. Oocyte- Secreted Factors Enhance Oocyte Developmental Competence
3.6 Acknowledgements
TS Hussein is supported in part by the Faculty of Health Sciences, University of
Adelaide. This project was supported by a National Health and Medical Research
Council (NHMRC) Program Grant (250306) and the Research Centre for Reproductive
Health, University of Adelaide. The authors would like to thank Dr. Olli Ritvos
(University of Helsinki) for generously donating the GDF-9 and BMP-15-expressing cell lines. The authors would like to thank Samantha Schulz, Fred Amato, Karen Kind,
Lesley Ritter, Alexandra Harvey and Darryl Russell for helpful technical and editorial suggestions.
tt2 Chapter 3. Oocyte-Secreted Factors Enhance Oocyte Developmental Competence
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It9 Chapt er 4
Temporal Effects of Oocyte-Secreted Factor(s)
During In Vitro Maturation on Bovine Oocyte
Developmental Competence
Tamer S. Hussein, Robert B. Gilchrist and Jeremy G. Thompson
Research Centre for Reproductive Health, Deparlment of Obstetrics and Gynaecology,
The University of Adelaide, The Queen Elizabeth Hospital, Australia.
To be submitted to Biology of Reproduction, December 2006 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovine Oocyte Developmental Competence
4.1 Abstract
Communication between the oocyte and its companion cumulus cells (CCs) is crucial to establish and maintain a highly specialised cumulus-oocyte complex microenvironment. Here we investigated the temporal effect of oocyte-secreted factors (OSF) in enhancement of oocyte developmental competence. Bovine cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries and matured in serum-free medium. COCs were either co-cultured with denuded oocytes (DOs) or treated with specific oocyte-secreted factors, (OSFs); recombinant bone morphogenetic protein-15 (BMP-15) or growth differentiation factor 9 (GDF-9) beginning from either 0 or th of ìn vitro maturation (IVM). To generate the th DO group, COCs were cultured intact for the first th of IVM then denuded. Following maturation, embryos were fertilized and cultured in vitro and blastocyst development and cell number were assessed on Day 8. Co-culturing intact COCs with DOs from 0 or from t hours did not affect cleavage rate, but increased (P<0.05) the number of oocytes that had reached the blastocyst stage on day 8 (51% and
61ol0, respectively), compared with COCs cultured alone (4I%o). Maturing oocytes with intact cumulus for the first t hours prior to denuding, and then co-culture with COCs, significantly (P<0.05) improved the blastocyst rate from l3%o (DOs, control) to 25%o. OSFs also improved embryo quality, as co-culture of COCs with DOs from 0 or t hour of IVM (P<0.05) increased blastocyst total and trophectoderm cell numbers, compared to blastocysts developed from control COCs. Addition of GDF-9 or BMP-I5 at 0 hour to rnafuring COCs enhanced (P<0.05) their development to the blastocyst stage from 40% (controls) Io 5l%o and 47o/o, respectively. However, treatment of COCs with GDF-9 or BMP-15 from t hour of IVM did not significantly increase blastocyst yields compared to the control. These results provide evidence that the temporal interaction between paracrine factors secreted by the oocyte and the loss of gap-junction communication is a vital determinant of oocyte developmental competence.
t2r Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence
4.2 Introduction
From the early stages of follicular growth, intercellular communications between all cell types within the ovarian follicle is important for the acquisition of oocyte developmental competence, which involves the ability of the oocyte to produce a normal and viable embryo after fertihzation (de Loos et al., 1991). During the antral phase and when the follicular antrum is forrned, oocyte-secreted factors (OSFs) play a crucial role in separating granulosa cells (GCs) into two anatomically and functionally distinct sub- types: mural granulosa cells (MGC), the cells lining the follicle wall; and cumulus cells
(CCs), the cells intimately surrounded the oocyte. The oocyte maintains intimate contact with CCs via an extensive network of gap-junctions, forming the cumulus-oocyte complex (COCs) which is relatively isolated and functionally distinct from the rest of the follicle (Eppig et al., 1997; Li eT a1.,2000). The close association between the oocyte and
CCs is fundamental to the maintenance of oocyte health and cumulus cell development
(Buccione et al., 1990; Gosden eTal.,1997).
It is clear that CCs can influence the quality of the oocyte obtained at ovulation and, as a result, the quality of embryo obtained. CCs play a major role in regulation of oocyte growth by direct transfer, via gapjunctions, of small molecules and substances that are responsible for the maintenance of meiotic arrest (Eppig,1982; Moor et al., 1980; Sirard and Bilodeau, 1990). Moreover, CCs play a crucial role in promoting oocyte maturation and acquisition of full embryonic developmental competence (Tanghe et a1.,2002). CCs also play important roles in oocyte protein phosphorylation and support oocyte cytoplasmic maturation needed for development of oocytes afler fertilization (Cecconi et
t22 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence al., 1991;(Fulka et al., 1998;Ka et a1.,1991). Furthermore, femoval of CCs from bovine oocytes prior to fertilization negatively affects embryo development (Fatehi et a1.,2002;
Zhang et al., 1995).
Gap junction communication between the oocyte and surrounding CCs is important to regulate the resumption of oocyte meiosis (Carabatsos et al., 2000; Kidder and Mhawi,
2002).In addition, it is highly likely that gap junctional communication is also involved in the process of oocyte maturation and therefore also the acquisition of developmental competence, Maturation of the fully-grown oocyte is associated with the loss of CC- oocyte gap junctional communication (Hyttel, 1987). In bovine COCs, the level of gap- junction communication between the two cell types dramatically decreases upon release of the COCs from the follicular environment, and continues to drop until t h of culture, at which point levels are effectiv ely zero and do not decrease further (Thomas et al., 2004) .
It has become increasingly evident that paracrine factors secreted from the oocyte potently regulate many functions of CCs, including proliferation, steroidogenesis, differentiation, apoptosis and expansion (Eppig, 2001; Gilchrist et aL.,2004a; Hussein et al., 2005). Although the effects of these oocyte-secreted factors (OSFs) have been extensively studied, their identities are still emerging. Members of the transforming growth factors-B (TGF- B) superfamily are candidate OSFs due to their abilify to mimic the actions of an oocyte on cumulus cells in vitro (Gilchrist et a1.,2004); in particular growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15).
For example, GDF-9 is known to stimulate cumulus cell proliferation and cumulus
123 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence expansion (Dragovic et al., 2005, Gilchrist el aL.,2004), and is involved in maintenance of cumulus cell amino acid transport to the oocyte (Marin Bivens et aL,2004; Sugiura and Eppig, 2005). GDF-9 also induces expression of several genes in CCs, including hyaluronic acid synthase 2 (HAS2), cyclooxygenase 2 (COX2: PTGS2) and gremlin
(GREMI) (Elvin et al., 1999; Pangas et al., 2004). BMP-15 reduces cumulus cell apoptosis by maintaining a localized gradient of anti-apoptotic factors within the COCs
(Hussein et a1.,2005). All the above findings most likely influence oocyte maturation, fertilization and subsequent embryo production (Downs et al., 1988; Lee el al., 200I;
McKenzie eTa1.,2004; Tanghe et a1.,2003).
The mechanisms by which the oocyte acquires developmental competence during its follicular development is not entirely known. However, it is most feasible that the close association between oocytes and their companion CCs is crucial in its acquisition and that the status of oocyte maturation is reflected in the biochemistry of the surrounding
CCs. To clariff the importance of the contribution of the oocyte to subsequent embryo quality, it is important to illustrate the functional coupling between the oocyte and CCs during the period of IVM, in particular the nature and relative importance to developmental competence of coupling prior to and following gap-junction breakdown.
We hypothesized that the temporal relationship between paracrine factors, secreted by the oocyte, and/or paracrine signaling interplay from CCs to the oocyte in the intact COC, and the loss of CCs-oocyte gap junction communication are determinants of the acquisition of developmental competence of the oocyte.
124 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence
4.3 Materials and Methods
Unless otherwise specified, all chemicals and reagents were purchased from Sigma (St
Louis, MO).
