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Studies on apoptosis in bovine follicles and embryos Yang, Ming Yuan 2000

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STUDIES ON APOPTOSIS IN BOVINE F O L L I C L E S AND EMBRYOS by MING YUAN YANG M.Sc, University of British Columbia, 1996 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSPHY in THE FACULTY OF GRADUATE STUDIES (Faculty of Agricultural Sciences) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 2000 © Ming Yuan Yang UBC Special Collections - Thesis Authorisation Form httpyAvww.library.ubc.ca/spcollAhesauth.htmI In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e re q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f A n i m a l S c i e n c e The U n i v e r s i t y o f B r i t i s h Columbia Vancouver, Canada Date July 25, 2000 lofl 7/24/00 4:17 P M ABSTRACT The aims of this thesis were to further study the involvement of apoptosis in bovine ovarian follicles and embryos. In the first part of the thesis, studies in vitro and in vivo were carried out to investigate whether apoptosis is the underlying mechanism of follicular atresia and to characterize its regulation by hormones. Studies in vitro demonstrated that granulosa cells (GCs) in follicles undergoing atresia display both morphological and biochemical characteristics of apoptosis. Apoptosis was observed mainly in GCs and in scattered theca cells. Apoptosis was not evident in cumulus cells. These results suggest that apoptosis is the most common pathway of GC deletion which leads to the destruction of the GC layer and finally triggers follicular atresia. Apoptosis was also detected in GCs of some morphologically healthy follicles, indicating that apoptosis is detectable before other morphological and biochemical signs of degeneration appear. A system for culturing GCs in vitro was developed to study the hormonal regulation of apoptosis. Using this culture system, it was observed that the rate of DNA fragmentation in cultured GCs from small follicles was higher than that from medium and large follicles. Follicle stimulating hormone (FSH) and insulin-like growth factor-I (IGF-I) attenuated spontaneous apoptotic cell death in cultured GCs, suggesting that they are follicle survival factors. An in vivo model in which the proestrus dominant follicle (DF) is maintained for 9 days was used to further study the precise regulation of the atresia of the nonovulatory DF induced by high concentration of progesterone (P4). With the time of P4 administration, DFs gradually underwent atresia and exhibited different levels of apoptosis in GCs, confirming our previous findings. P4 did not suppress apoptotic death in cultured GCs, suggesting P4-induced atresia of non-ovulatory bovine DFs is probably via regulation of luteinizing i i hormone (LH). In addition, it was demonstrated that follicular atresia was related to a shift in the ratio of death repressor (Bcl-2) to death inducer (Bax) protein expression. Using an in vitro embryo production system, apoptosis was found to be related to bovine oocyte degeneration and embryo fragmentation, suggesting the existence of a natural preprogrammed cell death mechanism which can respond to external stimuli and/or internal defects in bovine oocytes and embryos. The ratio of Bcl-2 and Bax protein expression was demonstrated to be an important determinant of developmental competence for both oocytes and embryos. This thesis provides important insight into the mechanisms of follicular atresia and early embryonic loss. Evaluation of the nature of these events would increase our understanding of the limitations with follicular dynamics and early embryonic development. i i i TABLE OF CONTENTS Abstract ii Table of Contents iv List of Figures vii List of Tables x List of Abbreviations xi Acknowledgments xii Dedication xiv CHAPTER 1: GENERAL INTRODUCTION 1 CHAPTER 2: LITERATURE REVIEW 7 I Ovarian Follicular Development 7 2.1. The ovarian follicles 7 2.2. Follicular dynamics 8 2.3. Follicular atresia 12 2.3.1. Morphological changes associated with atresia 13 2.3.2. Biochemical changes associated with atresia 13 II Apoptosis 14 2.4. Apoptosis vs. necrosis 15 2.5. Triggers of apoptosis 17 2.6. Gene regulation of apoptosis 18 III Apoptosis: An Underlying Mechanism of Follicular Atresia 20 2.7. Apoptosis in follicular atresia 20 2.8. Hormonal cotrol of follicular atresia 21 iv 2.8.1. Role of Gonadotropins 22 2.8.2. Role of steroid hormones 22 2.8.3. Role of insulin-like growth factors 24 2.8.4. Role of other hormones 26 2.8.5. Gene regulation of follicular atresia 28 IV Apoptosis in Oocyte and Embryo Degeneration 30 2.9. Oocyte degeneration 30 2.10. Oocyte classification schemes 30 2.11. Embryo fragmentation 31 2.12. Involvement of apoptosis during in vitro embryo development . 34 CHAPTER 3: MORPHOLOGICAL AND BIOCHEMICAL IDENTIFICATION OF APOPTOSIS IN SMALL, MEDIUM, AND LARGE BOVINE FOLLICLES AND THE EFFECTS OF FSH AND IGF-I ON SPONTANEOUS APOPTOSIS IN CULTURED BOVINE GRANULOSA CELLS 35 Abstract 35 Introduction 36 Materials and Methods 39 Results 45 Discussion 55 References 62 CHAPTER 4: INVOLVEMENT OF APOPTOSIS IN THE ATRESIA OF NON-OVULATORY DOMINANT FOLLICLES DURING THE BOVINE ESTROUS CYCLE 68 Abstract 68 Introduction 70 Materials and Methods 73 v Results 80 Discussion 92 References 96 CHAPTER 5: EXPRESSION OF BCL-2 AND BAX PROTEIN IN RELATION TO THE QUALITY OF BOVINE OOCYTES AND EMBRYOS PRODUCED IN VITRO 102 Abstract 102 Introduction 103 Materials and Methods 106 Results 112 Discussion 117 References 124 CHAPTER 6: GENERAL DISCUSSION 128 SUMMARY OF THESIS RESEARCH FINDINGS 132 REFERENCES 134 vi L I S T O F F I G U R E S Figure 2.1 Architecture and classification of ovarian follicles during Folliculogenesis 9 Figure 2.2 Growth and development of ovarian follicles during estrous cycle of cattle 11 Figure 3.1 Morphological changes of bovine follicles with the progression of follicular atresia 47 Figure 3.2 Bovine granulosa cells with characteristic morphologically features of apoptosis 48 Figure 3.3 In situ 3' end-labeling (TUNEL) of apoptotic cells and/or apoptotic Bodies in bovine granulosa layer 49 Figure 3.4 Disseminated pattern of apoptotic cells and/or apoptotic bodies in atretic bovine follicles 50 Figure 3.5 Internucleosomal DNA fragmentation in pooled granulosa cells obtained from morphological atretic and healthy small (< 4 mm), medium (5-8), and large (> 8 mm) bovine follicles 52 Figure 3.6 Spontaneous onset of apoptosis in cultured bovine granulosa cells obtained from small (< 4 mm), medium (5-8 mm), and large (> 8 mm) ~ bovine follicles 53 Figure 3.7 Effects of FSH on apoptosis in cultured bovine granulosa cells 54 Figure 3.8 Effect of IGF-I and IGF-I plus FSH on apoptosis in cultured bovine granulosa cells 56 vii Figure 4.1 Diameter changes of norgestomet (synthetic progestin)-maintained dominant follicles in controls and in cows treated with progesterone for 24, 48, and 72 h 81 Figure 4.2 Morphological changes of norgestomet (synthetic progestin)-maintained dominant follicles after 24, 48, and 72 h of progesterone treatment 83 Figure 4.3 Concentrations of progesterone and estradiol-17p in the follicular fluid of norgestomet (synthetic progestin)-maintained dominant follicles from controls and cows treated with P4 for 24, 48, and 72 h 84 Figure 4.4 Effects of in vivo progesterone treatment on apoptosis in the norgestomet (synthetic progestin)-maintained dominant follicles 86 Figure 4.5 Analysis of intemucleosomal DNA fragmentation in norgestomet (synthetic progestin)-maintained dominant follicles from control and cows treated with Progesterone for 24 and 48 h 87 Figure 4.6 Expression of Bcl-2 and Bax protein in norgestomet (synthetic progestin)-maintained dominant follicles from control and cows treated with P 4 for 24 and 48 h 89 Figure 4.7 Effects of P4 on apoptosis in cultured bovine granulosa cells 90 Figure 4.8 Expression of Bcl-2 and Bax protein in small (< 4 mm), medium (5-8 mm), and large (> 8 mm) bovine follicles 91 Figure 5.1 Classification of immature bovine cumulus-oocyte complexes 108 Figure 5.2 Morphology of abnormal immature bovine oocytes following 48 h in vitro culture 113 viii Figure 5.3 Percentage of morphologically abnormal oocytes originating from grade I and IV immature bovine oocytes following 48 h in vitro culture 114 Figure 5.4 Cleavage and blastocyst formation rates obtained from grade I immature bovine oocytes following in vitro maturation, in vitro fertilization and in vitro embryo culture 115 Figure 5.5 Morphology of normal and degenerated bovine embryos produced in vitro. 116 Figure 5.6 TUNEL assay of fragmented immature bovine oocytes and in vitro produced bovine embryos 118 Figure 5.7 Bcl-2 and Bax expression in immature bovine oocytes and in vitro produced bovine embryos 119 ix L I S T O F T A B L E S Table 2.1 Oocyte quality as determined by morphological examination 32 Table 2.2 Effects of oocyte type on fertilization rate and embryo yield. ... 33 x LIST OF ABBREVIATIONS bp = base pair °C = Celsius Ca 2 + = calcium CL = corpus luteum Ci = curie d =day DF = dominant follicle DMEM = Dulbecco's modified eagles medium DNA = deoxyribonucleic acid E 2 = estradiol-17p EDTA = Ethylenediaminetetraaceticacid EGF = epidermal growth factor FF = follicular fluid FSH = follicle-stimulating hormone g = gram GnRH = gonadotropin-releasing hormone h = hour hCG = human chorionic gonadotropin IGF(s) = insulin-like growth factor(s) IGFBP(s) = insulin-like growth factor binding proteins i.m. = intramuscular RJ = international unit IVC = in vitro culture IVF = in vitro fertilization IVM = in vitro maturation Kda = kilodaltons 1 = liter LH = luteinizing hormone MHz = megahertz ml = milliliters min = minutes n - sample size ng = nanogram p = probability P 4 = progesterone PBS = phosphate buffered saline PMSG = pregnant mare serum gonadotropin RIA = radioimmunoassay RT = room temperature SDS = sodium dodecyl sulfate Tris = Tris(hydroxymethyl)aminomethane UV = ultraviolet v/v = volume per volume w/v = weight per volume TGF-ct, -p = transforming growth factor-a, -p vs = versus xii A C K N O W E D G E M E N T S I would like to express my deepest gratitude to my research supervisor, Dr. R. Rajamahendran, for providing me an opportunity to work independently and for his guidance, encouragement, and constructive criticisms throughout the graduate program. Sincere thanks are extended to Drs. K.M. Cheng, P.C. Leung, C. Maccalman and T. Yoganathan who served as members of my supervisory committee and provided precious suggestions and guidance. Special thanks go to Drs. J.R. Thompson, R. Blair, J. Shelford, S. Samuels, X. Lin, for their guidance and help. I am grateful to all the technical help offered by G. Galzy, S. Leung, K. Sivakumaran, A. Luz, and S. Motani. I am thankful to all the Dairy Research Unit personnel, especially J. Paul and T. Cathcart for their contributions tendered during the field experiments. I appreciate the friendship and support of my fellow graduate students working in the Faculty of Agriculture Science and Biotechnology Laboratory, especially Lisa Stephens, J. Kurtu, M. Mohan, G. Gnanaratnam, M. Pavneesh, A. Muhammad, R. Nagaraja, M. Manikkam, H. Ken, JJ. Lee, R. Chong, N. Kazumi, H. Mohamed, D. Ambrose, C. Taylor, P.X. Wang. I am thankful to the National Hormone and Pituitary Program, USDA for providing FSH and LH for my research. I want to acknowledge my appreciation to my parents and my sister, Dr. C.Z. Yang for their encouragement and support. Lastly, I greatly appreciate the incredible support, understanding, and love of my wife, Yupu. This achievement is also theirs. xiii % ntcf, evc^e, tyufiu ^ u, fan fan, fatfr, tutdeMtOMdwy, and, love. CHAPTER 1 GENERAL INTRODUCTION The growth and development of an ovarian follicle proceeds uninterruptedly until the follicle either ovulates or undergoes a degenerative process known as atresia. It has long been recognized that the vast majority (> 99%) of follicles present at birth eventually become atretic with significantly less (< 1%) actually achieving ovulation. Despite the overwhelming occurrence of atresia in the ovary, the basic mechanisms of follicular atresia and the related molecular changes are still largely unknown. In cattle, the growth of antral follicles occurs in a wave-like pattern, with a wave being characterized by the recruitment of a pool of small antral follicles and the emergence and differential growth of a single dominant follicle (DF) while the remainder of its cohorts undergo atresia. Either two or three waves of follicular growth occur during each estrous cycle and thus there are two or three DFs capable of ovulation. However, only the DF present at the time of corpus luteum regression ovulates whereas the DFs that develop during the luteal phase never ovulate, but undergo atresia (Savio et al., 1988; Sirois and Fortune, 1988; Taylor and Rajamahendran, 1991; Roche, 1996). What causes the regression of the non-ovulatory DF? What is the mechanism of the induction of DF atresia? Embryo production in vitro has become an increasingly important scientific endeavour as well as one with considerable commercial application. In many laboratories, embryos are routinely obtained from oocytes after in vitro maturation (IVM), in vitro 1 fertilization (IVF), and in vitro embryo culture (IVC). Therefore, the proper selection of developmentally competent oocytes is crucial for successful in vitro embryo production. Previous studies have indicated that classification of bovine oocytes, based on visual assessment of the compactness of the cumulus investment as well as the homogeneity and transparency of the ooplasm, can be used to select immature oocytes for optimum maturation, fertilization, and development in vitro (Madison et al., 1992; Brackett and Zuelke, 1993). A clear relationship between oocyte morphology and embryo yield after IVM/IVF/IVC has recently been established (Lonergan et al., 1992). However, the mechanisms underlying the visual assessment and selection of immature oocytes are unknown. The quality of mammalian preimplantation embryos obtained under culture conditions in vitro is variable. When compared to embryos derived in vivo, embryos produced in vitro from several mammalian species exhibit retarded developmental progress (Dobrinsky et al., 1996; Du et al., 1996; Long et al., 1998). In addition, the appearance of cellular fragmentation during early embryo development in vitro has been commonly observed in pigs (Long et al., 1998) and humans (Jurisicova et al., 1995). These embryos contain irregularly sized blastomeres, multiple cellular fragments, and development will frequently become arrested with subsequent degeneration (Hardy et al., 1989). Moreover, fragmented embryos have an increased sensitivity to cryopreservation and manipulation, and therefore, are associated with poor survival following cryopreservation and with decreased pregnancy rates following embryo transfer (Hardy et al., 1989; Jurisicova et al., 1995). What is the mechanism of embryo fragmentation? 2 Apoptosis is an active, genetically governed process whereby cells die in a controlled fashion triggered by changes in specific physiological stimuli. Associated with apoptosis are a number of characteristic morphological features including condensation of chromatin around the periphery of the nucleus, endoplasmic reticulum swelling, cytoplasmic blebbing, formation of apoptotic bodies, and the subsequent phagocytosis of these bodies by neighbouring cells. A unique biochemical event in apoptosis is that endogenous nucleases digest DNA from apoptotic cells into oligonucleosomal fragments (multiples of 185-200 bp) which give the appearance of a DNA ladder after electrophoresis (Ellis et al., 1991; Raff, 1992; Cory and Adams, 1998). Every cell has an apoptotic cell death pathway. It has been proposed that the propensity to apoptosis is continuously counterbalanced in the cell by genes stimulating cell survival and proliferation. Upon induction by an appropriate stimulus, the cell activates or inactivates the repression of gene products responsible for control of the suicidal mechanism (Raff, 1992; Cory and Adams, 1998). Studies from extragonadal cell systems have shown that among the numerous proteins and genes involved, members of the Bcl-2 family of genes play key roles in regulating apoptosis. Recent studies, mostly from rodents, have demonstrated that apoptosis is responsible for follicular atresia (Tilly et al., 1991; Billig et al., 1994; Chun et al., 1994). However, research on follicle apoptosis is just beginning, and most of the data are descriptive. Knowledge of the morphological changes and basic molecular mechanisms, i.e., what are the modulating factors that regulate apoptosis during follicular selection, dominance and atresia and which genes are involved, are still lacking or very preliminary, especially in cows. In addition, although it has been shown that fragmented human embryos exhibit typical morphological changes that conform to the general criteria of apoptotic cell death 3 (Jurisicova et al., 1995), the occurrence and regulation of embryonic apoptosis and its relation to decreased developmental competence and embryonic loss has not been determined. Therefore, this thesis aims to examine the involvement and regulation of apoptosis and the associated molecular changes during follicular dynamics, oocyte maturation in vitro and embryo production in vitro. Much of the work is either published, in press, or submitted for publication. As such each chapter, save the general introduction, literature review and general discussion (Chapters 1, 2 and 6), constitutes an independent research paper: In antral follicles, the earliest and most prominent feature of atresia is the death of granulosa cells (GCs), leading to almost total destruction of the follicle (Rajakoski, 1960). Given that atresia is the consequence of individual cellular components of the follicle dying, one approach to further elucidate the mechanism underlying follicular atresia would be to examine the nature of the death in follicle cells. There have been some studies suggesting that apoptosis is responsible for the death of GCs. However, no comprehensive information on the morphological and biochemical changes of GCs and other follicular cells during the process of apoptosis has been reported. In addition, hormone regulation of apoptosis at different follicle developmental stages is not clear in cows. Therefore, the specific objectives in Chapter 3 were: 1) to determine whether the death of bovine follicular cells occurs by apoptosis during follicular atresia by displaying the morphological and biochemical characteristics of apoptosis; 4 2) to establish a model system in vitro to analyze the role of follicle-stimulating hormone and insulin-like growth factor-I in modulating apoptosis in granulosa cells at different stages of follicular development. If results from Chapter 3 suggest that apoptosis in GCs is regulated by hormones, what is the molecular mechanism responsible for transducing the signals? In addition, it has been suggested that high concentration of progesterone (P4) may play an important role in initiating atresia of non-ovulatory DFs that develop during the luteal phase in the bovine estrous cycle. DF atresia during the luteal phase of the bovine estrous cycle can be mimicked using an in vivo model developed in our lab (Taylor and Rajamahendran, 1991). Hence, in Chapter 4, it is of great interest to examine whether apoptosis is the underlying mechanism of the P4-induced atresia of non-ovulatory DFs and to study the biochemical and molecular pathways used by P4 to control the initiation of apoptosis. Therefore, the specific objectives in Chapter 4 were: 1) to investigate whether apoptosis is associated with P4-induced atresia of non-ovulatory DFs using a norgestomet (synthetic progestin)-maintained DF in vivo model; 2) to determine if atresia of the DFs induced by P4 is associated with alterations in Bcl-2 and Bax expression using a norgestomet-maintained DF in vivo model; 3) to test if P4 has a direct effect on apoptosis in bovine follicles using an in vitro culture model; 4) to study the pattern of expression of Bcl-2 and Bax in follicles at different developmental stages. 5 Oocytes and embryos that undergo degeneration show similar morphological changes characteristic of apoptosis. In order to understand the mechanisms underlying their degeneration and the limitations associated with current in vitro embryo production techniques, it is important to study the occurrence and regulation of apoptosis during oocyte and embryo development. Therefore, the specific objectives in Chapter 5 were: 1) to investigate whether differences in the developmental capacity of different quality of immature oocytes and subsequent embryo fragmentation are associated with apoptosis; 1) to characterize the changes in expression of Bcl-2 and Bax in relation to their regulation of apoptosis during oocyte maturation and early embryo development and their relationships to oocyte and embryo quality. 