4.3.1 Collection of oocytes and culture conditions
Bovine ovaries were collected from local abattoirs and transported to the laboratory in warm saline (30-35 'C). COCs were aspirated from antral follicles (3 to 8mm diameter) using an l8-gauge needle and a 10-ml syringe containiîg - 2 ml aspiration medium
(Hepes-buffered Tissue Cultured Medium-l99; TCM-199, ICN Biochemicals, Irvine,
CA, USA) supplemented with 50 pglml kanamycin, 0.5 mM sodium pyruvate, 50 pglml heparin and 4 mg/ml fatty acid-free bovine serum albumin (FAF-BSA; ICPbio Ltd,
Aukland, NZ). Intact COCs with compact cumulus vestments > -5 cell layers and evenly pigmented cytoplasm were selected under a dissecting microscope and washed twice in
Hepes-buffered TCM-199 and once in Hepes-buffered TCM-I99 supplemented with 10% fetal calf serum (FCS) (Invitrogen, Carlsbad, CA). The basic medium for oocyte maturation was Bovine VitroMat (Cook Australia, Eight Mile Plains, Qld, Australia), a medium based on the ionic composition of bovine follicular fluid (Sutton-McDowall et al., 2005). All IVM treatments were supplemented with 0.1 IU/ml FSH (Puregon,
Organon, Oss, Netherlands). Complexes were cultured in pre-equilibrated 200 pl drops overlaid with mineral oil and incubated at 39"C with 5% CO2 in humidified air for 24 hours.
12s Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence
4,3.2 Treatment of cumulus-oocyte complexes
4.3.2.1 Generation of denuded oocytes
Denuded oocytes (DO) were generated by removing CCs from COCs by vortexing for -
4 minutes in 2 ml Hepes-buffered TCM-199. Any remaining CCs were removed by repeated passage of the oocytes through a fine-bore fire-polished glass pipette in Hepes- buffered TCM-199. To determine whether removing CCs from COCs by vortexing has an adverse influence on oocyte quality, two methods of denuding were compared
(vortexing and manual denuding using fine-bore fire-polished glass pipette).
Development rates to blastocyst were not altered by the methods used to denude COCs
(see Appendix 1.4).
4.3.2.2 Growth factors
Recombinant mouse GDF-9 and recombinant ovine BMP-I5 were produced and partially purified in-house as previously described (Gilchrist et a1., 2004b; Hickey et al., 2005;
Hussein eT a7., 2005) using transfected 293 hwan embryonic kidney cell lines (293H) originally donated by O. Ritvos (University of Helsinki). Control conditioned medium
(hereafter designated '293H') was also produced from untransfected 293H cells and partially purified (Hickey et al., 2005).
4.3.3 In vitro fertilization ønd embryo culture
In vitro production of embryos was undertaken using defined, serum-free media (Bovine
Vitro series of media, Cook Australia). Frozen semen from the same bull of proven
r26 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence fertility was used in all experiments. Briefly, thawed semen was layered over a discontinuous (45o/o:90%) Percoll gradient (Amersham Bioscience) and centrifuged (RT) for 20-25 mins at 700 g. The supernatant was removed and the sperm pellet was washed with 500 ¡rl Bovine VitroWash (Cook Australia) and centrifuged for a further 5 minutes aÏ 200 g. Spermatozoa were resuspended with IVF medium (Bovine VitroFert, Cook
Australia), then added to the fertilization media drops (Bovine VitroFert, supplemented with 0.01 mM heparin, 0.2 mM penicillamine and 0.1 mM hypotaurine) at a final concentration of 1 x 106 spermatozoalml. COCs or DOs were inseminated at a densify of
10 pl of IVF medium per COC/DO for 24 h, at 39oC in 60/o CO2 in humidified air.
In treatment groups where the COC remained intact at the time of fertilisation (see section 5.1, Experimental Design) CCs were removed by gentle pipetting 23¿4 h post insemination. Five presumptive zygotes were transferred into each 20pl drop of pre- equilibrated Cook Bovine VitroCleave medium (Cook Australia) and cultured under mineral oil at 38.5'C inTo/o C,2,60/o CO2,balance Nz, for five days (day 1 to day 5)
On Day 5, embryos were transferred in groups of 5-6 to 20 pl drops of pre-equilibrated
Bovine VitroBlast (Cook Australia) at 38.5'C overlaid with mineral oil and cultured to
Day 8. Embryos were assessed for quality at Day 8 according to the definitions presented in the Manual of the International Embryo Transfer Society (Stingfellow, 1998) and assessments were performed independently and blinded by an experienced bovine embryologist,
127 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovine Oocyte Developmental Competence
4.3.4 Diffirential staining
Cell counts were performed using a modified version of the technique described by
Fouladi-Nashta et al. (2005). Briefly, expanded/hatched blastocysts were placed into acid I Tyrode's solution to remove 1he zona, followed by a brief wash in 4 mg mL poly-vinyl alcohol (PVA) in phosphate-buffered saline (PBS/PVA). Zona-free embryos were then incubated in 10 mM trinitrobenzene sulfonic acid (TNBS) in PBSÆVA at 4'C for 10 min. Following this, embryos were subsequently incubated with 0.1 mg ml,-r anti- dinitrophenol-BSA antibody (Molecular Probes, Eugene, OR, USA) at 37"C for 10 min.
Following complement-mediated lysis using guinea-pig complement, embryos were washed and incubated in 10 pg ml.-l propidium iodide for 20 min at 37'C (to stain the
I trophectoderm), followed by 4 pg mL bisbenzimide (HoechsT33342; Sigma-Aldrich) in
100% ethanol aI 4"C overnight (to stain both the inner cell mass (ICM) and trophectoderm). Embryos were then whole mounted in a drop of 80% glycerol in PBS on microscope slides and coverslips were sealed with nail polish. Embryos were then examined under a fluorescence microscope (Olympus, Tokyo, Japan) at 400x equipped with an ultraviolet filter and a digital camera attached to determine total and compartment cell counts where inner cell mass (ICM) nuclei appeared blue and trophectoderm (TE) nuclei stained pink.
4.3. 5 Experimental design
4.3.5.1 Experiment 1: Temporøl effects of OSFs on oocyte developmentøl competence
.following co- culture of intøct COCs with DOs at either 0 or thour of IVM
t28 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovrne Oocyte Developmental Competence
This experiment was designed to assess embryo developmental competence after co- culture of COCs with DOs at either 0 or t hour of IVM and to determine the temporal effect of native OSFs on oocyte developmental competence. COCs were randomly allocated into 4 treatment groups during IVM. Treatment (l) 20 denuded oocytes were cultured in 200 pl IVM media for 24 hours (DO; Fig 1). Treatment (2) 20 cumulus oocyte complexes were cultured in 200 ¡rl IVM media for 24 hours (COC; Fig 1).
Treatment (3) 20 cumulus oocyte complexes were co-cultured from 0 lo 24 hours with
100 denuded oocytes in 200 pl IVM media, after which the 20 complexes (COC (0h); Fig
1) and 20 denuded oocytes (DO (0h); Fig 1) were removed and fertilized. Treatment (4),
2O-cumulus oocyte complexes were matured in 200 pI IVM medium for the first t hours as intact COCs. In parallel, 100 COCs were matured for t hours, then denuded and the
100 DOs were transferred to mature with the 20 COCs for the remaining 15 how (9-24 hour COC + DO) of IVM. As for treatment 3, the 20 complexes (COC (9h); Fig 1) and
20 of the denuded oocytes (DO (9h); Fig 1) were then removed and fertilised as described above. All complexes were fertilized after 24 h of IVM and the number and quality of blastocysts formed was assessed on day 8 of culture. Seven replicate experiments were performed.
Culturing one COC together with 5 DO in a 10 ¡r1 drop, gives a concentration of 0.5
DO/pl, which is the range used previously to examine the influence of oocyte-secreted factors (Hussein et a1.,2005), (Chapter 2),
r29 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovrne Oocyte Developmental Competence
4.3,5.2 Experiment 2: Assessment of oocyte developmental competencefollowing
treøtment of COCs with GDF-9 or BMP-I5 øt either 0 or t hours of IVM.
This experiment was conducted to determine whether treatment of COCs with exogenous recombinant OSFs, GDF-9 or BMP-15 from 0 or t hours of IVM (0-24 or 9-24 h) improves subsequent oocyte developmental competence. COCs were cultured in the base
IVM medium described above, with the following additional treatments 1) none
(control), 2) 175 ng/ml GDF-9 from 0 hour (0-24 h), 3) 175 nglml GDF-9 from t hour
(9-24 h), 4) \0%v/v BMP-15 from 0 hour (0-24 h), 5) 10% v/v BMP-I5 from t hour (9-
24 h), 6) l0% vlv 293H from 0 hour (0-24 h) and 7) 10% vlv 293H from t hour (9-24 h).
After IVM, all complexes were fertilized and blastocyst formation was assessed on day 8.
Four replicates of these experiments were performed using 30 COCs per treatment group per replicate experiment.
4. 3. 6 Støtistical analyses
All replicated proportional development data were arc-sine transformed prior to analysis.
Statistical analyses were carried out by ANOVA using SigmaStat software (SPSS Inc,
Chicago, IL), and significant differences between means were determined using Tukey-
K¡amer post-hoc test for comparison of multiple means. Differences were considered statistically significant at p< 0.05.
130 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovine Oocyte Developmental Competence
Treatment 1 Treatment 2 Treatment 3 Treatment 4 0h- 0h 0h- 0h-
Denuded oocyte complex o Ê-
o 24h 24h 24h - 24h - + DO coc coc DO coc (eh) Do (sh)
IVF
Figure 1. Diagrammatic illustration of the experimental design of the co-culture system of cumulus-oocyte complexes in the presence or absence of denuded oocytes during IVM. Complexes and oocytes were divided into 6 treatment groups after IVM. Denuded oocyte (DO), Cumulus-oocyte complex (COC), cumulus-oocyte complexes co-culture with denuded oocytes from 0 to 24 hour COC (0h), denuded oocytes co-cultured with cumulus-oocyte complexes from 0 to 24 hour DO (0h), cumulus-oocyte complexes for 9 hour, then co-cultured with denuded oocyte for the last 15 hour of IVM COC (9h), cumulus-oocyte complexes for t hour prior to denuding, then co-culture with cumulus- oocyte complexes for the last 15 hour of IVM DO (9h).
131 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovlne Oocyte Developmental Competence
4.4 Results
4.4.1 Experiment 1: Temporal effects of OSFs on oocyte developmental competence
following co- culture of intact COCs with DOs at either 0 or t hour of IVM.
Exposure of intact COCs to native OSFs from DOs from 0-24 hour significantly increased (P<0.05) the number of oocytes that reached the blastocyst stage at day 8 post insemination (51%), compared with COCs cultured alone (4I"/o) (Table 1). However, maturation of oocytes with intact cumulus cell communication during the first t hours of
IVM, and subsequently co-culturing with denuded oocytes, cultured as intact COCs for the flrrst th of IVM then denuded, for the last 15 hours of IVM, resulted in significantly more blastocysts on day B (COC (9h); 6l%). Removal of cumulus cells before IVM significantly decreased the number of oocytes that reached the blastocyst stage at day 8 post-insemination, compared with intact COCs (13% vs 4I%o respectively, P<0.05; Table
1). Maturing oocytes with intact cumulus for the first t hours prior to denuding significantly improved the blastocyst rate compared with oocyte denuded before IVM
(DO (9h) vs DO, 25Yo vs l3o/o, respectively; P<0.05; Table 1). The presence of cumulus cells (from neighboring COCs) did not improve the developmental capability of DOs
(DO (0h)) whereby blastocyst rates were similar to DOs cultured alone (DO) (Table 1).
Cleavage of oocytes was not significantly different between denuded oocyte treatments, or between cumulus-oocyte complex treatments (Table 1), but denuding in general lowered subsequent fertilization rates, However, the incidence of polyspermy (as assessed in a separate cohorts of oocytes stained with, bisbenzrmide, Hoescht 33342) was not different between denuded oocytes and cumulus-oocyte complexes (data not shown).
t32 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovlne Oocyte Developmental Competence
Co-culturing COCs with DOs from 0 hour (0-24h; COC (0h)) or t hour (9-24 h; COC
(9h)) significantly increased the cell numbers of the ensuing blastocysts, with more total and trophectoderm cell numbers, compared to control COCs (P<0.05, Table 2).
Furthermore, the presence of cumulus cells (from neighboring COCs) improved subsequent embryo quality of the denuded oocyte (DO (0h), DO (9h) respectively) as evident by increased total, irurer cell mass and trophectoderm cell numbers, compared to control DO (P<0.05, T able 2).
4.4.2 Experiment 2: Assessment of oocyte developmental competence following
treatment of COCs ,eith GDF-| or BMP-I5 at either 0 or t hour of IVM
Addition of GDF-9 or BMP-15 from 0 hour to maturing COCs enhanced (P<0.05) their development to the blastocyst stage (51% and 47Yo, respectively), compared to control
COCs (40%) (Table 3). However, addition of GDF-9 or BMP-15 from t hour to maturing
COCs did not increase the number of oocytes that reached the blastocyst stage, compared to control COCs (Table 3). Consistent with Chapter 3, conditioned medium from the parent 293H cell line, when added from 0 hour, adversely affected oocyte developmental potential, lowering blastocyst rates by l2Yo compared to control COCs (P<0.05).
However, there was no adverse effect on oocyte developmental potential when 293H conditioned medium was added from t hour. FurtheÍnore, embryo cleavage was not significantly affected by any of the oocyte treatments.
133 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence
Table 1. Temporal effects of OSFs on oocyte developmental competence following co- culture of intact COCs with DOs at either 0 or t hour of IVM.
Number o/o Cleavage %o Blastocysts Co-culture Treatments of rate from treatments oocytes cleaved DO 176 58.7 + 4.6^ 12.7 +0.8^
DO (0h) coc (0-24h) 191 69.7 +2.0^6 15.4 + 1.6ub
DO (eh) coc (e-24h) 178 76.1+3.5ub 25.0 + 1.5b COC r63 92.6 t 2.7b" 40.7 t 1.4" coc (0h) DO (0-24h) t66 89.1, +2.6" 50.6 + 1.9d coc (eh) DO (e-24h) 158 91,8 + 1.8" 61.3 + 1,9' u-'Values with different superscripts within the same column represent a statistically significant difference (P<0.05). Values are expressed as (mean + SEM).
Table 2. Number of total, inner cell mass and trophectoderm cells (mean + SEM) of Day
B expanded and hatched blastocysts following co-culfure of intact COCs with DOs during IVM.
Treatments Blastocyst Total cells ICM TE (n) Mean Proportion Mean Proportion Cells Cells
DO l2 118.5 + 2.4^ 32.2 + 1.2^ 27.3 + 7.1^ 86.3 +2.6^ 72.7 + l.l^
DO (0h) r9 137.2 + t3b 39.7 + r.2b 2g.g + 0.7ub 97.5 +0.9b 77.1+0.7^b
Do (eh) 31 138.0 + 1.lb 36.7 +0.9b 26.5 + 0.5^ 101.3 + 0.7b 73.5 + 0.5"
COC 40 148.3 + t.2" 50.0 + 1.3' 33.6 + 0.6' 98.2 + 0.8" 66.4 t 0.6" coc (0h) 51 156.1 + 1.3d 49.4+Lf 31.5 + 0.5d 105.7 + 1.3b 6g.5 + 0.5"d coc (eh) 61 159.9 + 0.9d 50.2 + 0.9" 3r.2 + 0.4bd 109.8 + 0.4" 6g.g + 0.4bd u-o Values with different superscripts within the same column represent a statistically significant difference (P<0.05). Values are expressed as (mean + SEM).
t34 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence
Table 3. Development of in vitro-produced embryos following co-culture of COCs with GDF-9 or BMP-15 either at 0 or t hour of IVM.