6 CHARPTER 2 LITERATURE REVIEW This review summarizes ovarian follicular dynamics in cattle, current knowledge of the molecular mechanism of apoptosis, and its role in ovarian follicular atresia and in oocyte and embryonic demise in the bovine. I OVARIAN FOLLICULAR DEVELOPMENT 2.1. The Ovarian Follicles Ovarian follicles form early in embryonic development. The primary sex organs develop from ridges on the ventromedial surface of the mesonephric kidneys. The primordial germ cells arise from endodermal cells of the yolk sack and follicular cells primarily formed from mesonephric cells. Germ cells (i.e. oogonia) undergo mitoses and then meioses after arriving at the gonadal ridge. However, meiosis does not progress to completion in the embryonic ovary and germ cells arrest in diplotene stage of prophase I in most mammalian species. In cattle, most primary oocytes reach first meiotic arrest by day 170 to 175 of embryonic life and remain in this stage until puberty (Ohno and Smith, 1964). Follicle formation starts immediately after the first primary oocyte reaches the diplotene stage. In cattle, the first primordial follicles, a single oocyte surrounded by squamous, often irregular shaped epithelial cells that more or less have separated the oocyte completely from cortical connective tissue, are observed around day 90 to 100 of embryonic life. During early postnatal life, most oocytes are surrounded by somatic follicular cells to 7 become primordial follicles, whereas those remaining naked undergo degeneration (Erickson, 1966; Marion and Gier, 1971). Follicular development starts with recruitment of small fractions of the primordial follicle population. Once recruited to grow, the epithelial cells form a single layer of cuboidal cells which proliferate by mitosis and form a stratified follicular epithelium or granulosa layer with gap junctions. The follicle is termed a secondary follicle once cells surrounding the oocytes divide and form several layers. While these modifications take place, stroma cells immediately around the follicle differentiate to form the theca folliculi, which subsequently differentiate into theca interna and theca externa. As the follicle grows due to an increase in size and number of granulosa cells (GCs), accumulation of liquor folliculi appears between the GCs. When the cavities that contain liquor folliculi coalesce and form a cavity, the antrum, the follicle is termed a tertiary follicle. In the tertiary follicle, cells of the granulosa layer form a small pedicle of cells, the cumulus oophorous, which contains the oocyte and protrudes towards the interior of the antrum. The tertiary follicle is also called a Graafian follicle which reaches its maximal diameter and is responsive to the preovulatory surge of gonadotropins (Fig. 2. 1; Marion and Gier, 1971; Erickson et al., 1985). 2. 2. Follicular Dynamics Utilizing ultrasound imaging, researchers confirmed the hypothesis first proposed by Rajakoski (1960) that ovarian follicular growth in cattle occurs in waves. Two or three waves of follicular growth occur during each estrous cycle in heifers, and predominantly 8 PRIMOROIAL FOLLICLE PRIMARY FOLLICLE 6E -BASEMENT LAMINAE OICTYATE OOCYTE — GRANULOSA CELLS SECONDARY FOLLICLE EARLY TERTIARY FOLLICLE BASEMENT LAMINAE GRANULOSA CELLS •FULLY GROWN OOCYTE ZONA PELLUCIOA BASEMENT LAMINAE GRANULOSA CELLS ZONA PELLUCIOA FULLY GROWN OOCYTE PRESUMPTIVE THECA THECA EXTERNA BASEMENT LAMINAE STEROID SECRET-INS CELLS ANTRUM BLOOO VESSEL , ZONA PELLUCIOA % FULLY GROWN OOCYTE MULTIPLE LAYERS OF GRANULOSA CELLS THECA INTERNA HISTOLOGIC ARCHITECTURE. OF GRAAFIAN FOLLICLE MEMBRANA GRANULOSA CELLS THECA INTERNA LOOSE CONNECTIVE TISSUE CORONA RAOIATA GRANULOSA CELLS \ BASAL LAMINA THECA INTERSTITIAL CELLS CAPILLARIES ZONA PELLUCIOA CUMULUS OOPHOROUS GRANULOSA CELLS THECA EXTERNA Fig 2.1. Architecture and classification of ovarian follicles during folliculogenesis (from Erickson, 1986). Recruitment occurs within the pool of nongrowing primordial follicles. Once the oocyte is surrounded by a single layer of cuboidal cells, it is reactivated and completes its growth and zona pellucida formation at the primary-secondary follicle stage. Growth of the preantral follicle is a consequence of granulosa proliferation and ends with cavitation or antrum formation in the early tertiary stage. In response to FSH stimulation, follicular fluid accumulates in the antrum and the early tertiary follicle becomes a Graffian follicle. 9 two-waves of follicular growth occur in cows. (Fig. 2. 2; Ireland and Roche, 1987; Savio et al., 1988; Sirois and Fortune, 1988; Taylor and Rajamahendran, 1991; Adams et al., 1992). According to these studies, a wave of follicular growth is characterized by proceeding through stages of recruitment, selection, and dominance (Ireland and Roche, 1987). Recruitment is the process whereby a cohort of antral follicles begins to grow under gonadotropin stimulation. Selection is a process by which one single follicle is chosen and avoids atresia. Dominance is the mechanism(s) by which a selected follicle with the potential competence to achieve ovulation and inhibit further growth of other follicles in the same cohort. In each wave of follicles, coincident with a transient increase of follicle stimulating hormone (FSH; Turzillo and Fortune, 1990), a cohort of antral follicles (2-4 mm) is recruited at very early stages of the estrous cycle. From these, one is selected to continue growing between day 2 and day 3 of the cycle to become the dominant follicle by day 4 or 5, whereas the remainders of the follicular cohort fail to grow further and become atretic. Furthermore, once a follicle becomes selected, it also inhibits further recruitment of a new cohort of follicles (Adams et al., 1992). The dominant follicle reaches its maximum size on day 7 or 8 of the estrous cycle. Then it maintains its morphological and functional dominance until about day 11, then gradually starts to decline in size, and is no longer identifiable by day 14. A dominant follicle from the second wave is identifiable by day 14. If progesterone (P4) level in the circulation begins to decrease due to spontaneous regression of the corpus luteum (CL) while the second wave dominant follicle is in its growth phase, then the second wave dominant follicle invariably goes on to ovulate. If, on the other hand, P 4 remains elevated after the second wave dominant follicle has attained its maximum size, the 10 a u >• u CO 3 o r r CO UJ UJ I o z £ • 8 I < z >-Q rr 3 3 U o u. • • N B W t- »• — CO Zn O P 3 °°o° ° o 0 o f o o o l o o o o o Q o O 0 O Q ° „ o o o ° °2> o c r ° o o ° b o ° o ° o o o ° O o o ° o o 0 o o ° o o t • SSV-JO c SSV10 (nn) «3i3Hvia 3iomod t t t t S SSV10 l 5SV13 UJ -J o >• u CO 3 o cc t-co UJ UJ I H U. O CO < Q tS OH o o o l U o . T3 <D +j ^ cd £ 3 P o § I co C <D O CD *0 3 c O CO S CD -P ( D C H O s « CA CO cd U o II CN co 0 o co co 03 o > CD O •s ° •S a si <u o T3 cd .g K *0 9 CD a o o CD O O a o <D ta o •3 .g <D & co CD CO CO cd 3 t> CD c o -»—I cd 3 o CD * - C ^ O CD CD CO rH 1 S cd « S CD to CD "C XI c3 CD WD o o e I d A w CO C co § ^2 u U I S S3 £ 1 2 3 ° .8 m 11 dominant follicle begins to regress to be replaced by a third wave of follicular growth. In a bovine estrous cycle with three waves of follicle development, the diameter of the second wave dominant follicle is smaller than the size of the first dominant follicle, and the estrous cycle is extended because the third dominant follicle requires additional time to complete development before ovulation (Savio et al., 1988; Taylor and Rajamahendran, 1991). It is still not clear what factor(s) cause regression of these anovulatory dominant follicles, which appear during the first and second wave of follicular growth in each estrous cycle in cattle. Hormones, especially progesterone, may be involved in this regression. 2.3. Follicular Atresia The Greek word atresia (a = not; tresia = perforated) is usually used to describe the closure of a natural opening. In ovarian physiology, atresia refers to the degenerative process by which follicles are regress before reaching ovulation. Two major stages of cell degeneration can be distinguished during follicle formation and growth: degeneration of germ cells (attrition), which accounts for the largest loss of oocytes and occurs mainly prenatally, and follicle degeneration (atresia), which occurs during postnatal reproductive life. Greater than 99% of bovine follicles present at birth undergo atresia (Erickson, 1966). Therefore, follicle atresia during postnatal life has been the focus of most investigations. Although the major atresia occurs at the preantral and antral stages of follicular development (Hsueh et al., 1994), follicles can become atretic at any stage of growth. Growing and dominant follicles will become atretic if the hormone milieu is not appropriate for further growth or ovulation. 12 2. 3.1. Morphological Changes Associated With Atresia Degenerative changes in GCs are the first morphologically recognizable sign of follicular atresia in many species including the rat (Braw and Tsafriri, 1980), the sheep (Hay et al., 1976), and the human (Himelstein-Braw et al., 1976). At the early stage of atresia, there are a small number (< 10%) of GCs with pyknotic nuclei usually close to the follicular antrum, whereas some of the GCs are still in mitosis. As atresia progresses, the number of pyknotic GCs increase, the basement membrane loses its integrity, leukocytes infiltrate the granulosa layer, and meiotic-like changes occur in the oocyte. Rat follicles at this atretic stage cannot be rescued by pregnant mare serum gonadotropin (PMSG) treatment (Hirshfield, 1989). The advanced atresia is characterized by a massive reduction of GCs, none of them in mitosis, and the collapse of the follicle. The oocytes may shrink, become necrotic and these events often occur simultaneously with or are followed by fragmentation of the oocyte (Tsafriri and Braw, 1984). During atresia, the changes of theca layer vary among different species. In humans, rats, and rabbits, theca cells undergo extensive hypertrophy during atresia (Himelstein-Braw et al., 1976; Erickson et al., 1985; Hsueh et al., 1994). However, theca cells of hamster follicles do not exhibit marked morphological changes despite total collapse of the granulosa layer (Hubbard and Greenwald, 1985). In sheep, theca cells, obtained from follicles at advanced atretic stage, undergo nuclear condensation and degeneration similar to those observed in GCs (O'shea et al., 1978). 2. 3. 2. Biochemical Changes Associated With Atresia With atresia there is a decline in estrogen synthesis concomitant with increased progesterone production (Uilenbroek et al., 1979). The shift in the estrogen to progesterone ratio has been attributed to a decrease in C 1 7 jo'lyase activity, leading to a decrease in 13 androgen substrate for GC aromatization, and a loss of aromatase activity (Uilenbroek et al., 1979; Braw et al., 1981; Tilly, 1992). Follicular atresia is also correlated with reduced DNA synthesis of GCs (Greenwald, 1989), supression of the expression of a gap junction protein connexin 43 (Wiesen and Midgley, 1994), and decreased expression of the messenger RNA for aromatase and gonadotropin receptors (Tilly, 1992). Moreover, increased expression of several genes, including the IGF-binding proteins (Manikkam and Rajamahendran, 1997), sulfated glycoprotein-2 (Kaynard et al., 1992) and angiotensin-II receptor (Daud et al., 1988), has also been found during follicular atresia. Clearly, these morphological and biochemical changes associated with atresia provide useful markers for identifying the phenomenon; However, they do not disclose the mechanisms underlying this process. Given that atresia is the consequence of individual cellular components of the follicle dying, one approach to further elucidate the mechanism underling follicular demise would be to examine the nature of death in follicular cells (Hughes and Gorospe, 1991). II APOPTOSIS Death, along with growth and differentiation, is a critical part of the life cycle of a cell. The traditional concept of cell death is that a pathological process occurrs when cells are subjected to an injurious environment. This "accidental" cell death is called necrosis (Walker et al., 1988). It is only in the past few years, however, that attention has been focused on the physiological occurrence of cell death and its role in homeostasis. It has been found that this type of "natural" death, which is now termed apoptosis, is a widespread phenomenon that plays a crucial role in many physiological and pathological processes. 1 4 Apoptosis take place during embryogenesis as a force in sculpting the developing organism and in the course of normal tissue development maintaining homeostasis. It also serves as a defense mechanism to remove unwanted and potential dangerous cells, such as cells that have been infected by virus and tumour cells. In addition to the beneficial effects, the inappropriate activation of apoptosis may contribute to the pathogenesis of many diseases, such as cancer, neurodegenerative disorders, and resistance to chemotherapy. Apoptosis is an ancient Greek word meaning "falling off of petals from flowers, or leaves from trees. The concept of apoptosis was first proposed to describe the unique process of cell death by Kerr and co-workers in 1972 (Kerr et al., 1972). 2. 4. Apoptosis vs. Necrosis Based on morphological and biochemical differences, there are two pathways by which cell die: necrosis and apoptosis. Necrosis is a pathological process and occurs in response to a wide variety of harmful conditions and toxic substances (Walker et al., 1988). A cell undergoing necrosis typically exhibits distinctive morphological and biochemical characteristics. The earliest changes include swelling of the cytoplasm and organelles, especially the mitochondria, as the result of loss of control of selective permeability of the plasma membrane. These changes ultimately lead to the rupture of the plasma membranes, allowing the cellular contents to leak out into the extracellular space. Thus, necrosis typically affects groups of contiguous cells, and an inflammatory reaction usually develops in the adjacent viable tissue in response to the released cellular debris (Wyllie, 1981). There are only slight changes in the nucleus during necrosis and it involves no mRNA or protein synthesis. DNA exposed by proteolytic digestion of histones is cleaved by lysosomal deoxyribonuclease into fragments 15 displaying a continuous spectrum of sizes. Also, ATP levels are usually decreased in this "passive" cell death process (Duvall and Wyllie, 1986). As opposed to the injury-induced death of necrosis, apoptosis is an active, intrinsic, genetically governed process of selective cell deletion that take place in tissues underoging developmental changes or responding to alternations in physiological stimuli (Wyllie, 1981). Ultrastructurally, the earliest changes of apoptosis include the loss of the cell-cell junctions and other specialized plasma membrane structures such as microvilli. The chromatin rapidly forms dense and crescent-shaped aggregates lining the nuclear membrane. At the same time cytoplasm condenses. As the process continues the nucleus coalesces into one large mass and then breaks into several fragments. The cytoplasmic condensation increases and plasma membrane shows shrinkage and blebbing. Subsequently, the cell breaks up into several membrane-bounded apoptotic bodies that contain a variety of intact cytoplasmic organelles and some nuclear fragments. Apoptotic bodies vary greatly in size, and the number of apoptotic bodies produced from a dying cell is related to the size of the cell. Apoptotic bodies are quickly phagocytosed by nearby cells where they are degraded. Because phagocytosis usually takes place before the integrity of the plasma membrane is lost, there is no leakage of cytoplasmic components, and therefore, no inflammation reaction is induced (Schwartzman and Cidlowski, 1993, Vinatier et al., 1996). However, in some instances apoptotic bodies are not subjected to phagocytosis but instead are extruded into an adjacent lumen and undergo "secondary necrosis", a process that apoptotic bodies show progressive dilation and degradation of cytoplasmic organelles (Don et al., 1977). The process of apoptosis occurs extremely rapidly. Once initiated, cellular fragmentation is completed within several minutes (Matter, 1989). The short duration, along with the lack of inflammation, makes it difficult to observe the death process. For these 16 reasons, quantification of apoptosis occurring in tissues is often difficult. However, advances in biochemical studies have made the study easier. In 1980, Wyllie identified the presence of low molecular weight DNA in apoptotic cells for the first time. The DNA degradation occurred in a very specific pattern producing fragments of DNA that were multiples of 180-200 bp, the size of nucleosomes. This phenomenon is most often analyzed by agarose gel electrophoresis which measures DNA fragmentation in nuclear extracts showing the typical "DNA ladder" configuration contrasting with the migration of the randomized breakdown observed during necrosis. This suggests that the digestion involve nuclease activity without concurrent protease activity. A Ca27Mg2+-dependent endonuclease capable of generating characteristic apoptotic chromatin cleavage has been demonstrated constitutively within nuclei from a variety of cell types. Also, internucleosomal chromatin degradation has also been detected in nearly all observations of apoptotic cells. All these indicate that there may be a common mechanism by which apoptosis occurs in different cell types and DNA fragmentation is a reliable diagnostic for the occurrence of apoptosis (Schwartzman and Cidlowski, 1993, Vinatier et al., 1996). 2. 5. Triggers of Apoptosis With more studies on apoptosis, an increasing number of triggers of apoptosis have been reported and can be divided into several general groups in connection with physiological cell death in mammalian cells (Thompson, 1995; Jurisicova et al., 1995; Vinatier, 1996). The largest group includes growth factors and hormones. Lack of, or overexposure to them will trigger apoptosis by binding to their respective membrane-bound or intracellular receptors and eliciting changes in the intracellular environment. Another group is cytokines. 17 In this case, target cells undergo apoptosis in response to intracellular changes caused by interaction with cytotoxic cells (Thompson, 1995). The third group is reactive oxygen species (ROS) such as H 20 2 , which are by-products generated in cells through normal metabolic activity, hormone-mediated phospholipase activity and lipid peroxidation. Factors involved in destabilizing the optimal redox state of a cell, such as an increase in ROS or a decrease in intracellular antioxidants, will lead to a redox imbalance and cell death (Buttke and Sandstrom, 1994). By adjusting culture conditions, the negative effects of the in-vitro environment may be reduced or eliminated. For example, glutathione may prevent the induction of cell death triggered by ROS. In addition, certain antioxidants can prevent cell suicide caused by cytokine deprivation. However, although ROS-generating agents are potent inducers of cell death in many situations, they are not common to all cases of apoptosis. For example, it has been shown that cells grown under anaerobic conditions will die when induced with staurosporine, but are resistant to cell death induced by ROS-generating agents (Jacobson and Raff, 1995). Finally, calcium disturbances also have a regulatory function in apoptosis by controlling cell cycle. A sustained increase in intracellular Ca 2 + is one of the first detectable events observed in cells undergoing apoptosis after exposure to a variety of different stimuli (Cohen and Duke, 1996). However, similar to ROS, increased calcium does not seem to be a universal requirement for initiation of cell death (Cohen and Duke, 1996). 2. 6. Gene Regulation of Apoptosis The requirement for active RNA and protein synthesis during apoptosis of thymocytes (Cohen and Duke, 1984) and other cells (Oppenheim et al., 1990) has led many investigators to seek the genes and their products that regulate apoptosis. To date, a number 18 of genes have been identified as being involved in the apoptosis in different tissues and species. These genes either promote or inhibit apoptotic cell death machinery. From these studies, basic mechanisms have emerged. Although the array of potential apoptotic signals is vast and varies with cell types, all apoptotic pathways appear to terminate in the activation of a series of cytosolic proteases, the caspases (Salveson and Dixit, 1997; Thornberry and Lazebnik, 1998). The caspase family was originally discovered following a search for mammalian homologs of ced-3, a cell death gene described in the nematode worm Caenorhabditis elegans. The first mammalian caspase identified was interleukin-1-conveting enzyme (ICE), now known as caspase-1. Numerous other caspases (Caspase-2 to Caspase-13) have been discovered and has been given a variety of names. Caspases are synthesized as inactive precursors that must be cleaved autocatalytically or by other caspases for activation. Triggering of apoptosis results in a cascade of caspase activation, in which the last caspases to be activated are those that digest cellular substrates resulting in morphological changes and death of the cell. Caspase activation can be regulated in different ways. The regulation is simple in Caenorhabditis elegans, where apoptosis requires only the caspase, ced-3, and its activator ced-4, whose action can be counteracted by ced-9 (Hengartner et al., 1992), the nematode counterpart of Bcl-2 (B-cell lymphoma/leukemia-2 gene) which blocks apoptosis in many cells when overexpressed (Vaux et al., 1992). In mammalian species, the pathway is much more complicated and is not completely clear. Mammals possess multiple capsases and Bcl-2 homologues. At least 15 mammalian Bcl-2 family members have been identified and categorized into two subgroups: those that exert anti-apoptotic effects and those that are pro-apoptotic. Although anti-apoptotic homologues can form dimers with pro-apoptotic members, it is controversial whether or not dimerization is required for activity. What is clear, however, is that in most cases the ratio of pro-19 apoptotic to anti-apoptotic Bcl-2 homologues within a cell determines whether the cell will live or die. Examination of caspases in mammalian cells indicates that Bcl-2 inhibits apoptosis through blocking activation of caspases whereas Bax (Bel-associated x gene), one of the pro-apoptotic members, activate caspases and thus induce death (Reed, 1994; Allen et al., 1998). These suggest that the Bcl-2 family of proteins may regulate caspase activity induced by various stimuli (including apoptotic signaling originating due to changes in plasma membrane receptors like FAS/FASL and transcription factors like p53) and act as an active checkpoint of apoptosis. (Allen et al., 1998; Thornberry and Lazebnik, 1998). I l l Apoptosis: an Underlying Mechanism of Follicular Atresia 2. 