Number o/o Cleavage %o Blastocysts OSFs Treatments of rate from exposure oocytes cleaved Control t2l g2.g + 1.3ub 39.5 + 1.3"
GDF-e (0h) 0-24h 119 g0.g + 1.7ub 50.8 + 1.gb GDF-e (9h) 9-24h r27 84.3 + 1.1" 41.3 + 1.0u"
BMP-15 (oh) 0-24h 118 93.0 +2.4ub 47.0 + 1,0b"
BMP-15 (9h) 9-24h 129 g2.3 + 0,9"b 39.8 + r.4^
2e3H (0h) 0-24h 119 73.3 +2.4b 2g.0 + 0.9d
2e3H (eh) 9-24h r23 g0.0 + 3.5ub 36.0 + L6^ u-o Values with different superscripts within the same column represent a statistically significant difference (P<0.05). Values are expressed as (mean + SEM).
135 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence
4.5 Discussion
The results of the present study provide evidence that the developmental competence of oocytes matured as COCs is dependent on the native, uncharacterized mix of growth factors secreted from the oocyte. Oocyte quality, or developmental competence, is acquired during folliculogenesis as the oocyte grows and is dependent on cumulus cells to provide regulatory signals. On the other hand, oocytes secrete paracrine factors that regulate development of their surrounding cumulus cells and promote the cumulus cell phenotype. These effects of the oocyte contribute to the initiation and transmission of the regulatory signals by cumulus cells, which in turn contributes to the acquisition of the oocyte's own developmental competence.
The present study demonstrates that the capacity of IVM oocytes to reach the blastocyst stage can be substantially improved by treating intact cumulus-oocyte complexes during
IVM with native or recombinant OSF. Native OSFs affect oocyte quality throughout the oocyte maturation period, however what is particularly notewortþ is that OSF, have an optimal effect from t hour of IVM. In stark contrast, the major effect of exogenous recombinant BMP-15 and GDF-9 one oocyte developmental competence occurs during the first t hour of IVM. These results suggest that there is a temporal effect of native or recombinant OSF in enhancement of oocyte developmental competence. Furthermore, in this study, exposure of COC to native OSF improved subsequent embryo quality by affecting blastocyst formation and cell allocation to the trophectoderm or inner cell mass, evident as an increase in the total and trophectoderm cell numbers, where inner cell mass was not affected. Taken together, these results suggest that paracrine signaling between
136 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovlne Oocyte Developmental Competence
the oocyte and cumulus cells may contribute to the healthy development of the
subsequent embryo by improving post-implantaion developmental potential.
In this study it is notable that maturation of the oocyte as an intact complex for the first
th, prior to denuding resulted in increased blastocyst yield for the oocytes. This suggests
that direct transfer of molecules from CCs to oocytes via gap-junction channels accounts
for some enhancement of oocyte maturation and the acquisition of developmental
competence, as the oocyte which were cultured as an intact COCs form 0 to th prior to
denuding would be no different to the oocyte denuded at the start of IVM, if they did not.
Consistent with this suggestion, several studies have shown that gap-junction
communication between the oocyte and cumulus cells during early maturation is
important for the promotion of oocyte growth and development in vitro (Btccione et al.,
1990; Carabatsos et a1.,2000; Kidder and Mhawi, 2002).
Maturation of the oocyte is associated with the loss of cumulus cell-oocyte gap junctional
communication. Several studies have suggested that the loss of gap junctions between the
cumulus cells and the oocyte occurs at a time parallel with meiotic resumption (Dekel et
41., 1981; Hyttel, 1987; Sherizly et al., 1988). In bovine oocytes, gap junctional
communication ceases afLer t h of in vitro cultare (Thomas eL al., 2004). In the present
study, the loss of gap junction communication after t hours may also initiate changes within the denuded oocyte, which were cultured as intact COCs from 0 to th, involving differences in the quality or quantity of paracrine factors secreted from these denuded oocyte that have enhanced the blastocyst yield of the intact COCs cultured in their
137 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence presence. It also demonstrates that the quality of paracrine factors may depend on initial gap junctional coupling and suggests that an interaction between paracrine signaling and gap junctional communication may mediate the effect on subsequent embryo development (Gittens et al., 2005).
Our data also demonstrates that a mechanism other than the gap junction communication between CCs and oocytes is involved in enhancement of oocyte quality as co-culturing intact COCs with DOs from 0 or from t hours markedly increased the proportion of oocytes in intact complexes that reached the blastocyst stage, compared with COCs cultured alone. This data, for the first time, reveals ThaT a significant proportion of the acquisition of developmental competence is also derived from a paracrine signaling loop between the oocyte and surrounding cumulus cells. Furthermore, this loop is potentially unidirectional within the cumulus mass, so that the paracrine signaling by cumulus cells is directed towards the oocyte at the centre of the COC. This explains why oocytes within intact COCs have significantly higher levels of competence than denuded oocyte when intact COCs co-cultured with denuded oocyte from 9 to 24h of IVM, which were cultured as intact COCs from 0 to th, even though both types of oocytes have been exposed to cumulus cells throughout maturation.
Evidence exists, now from many different groups, of the roles of BMP-15 and GDF-9 in regulating cumulus cell phenotype (Gilchrist eT a1.,2004a; Juengel and McNatty, 2005).
Adding to this evidence, this study suggests that both exogenous GDF-9 and BMP- l5 are important for promoting the secretion of paracrine signaling within the COC. In the
138 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence curent study, it is interesting to note that adding exogenous recombinant BMP-15 or
GDF-9 to the medium from the start of IVM, but not from t hour of IVM, markedly increased the proportion of oocytes in intact complexes that reached the blastocyst stage.
This suggests that the exogenous recombinant BMP-15 and GDF-9 have their effects within the first t h of IVM in influencing oocyte developmental competence, and also this contrast the results described for native OSFs. This suggests that native OSFs that can affect oocyte quality throughout the oocyte maturation period, which may include endogenously derived BMP-15 and GDF-9, are altered in either quality or quantity.
These data provide evidence that endogenous BMP-15 and GDF-9 concentrations are important for subsequent oocyte quality; however, they may account for only part of the total influence of native oocyte secreted factors on oocyte quality. If other oocyte- secreted factors are responsible for enhancing oocyte quality, they remain to be determined. Candidate molecules may include other members of TGF- B superfamily, such as TGFps, activin and BMP-6, despite the fact that those molecules activate two divergent signaling pathways (Shimasaki eT aL.,2004).
It is not yet clear which oocyte and cumulus cell functions during maturation are involved in the enhancement of oocyte competence to reach the blastocyst stage, even though it is now widely accepted that oocyte secreted factors regulate a broad range of cumulus cell functions (Eppig,2001; Gilchrist et al., 2004a). One suchpossibility is the regulation of cellular metabolism and/or amino acid transport from the cumulus cells to the oocyte via gap junctions that are essential for the development of the oocyte. For example, it has recently been demonstrated that oocyte secreted factors regulate cumulus
139 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence cell amino acid and energy substrate uptake and transport to the oocyte (Eppig et al.,
2005; Sugiura and Eppig, 2005). Oocyte secreted factors may also promote cumulus cell secretion of soluble factors, which benefit oocyte quality either by inducing developmental competence or by removing inhibitory factors that suppress development.
In addition, there is some evidence that factors secreted by cumulus cells during fertilization arc very important in promoting embryonic development (Ball et al., 1983;
Tanghe et al., 2003).
Collectively, the evidence presented in this study demonstrates for the first time that there are partitioned levels of paracrine and gap-junction signaling effects, prior to and following gap-junction breakdown, that influence the acquisition of developmental competence of the oocyte. This study, therefore, suggests that the interaction between paracrine signaling and gap junctional communication is important in mediating the optimal subsequent embryo development.
4.6 Acknowledgements
TS Hussein is supported in part by the Faculty of Health Sciences, University of
Adelaide. This project was supported by a National Health and Medical Research
Council (NHMRC) Program Grant (250306) and the Research Centre for Reproductive
Health, University of Adelaide. The authors would like to thank Dr. Olli Ritvos
(University of Helsinki) for generously donating the GDF-9 and BMP-15-expressing cell lines. The authors would like to thank Samantha Schulz, Fred Amato, Alexandra
Harvey, Lesley Ritter and Karen Kind for helpful technical and editorial suggestions.
t40 Chapter 4. Temporal Effects of Oocyte-Secreted Factor(s) During In Vitro Maturation on Bovtne Oocyte Developmental Competence
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146 Chapter 5
Final Discussion Chapter 5. Final Discusssron
Final Discussion
In recent years a new paradigm has emerged in reproductive biology. It has become apparent that the oocyte is an essential controller offolliculogenesis, and now there is an extensive interest in paracrine factors secreted by the oocyte and their role in maintaining and promoting the cumulus cell phenotype. Oocytes regulate cumulus cell functions via the secretion of paracrine factors acting in a mitogenic manner by promoting cumulus cell proliferation and expansion, while suppressing steroidogenesis and luteinizing hormone receptor expression (reviewed; Gilchrist et a1.,2004a). Prior to this study, there was no data available as to whether these oocyte factors may also regulate and maintain the low incidence of cellular apoptosis within cumulus cells by acting in an anti-apoptotic manner, and whether these oocyte factors enhance oocyte developmental competence during in vitro maturation. The results from this thesis lead to an important new concept that is now emerging, which is that the oocyte secretes paracrine factors that determine the distinctive phenotype of the cumulus-oocyte complex by acting in an localised
manner, establishing a morphogenic gradient of OSFs within the COC, and thereby
regulating its own microenvironment. The capacity of an oocyte to regulate the COC
microenvironment by oocyte-secreted factors (OSFs) is essential for appropriate oocyte
maturation and is a vital component of oocyte developmental competence.
The first major aim of this thesis was therefore to determine the effect of oocyte-secreted
factors on cumulus cell apoptosis. In particular, this study examines the nature of the
paracrine network of BMP growth factors and their binding proteins regulating cumulus
cell apoptosis. This study demonstrates by various means that oocytes actively prevent
148 Chapter 5. Final Discusssron cumulus cell apoptosis by establishing a morphogenic gradient of oocyte-secreted factors. Firstly, the reduction in cumulus cell apoptosis was assessed by two different methods; using TLINEL together with quantitative confocal microscopy, and also by examining the expression of key proteins regulating apoptosis (anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins) by Western blot. Secondly, the anti-apoptotic actions of oocytes followed a gradient from the site of the oocyte(s). Thirdly, oocyte-secreted factors were able to protect cumulus cells from apoptosis induced by an apoptotic insult
(staurosporine) (Chapter 2). Moreover, results from the present study provide multiple lines of evidence that BMP signaling, in particular BMP-15 and BMP-6, and not GDF-9, signaling prevents cumulus cell apoptosis, as confirmed through experimental neutralization, using neutralizing antibodies and binding proteins, To antagonize the anti- apoptotic effects of these growth factors and oocytes on cumulus cells (Chapter 2). The evidence presented in this study demonstrates, for the first time, that oocytes are responsible for the low incidence of apoptosis within cumulus cells, through the establishment of a paracrine network of BMP growth factors and their binding proteins
(model Figure 10, Chapter 2). This finding adds further support to the emerging concept that oocyte secreted paracrine factors are crucial to establish and maintain an immediate highly specialized COC microenvironment which is distinct from that of the rest of the follicle (Eppig,2001; Gilchrist et al., 2004a).
The above concept led to the second major hypothesis of this thesis, that the capacity of oocytes to secrete paracrine factors and/or maintain a paracrine signaling loop from cumulus cells to the oocyte in the intact COC complex, is a function of high quality
149 Chapter 5. Final Discussston oocytes and is a determinant of the acquisition of oocyte developmental competence
(model Figure 4, chapter 3). In order to test this hypothesis, a unique co-culture system was designed and developed for exposing intact COCs to additional OSFs during in vitro maturation. In particular, experiments were designed to test this hypothesis using two different methods: (I) oocytes were exposed to an uncharacterised mix of native OSFs by co-culturing intact COCs with DO, from either the start of IVM, or from t hour of IVM,
(II) COCs were treated during IVM with exogenous recombinant BMP-15 and/or GDF-9, from either the start of IVM, or from t hour of IVM. This novel co-culture system was designed to illuminate a new perspective technology to develop an IVM system that enhances IVM oocyte developmental potential.
Results from Chapter 3 demonstrated that oocyte-secreted factors enhance oocyte developmental competence during in vitro maturation, whether in their native form as an uncharacterized mix of growth factors secreted by the oocyte, or as recombinant
exogenous BMP-15 and GDF-9 (Chapter 3), Also, exposure of COCs to oocyte-secreted
factors improved subsequent embryo quality as evident by increased total and
trophectoderm cell numbers. These results provide evidence towards a new paradigm in
oocyte biology and have significant insights into emerging concept that the secretion of
paracrine factors by oocytes is a crucial for the capacity of an oocyte to regulate its own
microenvironment, which in turn is important determinant of oocyte developmental
competence.
150 Chapter 5. Final Discussston
Experiments were planned to investigate the temporal relationship of the time of loss of cumulus-oocyte gap junction communication and secretion of paracrine signals with the change in oocyte developmental competence (Chapter 4). It is interesting to note that the major effect of exogenous recombinant BMP-15 and GDF-9 on oocyte developmental competence occurs during the first t hour of IVM. However, native OSFs affect oocyte quality throughout the oocyte maturation period and has an optimal effect from t hour of
IVM (Chapter 4). Endogenously derived BMP-15 and GDF9 have been proposed to interact synergistically and act as either homodimers and/or heterodimers, which may have different biological activities (McNatty et a1.,2005a; McNatty et al., 2005b). It is possible that the difference in the effect of endogenous OSFs on oocyte developmental competence, prior to and following t hour of IVM, may relate to the formation of homodimers and/or heterodimers. As a potential scenario, it may be that homodimers of
GDF-9 and BMP-15 are secretedby oocytes during the first t hour of IVM, resulting in less potent effects on developmental competence to those observed from t hour of IVM, when these growth factors may form heterodimers.
There are two divergent signaling pathways activated by oocyte-secreted factors; the
SMAD 213 pathway and SMAD 1/5/8 pathway (Shimasaki et al., 2004). Results from this
thesis have illustrated that the stimulation of the SMAD 213 signaling pathway, as
exemplified by GDF-9, and the SMAD 1/5/8 signaling pathway, as exemplified by BMP-
15, in cumulus cells are involved in regulating oocyte developmental competence. This
suggests that activation of the SMAD 1/5/8 pathway by exogenous BMP-I5 and BMP-6
transmits the anti-apoptotic actions of the oocyte by providing a protective mechanism
151 Chapter 5. Final Discusssron against cumulus cell apoptosis (Chapter 2), and activation of the alternate SMAD 2/3 pathway by exogenous GDF-9 conveys the oocyte's mitogenic signal by promoting cumulus cell proliferation and growth (Gilchrist et al., 2004a). However, endogenous
BMP-15, BMP-6 and GDF-9 oocyte proteins account for only part of the total influence of native oocyte secreted factors on cumulus cell apoptosis or oocyte quality. Those oocyte secreted factors responsible for the remaining portion of prevention of cumulus cells apoptosis and enhancement of oocyte quality remain to be determined. Candidate molecules may include other members of TGF- B superfamily, such as TGFBs and activin. Putative oocyte-secreted heterodimers may play some role in regulating these processes such as BMP-15 and GDF-9, which are known to interact synergistically to regulate some granulosa cell functions (McNatfy et a1.,2005a; McNatty et al., 2005b).
The work presented in this thesis investigates the nature of cellular interactions within the ovarian follicle that are responsible for maintaining the cumulus cell phenotype and establishing a highly specialized and distinctive COC microenvironment and sheds new light on the importance of oocyte-cumulus cells interaction during the final stage of development. These findings have far-reaching implications for improving the efficiency of oocyte maturation technologies in domestic species and in human infertility treatment, and support the role of oocyte-secreted factor production by the oocyte as a potential diagnostic marker for oocyte quality.