7. Apoptosis in Follicular Atresia The ovary is a dynamic tissue that undergoes continuous remodeling of its compartments throughout its lifespan. With each cycle, a cohort of follicles begins to develop, but only one will reach dominance and ovulation, while the majority of ovarian follicles will die through the process of follicular atresia. How does the ovary eliminate old atretic follicles without tissue damage or inflammation? Apoptosis, a process of physiological cell death, could be an efficient way. In 1978, O'Shea et al. pointed out that the degenerative changes occurring in ovine theca interna cells during atresia are identical to the characteristics of apoptotic changes based on a purely morphological study. Ten years later, Zelenik et al. (1989) identified a Ca27Mg2+-dependent endonuclease activity in rat ovaries, demonstrating for the first time that the ovary has the potential for apoptotic DNA fragmentation. After that, with the advance of molecular genetics, using different techniques and different animal models a clear association between apoptosis and follicular atresia has been established. GCs 20 harvested from immature, untreated rat ovaries consistently display laddering of their DNA (Hughes and Gorospe, 1992; Tilly et al., 1992): The results are in accordance with the presence of atretic follicles in prepubertal rat ovaries, suggesting that apoptosis is an ongoing process in ovarian GCs, mostly likely related to atresia. Tilly et al. (1991) identified internucleosomal DNA fragmentation in atretic pig follicles, but not in developing healthy follicles. However, in the sheep ovary, GC apoptosis is found in atretic as well as some morphologically healthy follicles (Jolly et al., 1997). Species difference may exist. So far, research on apoptosis in follicular artresia is just beginning, and most of the data are descriptive. Molecular mechanisms, i.e., which genes are involved and which are the modulating factors, are unknown or preliminary. 2. 8. Hormonal Control of Follicular Atresia Three theoretical models have been proposed for the determination of follicle fate: 1) The follicles undergoing atresia may be predetermined by inherent deficiency of the oocyte, follicle cells, or their immediate environment. Because the majority of follicles are capable of growth under appropriate hormonal stimulation, this mechanism is unlikely to be the basis for the atresia of most follicles. 2) Most, if not all, follicles are capable of reaching ovulation unless atresia is triggered by certain atretogenic stimuli, like GnRH-like substances and androgen discussed below. 3) The normal fate of all follicles is atresia. Only follicles reaching a specific stage of development, coinciding with critical hormonal signals, can escape atresia. As discussed below, gonadotropins, estrogens, and several growth factors are follicle survival factors. 2. 8.1. Role of Gonadotropins In addition to triggering the ovulatory process, gonadotropins are necessary for the growth and development of ovarian follicles. Hypophysectomy can result in the arrest of antral follicular growth (Gulyas et al., 1977). Also, atresia of large preovulatory follicles in rats and hamsters has been induced after abolishment of gonadotropin surges by hypophysectomy or administration of antibodies to gonadotropins (Braw et al., 1981; Terranova, 1981). Furthermore, early atretic follicles can be rescued by the administration of exogenous gonadotropins (Braw and Tsafriri, 1980). Measurement of DNA fragmentation indicated that treatment of hypophysectomized immature rats with FSH decreases follicular apoptosis (Billing et al., 1994). Preovulatory follicles contain predominantly intact high molecular weight DNA. However, if preovulatory follicles are cultured in serum-free medium, a time-dependent spontaneous onset of apoptosis will occur. Of interest, treatment with either FSH or human chorionic gonadotropin/luteinizing hormone (hCG/LH) prevents the spontaneous onset of apoptosis (Chun et al., 1994). All together, it underscores the role of gonadotropins as follicle survival factors through preventing apoptosis. 2. 8. 2. Role of Steroid Hormones In the ovary, steroid hormones are important intra-ovarian regulators of follicular development and atresia. It has also long been known steroid hormones can regulate the synthesis and secretion of pituitary gonadotrophins through feedback on the hypothalamic-pitutitary axis. Progesterone exerts a negative effect on LH secretion by decreasing the LH pulse frequency and the effect of P4 is probably primarily at the level of the hypothalamus. This 22 has been demonstrated in various species including cows (Ireland and Roche, 1982; Roberson et al., 1989). The negative effect of P4 does not appear to extend to FSH. Treatment of ovariectomized ewes with P4 does not decrease circulating FSH (Nett et al., 1981; Ireland and Roche, 1982). Feedback of estrogen (E2) on the hypothalamic-pitutitary axis is more complicated than that of P4. Estrogen has been shown to have both negative and positive feedback effects (Roche et al., 1970; Gregg and Nett, 1989; Leung and Steele, 1992). Negative effects of E 2 would appear to be due to a decrease in GnRH secretion from the hypothalamus leading to decreased synthesis and secretion of gonadotropins. The positive feedback effect of E 2 that occurs during gonadotropin surges is probably regulated at both the hypothalamic and pituitary levels. Immediately preceding the gonadotropin surge, E 2 increases GnRH secretion from the hypothalamus by increasing both GnRH pulse frequency and amplitude, resulting in a GnRH surge which in turn stimulates the gonadotropin surge. Also, E 2 acts at the level of the pituitary to increase the number of GnRH receptors, thus making the pituitary more sensitive to GnRH (Leung and Steele, 1992). Steroid hormones also have direct effects on the ovary. Treatment with E 2 increases follicular growth and the division of GCs (Williams, 1940; Payne and Hellbaum, 1955). In contrast, androgen not only inhibits aromatase activity and stimulate P4 production in GCs (Hillier et al., 1980), but also decreases ovarian weight and induces follicular atresia by increasing the number of pyknotic GCs and degenerated oocytes in rats (Azzolin and Saiduddin, 1983). It has also been shown that P4 inhibits GC aromatase activity in cows (Manikkam and Rajamahendran, 1997). Furthermore, atretic follicles exhibit decreased E 2 production and a lower E 2 : P4 (or androgen) ratio in the follicular fluid, suggesting the importance of local E 2 for the maintenance of healthy follicles and a possible role for P4 or 23 androgen in the progression of the atresia process (Carson et al., 1981, Manikkam and Rajamahendran, 1997). Gonadal steroids are efficient regulators of ovarian apoptotic cell death. Billig et al. (1993) demonstrated when immature, hypophysectomized rats were treated with an estrogen for two days, and when followed by estrogen removal, apoptotic DNA fragmentation increased in the ovary and the apoptosis occurred only in GCs. In contrast, replacement of estrogen completely prevented the ovarian weight loss and increases in GC apoptosis. On the other hand, testosterone treatment increased apoptosis in GCs. The specificity of the testosterone action was further demonstrated by the lack of effect of P4 on ovarian apoptosis. However, Peluso and Pappalardo (1994 and 1999) showed that P4 decreased apoptosis in cultured GC. Progesterone stimulates the synthesis of basic fibroblast growth factor (bFGF) in small GCs. bFGF then activates its receptors within large GCs, and this initiates a signal transduction pathway that maintains large GC viability (Peluso and Pappalardo, 1999). The effects of P4 on ovarian apoptosis remain controversial. 2. 8. 3. Role of Insulin-like Growth Factors (IGFs) The insulin-like growth factors (IGF-I and IGF-II) comprise another class of intra-ovarian regulators. The IGFs exert their effects by binding and activating specific cell surface receptors; IGF-I preferentially binds to the type-I receptor while IGF-II preferentially binds to the type-II receptor. However, IGF-I, IGF-II and insulin have similar structures and thus result in cross reactivity between the IGFs, insulin and their respective receptors (Giudice, 1992). Hammond (1981) showed that concentration of immunoreactive IGF-I was much higher in the follicular fluid than in serum, suggesting for the first time that IGFs may be 24 produced in the ovary. The presence and functions of IGFs have then been demonstrated in the ovary of many species (Giudice, 1992). IGF-I stimulates GC proliferation and promote GC steroidogenesis (Olsson et al., 1990; Spicer et al., 1993). In addition, IGF-I acts to amplify the actions of gonadotropins. IGF-I increases the FSH-stimulated induction of GC LH receptors (Adashi et al., 1985), FSH-stimulated inhibin synthesis (Bicsak et al., 1986), as well as FSH-stimulated GC P4 and E 2 production, suggesting IGF-I plays an important intra-ovarian role in regulating follicular development and atresia. Less is known about the effects of IGF-II, although it does stimulate GC P4 production (Veldhuis et al., 1985). IGFs in biological fluid are bound to IGF binding proteins (IGFBPs). There are six IGFBPs that have been cloned and sequenced in the rat and human (Shimasaki and Ling, 1991, Giudice, 1992). Although circulating IGFBPs prolong the half-life of IGFs, these preotins mostly inhibit the actions of IGFs. High levels of IGFBPs have been detected in atretic follicles. FSH treatment decreases secretion of IGFBP- 4 and -5 by rat GC (Giudice, 1992). Furthermore, the binding of endogenously produced IGF-I by exogenously added IGFBP-3 results in the suppression of gonadotropin stimulation of follicular growth and subsequent ovulation (Giudice, 1992). In preovulatory follicle culture, similar to FSH and hCG, IGF-I has been shown to prevent the spontaneous onset of apoptosis (Chun et al., 1994). However, the suppressive effect of hCG on follicle DNA fragmentation is prevented by co-treatment with IGFBP-3, and treatment with FSH and IGFBP-3 does not suppress apoptosis (Chun et al., 1994). All these data together suggest that IGF-I may be a survival factor for ovarian follicle, and part of the physiological effects of FSH may be mediated by IGF-I. 25 2. 8. 4. Role of Other Hormones Gonadotropin-releasing hormone (GnRH) has direct inhibitory effect on follicle differentiation. Indeed, the highest expression of GnRH receptors was recently demonstrated in atretic follicles (Whitelaw et al., 1995). Treatment with a GnRH agonist has been shown to directly induce ovarian apoptotic DNA fragmentation, and apoptotic cell death is confined to the GC due to activated endonuclease activity (Billig et al., 1994; Erickson et al., 1994). Because GnRH is known to increase intracellular Ca 2 +, The action of GnRH on endonuclease activity has been thought to be mediated through a Ca2+-dependent pathway (Billig et al., 1994; Erickson et al., 1994). Treatment of cultured GC with epidermal growth factor (EGF), transforming growth factor-a (TGF-a), and basic fibroblast growth factor (bFGF) inhibit the spontaneous apoptosis occurring in cultured GC or follicles. However, inclusion of tyrosine kinase inhibitor (genistein) completely blocks their suppressive effects on apoptosis (Tilly et al., 1992). EGF appears to be produced in theca cells. So far, its function in thecal cells is not clear. In GC, EGF stimulates growth as well as increase in FSH binding. TGF-a belongs to the TGF family. It binds to receptors for EGF, producing responses similar to that seen for EGF. High affinity receptors for EGF/TGF-ct and bFGF have been localized to GC (Hsu et al., 1987). Therefore, it has been suggested that GC apoptosis is prevented by the paracrine and/or autocrine action of EGF, TGF-a, and bFGF. Inhibin and activin are peptide hormones that can regulate gonadotropin secretion (Ling et al., 1986; Vale et al., 1986). Inhibins are heterodimers with a common a-subunit 26 and one of the two p-subunits (P A or P B). In contrast, activins are formed by the dimerization of inhibin p-chains (pApA, PAPB> a n d PBPB)- Inhibins are potent inhibitors of pituitary FSH release through negative feedback. It has been found that circulating inhibin and FSH are generally inversely related (Russell et al., 1994). Injection of recombinant human inhibin into the ovarian bursa of immature rats increases the number of medium-sized antral follicles. In contrast, activins stimulate the release of pituitary FSH without affecting LH. Injection of recombinant human activin cause follicular atresia (Woodruff et al., 1990). Moreover, inhibin production by GC is stimulated by gonadotropins (Bicsak et al., 1986). Thus, the effect of gonadotropins on follicular development and atresia may involve paracrine actions of inhibins and activins. Recent studies suggested that cytokines may exert regulatory roles in the ovary. Tumor necrosis factor-a (TNF-a), a cytokine expressed in the oocyte and GCs, signals through type I TNF-a receptor that contains a death domain and stimulates apoptosis in cultured ovarian follicles. Furthermore, the action of TNF-a on follicular apoptosis is mimicked by its second messenger, ceramide (Kaipia et al., 1996). Fas is a 45-kDa transmembrane receptor that induces apoptosis when activated by Fas lig'and (FasL). Both Fas and (FasL) have been detected in the ovary (Nagata, 1997). In addition, it has been demonstrated that the immunoreactivities of rat GC to both Fas and FasL is consistent with the localization of apoptosis and is well correlated with follicular atresia. Immunoreactivity was more intense in atretic follicles. In contrast, Fas and FasL were either undetectable or low in the GC of healthy follicles (Kim et al., 1998). Therefore, the activation of the Fas and FasL system may play an important role in the initiation of follicular apoptosis and follicular 27 atresia. Moreover, cloning of the Fas antigen reveals its similarity to the TNF-oc receptor, whereas isolation of FasL indicates that it is a protein homologous to members of the TNF-a family (Suda et al., 1993). Interleukin-6, another cytokine produced by GC, also induce DNA fragmentation in cultured GC, suggesting its role as a potential atretogenic factor (Gorospe etal., 1992). 2. 8. 5. Gene Regulation of follicular Atresia It is clear that gonadotropins, steroid hormones, and various growth factors play important roles in the regulation of follicle apoptosis and atresia. However, the mechanisms by which hormonal factors regulate apoptosis are not well understood, and only limited data on genes involved in the control of apoptosis in ovary have been reported. Although the decreased expression of certain ovarian genes, such as aromatase and gonadotropin receptors, as well as the increased expression of other ovarian genes such as IGFBPs, are associated with the initiation of follicle apoptosis and atresia (Tilly et al., 1992), most of these genes probably only play a role in the facilitation or suppression of apoptosis, but are not obligatory in the control of follicular growth or demise. It has been shown that proteins of the Bcl-2 gene family and caspase gene family are likely to be direct regulators of apoptosis onset in many cell types. Although Bcl-2 protein was not found in the human ovary (Reed et al., 1991), a recent report has suggested that Bcl-2 mRNA is expressed in rat ovaries. Additionally, the inhibitory effect on apoptosis of gonadotropins is associated with a marked reduction in the expression of Bax, another member of Bcl-2 gene family that antagonizes Bcl-2 activity (Tilly et al., 1995). Furthermore, ablation of Bcl-2 expression decreases the number of oocytes and primordial follicles in rats (Ratts et al., 1995). 28 The tumor suppressor gene, p53, is expressed in GCs. Apoptosis observed in antral follicles induced in serum-free medium is associated with a significant increase in p53 mRNA levels compared to those in healthy follicles without apoptosis, suggesting a correlation between apoptosis and expression of p53 in the ovary (Tilly et al., 1995). The mechanism involved in the induction of apoptosis by p53 in follicle is not clear. However, it has been shown that concomitant with reduced levels of p53, expression of Bax is also markedly reduced in rat GC after treatment with gonadotropins (Tilly et al., 1995; Xiao et al., 1999). It is possible that Bax gene may be a target for p53 transcription and thus loss of p53 may contribute to decreased expression of Bax in ovary, as has been reported for other cell types (Miyashita and Reed, 1993). Several genes of the caspase family are expressed in the ovary (Flaws et al., 1995; Exley et al., 1999). Gonadotropin treatment of rat follicles significantly decreases the levels of transcription of Caspase-1 and -3, suggesting these genes are involved in apoptosis. Recently, a novel family of intracellular proteins, inhibitors of apoptosis proteins (IAPs), has been reported to suppress apoptosis (Liston et al., 1996). Five IAPs have been identified in the mammal: neuronal apoptosis inhibitory protein (NAIP), X-linked inhibitor of apoptosis protein (XIAP), human inhibitor of apoptosis protein-1 (HIAP-1), human inhibitor of apoptosis protein-2 (HIAP-2), and survivin (Liston et al., 1996). These proteins have been detected in whole ovarian extracts and XIAP and HIAP-2 expression are closely related to GC apoptosis. The mechanism(s) by which IAPs regulate apoptosis is not clear (Xiao et al., 1999). 29 IV APOPTOSIS IN OOCYTE AND EMBRYO DEGENERATION 2.9. Oocyte Degeneration Besides follicular degeneration (atresia), another major stage of cell degeneration can be distinguished during follicle formation and growth is degeneration of germ cells (attrition), which accounts for the major loss of oocytes. In the 20-week-old human female embryo, the number of germ cells is around 5 million, and this number is reduced to about 1 million by the time of birth (Siracusa et al., 1985). Shortly after birth, most oocytes are surrounded by follicular cells to form the primordial follicles; those (50-70% in rats) that remain naked usually degenerate (Ohno and Smith, 1964). Furthermore, depending on the species, the period during which mammalian oocytes are capable of being fertilized is thought to be only 8-30 hr, oocytes which are not fertilized undergo degeneration (Odell and Moyer, 1971; Siracusa et al., 1985). Little is know about the mechanisms underlying the degeneration of unfertilized occytes. 2.10. Oocyte Classification Schemes The development of in vitro techniques for the laboratory production of animal embryos has excellent potential, both for basic research and for practical applications. In many laboratories, embryos are routinely obtained from oocytes after in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro embryo culture (IVC). Therefore, the proper selection of developmentally competent oocytes is crucial for successful in vitro embryo production. The possibility of selecting good oocytes based on morphology was initially investigated for cattle in 1979 (Leibfried and First, 1979). Since that time, many reports have indicated that classification of bovine oocytes, based on visual assessment of the compactness of the cumulus investment as well as the homogeneity and transparency of the ooplasm, can be 30 used to select immature oocytes for optimum maturation, fertilization, and development in vitro (Loos et al., 1989; Younis et al., 1989; Lonergan et al., 1992; Madison et al., 1992; Brackett and Zuelke, 1993). Some of the classification schemes suggested by researchers for cumulus oocyte complex (COC) classification are shown in Tale 2.1. In addition, a clear relationship between oocyte morphology and embryo yield after IVM/IVF/IVC has also been established (Lonergan et al, 1992; Table 2.2), indicating an oocyte classification scheme with a good predictive value is of considerable importance in embryo production system. Bovine oocytes can be broadly categorized into those which are acceptable and those that are not (see Tale 2.1). The authors have shown that unacceptable-quality oocytes should not be selected for IVM because of the decreased capacity to mature and very low embryo production rate in vitro. Reasons for the significant differences in fertilization rate and embryo yield caused by different oocyte types used in IVM are unknown. Related to physiological conditions, one possible explanation may be those oocytes are at different levels of degeneration through apoptosis. 2.11. Embryo Fragmentation In spite of the above selection criteria, the quality of mammalian preimplantation embryos obtained under in vitro culture conditions are variable. Furthermore, the appearance of cellular fragmentation during early embryo development in vitro has been commonly observed in pigs (Long et al., 1998) and humans (Hardy et al., 1989; Jurisicova et al., 1995). These embryos contain irregular sized blastomeres, multiple cellular fragmentation, and will often arrest in development with subsequent degeneration (Kruip and den Daas, 1997). Fragmented embryos reach the blastocyst stage less frequently (Jurisicova et al., 1995). Moreover, fragmented embryos have an increased sensitivity to damage due to 31 Table 2.1. Oocyte Quality as Determined by Morphological Examination Category Cumulus-oocyte-complexes (COCs) Criteria as employed by Loos et al. (1989) 1. Compact multilayed cumulus investment; homogeneous ooplasm; total COC light. 2. Compact multilayed cumulus investment; homogeneous ooplasm but with a coarse appearance and a darker zone at the periphery of the oocyte; total COC slighter darker. 3. Less compact cumulus investment; ooplasm irregular with dark clusters; total COC darker than 1 or 2 above. 4. Expanded cumulus investment; cumulus cells scattered in dark clumps in a jelly matrix; ooplasm irregular with dark clusters; total COC dark and irrgular. t Criteria as employed by Younis et al. (1989) • Selected: Homogeneous-appearing ooplasm and compact cumulus cells tightly adherent to the zona pellucida. • Unselected: other categories with incomplete cumulus complements and heterogeneous ooplasm. Criteria as employed by Lonergan et al. (1992) Acceptable: 1) Oocytes showing many tight layers of cumulus cells. 2) Oocytes showing three to four cumulus cell layers. 3) Oocytes showing two to three cumulus cell layers. Unacceptable: 1) Oocytes with expanded cumulus cells, with the cells scattered in dark clumps in a j elly-like matrix. 2) Denuded oocytes. 32 cryopreservation and manipulation, and therefore, are associated with poor survival following cryopreservation and with decreased pregnancy rates following embryo transfer (Jurisicova et al., 1995; Kruip and den Daas, 1997). Recent studies using pig and human fragmented embryos have revealed that some of the fragmented cells have condensed cytoplasm, chromatin, and membrane-bounded structures. These observations strongly suggest the occurrence of apoptosis in fragmented embryos (Hardy et al., 1989; Jurisicova et al., 1995; Long et al., 1998). Therefore, apoptosis may contribute to the abnormalities observed during embryo development. 2.12. Involvement of Apoptosis During in vitro Embryo Development Embryos can fragment and die through apoptosis, suggesting a natural 'pre-programmed' response to external stimuli or internal defects. Several studies have shown that nearly all in vitro produced blastocysts display more apoptotic cells than do in vivo derived embryos at a similar stage (Dobrinsky and Johnson, 1996; Du et al., 1996; Long et al., 1998). This may be the reason for in vitro produced embryos from several mammalian species demonstrating higher rate of embryo fragmentation and retarded developmental progress when compared to in vivo derived embryos (Long et al., 1998). All these data indicate that the in vitro embryo production process may be at least partially to be blamed for triggering apoptosis and embryo fragmentation. For example, state of oocytes, exposure to excessive number of spermatozoa, and inappropriate culture conditions may all contribute to excessive activation of apoptosis. However, all these remain to be fully investigated. Very little is known about the mechanism by which apoptosis is executed and controlled during embryo development. 34 'CHAPTER 3 MORPHOLOGICAL AND BIOCHEMICAL IDENTIFICATION OF APOPTOSIS IN SMALL, MEDIUM, AND LARGE BOVINE FOLLICLES AND THE EFFECTS OF FSH AND IGF-I ON SPONTANEOUS APOPTOSIS IN CULTURED BOVINE GRANULOSA CELLS ABSTRACT The first objective of this study was to determine whether the death of bovine granulosa cells (GC) isolated from small (< 4 mm), medium (5-8 mm) and large (> 8 mm) follicles during follicular atresia occurs by apoptosis. The second objective was to establish an in vitro model system to elucidate the developmental (GC from follicles of different sizes) and hormonal [follicle stimulating hormone (FSH) and insulin-like growth factor-I (IGF-I)] regulation of bovine GC apoptosis during follicular atresia. Bovine ovaries were obtained from a nearby slaughterhouse. Follicles were classified by morphometric criteria as healthy or atretic. Apoptosis in GC from follicles of different sizes was analyzed by both morphological and biochemical methods. Bovine GC were cultured for 48 h at a density of 5 x 106 cells/ml in serum-free medium at 39 °C to determine the effects of FSH and IGF-I on apoptosis. The results showed that apoptosis occurred in GC from all sizes of follicles. Apoptosis in GC was also detected in some healthy follicles. Degenerate GC displayed the morphological characteristics of apoptosis, including nuclei with marginated chromatin, a single condensed nucleus, multiple nuclear fragments and/or membrane-bounded structure containing variable amount of chromatin and/or cytoplasm (apoptotic bodies). All GC 1 Yang M Y and Rajamahendran R, Biol Reprod 2000; 62: 1209-1217. 35 classified as apoptotic based on their morphology contained fragmented DNA measured by TUNEL technique. Cells that had undergone apoptosis were observed mainly in GC and in scattered theca cells. Throughout the GC layer, apoptotic cell death was more prevalent among antral GC than among mural GC. Interestingly, morphological results showed that no apoptosis occurred in cumulus cells. A time-dependent, spontaneous onset of apoptosis occurred in GC from small, medium and large follicles during in vitro serum-free culture. The rate of DNA fragmentation in the culture of GC from small follicles was higher than that from medium and large follicles. FSH attenuated apoptotic cell death in GC from medium follicles more effectively than those from small follicles. IGF-I also suppressed apoptosis in cultured GC from small follicles. In conclusion, this study showed that: 1) GC death during bovine follicular development and atresia occurs by apoptosis; 2) apoptosis occurs in GC and theca cells, however, apoptosis does not occur in cumulus cells even in atretic antral follicles; 3) GC from all small, medium, and large follicles undergo spontaneous onset of apoptosis when cultured under serum-free conditions; and 4) FSH and IGF-I can attenuate apoptosis in cultured bovine GC. I N T R O D U C T I O N During bovine ovarian follicular development, only limited numbers of follicles (« 1%) are selected for ovulation whereas the remaining undergo atresia at various stages of follicular development (Mariana et al., 1991). Despite the overwhelming occurrence of follicular atresia in the ovary, the cellular and molecular mechanisms underlying this phenomenon still remains poorly understood although many studies on the onset and progression of follicle atresia have been done (Kaipia and Hsueh, 1997). 36 Previous studies have suggested that degenerative changes associated with atresia appear initially in the granulosa cell (GC) layer. The death of GC lead to almost total destruction of the GC layer lining the inner follicular wall and trigger the atresia of the follicles (Hughes and Gorospe, 1991; Rajakoski, 1996). Recent studies have demonstrated that the death of GC during follicular atresia in ewe, pig, chicken, cow, and rodent ovaries occurs by apoptosis — a physiological, active, and genetically governed process whereby cells die in a controlled fashion triggered by changes in the levels of specific physiological stimuli (Arends et al., 1990; Hughes and Gorospe, 1991). A unique biochemical event in apoptosis is the activation of a Ca27Mg2+-dependent endogenous endonuclease. The enzyme cleaves genomic DNA at internucleosomal regions resulting in DNA fragments in the size of 180-200 bp. When separated by agarose gel electrophoresis, DNA fragments can be visualized as a distinctive ladder pattern. The presence of this DNA pattern in cells is considered a hallmark indicator of apoptosis (Arends et al., 1990; Schwartzman and Cidlowski, 1993; Hsueh et al., 1994). Cells undergo apoptosis under various physiological and experimental conditions and show distinctive morphological features, including condensed cytoplasm and nuclear chromatin coalesced into one or several large masses. As apoptosis continues, the nucleus breaks into several fragments, then the cell breaks up into several membrane-bound smooth-surfaced apoptotic bodies that contain a variety of intact cytoplasmic organelles and some nuclear fragments. Apoptotic bodies are typically phagocytosed by nearby cells or macrophages, or are extruded into body cavities (Arends et al., 1990; Schwartzman and Cidlowski, 1993; Hsueh et al., 1994). In cows, apoptosis has been demonstrated in GC from follicles (> 4 mm only) and CL by the DNA ladder pattern [Imig et al., 1993; Jolly et al., 1994; Rueda et al., 1997). It is known that although atresia occurs at all stages of follicle development (Mariana et al., 37 1991), the majority of follicles undergo degeneration at the early antral stage rather than the preantral and preovulatory stages (Hirshfield, 1991). Considering that atresia is a stage-dependent process, the study of apoptosis in GC from follicles at different developmental stages is important. To our knowledge, no such study has been reported. In addition to the detection of oligonucleosomes in isolated DNA, the occurrence of apoptosis may be inferred from the characteristic morphological appearance of degenerating cells, together with the detection of fragmented DNA in single cells in situ through the use of 3' end labeling technique (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling; TUNEL) (Gavrieli et al., 1992; Palumbo and Yeh, 1994). TUNEL is a direct and specific method for the in situ visualization of apoptosis at the single-cell level, while preserving tissue architecture (Gavrieli et al., 1992). Previous histological studies have identified morphological changes of atretic follicles, including the degeneration and detachment of the GC layer from the basement membrane and the presence of pyknotic nuclei (Carson et al., 1981; Hughes and Gorospe, 1991; Tilly et al., 1992). These morphological features are similar to, but not necessarily specific for apoptotic cell death. In cows, there is no report on whether the changes in the morphological features of follicular cells during atresia conform to the distinctive morphological features of apoptotic cell death. Also it is not clear whether cells or cell debris classified as apoptotic on the basis of their morphological appearance contain fragmented DNA. In addition, it is not known what cell type(s) are involved in bovine follicle apoptosis. During follicular development, FSH and insulin-like growth factor-I (IGF-I) have been considered follicle survival factors capable of stimulating estrogen production in vivo and in vitro (Adashi et al., 1985; Manikkam and Rajamahendran, 1997). Considerable evidence has indicated that the occurrence of apoptosis in individual atretic follicles was 38 correlated with decreased levels of intrafollicular estrogen [Carson et al., 1981; Tilly et al., 1992) and aromatase mRNA (Tilly et al., 1992). A recent study has found that both FSH and IGF-I attenuated apoptosis in cultured porcine GC (Guthire et al., 1997). In contrast, in rats, FSH and IGF-I did not attenuate apoptosis in isolated GC in culture. Apoptosis in rat GC was attenuated by FSH and IGF-I only when they were added to cultures of intact preovulatory follicles (Tilly et al., 1992). In the bovine, the roles of FSH and IGF-I on apoptosis in follicles are unknown. The aims of the present study were 1) to determine whether the death of bovine GC from [small (< 4 mm), medium (5-8 mm) and large (> 8 mm)] follicles during follicular atresia occurs by apoptosis in the bovine by showing: i) DNA fragments extracted from GC form a distinctive ladder pattern when separated electrophoretically; ii) morphological changes in GC during atresia conform with general criteria of apoptotic cell death tested using tissue sections stained with hematoxylin and eosin; and iii) cells classified as apoptotic based on their morphology contain fragmented DNA as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique; and 2) to establish an in vitro model system to analyze the role of FSH and IGF-I in modulating apoptosis in GC at different follicle developmental stages. MATERIALS AND METHODS Collection of Ovaries and Classification of Follicles Ovaries were collected, and follicles were classified as previously described (Yang and Rajamahendran, 1998). Briefly, ovaries were collected at a local slaughterhouse and transported to the laboratory (within 2 h of slaughter) on ice in chilled collection medium composed of Dulbecco's Modified Eagle Medium (DMEMVHam's F-12 (1:1) containing 39 0.1% (w/v) BSA, L-glutamine, and 50 pg/ml gentamicin (Sigma Chemical Co., St. Louis, MO). The follicles were classified into three groups based on surface diameters: small (< 4 mm), medium (5-8 mm) and large follicles (> 8 mm diameter). Follicle-size categories were selected on the basis of reported gonadotropin dependence and changes in the expression of steroidogenic enzyme and luteinizing hormone (LH) receptor mRNAs (Xu et al., 1995). Studies have shown that preantral follicles can grow to the antral stage without gonadotropin support. At 4 mm, follicle growth will be halted if FSH is suppressed. From around 8 mm, follicles express LH receptors on GC and require pulsatile LH stimulation to continue growing (Xu et al., 1995). Small, medium, and large follicles were then classified as healthy or atretic according to previously established morphological criteria (Metcalf, 1982). Healthy follicles had vascularized (pink- or red-colored) theca interna, clear amber follicular fluid (FF) with no debris, and contained > 25 % of the maximum number of GC that could be present for a given follicle size (> 25% Gm a x). Follicles that did not satisfy any one of these criteria were classified as atretic. Morphological Analysis Ovaries (n = 26), containing the follicles of interest (healthy or atretic), were excised with a scalpel and fixed in 4% neutral buffered formalin for 48 h. Fixed tissues were washed in PBS solution, dehydrated through a graded series of ethanol (70-100%), cleared in xylene, embedded in paraffin, and sectioned (5 pm). The sections (n = 50 from 26 ovaries) were deparaffinized in xylene, rehydrated through a graded series of ethanol (100-50%), and then stained with hematoxylin and eosin (H & E) to identify apoptotic cell death. 40 Histological Assessment of Different Types of Apoptotic Cell Death Morphological criteria of apoptotic cells and bodies described previously were used (Gavrieli et al., 1992; Jolly et al., 1997). Briefly, apoptotic cells were defined as cells with nuclei containing condensed chromatin that had either aggregated in large compact granular masses that abut on the nuclear membrane (marginated chromatin), had shrunken into a single regular shaped, dense, homogeneously staining mass (pyknotic appearance), or had fragmented into multiple densely staining masses (multiple fragments). The masses, which appeared to originate from a single cell, clustered together and were situated among and apparently not internalized by neighboring viable cells. Apoptotic bodies are remnants of apoptotic cell death. They are defined as discrete membrane-bounded structures with roughly spherical or ovoid shape containing variable amounts of condensed chromatin and/or cytoplasm dispersed in the intercellular spaces, and are either extruded into an adjacent lumen or, commonly, phagocytosed by resident tissue cells. An oil-immersion objective (lOOx) was used to observe cell structure. In Situ 3' End-labeling: Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End-labeling (TUNEL) The internucleosomal DNA fragmentation in follicles was detected using nonradioactive labeling of DNA 3'-ends (TUNEL) described by Gavrieli et al. (1992) using a FragELTM kit (Oncogene, Cambridge, MA). Briefly, interested tissue sections (n = 25) were deparaffinized and rehydrated. Sections were then washed in tris-buffered saline (TBS) and treated with 20 pg/ml proteinase K for 20 min at room temperature. Tissues were treated with 3% H 2 0 2 for 5 min to inactivate endogenous peroxidase, and their DNA was labeled at 3'-ends with biotin-deoxyUTP by incubation with the reaction buffer containing terminal 41 deoxynucleotidyl transferase enzyme for 1.5 h at 37 °C. The sections were further incubated with streptavidin-horse radish peroxidase conjugate to detect biotinylated nucleotides for 30 min at room temperature. Diaminobenzidine reacted with the labeled samples to generate an insoluble colored substrate at the site of DNA fragmentation. Finally, sections were counterstained with methyl green to aid in the morphological evaluation and characterization of normal and apoptotic cells as described previously. Negative control sections were processed identically except that the labeling enzyme (terminal deoxynucleotidyl transferase enzyme) was omitted. An oil-immersion objective (lOOx) was used to observe cell structure. Recovery of GC For each follicle size category [healthy and atretic small (n = 150), medium (n = 50), and large (n = 40) follicles], follicles were punctured with an 18-gauge needle and FF was aspirated. Granulosa cell collection medium, Ca27Mg2+-free buffer (20mM Tris, 140 mM NaCl, 2 mM EDTA, pH 7.4), was flushed in and out of the follicles repeatedly. The follicles were then cut into hemispheres, and the interior walls were gently scraped with an inoculating loop to remove GC, leaving the basement membrane and theca cells intact. Samples of medium obtained from different categories of follicles were placed in 15-ml centrifuge tubes. The GC were harvested by centrifuging at 400 x g for 10 min. Cells were washed three times with collection medium, and cell number and viability were determined using a hemocytometer and trypan blue dye exclusion method. Granulosa cells from both healthy and atretic small, medium, and large follicles were then suspended in 1 or 2 ml collection medim and snap frozen to -70 °C for subsequent DNA extraction (Tilly and Hsueh, 1993). 42 DNA Extraction The DNA isolation procedure was adapted from Tilly and Hsueh (1993). Cells were snap-frozen and stored at -70 °C to prevent nonspecific activation of DNase. Cells were first disrupted by addition of homogenization buffer and repeated passage through a pipette. Homogenates were then lysed. The DNA was extracted by the phenol/chloroform/isoamyl alcohol (25:24:1, v:v:v) method and quantitated by absorbance at 260 nm. Agarose Gel Electrophoresis The DNA was separated (10-20 pg/lane) according to size in a 2% agarose gel by electrophoresis. Gels were stained with ethidium bromide and washed in double distilled H 20. The DNA fluorescence was viewed with an UV transilluminator, and the gels were photographed (Tilly and Hsueh, 1993). DNA extraction and agarose gel electrophoresis were carried out ten times. GC Culture Conditions To establish an in vitro model system, 5 x 106 viable GC from healthy small (n = 90), medium (n = 45), and large (n = 35) follicles were cultured in 6-well plates (n = 3 culture wells for each follicle size) with 2 ml DMEM/Ham's F-12 supplemented with 100 U/ml penicillin and 100 pg/ml streptomycin sulfate. The cells were incubated in a humidified 5% C0 2 atmosphere at 39 °C. After 16 h culture, dead cells were washed off. Only viable GC were left and were attached tightly to the culture plates. These GC were cultured for another 48 h in serum-free DMEM/Ham's F-12 medium. Granulosa cells from follicles of different 43 size were collected from the wells and snap-frozen at 0, 24, and 48 h (i.e., 16, 40, and 64 h from the beginning of culture) for DNA extraction. Three trials were conducted. Treatment of Cultured GC Granulosa cells from healthy small (n = 90) and medium (n = 45) follicles were cultured in the absence or presence of bFSH (1 ng/ml) (n = 3 culture wells per treatment) for 48 h in a humidified 5% C0 2 atmosphere at 39 °C. Bovine FSH was obtained from the USDA National Hormone and Pituitary Program (Bethesda, MD). To study the effects of IGF-I on apoptosis, GC from healthy small follicles (n = 90) were cultured with either 10 or 100 ng/ml IGF-I (Sigma Chemical Co., St. Louis, MO) with and without FSH (1 ng/ml) (n = 3 culture wells per treatment) for 48 h in a humidified 5% C0 2 atmosphere at 39 °C. After culture, GC were harvested, snap-frozen, and stored at -70 °C until processed for DNA extraction. The doses of FSH and IGF-I used in the cultured system were determined based on their effects on GC steroidogenesis in vitro (Adashi et al., 1985; Yang and Rajamahendran, 1998). Each experiment was repeated three times. DNA 3'-End Labeling and Quantification DNA 3'-end labeling is a simple and sensitive autoradiographic method for qualitative and quantitative analysis of apoptosis in minute quantities of tissues and cultured cells. Five hundred nanograms of DNA from samples were labeled at the 3'-end with [a32P]dideoxy-ATP (3000 Ci/nmol; Amersham, Arlington Heights, IL) using 25 U terminal transferase enzyme (Boehringer Mannheim, Laval, QC) as previously described (Tilly and Hsueh, 1993). After separation of the labeled DNA samples through 2% agarose gels, gels 44 were dried for 2.5 h without heat in a slab-gel dryer and exposed to Kodak X-OMAT films (Eastman Kodak Co., Rochester, NY) at -70 °C for 2 h. The amount of radiolabeled ddATP incorporated into low-MW DNA fraction (< 1 kb) for each sample was quantitated by cutting the individual lane containing these fractions from dried gels with a scalpel and measuring the radioactivity of these when immersed in a liquid scintillation cocktail (SCINT-A XF, Packard Instrument Company, INC., IL) in a p counter (LS 6500 scintillation system, Beckman, CA). The extent of radiolabeled incorporation into low-MW fractions was used to provide a quantitative estimate of the degree of internucleosomal DNA fragmentation among samples. Data Analysis Experiments were repeated by a minimum of three times. Representative photomicrographs and autoradiograms are presented. Quantitative data represent the mean ± SEM of three cultures expressed as percent changes compared to the molecular weight standard. The effects of cell types (GC from different size follicles), time/treatment, and cell type-by-time (or treatment) interactions on GC apoptotic death were analyzed using two-way ANOVA. One-way ANOVA was performed on data from IGF-I treatment followed by Student's t test. Ap value of < 0.05 was considered significant. RESULTS Morphological and In Situ 3' End-labeling Evidence of GC Apoptosis Follicles classified as healthy had an intact and well organized GC layer, with few pyknotic cells observed under higher magnification (Fig. 3.1a). In early atretic follicles, the GC layer had thinned considerably, and in some cases became either partially or completely 45 detached from the basement membrane (Fig. 3.1b), with a moderate number of degenerate cells and/or atretic bodies observed. With the progression of atresia, the GC layer was totally disorganized and numerous degenerate cells and/or atretic bodies were widespreaded in the follicle (Fig. 3.1c). Finally, with advanced atresia, the GC layer was virtually absent (Fig. 3.Id). Apoptotic cells and apoptotic bodies were evident in the GC of both morphologically healthy and atretic follicles (Fig. 3.2). These included cells with nuclei containing marginated chromatin (Fig. 3.2a), cells with a single small densely staining nucleus (Fig. 3.2b), cells with multiple densely staining nuclear fragments (Fig. 3.2c), and membrane-bounded structures containing condensed chromatin and/or cytoplasm (apoptotic bodies; Fig. 3.2d). Cells and subcellular structure with the above-mentioned apoptotic morphological features were found, through the TUNEL method, to contain fragmented DNA (Fig. 3.3, a, b, c, and d). Dead cells with morphological features of necrosis (swollen, irregular shape, and/or karyolysis), caused during staining, also were observed (Fig. 3.3, a, b, c, d, and e). In negative control (Fig. 3.3e), no labeling was observed indicating that the labeling procedure was specific. Within the membrana granulosa, apoptotic cells and/or bodies were more prevalent among antral GC than among mural GC (Fig. 3.4a). Many more apoptotic cells and/or apoptotic bodies within and/or along the antral border of the membrane granulosa were found in the atretic follicles than in the healthy follicles (not shown). Apoptotic cells and apoptotic bodies were mainly found in GC as compared with theca cells (Fig. 3.4b). Apoptosis of cumulus cells collected from morphological healthy and atretic follicles was also examined. No apoptotic cells or bodies were detected in oocyte-cumulus cell complex as opposed to the apoptotic cells and/or bodies observed within the GC layer. 