The outcomes of the research presented in this thesis have made a substantial contribution to our understanding of the mechanisms regulating oocyte maturation and
152 Chapter 5. Final Discusssion the acquisition of developmental competence. Results from the present study demonstrate a means of improving oocyte maturation during IVM where oocyte-secreted factors may prove to be a beneficial component of oocyte IVM culture systems, increasing the quality and quantity of in vitro embryo production.
Future directions
The novel IVM system that was developed in this thesis is based on a serum free medium, and the substantial increase in embryo production efficiency in this system has commercial and clinical applications including cloning, production of transgenic animals and treatment of human infertility using IVM. Furthermore, these results are the first to demonstrate the concept of using OSFs as IVM media additives to improve embryo developmental potential. Further application of this technology will require the production of purified GDF-9 and/or BMP-15, as these growth factors in this study were prepared in partially purified form, and the impurities present in the conditioned medium
from the untransfected parent cell lines had a marked inhibitory effect on oocyte
developmental competence. Also, as these OSFs are recently discovered little is known
of their biochemistry and molecular signaling and the ability to produce stabilised forms
of these factors for clinical and commercial application to IVM technology would be of
benefit.
As a part of future work, the contribution of other oocyte-secreted factors accountable for
regulation of cumulus cell apoptosis and enhancing oocyte quality should be further
153 Chapter 5. Final Discusssron investigated. In particular, it would be interesting to know if these OSF molecules interact synergistically by forming heterodimers. Categorization of the signaling pathways that the heterodimers use would be extremely beneficial to the future of this work, Following the work examining cumulus-oocyte interactions presented in this thesis, future work should investigate diffusible factors produced by cumulus cells and/or crosstalk between the oocyte and cumulus cells in the regulation of oocyte developmental competence. Furthermore, the interaction between paracrine signaling and gap junctional communication needs to be further investigated as it presents a new paradigm in the oocyte CCs regulatory loop with potential effects on oocyte maturation, ovulation, and developmental competence of the ensuing embryo, which may mediate the effect on subsequent embryo development.
Future directions of this study may also include design of a co-culture system of intact
COCs with denuded oocytes, where the oocyte in both the intact andlor denuded state are in different stages of meiosis (this can be achieved using meiotic resumption inhibitors -
such as milrinone) to determine if there is relationship between the different stages of oocyte meiosis and secretion of additional or different oocyte secreted factors that may affect oocyte quality. Investigation of the developmental capacity acquired by oocytes matured in vitro in the presence of oocyte-secreted factors combined with gap junction blockers, such as carbenoxolone, would provide additional information as to the role of
gap junctional communication. It would also be of interest to examine whether
endogenous GDF-9 and BMP-15 are less effective at influencing oocyte quality from 9 hour of IVM, which would be achieved through neutralization experiments specifically
t54 Chapter 5. Final Discusssion
antagonizing the endogenous effect of GDF-9 and BMP-15 on oocyte subsequent development.
Finally, in vitro oocyte maturation (IVM) conditions have remained relatively unchanged over the past few decades. To date there are no deflrnitive, non-invasive measures for developmental potential of the immature oocyte, with development to the blastocyst stage the current best indicator of oocyte quality. This situation could be improved by investigating and examining the influence of oocyte secreted factors on cumulus cells,
focusing on gene anays of cumulus cells, metabolic profiles and energy substrate usage
of curnulus oocyte complexes and meiotic status of the oocyte . These approaches could be combined to design a culture system that may improve the developmental capacity of
in vitro matured oocytes.
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179 References
Zuell Reprod 43,784-7 . 180 Appendices Appendices Appendix L: Additional Experiments 1.1 The influence of BSA, FCS and PVA on cumulus cells øpoptosis Preliminary experiments were perfonned to compare the effects of different protein sources on cumulus cell apoptosis. COCs were cultured in 50 prl drops of base medium (BTCM-199), with the following additional supplements 1) none (control), 2) 4 mglml BSA, 3) 10% FCS, 4) 4mglml BSA and 10% FCS, and 5) 0.3 mg/ml PVA. After 24 h culture, cumulus cell apoptosis was assessed using TUNEL. As shown in Fig 1, there was no obvious difference in cumulus cell apoptosis whether the base medium was supplemented with BSA and FCS, alone or in combination, compared to the control. However, supplementing the base medium with PVA reduced cumulus cell apoptosis, compared to the control and other supplements. 35 t'-----r coc + BTcM-199 ã30 ;o\ '-tn 25 o o. 8- zo E15 tt, E10 E o5 0 control BSA FCS BSA + FCS PVA Figure 1. Cumulus cell apoptosis for COC complexes cultured in a base medium supplemented with different proteins sources (BSA, FCS and PVA). r82 Appendices 1.2 Dose response of sluurosporine on cumulas cell apoptosis Staurosporine induces apoptosis via a cellular signaling cascade (to date uncharacterized) as opposed to causing indiscriminate DNA damage (Weil et a1.,1996; Yuan et a1.,2004). A preliminary experiment was conducted to determine the apoptotic effect of staurosporine on cumulus cells. COCs were treated with an increasing concentration of staurosporine (0.1-100 ¡rM). COCs were also cultured alone as a negative control. As expected, cumulus cell apoptosis was induced in a dose-dependent manner by treating COCs with increasing concentration of staurosporine (Fig 2). 120 coc ---o- GOC + STS 100 s .9 8Bo oCL CL f60 õ C) o =40 E o 20 0 0 0.1 5 10 25 50 100 srs (pM) Figure. 2Effect of staurosporine (STS) on cumulus cell apoptosis. COCs were cultured alone or with an increasing dose of staurosporine (0. 1- I 00 ¡rM). 183 Appendices 1.3 Assessment of fhe bioactivity of recombinønt ovine BMP-15 Recombinant ovine BMP15 was produced in house using transfected 293H human embryonic kidney cell lines (293H). BMP-15 underwent pafüal purification using hydrophobic interaction chromatography (HIC). In addition, control conditioned medium (293H) was produced by culturing untransfected 293H cells. This was also partially purified using HIC. Bioactivity of BMP-15 is likely to vary between batches. In order to assess the bioactivity of BMP-15, a novel assay (TUNEL and laser confocal scanning microscopy followed by image analysis) was developed and validated for the quantitative measurement of cumulus cell apoptosis. The bioactivities of 4 different batches of BMP- 15, produced throughout the year, were assessed using the above assay to examine their effects on cumulus cell apoptosis. OOX complexes were treated with 4 different batches of I0% BMP-15 or with l0% 293H. OOX complexes and COCs were also cultured alone as negative and positive controls respectively. As expected, intact COCs had low cumulus cell apoptosis and the negative control OOX had the highest incidence of apoptosis. The 4 different batches of BMP-15 were able to reduce cumulus cell apoptosis (Fig 3). However, each batch reduced apoptosis to a different degree, indicating that different batches of BMP-I5 do vary in their bioactivity, at least in terms of the ability to inhibit apoptosis' 184 Appendices 40 10%293H oox rssr 10%BMP-15 r coc \o-ù30 at, tD o a. o a. oo ø J =Ero o 0 2e3H oox (1) (21 (3) (4) coc Different Batches of BMP-15 Figure. 