46 ™ V7- '• ;' O H ft c « >~> ^ o £ 5J 1 3 g '3 o O - —H I o ft c£ -a 03 CJ G C3 CH X) £ CO t-t ._ . • — i HH cn P s CJ £ e -a d 03 U ft >. g . iS oV oo O CO JD o eg -a o 09 a o • -H H-CJ CJ co ca C O 2 CJ '-HH o e C J £ o co 03 CD X! g O o 13 CD 3 .N O a Xj SJ X; QO CD cS CO O o co '•3 x '% CD O o . 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CO CJ 1 3 3 ^ 3 £ o • o CD O H ^ CJ > a CJ CO CJ H -Q H C J psS cS Xi H-i 1 03 CJ x 3 X •2 CJ -a "co a o CJ g o X co C O C J '-3 5 CJ C J CL) ts HH CJ fi CD , CO a-CD HH 13 O CJ CD CD If f O CO O CD BC T3 • r - fi fe cS 3 C CJ CO I J CD ts CD C -a © £ T 3 47 48 jam* W am 1 # n C8 59 ' I •3 o a , o a* 03 ~ O H C O c/3 CO S u o o 5P § I O cn c o o 00 a rt d bo o o ^ o E c*-i -C O £ 00 G tu "S c/3 C/3 c u - a c3 c " 5 I J5 I '3 03 c/3 a s o C+H o OC' I O CU | | cn c S 2 I OB T3 0 <U 1 s I s I I « _ > — 1 c u <L> CO C/3 co <L> o * g • GO CJD > • r - o 6 a 8 M -a S ° i o cu c/3 •5b e 1 2 S 1 • g> § .3 U 2 <u ' cS > c3 g 00 t> cu .53 Z . s r C/3 < z P - a CD o I - a 3 C3 09 00 C3 C o 6 ; 00 « cn c3 1> <L> * s * U_i 00 ^ o « co a> * .2 9 »^  -s: fl 3 o «5 a g i n fl a a c co 49 50 Internucleosomal DNA Fragmentation in Pooled GC from Healthy and Atretic Small, Medium, and Large Follicles Regardless of follicle size, distinct ladder-like patterns of internucleosomal DNA fragmentation, characteristic of apoptosis, were apparent in pooled GC collected from atretic small, medium, and large follicles (Fig. 3.5a). Evidence of internucleosomal DNA fragmentation was also detected in some pooled GC collected from healthy follicles (Fig. 3.5b). Spontaneous Onset of Apoptosis in Cultured GC from Follicles at Different Developmental Stages Granulosa cells extracted at 0 h contained predominantly intact high mol wt (MW) DNA. However, the spontaneous onset of apoptosis, as evidenced by internucleosomal fragmentation of DNA into 185-bp multiples (Fig. 3.6a), was clearly visible by 24 and 48 h of culture, and the extent of DNA cleavage into low-MW fragments increased in a time-dependent manner throughout the 48 h of culture of GC from all small, medium, and large follicles (vs 0 h, p < 0.05). When comparing the increasing rate of apoptosis of GC from follicles of different sizes in serum-free culture, it was found that GC from small follicles had the highest rate (48 h vs 0 h, p < 0.05, Fig. 3.6b). Suppression of Spontaneous Onset of Apoptosis by FSH and IGF-I in Cultured GC FSH (1 ng/ml) inhibited spontaneous apoptotic DNA fragmentation in cultured GC from both small and medium follicles (p < 0.05). The inhibitory effect was more apparent on GC from medium follicles than from small follicles (Fig. 3.7). Treatment with 10 ng/ml of IGF-I suppressed apoptosis in cultured GC from small follicles (p < 0.05), while higher 51 (0 • U I U S A O B U B L | 0 p|0|) O u n ^ q . i W N Q M N M O T I S y « <+H P H '53 a o ! fe - H p d i © 2 "S o cS > c3 T3 Xi c3 tS K fi ccj a -a. 03 3 53 "3 S. o CM p a o o o N o B * m (|0J|U03 »A|;3»d t A cButqa %) Sujjsqai VHQ MN *<n 00 0 0 U Cu cci J S CCJ cr "3 54 concentrations of IGF-I (100 ng/ml) stimulated it. However, FSH (1 ng/ml) prevented the stimulatory effect of the high dose of IGF-I (100 ng/ml) (p < 0.05) (Fig. 3.8). DISCUSSION Atresia, the degeneration of ovarian follicles, occurs at all stages of follicular growth and development. However, at the early antral stage, follicles are most susceptible to atresia (Tilly et al., 1991). Recent studies suggest that apoptosis is the molecular mechanism underlying follicular atresia (Arends et al., 1990; Hughes and Gorospe, 1991; Kaipia and Hsueh, 1997). Considering the massive atresia of follicles at the penultimate stage of follicle growth under physiological conditions, studies of apoptosis in small, medium and large follicles (different developmental stage) are important. Internucleosomal DNA fragmentation has been considered to be characteristic of apoptosis and is one of the earliest events (Schwartzman and Cidlowski, 1993). In the present study, oligonucleosome formation was first detected in GC isolated from all small, medium, and large follicles classified as atretic. These results provided biochemical evidence that GC death during ovarian follicular atresia in cows occurs by apoptosis, consistent with the findings in other studies to date in cows (Imig et al., 1993; Jolly et al., 1994), ewes (Jolly et al., 1997) and rats (Hsueh et al., 1994). Internucleosomal DNA fragmentation was also detected in GC isolated from healthy follicles, indicating that apoptosis in the bovine may occur' very early in the atretic process before other morphological and biochemical signs of degeneration or dysfunction are evident. Therefore, the morphological criteria used previously (Metcalf, 1982) for classifying healthy and atretic follicles are not very accurate. My findings are consistent with one study in cows in which follicles were collected during the luteal phase (Jolly et al., 1994) and a study in ewes (Tilly 55 Bu||«qt-| VNQ MM *<>l C cu Cu) o a 1 fa t u a c u =3 l N > - cu — C3 and Hsueh, 1993). However, in another study on cows (Hsueh et al., 1994), oligonucleosomes were not detected in dominant preovulatory follicles recovered during a prostaglandian-induced follicular phase. Also, in all previous reports on rats, pigs and chickens, the presence of oligonucleosomes has been confined solely to DNA isolated from atretic follicles. In addition to the detection of oligonucleosomes in extracted DNA, the occurrence of apoptosis may also be inferred from the characteristic morphological appearance of degenerating cells, together with the detection of fragmented DNA in single cells in situ using TUNEL (Gavrieli et al., 1992; Palumbo and Yeh, 1994). Most previous studies [Imig et al., 1993; Jolly et al., 1994; Jolly et al., 1997) were done using pooled DNA extracted from ovaries or GC homogenates; therefore, morphological changes in GC undergoing apoptosis were unknown. Although the descriptions of nuclear pyknosis and the subsequent formation of atretic bodies are similar to the descriptions of cellular and nuclear changes that occur during apoptotic cell death [Kerr et al., 1972; Wyllie et al., 1980; Majno and Joris, 1995), the relationship between the prevalence of pyknosis (conventional histological staining) and apoptosis in GC is not clear in cows. Using classic histological techniques and in situ 3' end labeling (TUNEL) which can detect apoptosis precisely at the single cell level without disruption of the tissue morphology (Gavrieli et al., 1992; Palumbo and Yeh, 1994), the present study related specific morphological features of granulosa cell death in follicular atresia (nuclear pyknosis, karyorrhexis, and formation of atretic bodies) to the physiological process of apoptosis. The relationship was supported by a combination of biochemical evidence, classic histological evidence and in situ histochemical evidence of DNA fragmentation (a hallmark feature of apoptosis). Different cell appearances were observed in atretic and healthy follicles classified by morphological criteria, including cells with a single 57 shrunken and dense nucleus (pyknotic appearance) and cells with marginated chromatin and/or nuclear fragmentation. In agreement with our histological study described above, TUNEL confirmed the presence of fragmented DNA in cells with those morphological appearances in atretic and healthy follicles. These results are consistent with the results of studies in ewes (Tilly and Hsueh, 1993), but are in contrast to studies in rats, pigs, and chicken ( Hirshfield, 1991; Schwartzman and Cidlowski, 1993; Hsueh et al., 1994) which suggests that apoptosis occurs only in atretic follicles. The reason may be the different criteria used in different studies to classify follicles as healthy or atretic. Lussier et al. (1987) reported a mean prevalence of pyknotic cells of 0.13-0.67% in granulosa cell layer considered to be intact and normal in antral follicle. However, in another study in cows (Ireland and Roche, 1987), pyknotic cells were observed in GC layer in 30-60% of estrogen-active large follicles. Thus, the mere presence of pyknotic cells in granulosa cell layer does not imply that they are atretic. The actual relationship between the prevalence of pyknotic cells in the GC layer and their ability to maintain GC function is not known. However, together, the morphological and biochemical results in the present study strongly indicate that apoptosis may occur to a certain level during normal follicle growth and development and apoptotic death of GC may be detectable before other morphological and biochemical signs of degeneration appear in cows. The present study provides evidence that cells involved in apoptosis during bovine follicular atresia were mainly GC, though occasional theca interna cells also underwent apoptosis. This is consistent with the study in ewes (Jolly et al., 1997) and the concept that during the atretic process GC become pyknotic and die, whereas, most of the theca cells are reincorporated into the ovarian intersititum (Palumbo and Yeh, 1994). Apoptotic cells appeared to be disseminated throughout the GC layer. However, a higher prevalence of 58 apoptotic cells and bodies was observed in the antral GC than in mural GC. This may be due to different steroidogenic activity between antral and mural GC in the bovine (Rouillier et al., 1996). In addition, a recent study suggests that the follicular basement membrane plays an important role in transmitting survival signals and in prevention of apoptosis (Amsterdam et al., 1998). In our study, no apoptotic cumulus cells in the oocyte-cumulus cell complex in atretic follicles were observed, even though scattered GC with condensed nuclei and DNA fragmentation were observed in the same follicle. To our knowledge, similar data have not previously been reported for cows. Cumulus cells are a subpopulation of GC that are extruded normally from the follicle along with the oocyte. Dimfeld et al. (1993) reported that in humans, cumulus cells have less steroidogenic capability compared with GC. The process of apoptosis in follicles is associated with decreased levels of aromatase mRNA and FSH- and LH-receptor mRNAs, and these decreased levels are consistent with the decreased response of GC to gonadotropins and decreased estrogen concentrations in FF (Tilly et al., 1992). Therefore, extra-follicular factors probably affect mural GC, antral GC, and cumulus cells differently. In addition, biochemical studies have provided evidence that cells undergoing apoptosis exhibit distinct morphological changes and DNA fragmentation that may be regulated by an endogenous neutral Ca2+/Mg2+-dependent endonuclease (Petisch et al., 1994). A recent study has shown that high activity of neutral Ca2+/Mg2+-dependent endonuclease was noted only in GC and not in cumulus cells in porcine atretic follicles (Manabe et al., 1996). Identification of endocrine or paracrine factors which modulate apoptosis in ovarian cells can provide a basis for elucidating the hormonal regulation of follicle atresia. To study the hormonal regulation of follicle atresia further, an in vitro model system based on serum-free culture of GC developed in the previous study (Yang and Rajamahendran, 1998) was 59 established to examine stage-dependent differences in the hormonal regulation of follicle apoptosis. Granulosa cells isolated from small, medium, and large follicles as classified by their gonadotropin dependence and changes in the expression of steroidogenic enzymes were used to represent different follicular developmental stages (Xu et al., 1995). A spontaneous onset of apoptotic cell death was observed from cultured GC in all classes. The reason for the spontaneous onset of apoptotic cell death during the culture may be that increases in nuclear cation (Ca27 Mg2+) levels in cultured GC activated the existing endonuclease, resulting in DNA fragmentation (Tilly et al., 1992). These results are similar to those in rats and pigs (Tilly et al., 1992; Guthire et al., 1997) and may indicate that survival factors are required by GC to overcome apoptotic cell death. With time of culture, the level of spontaneous onset of apoptotic cell death increased. The increased rate of spontaneous apoptosis in the culture of GC from small follicles was highest. This suggested that follicular apoptosis may be stage-specifically regulated and explains the reason why the antral transition is the "bottle neck" for developing follicles (Hirshfield, 1991). Preantral follicles, although responsive to gonadotropins, can grow until the antral stage without gonadotropin support. Once antral (4 mm onwards), follicular growth will be halted if FSH is suppressed (Xu et al., 1995). In the present study, FSH treatment significantly suppressed apoptosis in the culture of GC from medium follicles compared to the culture of GC from small follicles. This suggested that FSH is a follicle survival factor and also indicated again that follicular apoptosis is stage-specifically regulated. Likewise, Chun et al. (1996) reported that LH/hCG had a significant suppressive effect on apoptosis of preovulatory follicles, whereas only a marginal effect on apoptosis in the early antral follicles. LH/hCG receptors are predominantly in only theca cells of small antral follicles and in both granulosa and theca cells of preovulatory follicles (Adashi et al., 1985). In 60 addition to gonadotropins, the development of ovarian follicles is also controlled by locally produced intraovarian regulators such as growth factors and cytokines, that act in a paracrine/autocrine manner. Insulin-like growth factor-I can amplify the actions of gonadotropins (Adashi et al., 1985; Richards, 1994). In our study, IGF-I (10 ng/ml) attenuated apoptosis while IGF-I (100 ng/ml) increased apoptosis in cultured GC, and a combination treatment of FSH (1 ng/ml) and IGF-I (100 ng/ml) inhibited apoptosis. Our findings were similar to that in pigs in that both FSH and IGF-I (0-250 ng/ml) suppressed apoptosis in cultured GC (Guthire et al., 1997). However, in rats, FSH and IGF-I attenuated apoptosis in GC only when they were added to the culture of intact preovulatory follicles (Tilly et al., 1992). The pathways of FSH, IGF-I, IGFBPs and their interactions on regulation of apoptosis in cultured GC are controversial and remain to be elucidated (Amsterdam et al., 1996). Species differences may also exist. In our study, the mechanism for the high concentration of IGF-I (100 ng/ml) stimulating apoptosis in cultured GC is not clear. However, a previous study has shown that high concentration IGF-I (evident at > 30 ng/ml) inhibits estrogen production by GC from small bovine follicles (1-5 mm) (Spicer et al., 1994). In vivo, negative correlations were also found in some studies where follicles > 8 mm were collected (Spicer et al., 1988). In most mammals, follicular atresia is correlated with a decline in estrogen synthesis concomitant with increased progesterone production (Hsueh et al., 1994). Estrogens have been shown to inhibit ovarian GC apoptosis in rats (Billig et al., 1993). In summary, my data indicate that: 1) apoptosis may occur to a certain level during normal follicular growth and development and apoptotic death of GC may be detectable before other morphological and biochemical signs of degeneration appear in cows; 2) apoptosis occurs in GC and theca cell, however, apoptosis does not occur in cumulus cells 61 even in atretic antral follicles in bovine ovaries; 3) FSH and low concentration of IGF-I are follicle survival factors and follicle apoptosis is regulated differentially depending upon the stage of follicular development in cows. R E F E R E N C E S Adashi EY, Resnick CE, D'Ercole AJ, Svoboda ME, Van Wyk JJ. Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr Rev 1985; 6: 400-420. Amsterdam A, Dantes A, Hosokawa K, Schere-Levy CP, Kotsuji F, Aharoni D. Steroid regulation during apoptosis of ovarian follicular cells. Steroids 1998; 63: 314-318. Amsterdam A, Tal IK, Aharoni D. Cross-talk between cAMP and P53-generated signals in induction of differentiation and apoptosis in steroidogenic granulosa cells. Steroids 1996;61:252-256. Arends MJ, Morris RG, Wyllie AH. Apoptosis: The role of the endonuclease. Am J Pathol 1990; 136: 593-608. Billig H, Furuta I, Hsueh AJW. Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 1993; 133: 2204-2212. Carson RS, Findlay JK, Clarke IJ, Burger HF. Estradiol, testosterone, and androstenedione in ovine follicular fluid during growth and atresia of ovarian follicles. Biol Reprod 1981; 24: 105-113. 62 Chun SY, Eisenhauer KM, Minami S, Billig H, Perlas E, Hsueh AJW. Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor. Endocrinology 1996; 137: 1447-1456. Dirnfeld M, Goldman S, Gomen Y, Koifman M, Lissak A, Kraiem Z, Abramovici H. Functional differentiation in progesterone secretion by granulosa versus cumulus cells in the human preovulatory follicle and effect of different induction of ovulation protocols. Fertil Steril 1993; 60: 1025-1030. Gavrieli Y, Sherman Y, Bensasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119: 493-501. Guthire HD, Garrett WM, Cooper BS. Follicle-stimulating hormone and insulin-like growth factor-I attenuate apoptosis in cultured porcine granulosa cells. Biol Reprod 1997; 58: 390-396. Hirshfield AN. Development of follicles in the mammalian ovary. Int Rev Cytol 1991; 124: 43-101. Hsueh AJW, Billig H, Tsafriri A. Ovarian follicle atresia: a hormonally controlled apoptotic process. EndocrRev 1994; 15: 707-724. Hughes RM, Gorospe WG. Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Endocrinology 1991; 129: 2415-2422. Imig JL, Juengel BE, Salfen BE, Youngquist RS, Hamilton SA, Smith MF, Garverick HA. Evidence for a role of oligonucleosome formation (apoptosis) in follicular atresia in cattle. Biol Reprod 1993; 48 (Suppl 1): 120 (abstract 245). 63 Ireland JJ, Roche JF. Growth and differentiation of large antral follicles after spontaneous luteolysis in heifers: changes in concentration of hormones in follicular fluid and specific binding of gonadotropins to follicles. J Anim Sci 1983; 57: 157-167. Jolly PD, Smith PR, Heath DA, Hudson NL, Lun S, Still LA, Watts CH, McNatty KP. Morphological evidence of apoptosis and the prevalence of apoptotic versus mitotic cells in the membrane granulosa of ovarian follicles during spontaneous and induced atresia in ewes. Biol Reprod 1997; 56: 837-846. Jolly PD, Tisdall DJ, De'ath G, Heath DA, Lun S, Hudson NL, McNatty KP. Granulosa cell apoptosis, aromatase activity, cyclic adenosine 3',5'-monophosphate response to gonadotropins, and follicular fluid steroid levels during spontaneous and induced follicular atresia in ewes. Biol Reprod 1997; 56: 830-836. Jolly PD, Tisdall DJ, Heath DA, Lun S, McNatty KP. Apoptosis in bovine granulosa cells in relation to steroid synthesis, cyclic adenosine 3', 5'-monophosphate response to follicle stimulating hormone and luteinizing hormone, and follicular atresia. Biol Reprod 1994; 51: 934-944. Kaipia A, Hsueh AJW. Regulation of ovarian follicle atresia. Annu Rev Physiol 1997; 59: 349-363. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239-257. Lussier JG, Matton P, Dufour JJ. Growth rates of follicles in the ovary of the cow. J Reprod Fertil 1987; 81: 301-307. Majno G, Joris I. Apoptosis, oncosis, and necrosis: an overview of cell death. Am J Pathol 1995; 146:3-15. 64 Manabe N, Imai Y, Ohno H, Takahagi Y, Sugimoto M, Miyamoto H. Apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pig ovaries. Experientia 1996; 52: 647-651. Manikkam M, Rajamahendran R. Progesterone-induced atresia of the proestrous dominant follicle in the bovine ovary: changes in diameter, insulin-like growth factor system, aromatase activity, steroid hormones, and apoptotic index. Biol Reprod 1997; 57: 580-587. Mariana JC, Monniaux D, Driancourt MA, Mauleon P. Folliculogenesis. In: Cupps PT (ed), Reproduction in domestic animals, 4th ed. San Diego, CA: Academic Press. 1991; 119-171. Metcalf MG. Estimation of viability of bovine granulosa cells. J Reprod Fertil 1982; 65: 425-429. Palumbo A, Yeh J. In situ localization of apoptosis in the rat ovary during follicular atresia. Biol Reprod 1994; 51: 888-895. Petisch MC, Mannherz HG, Tschopp J. The apoptosis endonucleases: cleaning up after cell death? Trend Cell Biol 1994; 4: 37-41. Rajakoski E. The ovarian follicular system in sexually mature heifers with special reference to season, cyclical and left-right variation. Acta Endocrinol Suppl 1996; 52: 1-68. Richards JS. Hormonal control of gene expression in the ovary. Endocr Rev 1994; 15: 725-751. Rouillier P, Matton P, Sirard MA, Guilbault LA. Follicle-stimulating hormone-induced estradiol and progesterone production by bovine antral and mural granulosa cells cultured in vitro in a completely defined medium. J Anim Sci 1996; 74: 3012-3019. 65 Rueda BR, Tilly KI, Botros IW, Jolly PI, Hansen TR, Hoyer PB, Tilly JL. Increased bax and interleukin-ip-converting enzyme messenger ribonucleic acid levels coincide with apoptosis in the bovine corpus luteum during structural regression. Biol Reprod 1997; 56: 186-193. Schwartzman RA, Cidlowski JA. Apoptosis: the biochemistry and molecular biology of programmed cell death. EndocrRev 1993; 14: 133-151. Spicer LJ, Alpizar A, Stewart RE. Evidence for an inhibitory effect of insulin-like growth factor-I and -II on insulin-stimulated steroidogenesis by nontransformed ovarian granulosa cells. Endocrine 1994; 2: 735-739. Spicer LJ, Echternkamp SE, Canning SF, Hammond JM. Relationship between concentrations of immunoreactive insulin-like growth factor-I in follicular fluid and various biochemical markers of differentiation in bovine antral follicles. Biol Reprod 1988; 39: 573-580. Tilly JL, Billig H, Kowalski KI, Hsueh AJW. Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol Endocrinol 1992; 6: 1942-1950. Tilly JL, Hsueh AJW. Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation. J Cell Physiol 1993; 154: 519-526. Tilly JL, Kowalski KI, Johnson AL, Hsueh AJW. Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 1991; 129: 2799-2801. 66 Tilly JL, Lowalski KI, Schomberg DW, Hsueh AJW. Apoptosis in atretic ovarian follicles is associated with selective decreases in messenger ribonucleic acid transcripts for gonadotropin receptors and cytochrome P450 aromatase. Endocrinology 1992; 131: 1670-1676. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980; 68:251-306. Xu ZZ, Garverick HA, Smith GW, Smith MF, Hamilton SA, Youngquist RS. Expression of follicle-stimulating hormone and luteinizing hormone receptor messenger ribonucleic acids in bovine follicles during the first follicular wave. Biol Reprod 1995; 53: 951-957. Yang MY, Rajamahendran R. Effects of gonadotropins and insulin-like growth factor-I and -II on in vitro steroid production by bovine granulosa cells. Can J Anim Sci 1998; 78: 587-597. 67 'CHAPTER 4 INVOLVEMENT OF APOPTOSIS IN THE ATRESIA OF THE NON-OVULATORY DOMINANT FOLLICLE DURING THE BOVINE ESTROUS C Y C L E ABSTRACT To increase our understanding of the control of follicular dynamics in cattle, the present study was designed to 1) investigate whether apoptosis is responsible for atresia of the non-ovulatory dominant follicle (DF); 2) to determine if atresia of the non-ovulatory DF is associated with alterations in Bcl-2 and Bax expression; 3) to test if P4 has a direct effect on apoptosis in bovine follicles; and 4) to study the pattern of expression of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large). In experiment 1, sixteen cycling cows received a norgestomet ear implant at proestrus (Day 1) to mimic the sub-luteal phase for 9 days. The cows were assigned to either the control (n = 4) or P4-treated groups (n=12). Injections of P4 (150 mg, i.m.) were given on Day 3 (n = 4); on Days 3 and 4 (n = 4), and on Days 3, 4, and 5 (n = 4) of the implant period. Controls received injections of com oil on Days 3, 4, and 5. Unilateral ovariectomy was performed on Days 4, 5, and 6 to recover the DFs from cows treated with P4 for 24 h, 48 h and 72 h, respectively. DFs of cows in the control group were collected on Day 6. The onset of atresia of the DFs was assessed by ultrasound to determine DF diameters, histologically by light microscopic inspection of tissue sections and functionally by quantification of follicular fluid steroid hormone levels. Apoptosis was detected by DNA analysis and in situ TUNEL labeling. Expression of Bcl-2 and Bax proteins was examined by western blot "Yang M Y and Rajamahendran R, B i o l Reprod (accepted, M a y 2000) 68 analysis. The earliest signs of atresia were detected 24 h after P4 injection as evidenced by decreased diameter, by the degeneration and detachment of granulosa cells (GCs) from the basal lamina, and by a dramatically reduced ratio of estrogen to P4. Electrophoretic analysis of DNA extracted from DFs of cows treated with P4 for 24-h revealed a distinct ladder pattern of DNA fragments. In contrast, this pattern was not obvious in DFs from control cows. Similar results were also obtained from TUNEL analysis of DFs. Furthermore, both Bcl-2 and Bax were found to be present in all DFs. However, the ratio of Bcl-2 and Bax protein levels was significantly reduced by 24-h P4 treatment as compared to DFs from the control group (p < 0.05). In experiment 2, using ovaries obtained from a local slaughterhouse, the direct effect of P4 (4 ng/ml) on apoptosis of cultured GCs was investigated. In addition, the pattern of expressions of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large follicles) was studied. No increase in apoptotic DNA fragments was detected in GCs treated with P4. The ratio of Bcl-2 and Bax protein levels was variable in small follicles. However, Bax protein level was relatively higher than that of Bcl-2 in medium and large follicles. In conclusion, the present study suggests that apoptosis is the mechanism underlying the atresia of the non-ovulatory DF that develops during the luteal phase of the bovine estrous cycle. Progesterone does not have a direct effect on apoptosis in bovine GCs. Therefore, atresia of non-ovulatory bovine DFs is probably via regulation of LH by the negative feedback of P4. Furthermore, our results indicate that atresia in bovine follicles may be linked to a shift in the ratio of antiapoptotic protein (Bcl-2) and proapoptotic protein (Bax) expression and that this shift is mediated primarily through alterations in Bax. 69 I N T R O D U C T I O N The growth of antral ovarian follicles in cows occurs in a wave-like pattern, with a wave being characterized by the recruitment of a pool of small antral follicles and the selection and dominance of a single dominant follicle (DF) while the remainder of its cohort regresses (Ireland and Roche, 1987; Savio et al., 1988; Sirois and Fortune, 1988; Taylor and Rajamahendran, 1991). There are two or three waves of follicular growth in each bovine estrous cycle (Sirois and Fortune, 1990; Taylor and Rajamahendran, 1991; Adams et al., 1992). The DF from the first wave reaches its largest size on day 7 or 8 of the estrous cycle (day of estrus designated as day 0). The DF maintains its morphological and functional dominance until around day 11, then becomes atretic and begins to regress between days 11-14, to be replaced by a second wave of follicular growth. The number of waves of follicular growth during the estrous cycle is determined by the length of the luteal phase (Taylor and Rajamahendran, 1991; Adams et al., 1992). If progesterone (P4) levels in the circulation begin to decrease due to the spontaneous regression of the corpus luteum (CL) while the second wave DF is in its growth phase, then the second wave DF will ovulate. Alternatively, if P4 remains elevated in the presence of an active CL after the second wave DF has attained its maximum size, the DF undergoes atresia and the third wave of follicular growth will emerge (Taylor and Rajamahendran, 1991). What regulates this pattern and initiates the regression of non-ovulatory DFs is not clear. Evidence indicates that P4 may play a major role. It has been suggested that atresia of the non-ovulatory DF in bovine estrous cycles is induced by high levels of P4 via negative feedback regulation of release of luteinizing hormone (LH; Savio et al., 1993; Taylor et al., 1993). However, a direct action of P4 at the level of the ovary has not been ruled out. Furthermore, although some cellular and biochemical characteristics of atretic DF induced by P4 have been identified recently, 70 including decreased concentrations of follicular fluid estradiol-17p (E2), insulin-like growth factors -I and -II (IGFs -I and -II), reduced aromatase activity, and increased low molecular weight IGF binding proteins, these events only provide useful markers for identifying atresia of DF that develops during the luteal phase of the estrous cycle in cows (Manikkam and Rajamahendran, 1997). The molecular mechanisms underlying atresia of the non-ovulatory DF caused by a high level(s) of P4 is still unclear. Recent studies have suggested that follicle atresia in cows, chickens, pigs, and rodents is associated with apoptosis, which is an active, intrinsic, genetically governed process of selective cell deletion (Arends et al., 1990; Hughes and Gorospe, 1991; Tilly et al., 1991). Gonadotropins have been reported to play a critical role in preventing apoptosis in granulosa cells (GCs) of bovine and rat antral follicles (Chun et al., 1994; Yang and Rajahamendran, 2000). In addition, evidence that ovarian steroids inhibit (estrogens) or induce (androgens) apoptosis in ovarian GCs of the estrogen-implanted, hypophysectomized immature rats has been demonstrated (Billig et al., 1993). However, the effects of P4 on ovarian follicle apoptosis remain controversial. An in vivo study found treatment with P4 does not affect rat ovarian apoptosis whereas another in vitro study showed that P4 inhibited apoptosis in cultured rat granulosa cell (Billig et al., 1993; Peluso and Pappalardo, 1999). The effects of P4 on apoptotic cell death in bovine ovarian follicles have not been reported. Moreover, nothing is known of the underlying events associated with the initiation of apoptosis during atresia of the DF that develops during the luteal stage of the estrous cycle in cows. The initiation of apoptosis in various ovarian cell lineages probably depends upon cell-specific stimuli via hormonal signals, the absence or presence of which activates or stops repression of gene products responsible for the suicidal mechanism (Ellis et al., 1991; 7 1 Cory and Adams, 1998). Studies from extragonadal cell systems have shown that among the numerous proteins and genes involved, the Bcl-2 family of proteins constitutes a critical intracellular checkpoint of apoptosis within a distal common cell death pathway. At least 15 mammalian Bcl-2 family members have been identified and categorized into anti-apoptotic (Bcl-2, Bcl-w, Bcl-XL, A l , MCL-1) and pro-apoptotic (Bax, Bak, Bok, Bik, Blk, Hrk, BNIP3, Bim, Bad, Bid, Bcl-Xs) subgroups (Cory and Adams, 1998). The Bcl-2 protooncogene was originally identified from a human chromosomal translocation that predisposed affected individuals to malignant transformation of immune cells (Cleary et al., 1986). It has been found that Bcl-2 protein prevents apoptosis induced by a variety of stimuli and maintains cell survival by influencing the release of cytochrome c from mitochondria rather than altering proliferation (Yang et al., 1997). Bax, identified by coimmunoprecipitation with Bcl-2 protein, is the first pro-apoptotic homolog. When Bax is overexpressed in cells, apoptotic death in response to death signals is accelerated. In addition, Bax can heterodimerize with Bcl-2 and thus function to counter the effects of Bcl-2 on cellular survival (Oltvai et al., 1993). Therefore, the ratio of Bcl-2 to Bax expression is important in determining susceptibility to apoptosis (Oltvai et al., 1993). However, the roles of Bcl-2 and Bax in bovine follicular development and atresia remain to be elucidated. Therefore, the aims of the present study were 1) to test the hypothesis that apoptosis is the mechanism underlying the atresia of non-ovulatory DF that develops during the luteal phase of bovine estrous cycle; 2) to determine if atresia of DF is associated with alterations in Bcl-2 and Bax expression; 3) to test if P4 has a direct effect on apoptosis in bovine follicles; and 4) to study the pattern of expression of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large). 72 MATERIALS AND METHODS Experimental Protocol Two experiments were conducted. In experiment 1, an in vivo model of the norgestomet (synthetic progestin)-maintained DF was used to determine the mechanism underlying the atresia of the non-ovulatory DF that develops during a simulated luteal phase of the bovine estrous cycle, and to determine if atresia of the DFs is associated with alterations in Bcl-2 and Bax expression. Research from our laboratory has demonstrated that with a single norgestomet (Syncromate-B; Sanofi Inc., Overland Park, KS) implant insertion, the proestrus DF could be maintained healthy and functional for 9 days and was capable of ovulation after implant withdrawal. The time span of 9 days is long enough to subject the follicle to P4 treatments. In addition, the synthetic progestin (norgestomet) has not been reported to cross-react with P4, and it does not alter systemic P4 concentrations, hence, it is very useful for our study in which exogenous P4 was injected to mimic the mid-luteal phase of the bovine estrous cycle (Taylor et al., 1993; Manikkam and Rajamahendran, 1997). In experiment 2, using ovaries obtained from a local slaughterhouse, the direct effect of P4on bovine follicle apoptosis was investigated. In addition, the pattern of expressions of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large) was studied. Experiment 1 Animals and Treatments Sixteen cycling, dry Holstein cows were housed at The University of British Columbia Dairy Teaching and Research Unit and cared for according to the guidelines of the 73 Canadian Council of Animal Care. The cows were chosen for the experiment at the proestrus stage (day 19) of the estrous cycle. The proestrus phase was confirmed by ultrasonic detection of a large follicle and a regressing corpus luteum. During proestrus, each cow received a 6 mg norgestomet ear implant (Day 1 = the day of implant insertion), which was removed 9 days later. After implant insertion, the cows were randomly allotted to either the control (n = 4) group or the P4-treated (n = 12) groups. Cows in the P4-treated groups received i.m. injections of 150 mg P4 (Sigma, St. Louis, MO) in com oil on Day 3 (n = 4), on Days 3 and 4 (n = 4), and on Days 3, 4, and 5 (n = 4) of the implant period. Cows in the control group received injections of com oil on Days 3, 4, and 5. Unilateral ovariectomy was performed on Days 4, 5, and 6 of the implant period to recover the DFs from cows treated with P4 for 24 h, 48 h and 72 h groups, respectively. Unilateral ovariectomy was performed in the control cows on Day 6. After the initial ovariectomy, the remaining ovary was monitored daily by ultrasonography to observe follicular development, and the experiment was repeated. The turnover of follicles in the remaining ovary was similar to that observed for cows with both ovaries and did not influence the emergence and development of the next DF or its maintenance by progestin. Ovarian Ultrasonography The development of the DF in each cow was monitored daily by ultrasound imaging from the day of norgestomet implant insertion until the day of ovariectomy. Ultrasound examination was conducted as described by Rajamahendran and Taylor (1990) using a real-time B-mode linear array ultrasound scanner equipped with a 5 MHz rectal transducer. Appropriate images of DF were arrested on screen and follicular diameters were measured using a built-in caliper system. Permanent records were made using a video-processing unit 74 (Mitsubishi Electronics Co. Ltd., Tokyo, Japan). Processing of the Ovaries following Ovariectomy Ovariectomy was performed per vagina using an ecraseur (colpotomy) under epidural anesthesia (Drost et al., 1992) on designated days. Once removed, the ovaries were immediately placed in chilled Dulbecco's Modified Eagle Medium (DMEM) containing glucose, sodium pyruvate, and L-glutamine supplemented with 0.1% (w:v) bovine serum albumin (BSA) and 100 pg/ml streptomycin (Sigma, St. Louis. MO) on ice and processed in the laboratory within 1 h. The DF from each ovary was individually dissected free from extraneous tissue. Follicular fluid of the DF was aspirated with an 18-gauge needle and a 10-ml syringe, and stored frozen until analysis for P4. Thereafter, the DF was cut into two halves. One-half was fixed in 4% neutral buffered formalin for histological study, the other half was snap frozen and stored at -70 °C until processed for analysis of DNA integrity or Bcl-2 and Bax expression. Morphological Analysis The follicles (n=8) were fixed in 4% neutral buffered formalin for 48 h. Fixed tissues were washed in phosphate buffered saline (PBS) solution, dehydrated through a graded series of ethanol (70-100%), cleared in xylene, embedded in paraffin, and sectioned (5 urn). The sections were deparaffinized in xylene, rehydrated through a graded series of ethanol (100-50%), and then stained with hematoxylin and eosin (Ff & E) for morphological analysis. 75 Progesterone and Estradiol-17p (Ez) Analysis The concentrations of P4 and E 2 in the follicular fluid (n=20) were determined by radioimmunoassay (RIA) in samples (Yang and Rajamahendran, 1998), diluted (1: 10 to 1: 1000) in assay buffer, with commercially available solid-phase RIA kits (Coat-A-Count; Diagnostic Products Corp., Los Angeles, CA). The intra-assay coefficients of variation were 9.8% for P4 and 8.4% for E 2 . The sensitivity of the assay was 0.05 ng/ml and 0.01 ng/ml for P4 and for E 2 respectively. In Situ 3' End-labeling of DNA Fragments DNA fragments in DFs were identified by labeling free 3'-OFf DNA ends (TUNEL) using a in situ cell death detection kit (Boehringer Mannheim, ON). Briefly, tissue sections (n = 8) were deparaffinized and rehydrated. Sections were then washed in tris-buffered saline (TBS) and treated with 20 pg/ml proteinase K for 20 min at room temperature. Tissues were further treated with 3% H 2 0 2 for 5 min to inactivate endogenous peroxidase. Finally, the DNA strand breaks were labeled at 3'-ends with fluorescein dUTP by incubation with the reaction buffer containing terminal deoxynucleotidyl transferase enzyme for 1.5 h at 37 °C in the dark. Samples were analysed immediately under a fluorescent microscope. Negative control sections were processed identically except that the labeling enzyme (terminal deoxynucleotidyl transferase enzyme) was omitted whereas positive control sections were treated with DNase I (0.5 mg/ml). DNA Extraction and Analysis Genomic DNA was prepared from follicles as previously described (Yang and Rajahamendran, 2000). Briefly, follicles (n=9) were snap-frozen and stored at -70 °C to 76 prevent nonspecific activation of DNase. Cells were first disrupted by addition of homogenization buffer and repeated passage through a pipette. Homogenates were then lysed. Genomic DNA was extracted by the phenol/chloroform/isoamyl alcohol (25:24:1, v:v:v) method and quantitated spectrophotometrically at 260 nm (Yang and Rajahamendran, 2000). The DNA was separated (20-25 pg/lane) according to size in a 2% agarose gel by electrophoresis. Gels were stained with ethidium bromide and washed in double distilled H 20. The DNA fluorescence was viewed with an UV transilluminator, and the gels were photographed. The integrated optical density of the stained internucleosomal DNA fragments in each sample lane was measured by densitometry using a gel documentation system and IS-500 Digital Imaging System Software version 1.97 (Alpha Innotech Corporation, San Leandro, CA) as described (Guthrie et al., 1998). Analysis of Bcl-2 and Bax Expression Samples (n=9) were thawed and homogenized with ice-cold homogenization solution (pH 7.5) containing 100 mM Tris-HCL, 0.1% SDS, and protease inhibitors (1 mM phenylmethylsulfonylfluoride, 100 pM Pepstatin, 50 uM Leupeptin, and 50 pM Aprotinin). Homogenates were centrifuged at 15,000 g for 1 h at 4 °C. The protein content was determined with the DC protein assay (Bio-Rad, Hercules, CA). Proteins were solubilized in Laemmli's sample buffer, boiled for 5 min, and separated by electrophoresis on a discontinuous SDS gel system consisting of 6% polyacrylamide stacking and 12% separating components. Electrophoretic proteins were subsequently electrotransferred to nitrocellulouse membranes (Hybond-ECL; Amersham Life Science, ON) in 25 mM Tris-glycine buffer (pH 8.3) containing methanol and 0.1% SDS. The membranes were blocked 77 with Blotto (1 m M Tris containing 0.9% NaCl, 0.2% Tween 20, and 5% nonfat dried milk; pH 7.4) for 1 h at room temperature, incubated with Bcl-2 and Bax goat polyclonal antibody (1.4 pg/ml in Blotto; Sanata Cruz Biotechnology, CA) overnight at 4 °C, washed, and then incubated with HRP-conjugated secondary antibody (1:1000) in Blotto. Peroxidase activity was visualized with an enhanced chemiluminescence western blotting immunodetection kit (Amersham Life Science, Oakville, ON). Bcl-2 and Bax protein expression was determined densitometrically. Briefly, images were scanned using a flat bed scanner (Scan-Jet 4C) and quantitated by the IS-500 Digital Imaging System Software version 1.97 (Alpha Innotech Corporation, San Leandro, CA). Experiment 2 Collection of Ovaries and Classification of Follicles Ovaries were