3 Effect of different batches of BMP-15 on cumulus cell apoptosis. OOX complexes were cultured alone, or treated with 10% 293H or with 4 different batches of BMP- 1 5 : 1 ) 200 1 05, 2) 090604, 3) 27 0106, 4) 07 0404. 185 Appendices 1.4 Generation of denuded oocyte and oocyte developmentøl competence Denuded oocytes were generated by removing cumulus cells from COCs by vortexing for - 4 minutes in 2 ml Hepes-buffered TCM-199. To determine whether removing CCs from COCs by vortexing has an adverse influence on oocyte quality, two methods of denuding were compared (vortexing and manual denuding using f,rne-bore fire-polished glass pipette). Development rates to the blastocyst stage were not altered by the methods used to denude COCs (Table 1). This suggests that vortexing has no adverse effect on production ofparacrine factors from the denuded oocytes. Table 1. Effect of removing cumulus cells from COCs by vortexing or manual denuding on oocyte developmental competence Number o/o Cleavtge 7o Blastocysts Treatments of rate from oocytes cleaved Vortex 55 83.5 r 8.5 28.5 r 0.5 Manual 57 75.5 r 1.5 29.5 ! 4.5 Values are expressed as (mean + SEM). 186 Appendices Appendix 2: TUNEL Assay I. Reagents and Solutions Washing Buffer: 1% Bovine serum Albumin in PBS Dissolve 1g BSA in 100m1PBS leave for 15min at room temp store 4oC Fixation Solution: 4%o paraformaldehyde in PBS pH7 '4 . Add 100m1 of PBS into a flask and heat to -60'C in water bath in fume hood . Add 49 PFA to 100ml PBS . Add dropwise (<2 drops) 1M NaOH until solution clears o Measure pH adjust to -7.4 o Filter solution .22pmfiter o Store at 4"C for 1 week Permeabilisation solution: 0.1% Tritonx-100 in0.l%o Sodium Cilrate o Add 100m1MQ water into flask ¡ Add 100p1TX-100 into 100ml dh20 . Weigh 0.19 Sodium Citrate ¡ Add to 100m1MQ/TX soln . Filter sterilise .2Z¡tmfllrter TUNEL Reaction: Roche: In Situ Cell Death Detection Kit Fluorescetn' o Bottle 1: Enzyme reaction (terminal deoxynucleotidyl Transferase) o Bottle 2: Label solution (nucleotide mixture in reaction mix) o Remove 40 pl from bottle 2X20¡tl each negative control into and Eppendorf tube on ice in dark o TIINEL reaction mix 1:9 ratio Bottle 1:Bottle 2 ie. 1Opl in 90ptl 187 Appendices a Require 20 ¡rl per 5 cell reaction RNAseA / Propidium Iodide solution: . RNAseA stock solution 10 mglml required at a working concentration of [0.lmg/ml] in a final volume of 500p1 o Aliquot 5 ¡.rl into an Eppendorf tube o PI stock: 1 mg/ml;require aftnal conc of 0.5 pglml . Dilute PI stock 10 pl [1 mg/ml] into 1990 pl RNAse buffer giving a conc of 5 p'glml ¡ Aliquot 50 ¡rl PI [5 pllml] to the Eppendorf tube containing RNAseA solution and add 445¡l RNAse buffer, giving a final conc of PI of [0.5pg/ml] RNAse Buffer . 0.8764g TRIS o 0.05849 NaCl ¡ 0,057 g MgCb Add these components to 80ml of water Bring pH to 8.0 and make up to a final volume of 100m1 DNAse I solution [0.05 U/¡tl] o Stock [1Ou/pl] o Add I pl of DNAsel [10U/pl] to 199 ¡rl PBS/I%BSA e Store 50pl aliquots at -20oC 188 Appendices II. WASHER Setup 1. Remove washer (discs) from ethanol 2. Melt paraffin wax 3. Apply a circle of paraffin around the chamber 4. Place a cover slip over the paraffin wax layer 5. Melt the paraffin using a warming plate (watch the melting point) 6. Remove the washer from the warm plate and allow to cool. il[. TUNEL PROCEDURE for COC/OOX 1. Remove cells after 24h matwation from a 4 well dish and wash 3 times in 50 pl drops of PBS/BSA 2. After a final wash transfer cells to 50 pl drops of 4o/o paraformaldehyde and incubate overnight at 4"C. 3. Wash 2X in 50pl drops of PBS/BSA. 4. Apply 2 ¡t"l Cell Tak (Becton Dickinson) to the coverslip and allow to dry (10-15 min) 5. Wash the coverslip 2X with 20¡rl PBS/BSA 6. Transfer (3-5) fîxed COC/OOX to the coverslip 7. Allow the COCs to adhere to the Cell Tak and aspirate excess PBS/BSA (do not allow the COC to completely dry) 8. Wash lX with 20 pl PBS/BSA 9. Apply 20 pl of permeabilisation solution and incubate for t h at room temperature. 10. 20 min prior to permeabilisation completion prepare for +VE control by adding 20 pl Dnase I, wash 1X with PBS/BSA 11. Wash all samples 2X with 20 ¡rl PBS/BSA 12. Apply 20 p,l of TUNEL reaction mixture (samples and +VE control) and 20 ¡rl label solution (-VE control), incubate at 37oC for I h in a humified atmosphere in the dark. 13. V/ash 2X with 20 pl PBS/BSA. 189 Appendices 14. Wash 1X with 20 pl RNAse buffer. 15. Apply 20 ¡,rl of RnaseAÆI solution and incubate for t h at room temperature in the dark. 16. Wash lX with 20 ¡rl PBS/BSA. 17. Apply 5 pl antifade (give details) in the centre of the slide 18. Examine using confocal microscope. 190 Appendices Appendix 3: Culture Media Stock Solutions for IVM/IVF/IVC A1l chemicals and reagents were purchased for Sigma (St Louis, MO, USA) unless otherwise stated. Stock A (xa) Stock HeParin 2337.6 mg NaCl 20 mgheParin 59.64 mg KCI Dissolve in 2 ml0.9% sterile saline 78.87 mg MgSOaolflrg 108.87 mg KH2PO4 Stock Hypotaurine (10 mM) Dissolve in 100 ml of Milli Q 10.9 mg hypotaurine Dissolve in 10 mI0.9% sterile saline Stock B 1.05 g NaHCO3 Stock Penicillamine (20 mM) 5 mg Phenol Red 29.84 mgpenicillamine Dissolve in 50 ml Milli Q Dissolve in 10 ml 0.9 % sterile saline Stock C (xl25) Stock S (x10) 5l mgNapyruvate 5.5 g NaCl Dissolve in 10 ml Milli Q 300 mg KCI 36 mgNaHzPO¿ Stock GL (x10) 723 mgMgSO+.7HzO 405 mg D-glucose 100 mg Kanamycin Dissolve in 100 ml Milli Q Dissolve in 100 ml Milli Q Stock SH SPAD (x100) 600 mg HEPES (free acid) 292 mg NaCl 650 mg HEPES (sodium salt) 300 mg CaClz.2HzO Dissolve in 25 ml Stock S Dissolve in 10 mlMilli Q 19t Appendices Stock CA (xf 0) Stock H 85.08 mg Ca flactate] 6 g HEPES (free acid) Dissolve in 12 ml Milli Q 6.5 g HEPES (sodium salt) 20 mgphenol red N-Acetyl-L-cysteine (xl 00) Dissolve in 200 ml Milli Q \ 6.32 mg N-Acetyl -L-cysteine Dissolve in 10 ml Milli Q TCM199 (x2) 1 sachet TCM199 (ICN Biochemicals) Cysteamine (xl00) 50 mg kanamycin sulphate 7 .7 7 mg Mercaptoethylamine Dissolve in 500 ml Milli Q Dissolve in 10 ml Milli Q Check osmolarity (- 480) Media for IVM HEPES buffered TCM199 + BSA (H199 + BSA) 50 ml TCMI99 (x2) 8 ml Stock H 2 ml Stock B 1 ml Stock C Add MilliQ to make up to 100 ml and add 4 mglml fatty acid free BSA (ICPbio Ltd, Auckland, NZ). Osmolarity:270 Osm Bicarbonate buffered TCM199 + BSA (BTCM-199 + BSA) 50 ml TCMI99 (x2) 10 ml Stock B I ml Stock C 192 Appendices Add MilliQ to make up to 100 ml and add 4 mglml fatty acid free BSA Osmolarity:270 Osm Bicarbonate buffered TCM199 + PVA (BTCM-199 + PVA) 50 ml TCMI99 (x2) 10 ml Stock B 1 ml Stock C Add MilliQ to make up to 100 ml and add 0.3 mg/ml polyvinyl alcohol Osmolarity:270 Osm Bovine Invito Maturation Medium (BFFM + 2.3 mM glucose) 25 ml Stock A 16 ml Stock B 800 pl Stock C 10 ml Stock CA 24.34 ml Stock GL 1 ml Glutamax I (Gibco Invitrogen Corporation, Carlsbad, CA, USA), 1 ml N-acetyl-cysteine 1 ml cysteamine I ml non-essential amino acids (100X, Gibco Invitrogen Corporation), 2 ml essential amino acids (50X, Gibco Invitrogen Corporation) Add Milli Q to make up to 100 ml and add4 mglml BSA. OsmolaritY:294 Osm t93 Appendices Media for IVF F-SOF Cook Bovine In Vitro Fert, Cook Veterinary Products IVF Media 10 mlF-SOF 100 pl Stock Penicillamine 100 pl Stock Hypotaurine 10 pl Stock Heparin 907o Percoll 4,5 ml Percoll 400 pl Stock SH 50 pl Stock B 50 pl 100X SPAD 457o Percoll Dilute 2 ml or 90% Percoll with 2 ml HSOF All solutions used to make Percoll gradients were warmed to room temperature before use. Sperm thawed in 37oC water. H-SOF Cook Bovine In Vitro Wash; Cook Veterinary Products. E-SOF Cook Bovine In Vitro Cleave; Cook Veterinary Products. L-SOF Cook Bovine Blast Media, Cook Veterinary Products. 