collected, and follicles were classified as previously described (Yang and Rajamahendran, 1998). Briefly, ovaries were collected at a local slaughterhouse and transported to the laboratory (within 2 h of slaughter) on ice in chilled collection medium composed of DMEM/Ham's F-12 (1:1) containing 0.1% (w/v) BSA, L-glutamine, and 50 pg/ml gentamicin (Sigma Chemical Co., St. Louis, MO). The follicles were classified into three groups based on surface diameters: small (< 4 mm), medium (5-8 mm) and large follicles (> 8 mm diameter). Follicle-size categories were selected on the basis of reported gonadotropin dependence and changes in the expression of steroidogenic enzyme and luteinizing hormone (LH) receptor mRNAs (Xu et al., 1995). Small, medium, and large follicles were then classified as healthy or atretic according to previously established morphological criteria with modification (Metcalf, 1982). Healthy 78 follicles had vascularized (pink- or red-colored) theca interna, and clear amber follicular fluid (FF) with no debris. Granulosa Cell Culture and P4 Treatment The GCs were harvested from large healthy follicles (n = 18) and cultured as previously described (Yang and Rajamahendran, 1998). Briefly, follicles were punctured with an 18-gauge needle and follicular fluid was aspirated. Granulosa cell collection medium, Ca27Mg2+-free buffer (20mM Tris, 140 mM NaCl, 2 mM EDTA, pH 7.4), was flushed in and out of the follicles repeatedly. The follicles were then cut into hemispheres, and the interior walls were gently scraped with an inoculating loop to remove GC, leaving the basement membrane and theca cells intact. The GCs were harvested by centrifugation and washed three times. Cell number and viability were determined using a hemocytometer and trypan blue dye exclusion. Viable GCs (2 x 106 cell/ml) were cultured in 6-well plates with 2 ml DMEM/Ham's F-12 supplemented with penicillin (100 U/ml) and streptomycin (100 pg/ml; Sigma, St. Louis, MO). The cells were incubated in a humidified 5% C0 2 atmosphere at 39 °C. After 16 h culture, dead cells were washed off. Only viable GCs were left and they were attached tightly to the culture plates. To study the effect of P4 on GC apoptosis, these GCs were cultured for 48 h in serum-free DMEM/Ham's F-12 medium with or without P4 (4 ng/ml; n =3 culture wells treatment/trial). Progesterone was dissolved in ethanol and then diluted in DMEM/Ham's F-12 to the desired final concentration. The final ethanol concentration in the cell cultures was 0.1% (vol/vol). The treatment concentration was based upon the in vivo circulating plasma P4 concentration that can induce atresia of DFs (Sirois and Fortune, 1990; 79 Taylor et al., 1993). After culture, GCs were harvested, snap-frozen, and stored at -70 °C until processed for D N A analysis as described in experiment 1. Analysis of Bcl-2 and Bax Expressionin in Small, Meidum, and Large Follicles Small (n = 12), medium (n = 6), and large (n = 6) follicles were dissected free from extraneous tissue. Protein levels of Bcl-2 and Bax in follicles of different sizes were analyzed by western blot as described in experiment 1. Statistical Analyses One-way analysis of variance was used for comparisons between more than two means, and when a significant difference was found, a Duncan's multiple range test was used to determine which means were significantly different. Student's t tests were used for comparisons between two means. Statistical significance was inferred at/? < 0.05. RESULTS Experiment 1 Effects of P 4 Treatment on DF Diameter Representative ovarian ultrasound photographs demonstrating the changes in diameter of a norgestomet-maintained DF over a 72 h period after P 4 treatment are shown in Fig. 4.1a. After 72 h of P 4 treatment, the diameters of the DFs decreased by 3.28 + 0.74 mm.The diameters of the DFs decreased by 0.6 ±0.17 mm and 2.65 + 1.29 mm in cows treated with P 4 for 24 and 48 h, respectively. On the contrary, the diameters of the DFs in control cows slightly increased by 0.4 ± 0.28 mm (Fig. 4.1b). 80 -a o H H xi CM ( S i 0 0 PH xi Cl c*2 & 3 O 2-* a c s P H OS o H •(-> C o o <r- O T - CM CO ^ " l O i i i i i (LULU) jejeuieia J Q JO saBueijo -D PH on .3 00 rr. P fl g^ Q 3 g .s •§ i-i ca CI_I 11 ° H I 2 o i bo « P § o C fl • cS c,_ C(-l O O M _ on CD on <U 00 <U 00 fl 00 cs « • X i o cu cu £ o o cu (Zl c3 OJ CU 1 X CS -a fl cn 0© CM 5U O C c3 X a ;-. cu cu h cu — Xi * a • i £ -a 14 CU CU +5 £ "5 cn fl O s CU -a cn -G & . •H on 00 CU ta CU c o O CO Q £ o C + H A CU > '5b cu — cu £ U .3 O X ! P< c_ T3 O P •c CCJ > o cu > C W cn cu CH E J O w cu fl cs fl oo tN c--S-l TT" CD CN O o o c 00 o I T ) c2 ta x <2 CU CO o CU c T3 -H -S JS OS 0J3 cn C .2 A, • f l fl o o CD ' f l 1 0 0 . fl on o § .£ bO X CD fl fl s» •£ "5b 81 Effects of P 4 Treatment on DF Morphology Representative photomicrographs (200x) demonstrating the morphological changes of norgestomet-maintained DFs after 24, 48, and 72 h of P4 treatment are shown in Fig. 4.2. In control follicles (Fig. 4.2a), the theca layer was distinct, and the granulosa layer appeared thick and well organized. A few pyknotic cells were observed under higher magnification. Twenty-four hours after P4 treatment (Fig. 4.2b), follicles exhibited features of atresia. The granulosa layer had thinned considerably and become disorganized. In some cases the granulosa layer had become either partially or completely detached from the underlying basal lamina. With the progression of atresia, by 48 h P4 treatment (Fig. 4.2c), only very few GCs could be observed. The granulosa layer was virtually absent by 72 h P4 treatment (Fig. 4.2d). Effects of P4 Treatment on Follicular Fluid Steroid Hormone Concentrations The effects of P4 treatment on concentrations of P4 and E 2 in the DFs of the treated groups and control are shown in Fig 4.3. No significant differences in follicular fluid P4 concentrations was observed among the groups (p > 0.05). However, there was a great decrease of follicular fluid E 2 concentrations when comparing control to treated groups (p < 0.01). 82 e cu - £ o co • — .? B to <a 00 . 2 O •2 1 1 £ >-> o « "fe u o d / — . oo ccj 00 C c 1 O X I « C N i ^ CD I ~ « " i o fi S H cu c3 HH 00 c3 I D el x I !! CO cu O CO i-cu £ / o £ £3 .2 co co }a £ P CO HH x rj ca-rl£ ^ 3 cc e E? § *§ 0 13 3 = 8 -•7* N CO 1 '£ u ^ g> £ .1) O CO 13 & C3 CJ '5b ~6 x o £ H—' C3 CJ £ ~ c u X oo aj G oo U 00 o 2 -t—' CO c o O H C n -o CN * 3 O , 11 60 g 0 3 1 c 8 x £ I G C c c u cu 1 3 o o C-J co 2£ x •* c ? HH (N 2 M •g 5 5 C co 2 & o Q Q £ v - t -» nJ co .—' CU co CL, O CJ ~*2 o 3 T-J-d <N • H — O 0 3 tS fa £ Q QJ y— X X X I . 1 3 c u G '3 fa £ « CJ >^  cc! OH C+H O X oo r|-— cu <fi IX Q 'CJ CU w CD cc! O JS o £ c OJ S £ ^ CD _>> (3 -*-> • ° X CD 2 6 £ 83 E •a to 150 i ? 100 o c o 0) Q) O 50 0 1200 1000 g- 800 600 I 400 fc 200 0 X control P 424h P 448h P472 h control P 4 24h P 448h P472 h Treatment Groups Fig. 4.3. Concentrations of progesterone (P4) and estradiol-17P (E2) in the follicular fluid of norgestomet (synthetic progestin)-maintained dominant follicles (n=20) from controls and cows treated with P4for 24, 48, and 72 h. Letters a, b denote significant difference (p <0.01). 84 Effects of P4 Treatment on Apoptosis DFs (In Situ 3' End-labeling, TUNEL) The effects of P4 treatment on apoptosis in DFs are shown in Fig. 4.4. DFs from the control group exhibited very few detectable levels of DNA fragment labeling (Fig. 4.4a). In contrast, DFs from 24-h P4-treated group showed heavily fluorescent signals indicating DNA fragmentation, confined mainly in the GCs and in scattered theca cells (Fig. 4.4b). A lower apoptotic signal was detected in theca cells of DFs from 48-h and 72-h P4-treated groups due to the lack of GCs (Fig. 4.4, c and d). Negative control sections did not fluoresce whereas an extreme intense fluorescent signal was observed in positive controls, demonstrating that this in situ labeling procedure is specific. Effects of P4 Treatment on Apoptosis in DFs (DNA Fragmentation Analysis) Results from above morphological studies demonstrated that the granulosa layer in DFs from the 72-h P4-treated group had totally disappeared. Therefore, the "DNA ladder" configuration, a characteristic biochemical feature of apoptosis, was examined only in DFs from control, 24-h P4-treated and 48-h P4-treated groups (Fig. 4.5). As shown, DFs from control group lacked a signal indicative of low molecular weight DNA. However, fragmented DNA extracted from DFs from 24-h P4-treated group, forming a distinctive ladder pattern, was clearly apparent. The ladder pattern became less distinct for DFs from 48-h P4-treated groups. 85 >? - : j • - . *• 9 > » C5 jf 0> xj f—1 cu XI CO O cu £ o oo • — O ••a O H O O H C o H-H n cu £ H-» ccS CU f N co - a o cu > - a cu 4—1 CU cu cu 13 co. ccS 0 0 x l C cu X cc! £ o co UH a c co „,H* fa a cu e s cu HH CO cu 00 p CO 00 o H o. o o. < c cu £ 00 — cs 13 cK B < cu "S Q -CO G O , co 03 HH CU I c a a "cu O > _ f i o ca +-< cu ta cu cu X oo 5 a H CO C o £ cu - a o o e cu cu C O E O a Q -o fi co 13 l I S x G co 00 _ - o co CU 1 I £ O 13 13 CU c "3 HH d "3 £ CO 03 CU HH CJ . G CO KS ca 00 _d "8 I CO CO cu HH ccS X I 1 CS d CJ £ 6. CS 00 <u — HH 2 ^  — o CJ £ o OH CHH o CO X & 00 o H cj £ o HH o x OH r r T f oi fe 00 cfc o 5 fe OH Q X C N 13 G cCJ cu _> HH CS 00 cu Z m op £ cu cu CZ. H H cu t CS CO _o 3 3 03 HH 0 0 ^ d is >. cu co cu £ o HH co 5 0 5 w 3 H 0 0 1 3 fi ^ ^ 00 _c "cu X JS cu CO 3 CU cu CO <£ cS CS 13 ."fi U X cS l e 2 * £3 CU .a x ' Effects of P4 Treatment on Bcl-2 and Bax protein Expression The effects of P4 treatment on the changes of expression of Bcl-2 and Bax proteins in DFs from control, 24-h P4-treated and 48-h P4-treated groups are shown in Fig. 4.6. Both Bcl-2 and Bax were present in the DFs. There was no difference in the expression of Bcl-2 with the progression of atresia induced by P4 treatment. In contrast, P4-treated DFs showed a marked increase in Bax expression. Densitometry revealed that the ratio of Bax/ Bcl-2 of DFs from cows treated with P4 was significantly increased compared with that of controls (p < 0.05). Experiment 2 Effects of P 4 on DNA Fragmentation in Cultured GCs Analysis of DNA fragmentation in GCs cultured for 48 h with or without P4 (4 ng/ml) demonstrated that treatment with P4had no significant effect on apoptosis (p.< 0.05; Fig. 4.7). In addition, P 4 treatment did not affect cell viability (control: 65 ± 4% vs. treatment: 61 ± 5%). Bcl-2 and Bax Expression in Small, Medium, and Large Follicles Bcl-2 and Bax expression in small, medium, and large follicles is show in Fig. 4.8. The ratio of Bcl-2 and Bax protein expression was variable in small follicles. In contrast, Bax protein expression was relatively higher than that of Bcl-2 in medium and large follicles. 88 CN i PQ x PQ o CD 00 o u a © 08 CN i O N CD CO CD bi) o a .£ -d o ^ J * CQ CN ,p CN P M CQ "5 O 3^ CD o rt CD co *•* CD co SH p w o CD -o —; ca oi £ fa 8 89 CCS CO 3 i o — n o 8 | cd ^b to G G ^ 2 t 0 0 ^ CD C J G CD CO CD s-G . T3 CD 3 CD C G O O '•S o | § I 8 < s CO g C3 1 — 1 CD S G co o 5 3 CD CD sa CD O co to CD CD OO .2 .fa H ^ S •S A l O CD i-T ° tD M "rt w g l <a -o . co * — DO 32. S V- <D 3 faux 90 DISCUSSION Evidence indicates that a high concentration of P4 may play an important role in initiating the regression of non-ovulatory DFs during the bovine estrous cycle (Sirois and Fortune, 1990; Taylor and Rajamahendran, 1991; Adams et al., 1992; Taylor and Rajamahendran, 1993). In the present study, the hypotheses that apoptosis is responsible for the atresia of DFs that develop during luteal phase and atreisa of these DFs are associated with alterations in Bcl-2 and Bax expression were investigated. To test this hypothesis, firstly, a model was used in which the proestrus DF can be maintained healthy and dominant for 9 days and the regression of the DF can be induced by P4 injections. Results from the ultrasound study showed that the maintenance and growth of DFs in control group and regression of DFs in P4-treated groups in this study were similar to those observed in other studies (Taylor et al., 1993; Manikkam and Rajamahendran, 1997). Histologically, our finding demonstrated that DFs exhibited morphological signs of atresia such as the granulosa layer had thinned considerably and became disorganized just 24 h after P4 injection. These features were further exacerbated with the duration of P4 treatment. A high E 2 : P4 (> 1) ratio in follicular fluid has been considered characteristic of healthy follicles, whereas a low E 2 : P4 (<1) ratio is indicative of the atretic status of the follicle (Ireland and Roche, 1983). Analysis of steroid hormone concentrations in the present study revealed that significant changes in the ratio of E 2 : P4 from high (> 1) to low (< 1) occurred 24 h after P4 injection, agreeing with the morphological changes during atresia. The decrease in E 2 production may be due to a reduction in the number of viable GCs and/or aromatase activity (Tilly et al., 1992). These observations demonstrated P4 treatment causes atresia of the DFs. Recently, it has been demonstrated that apoptosis is likely the underlying event associated with the initiation and progression of follicular atresia in all vertebrate species 92 studied to date (Tilly et al., 1991; Ellis et al., 1991; Kaipia and Hsueh, 1997; Cory and Adams, 1998). Examination of the varied examples of apoptosis shows that the mediators of apoptosis can be divided into several general classes. The largest group includes those mediators that most likely induce apoptosis by binding to their respective membrane-bound or intracellular receptors and eliciting changes in the intracellular environment. Hormones and growth factors that regulate apoptosis in particular cell types typify this group (Thompson, 1995; Vinatier et al., 1996). Sex steroids are important intra-ovarian regulators of follicular atresia (Harman et al., 1975). In the present study, in situ labeling and electrophoretic analysis of low molecular weight DNA of DFs revealed the presence of internucleosomal DNA fragmentation, characteristic of apoptosis. Consistent with previous studies (Hughes and Gorospe, 1991; Chun et al., 1994; Yang and Rajahamendran, 2000), the identification of much more internucleosomal DNA fragmentation in DFs from the 24-h P4-treated group than those from the control group suggests that apoptosis is the underlying mechanism of follicle degeneration during atresia. The process of apoptosis occurs rapidly and apoptotic bodies are typically phagocytosed by nearby cells (Bursch et al., 1990). Combined with the results from our histological study showing decreased numbers of GCs, this may explain why there was less internucleosomal DNA fragmentation in DFs from 48-h P4-treated group. Taken together, the above observations suggest that apoptosis in DFs is regulated in vivo by P4. How does P4 exert its effects on DFs? The effects of P4 on DFs could be at two levels. Firstly, via the hypothalamic-pituitary axis. It has long been known that gonadotropins are essential for the growth and development of ovarian follicles and their secretion can be modulated by negative feedback loops from P4. Previous studies, using different models in which DF are maintained in the absence of a CL using synthetic 93 progestins and low doses of circulating P4 (1- 2 ng/ml), have shown that treatments which induce the maintenance of DFs result in a persistent high-frequency and low-amplitude LH pulse pattern (Sirois and Fortune, 1990; Taylor and Rajamahendran, 1991). However, high levels of P4 (4-6 ng/ml) reestablish the wave-like growth of antral follicles and result in low-frequency LH pulses (Sirois and Fortune, 1990; Taylor and Rajamahendran, 1991). Based on these observations, it has been hypothesized that low-frequency LH pulses, characteristic of the luteal phase, fail to support thecal androgen production, thus impairing GC function and leading to atresia of the DF and a new wave of follicular growth (Savio et al., 1993). In addition, recent studies demonstrated that both FSH and LH/human chorionic gonadotropin inhibited apoptosis and thus are effective survival factors for preovulatory follicles in cows and rats (Chun et al., 1994; Yang and Rajahamendran, 2000). Alternatively, or coincidentally, P4 may act directly at the level of follicle. The supporting evidence comes from studies showing P4 suppressed the FSH stimulated E 2 production by rat (Schreiber et al., 1981) and goat GCs (Kharbanda et al., 1990). But, this concept remains controversial because some studies examining the effects of P4 on FSH-stimulated induction of aromatase mRNA in rat GCs failed to find any suppressive effect (Fitzpatrick and Richards, 1990). In the present study, P4 had no direct effect on apoptosis in cultured GCs. This is in agreement with the results from an in vivo study in which treatment with P4 had no effect on ovarian apoptosis in hypophysectomized rats (Billig et al., 1993). In contrast, a very recent study suggested that P4 can inhibit only large rat GCs from undergoing apoptosis indirectly by stimulating small granulosa cells to synthesize basic fibroblast growth factor (Peluso and Pappalardo, 1999). Even though the mechanism by which hormonal factors regulate apoptosis are not well understood, it has been proposed that the absence or presence of cell-specific stimuli 94 via hormonal signals may activate or stop repression of gene products responsible for the suicidal mechanism. As the Bcl-2 family of proteins constitutes a critical intracellular checkpoint of apoptosis within a common cell death pathway (Chao and Korsmeyer, 1998), we hypothesized that the underlying events involved in high concentration P4-triggered GC apoptosis and atresia of DFs that develop during the luteal phase of the bovine estrous cycle would involve members of this family. Analysis of Bcl-2 and Bax protein expression in the present study indicated that atresia of DFs triggered by in vivo injection of P4 to cows was associated with a marked increase in Bax protein level. Although Bcl-2 expression was not significantly decreased as expected, the elevation in Bax expression still effectively make the ratio of Bax to Bcl-2 in favor of higher death inducer levels. Therefore, our study supports the concept that pro-apoptotic proteins such as Bax and Bad accelerate cell death. Conversely, anti-apoptotic members such as Bcl-2 and Bcl-XL prevent cell death. Their ratio or balance decides the fate of a cell during development (Chao and Korsmeyer, 1998). However, in certain cases, just pro-apoptotic proteins are sufficient to cause apoptosis independent of additional signals (Allen et al., 1998). In addition, a similar expression pattern of Bcl-2 and Bax has been reported from rat follicles at the mRNA level (Tilly et al., 1995). In contrast, apoptosis in bovine endothelial cells was associated with altered Bcl-2 protein expression rather than Bax (Dhanabal et al., 1999). Follicles of different sizes (small, medium and large) were chosen to further study the pattern of Bcl-2 and Bax expression during follicular development. These data revealed that the ratio of Bcl-2 to Bax was variable in small follicles whereas the level of Bax protein was higher than that of Bcl-2 protein in medium and large follicles, suggesting that some of small follicles might be healthy and most of the medium and large follicles might be undergoing atresia. In a bovine follicular wave, cohorts of antral follicles (2-4 mm) are 95 recruited out of a pool of smaller antral follicles. After 2 to 4 days of recruitment, one follicle is selected. Before selection take place, several medium-size follicles (5-9 mm) can be detected by ultrasonic imaging. However, after selection, only the selected follicle continues to grow, whereas others become atretic (Roche et al., 1996). In addition, our results further confirm that an elevated ratio of Bax to Bcl-2 expression occurs in atretic follicles. In conclusion, our study suggests that apoptosis is the mechanism underlying atresia of non-ovulatory DF that develops during the luteal phase of the bovine estrous cycle. Progesterone (4 ng/ml) does not have a direct effect on apoptosis in bovine GCs. Therefore, atresia of non-ovulatory bovine DFs is probably via P4 negative feedback regulation on LH, reducing its inhibitory effect on apoptosis. Furthermore, our results indicate that atresia in bovine follicles may be linked to a shift in the ratio of antiapoptotic protein (Bcl-2) and proapoptotic protein (Bax) expression and that this shift is mediated primarily through alterations in Bax. REFERENCE Adams GP, Matted RI, Kastelic JP, Ko, JCH, Ginther, OJ. Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers. J Reprod Fertil 1992; 94: 177-188. Allen RT, Cluck MW, Agrawal DK. Mechanisms controlling cellular suicide: role of Bcl-2 and Caspases. Cell 1998; 54: 427-445. Arends MJ, Morris RG, Wyllie AH. Apoptosis: The role of the endonuclease. Am J Pathol 1990; 136: 593-608. 96 Billig H, Furuta I, Hsueh AJW. Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 1993; 133: 2204-2212. Bursch w, Paffe S. Putz B. Barthel G. Schulte Hermann R. Determination of the length of the histological stages of apoptosis in normal liver and in altered hepatic foci of rats. Carcinogenesis 1990; 11: 847-853. Chao DT, Korsmeyer SJ. Bcl-2 family: regulators of cell death. Annu Rev Immunol 1998; 16: 395-419. Chun SY, Billing H, Tilly JL, Furuta I, Tsafriri, Hsueh AJW. Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor-I. Endocrinology 1994; 135: 1845-1853. Cleary ML, Smith SD, Sklar J. Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t (14; 18) translocation. Cell 1986; 47: 19-28. Cory S, Adams JM. Matters of life and death: programmed cell death at Cold Spring Harbor. Biochimica et Biophysica Acta 1998: 1377: R25-R44. Dhanabal M, Ramchandran R, Waterman MJF, Lu H, Knebelmann B, Segal M, Sukhatme VP. Endostatin induces endothelial cell apoptosis. J Biolo Chem 1999; 274: 11721-11726. Drost M, Savio JD, Barros CM, Badinga L, Thatcher WW. Ovariectomy by colpotomy in cows. J Am Vet Med Assoc 1992; 200: 337-339. Ellis RE, Yuan JY, Horvitz HR. Mechanisms and functions of cell death. Annu Rev Cell Biol 1991: 7: 663-698. 