194 Appendices Appendix 4: Reagents 293 HEK 293H conditioned media (produced in house). Culturing untransfected 293 human embryonic kidney (HEK) cells (Gibco life Technologies, Paisley, UK) produced2g3H control conditioned media. Partially purified using HIC. Fractions dialysed against PBS and stored at -80"C. GDF-9 Bioactive recombinant mouse GDF-9 (produced in house). Recombinant human embryonic kidney-293H cells expressing mGDF-9 were donated by O. Ritvos (Helsinki). Partially purified using HIC. Fractions dialysed against PBS and stored at -80'C. BMP-I5 Bioactive recombinant ovine BMP-15 (produced in house), Recombinant human embryonic kidney-293H cells expressing oBMP-15 were donated by O. Ritvos (Helsinki). Partially purified using HIC, Fractions dialysed against PBS and stored at -80'C. BMP-6 Recombinant human BMP-6 (R&D systems, Minneapolis, MN). 50 pglml stock solutions prepared by dissolving 20 mg in 400 pl of 4 mM HCI containing 0.1% BSA. Stored at -20"C. BMP-7 Recombinant Human BMPT (R&D Systems, Minneapolis, MN) 10 ¡rglml stock solutions prepared by dissolving 10 pg in 1 ml 4 mM HCI containing 0.1% BSA. Stored al-20'C. 195 Appendices Follistatin Follistatin-288 (donated by S. Shimasaki, University of California San Diego, USA). 100 pglml stock preparedby % dilution of 400 pglml stock sterile saline containing 0.1% BSA. Stored at -20"C. Gremlin Recombinant Mouse Gremlin (R&D Systems, Minneapolis, MN) 5Opg/ml stock solutions prepared by dissolving 50 ¡rg in 1 ml 4 mM HCI containing 0.1% BSA. Stored aT-20"C. BMP-6 neutralizing antibody. Anti-Human BMP6- Antibody (R&D Systems, Minneapolis, MN) 500 ¡rglml stock solutions prepared by dissolving 500 pg in 1 ml sterile PBS. Stored at - 20'c. sB-431542 ALK4|í17 kinase inhibitor (donated by GlaxoSmithKline) 10 mM stock solution prepared by dissolving5.6T mg in 1.3 ml of DMSO. Stored at - 20"c. rhFSH Recombinant human follicle stimulating hormone (rhFSH, Organon, Netherlands). 10 IU/ml stock solution prepared by dissolvingT5IU in 7.5 ml sterile saline containing 0.1% BSA. Storedat -20"C in 0.1 and 0.5 ml aliquots (1 and 5 IU respectively). Mineral Oil Embryo grade (Sigma) store at room temperature in the dark. Maintain sterility, 196 Appendices Appendix 5: Blastocyst Scoring System Blastocyst scoring system used to select in vitro produced embryos for analysis (adapted from the Manual of the International Embryo Transfer Society). The assessment can be performed on a dissecting microscope. Initially blastocysts are categorized according to their developmental stage: lc one cell (oocyte/embryo); no cleavage has occurred and the embryo appears like a single cell CL cleaved embryo; one or more cleavage divisions has occurred CM compact morula: embryo consisting of at least 16 cells that has compacted away from the zona and cell definition is lost eB early blastocyst; the blastocoel cavity being less than half the volume of the embryo B blastyocyst; the blastocoel being greater than or equal to half the volume of the embryo XB expanded blastocyst; the blastocyst has initiated expansion, increasing the volume of the bastocoel, thereby increasing in diameter, accompanied by zona pellucida thinning HB hatched blastocyst; the blastocyst has completely escaped from The zona. For compact morula and blastocyst stages, embryos are then graded from 1-3, or as degenerating, depending on the development and appearance of the inner cell mass and trophectoderm. 197 Appendices I Excellent/Good. Symmetrical and spherical embryo mass with individual blastomeres (cells) that are uniform in size, color and density. The embryo is consistent with its expected stage of development. The embryo possesses a single, tightly packed inner cell mass with many cells, translucent trophectoderm consisting of many cells, and little cellular debris. Any irregularities should be relatively minor, consisting of less than I5Yo of the total embryonic mass. This judgment should be based on the percentage of embryonic cells represented by extruded material in the perivitelline space. The zona pellucida should be smooth and have no concave or flat surfaces that may cause the embryo to adhere to a Petri dish. 2 Fair. Moderate irregularities in overall shape of the embryonic mass or in size, color and density of individual cells. Loosely grouped inner cell mass with several cells or spatially not confined, translucent/slightly dark trophectoderm consisting of several-many cells. Some cellular debris, consisting of not greater than 50%o of cellular material. 3 Poor. Major irregularities in shape of the embryonic mass or in size, color and density of individual cells. Few cells in compartment, dark appearaîce, cellular debris consisting of greater than50%o of the total embryonic mass, 4 Dead or degenerating. Granular appearance of cytoplasm, or fragmenting cells. Non-viable. 198 Appendices Appendix 6: Published Version of Chapter 2 Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins. Tamer S. Hussein, David A. Froiland, Fred Amato, Jeremy G. Thompson and Robert B. Gilchrist Journal of Cell Science; ll8: 5257 -5268 STATEMENT OF AUTHORSHIP CONTRIBUTIONS Tamer S. Hussein Designed and carried out all experiments, interpreted data and wrote the manuscript. David A. Froiland Provided technical assistance, assisted with confocal microscopy and image analysis Fred Amato Purified the recombinant ovine BMP-15 and recombinant mouse GDF-9, assisted with Western blot. Jeremy G. Thompson Co-supervised the work, assisted with experimental design, data analysis and manuscript preparation. Robert B. Gilchrist Overall supervision of the work assisted with experimental design, data analysis, and manuscript preparation and acted as conesponding author. t99 T.S. Hussein, D.A. Froiland, F. Amato, J.G. Thompson and R.B. Gilchrist (2005) Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins. Journal of Cell Science, v. 118 (22), pp. 5257-5268, November 2005 NOTE: This publication is included in the print copy of the thesis held in the University of Adelaide Library. It is also available online to authorised users at: http://dx.doi.org/10.1242/jcs.02644 Appendices Appendix 7 z Published Version of Chapter 3 Oocytes-secreted factors enhance oocyte developmental competence' Tamer S. Hussein, Jeremy G' Thompson and Robert B' Gilchrist Developmental Biology ; 296 (2006): 514-521 STATEMENT OF AUTHORSHIP CONTRIBUTIONS Tamer S. Hussein Designed and carried out all experiments, interpreted data and wrote the manuscript' Jeremy G. Thompson Co-supervised the work, assisted with experimental design, data analysis and manuscript preparation. Robert B. Gilchrist Overall supervision of the work assisted with experimental design, data analysis, and manuscript preparation and acted as conesponding author' 200 T.S. Hussein, J.G. Thompson and R.B. Gilchrist (2006) Oocyte-secreted factors enhance oocyte developmental competence. Developmental Biology, v. 296 (2), pp. 514-521, August 2006 NOTE: This publication is included in the print copy of the thesis held in the University of Adelaide Library. It is also available online to authorised users at: http://dx.doi.org/10.1016/j.ydbio.2006.06.026