97 Fitzpatrick SL, Richards JS. Regulation of cytochrome P450 aromatase mRNA and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 1991; 129: 1452-1462. Guthrie HD, Garrett WM, Cooper BS. Follicle-stimulating hormone and insulin-like growth factor-I attenuate apoptosis in cultured porcine granulosa cells. Biol Reprod 1998; 58: 390-396. Harman SM, Louvet JP, Ross GT. Interaction of estrogen and gonadotropins on folliclular atresia. Endocrinology 1975; 96: 1145-1152. Hughes RM, Gorospe WG. Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Endocrinology 1991; 129: 2415-2422. Ireland JJ, Roche JF. Development of nonovulatory antral follicles in heifers: changes in steroids in follicular fluid and receptors for gonadotropins. Endocrinology 1983: 112: 150-156. Ireland JJ, Roche JF. Hypotheses regarding development of dominant follicles during the bovine estrous cycle. In: Roche JF, O'Callagan D (eds) Follicular growth and ovulation rate in farm animals. Martinus Nijhoff Publishers Dordrecht. 1987: pp 1-18. Kaipia A, Hsueh AJW. Regulation of ovarian follicle atresia. Annu Rev Physiol 1997; 59: 349-363. Kharbanda SM, Band V, Murugesan K, Farooq A. Modulation of steroid production in goat ovarian cells: effects of progestins and anti-progestins. Endocrine Res 1990; 16: 293-309. 98 Manikkam M, Rajahendran R. Progesterone-induced atresia of the proestrous dominant follicle in the bovine ovary: changes in diameter, insulin-like growth factor system, aromatase activity, steroid hormones, and apoptotic index. Biol Reprod 1997; 57: 580-587. Metcalf MG. Estimation of viability of bovine granulosa cells. J Reprod Fertil 1982; 65: 425-429. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vitro with a conserved homology, Bax, that accelerates programmed cell death. Cell 1993; 74: 609-619. Peluso JJ, Pappalardo A. Progesterone maintains large rat granulosa cell viability indirectly by stimulating small granulosa cells to synthesize basic fibroblast growth factor. Biol Reprod 1999; 60: 290-296. Rajamahendran R, Taylor C. Characterization of ovarian activity in postpartum dairy cows using ultrasound imaging and progesterone profiles. Anim Reprod Sci. 1990; 22: 171-180. Roche JF. Control and regulation of folliculogenesis - a symposium in perspective. Rev Reprod 1996; 1: 19-27. Savio JD, Boland MP, Hynes N, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle in heifers. J Reprod Fertil 1988; 83: 663-671. Savio JD, Thatcher WW, Badinga L, de la Sota RL, Wolfenson D. Regulation of dominant follicle turnover during the oestrous cycle in cows. J Reprod Fertil 1993; 97: 197: 203. Schreiber JR, Nakamura K, Erikson GG. Progestins inhibit FSH-stimulated granulosa estrogen production at a cAMP site. Mol Cell Endocrino 1981; 21: 161-170. 99 Sirois J, Fortune JE. Lengthening the bovine estrous cycle with low levels of exogenous progesterone: a model for studying ovarian follicular dominance. Endocrinology 1990; 127: 916-925. Sirois J, Fortune JE. Ovarian follicular dynamics during the estrous cycle monitored by real time ultrasonography. Biol Reprod 1988; 39: 308-317. Taylor C, Rajamahendran R, Walton JS. Ovarian follicular dynamics and plasma luteinizing hormone concentrations in norgestomet treated heifers. Anim Reprod Sci 1993; 32: 173-184. Taylor C, Rajamahendran R. Follicular dynamics, corpus luteum growth and regression in lactating dairy cattle. Can J Anim Sci 1991; 71: 61-68. Thompson CB, Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267: 1456-1462. Tilly JL, Kowalski KI, Johnson AL, Hsueh AJW. Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 1991; 129: 2799-2801. Tilly JL, Kowalski KI, Schomberg DW, Hsueh AJW. Apoptosis in atretic ovarian follicles is associated with selective decreases in messenger ribonucleic acid transcripts for gonadotropin receptors and cytochrome P450 aromatase. Endocrinology 1992; 131: 1670-1676. Tilly JL, Tilly KI, Kenton ML, Johnson AL. Expression of members of the Bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased Bax and constitutive Bcl-2 and Bcl-X long messenger ribonucleic acid levels. Endocrinology 1995; 136: 232-241. 100 Vinatier D, Dufour PH, Subtil D. Apoptosis: a programmed cell death involved in ovarian and uterine physiology. European J Obs & Gyn Reprod Biol 1996; 67: 85-102. Xu ZZ, Garverick HA, Smith GW, Smith MF, Hamilton SA, Youngquist RS. Expression of follicle-stimulating hormone and luteinizing hormone receptor messenger ribonucleic acids in bovine follicles during the first follicular wave. Biol Reprod 1995; 53: 951-957. Yang J, Liu X, Bhalla K. Prevention of apoptosis by bcl-2: release of cytochrome c from mitochondria blocked. Science 1997; 275: 1129-1136. Yang MY, Rajamahendran R. Effects of gonadotropins and insulin-like growth factor-I and -II on in vitro steroid production by bovine granulosa cells. Can J Anim Sci 1998; 78: 587-597. Yang MY, Rajahamendran R. Morphological and biochemical identification of apoptosis in bovine follicular cells and effects of follicle stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells. Biol Reprod 2000; 62: 1209-1217. 101 'CHAPTER 5 EXPRESSION OF BCL-2 AND BAX PROTEIN IN RELATION TO THE QUALITY OF BOVINE OOCYTES AND EMBRYOS PRODUCED IN VITRO ABSTRACT The objectives of this study were: 1) to investigate whether differences in the quality of immature oocytes and subsequent embryo fragmentation are associated with apoptosis; and 2) to study the pattern of Bcl-2 and Bax expression in oocytes and embryos to help elucidate their potential roles in the regulation of apoptosis during development. Bovine oocytes were obtained from slaughterhouse ovaries and divided into four grades (grade I -IV) based on their morphology. Oocytes of different grades were cultured in serum-free medium for 48 h. Embryos were produced only from grade I oocytes (highest quality) via IVM, IVF and rVC procedures. The morphological analysis of apoptosis in oocytes and embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expression of Bcl-2 and Bax in oocytes and embryos of different qualities and stages was determined using western blotting. The results showed that the number of morphologically abnormal oocytes with shrinkage and/or fragmentation of the ooplasm, which are typical features of apoptosis, was significantly higher in grade TV oocytes (denuded oocytes, the lowest quality) than in grade I oocytes after 48 h in vitro culture (p < 0.05). DNA fragmentation, a hallmark of the biochemical changes seen in apoptotic cell death, was observed in morphologically fragmented oocytes and embryos. The expression of Bcl-2 was high in good quality oocytes and embryos, low in ' Y a n g M Y and Rajamahendran R , A n i m Reprod Sc i (submitted) 102 fragmented embryos, and hardly detectable in denuded oocytes. In contrast, the expression of Bax was found in all types of oocytes and embryos with the highest expression in the denuded oocytes. This implies that the ratio of Bcl-2 to Bax may be used to gauge the tendency of oocytes and embryos towards either survival or apoptosis. Overall, our results show that apoptosis appears to be an underlying mechanism of bovine oocyte degeneration and embryo fragmentation. Interactions between the Bcl-2 family of proteins may play a critical role in preimplantation embryo development. These findings could have important implications for improving IVF and related techniques. I N T R O D U C T I O N In many laboratories, embryos are routinely obtained from oocytes after in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro embryo culture (IVC). Therefore, the proper selection of developmentally competent oocytes is crucial for successful in vitro embryo production. The possibility of selecting oocytes based on morphology was initially investigated for cattle in 1979 (Leibfried and First, 1979). Since that time, many reports have indicated that classification of bovine oocytes, based on visual assessment of the compactness of the cumulus investment as well as the homogeneity and transparency of the ooplasm, can be used to select immature oocytes for optimum maturation, fertilization, and development in vitro (Madison et al., 1992; Brackett and Zuelke, 1993). Furthermore, a clear relationship between oocyte morphology and embryo yield after IVM/IVF/IVC has recently been established (Lonergan et al., 1992). Bovine oocytes can be broadly categorized into acceptable-quality oocytes, which have a homogeneous ooplasm and at least two to three cumulus cell layers and unacceptable-quality oocytes, which have a heterogeneous ooplasm and expanded cumulus cells scattered in dark clumps in a jelly-like matrix or are totally 103 denuded (Leibfried and First, 1979; Lonergan et al., 1992; Madison et al., 1992; Brackett and Zuelke, 1993). Research has shown that unacceptable-quality oocytes should not be selected for IVM because of a decreased capacity to mature and a very low embryo production rate in vitro. In spite of the above selection criteria, the quality of mammalian preimplantation embryos obtained under in vitro culture conditions is variable. When compared to in vivo derived embryos, in vitro produced embryos from several mammalian species exhibit retarded developmental progress (Dobrinsky et al., 1996; Du et al., 1996; Long et al., 1998). The appearance of cellular fragmentation during early embryo development in vitro has been commonly observed in pigs (Long et al., 1998), cows (Matwee et al., 1999; Yang and Rajamahendran, 1999), and humans (Jurisicova et al., 1995). These embryos contain irregularly sized blastomeres, multiple cellular fragments, and development will frequently become arrested with subsequent degeneration (Hardy et al., 1989). Moreover, fragmented embryos have an increased sensitivity to cryopreservation and manipulation, and therefore, are associated with poor survival following cryopreservation and with decreased pregnancy rates following embryo transfer (Hardy et al., 1989; Jurisicova et al., 1995). Thus, investigating the mechanism of embryonic fragmentation is important. Apoptosis is a highly conserved and regulated "program" by which cells commit suicide under a variety of internal and external controls. It is the most common form of cell death and plays an important role in the development and differentiation of many multicellular organisms (Raff, 1992). Apoptosis is a morphologically and biochemically distinct physiological process triggered by changes in the levels of specific stimuli. The major characteristics of apoptotic death are nuclear and cytoplasmic condensation, endoplasmic reticulum swelling, and cytoplasmic blebbing. In addition, endogenous 104 nucleases digest DNA from apoptotic cells into oligonucleosomal fragments (multiples of 185-200 bp) which give the appearance of a DNA ladder after electrophoresis (Ellis et al., 1991; Raff, 1992; Cory and Adams, 1998). Recently, we have shown that fragmented bovine embryos exhibit typical morphological changes that conform to the general criteria of apoptotic cell death (Yang and Rajamahendran, 1999). However, the occurrence and regulation of embryonic apoptosis and its relation to decreased developmental competence and embryonic loss are not well understood. Every cell has an apoptotic cell death pathway. It has been proposed that the propensity to apoptosis is continuously counterbalanced in the cell by genes stimulating cell survival and proliferation. Upon induction by an appropriate trigger, the cell activates or stops the repression of gene products responsible for control of the suicidal mechanism (Ellis et al., 1991; Cory and Adams, 1998). Studies from extragonadal cell systems have shown that among the numerous proteins and genes involved, members of the Bcl-2 gene family play key roles in regulating apoptosis. At least 15 mammalian Bcl-2 gene family members have been identified and categorized into two subgroups, anti-apoptotic (Bcl-2, Bcl-w, Bcl-xL, A l , Mcl-1) and pro-apoptotic (Bax, Bak, Bok, Bik, Blk, Hrk, BNIP3, Bim, Bad, Bid, Bcl-xs) (Cory and Adams, 1998). The Bcl-2 protooncogene was originally identified from a human chromosomal translocation that predisopsed affected individuals to malignant transformation of immune cells (Cleary et al., 1986). It has been found that the Bcl-2 protein prevents apoptosis induced by a variety of stimuli and maintains cell survival by influencing the release of cytochrome c from mitochondria rather than by altering proliferation (Yang et al., 1997). Bax, identified by co-immunoprecipitation with the Bcl-2 protein, is the first pro-apoptotic homolog. When Bax is overexpressed in cells, apoptotic death is accelerated. It has been shown that Bax heterodimerizes with Bcl-2 and thus counters the effects of Bcl-2 on 1 0 5 cellular survival. (Oltvai et al., 1993). These investigators (Oltvai et al., 1993) also demonstrated that the ratio of the expression of Bcl-2 to Bax is the critical determinant of either cell survival or death. However, no literature has been found on the presence and function of the Bcl-2 family gene products in bovine oocytes and embryos at different developmental stages. Therefore, the present study was conducted to investigate whether differences in the developmental capacity of immature oocytes (grades I - IV) and embryo fragmentation are associated with apoptosis. In addition, the changes in expression of Bcl-2 and Bax in relation to their regulation of apoptosis during oocyte maturation and early embryo development and their relationships to oocyte and embryo quality were characterized by western blot analysis. Our findings could have important implication for IVF and related assisted reproductive techniques. MATERIALS AND METHODS Oocyte Collection and Grading Bovine ovaries were collected at a local abattoir and transported to the laboratory in physiological saline at 30 - 35 °C within 2 hours. Oocytes were aspirated from follicles (< 6 mm in diameter) with an 18-gauge needle and 10 ml syringe using aspiration media [phosphate buffered saline (PBS) supplemented with 0.3% bovine serum albumin (BSA) and 50 pg/ml gentamicin; Sigma, St Louis, MO]. Oocytes were then divided into four categories based on the compactness of the cumulus investment and homogeneity and transparency of the ooplasm: grade I) oocytes with homogeneous ooplasm and many tight layers of cumulus cells; grade II) oocyte with homogeneous ooplasm and two to three cumulus cell layers; 106 grade III) oocytes with heterogeneous ooplasm and expanded cumulus cells scattered in dark clumps in ajelly-like matrix; and grade IV) denuded oocytes (Fig. 5.1). Morphological Evaluation of Oocytes Following Short Time Culture Oocytes (grades I and IV, 50 of each) were incubated in tissue culture medium 199 (TCM 199; Sigma, St Louis, MO) at 38.5 °C in an atmosphere of 5% C0 2 . Morphological changes were observed after 48 h incubation. Oocytes showing shrinkage and/or fragmentation of the ooplasm were judged to be abnormal, and the percentage of abnormal oocytes in different grades was counted. In vitro Maturation Only oocytes of acceptable quality (grade I) were selected for IVM/IVF/IVC. Briefly, selected oocytes were washed three times with maturation medium, which consisted of TCM 199 supplemented with follicle stimulating hormone (FSH, 0.01 mg/ml; Folltropin V (20 mg/ml); Vetrapharm, ON, Canada), 5% superovulated cow serum (SCS), and 50 pg/ml gentamicin. After washing, cumulus oocyte complexes were placed in a dish containing maturation media and cultured for 22 - 24 h at 38.5 °C under 5% C0 2 in a conventional incubator. The medium was overlaid with mineral oil (Sigma, St Louis, MO). 107 108 Preparation of Sperm for In vitro Fertilization Frozen-thawed semen from a single bull was used for in vitro fertilization. Briefly, frozen semen was thawed in warm water (25 °C) and washed twice by centrifugation (500 g, 5 min) in Brackett and Oliphant's (BO) medium containing 2.5 mM caffeine (Caff-BO). The resultant sperm pellet was then suspended in Caff-BO-BSA medium, consisting of Caff-BO medium supplemented with 20 pg/ml heparin (Sigma, St Louis, MO) and 1% BSA, to a final concentration of 5 x 106 sperm/ml. A 100 pi aliquot of the sperm suspension was overlaid with mineral oil and preincubated for 1 h at 38.5 °C under 5% C0 2 . In vitro Fertilization and In vitro Embryo Culture Oocytes matured in vitro were washed three times in Caff-BO-BSA medium and transferred into sperm microdrops (5 x 106 sperm/ml; 20 oocytes per microdrop) for fertilization. After 16-18 h, sperm exposed oocytes were washed 4 times in culture medium containing TCM 199 supplemented with 5% SCS, 5 pg/ml insulin (Sigma, St Louis, MO), and 50 pg/ml gentamicin. Finally, sperm exposed oocytes were transferred into four-well multidishes and incubated in culture medium overlaid with mineral oil at 38.5 °C under 5% C0 2 . Sperm exposed oocytes were examined for initial cleavage after 48 h and for blastocyst development on days 7 and 8. Embryo Freezing and Thawing Embryos at the early blastocyst stage were pre-incubated (5-10 min) in freezing solution (1.5 M ethylene glycol + 0.2 M sucrose), then loaded into 0.25 ml french straws and placed in a programmable freezer (Bio-cool, FTS SYSTEM INC., NY). The straws were 109 maintained at 0 °C for 2 min, cooled to -6°C at a rate of l°C/min, seeded and held for 10 min then cooled again at the rate of 0.3 °C/min to -35 °C and finally plunged into liquid nitrogen. The straws were held at -196 °C for 1-3 months prior to thawing. The cryopreserved straws were placed in air for 5 s and then placed in a 37 °C water bath for 10 s for thawing. DNA Analysis Genomic DNA of degenerated oocytes (n=200) and embryos (n=150) was extracted by the phenol/chloroform/isoamyl alcohol method and analyzed by electrophoresis in a 2% agarose gel. Gels were stained with ethidium bromide and visualized under UV light. Molecular sizes of DNA fragments were estimated by comparing migration distance to a 100-bp DNA ladder (Yang and Rajahamendran, 2000). Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling (TUNEL) Fragmented bovine oocytes (n=35) and embryos (n=35) were washed three times in PBS (pH 7.4) containing polyvinylpyrolidone (PVP; 1 mg/ml) and fixed in 4% paraformaldehyde in PBS for a minimum of 1 h at room temperature or overnight at 4 °C. After fixation, the oocytes and embryos were washed in PBS/PVP and permeabilized by incubation in 0.5% Triton X-100 for 1 h at room temperature. Then, oocytes and embryos were washed twice in PBS/PVP and incubated in fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase enzyme (Boehringer Mannheim, QC, Canada) in the dark for 1 h at 37 °C. After being counterstained with 50 pg/ml RNase A in 10 mg/ml propidium iodide (PI) for 1 h at room temperature to label all nuclei, oocytes and embryos were washed 110 in PBS/PVP, mounted with slight coverslip compression, and examined under a fluorescent microscope. Western Blot Analysis Bovine oocytes of the highest quality [grade 1, in which cumulus cell layers were removed by placing oocytes into 0.3% hyaluronidase (Sigma)], oocytes of the lowest quality (denuded, grade 4), 2-8 cell stage embryos with "healthy" morphology, healthy blastocysts, fragmented blastocysts, and preserved blastocysts were washed three times in PBS and frozen at -74 °C until use. Samples were thawed and membranes disrupted by sonication. Protein preparations were solubilized in Laemmli's sample buffer (Laemmli, 1970), boiled for 5 min, and separated by electrophoresis on a discontinuous SDS gel system consisting of 6% polyacrylamide stacking and 12% separating components. Electrophoretic proteins were subsequently electrotrasferred to nitrocellulouse membranes (Hybond-ECL; Amersham Life Science, ON) in 25 mM Tris-glycine buffer (pH 8.3) containing methanol and 0.1% SDS. Blots were blocked in 1 mM Tris containing 0.9% NaCl, 0.2% Tween 20, and 10% nonfat dried milk (blocking buffer; pH 7.4) for 1 h at room temperature or overnight at 4 °C, then reacted using Bcl-2 and Bax rabbit polyclonal antibodies (Sanata Cruz Biotechnology, CA, USA; no cross-reaction between Bcl-2 and Bax) and visulized with an ECL western blotting immunodetection kit (Amersham Life Science, ON). Statistical Analysis The percentage of morphologically abnormal oocytes between grade I and grade IV oocytes after 48 h culture was compared using the chi-square analysis. Statistical significance was inferred atp < 0.05. i l l RESULTS Morphological Evaluation of Oocytes Following Short Time Culture Oocytes cultured in vitro showed an increase in shrinkage and/or fragmentation of the ooplasm with time (Fig. 5.2, a, b, and c). There was a significant difference in the percentage of morphological abnormal oocytes between class I and class IV oocytes after 48 h culture (11.3% vs 22.6%,p < 0.05, Fig 5.3). In vitro Embryo Production The cleavage and the blastocyst formation rates for grade 1 oocytes were 74% and 33%, respectively (Fig. 5.4). The frequencies of normal cleavage to the 2 to 8:cell stage on Day 2 post-insemination and blastocyst formation by Day 7-8 were the endpoints for the evaluation. Compared to normal embryos (Fig. 5.5, a and b), degenerated embryos typically exhibit irregular size blastomeres and cytoplasmic fragmentation (Fig. 5.5, c and d). DNA Analysis of Degenerated oocytes and blastocysts In the DNA analysis study, only high molecular DNA bands were observed. Typical 'ladder' pattern of DNA fragments as a result of nucleosomal cleavage, a biochemical hallmark of apoptosis, could not be demonstrated (not shown). TUNEL Assay DNA fragments (or apoptotic nicking of genomic DNA) in individual oocytes and embryos were further determined using a TUNEL assay, which utilizes the terminal transferase enzyme to add dUTP-FITC to the exposed 3'-hydroxyl ends of DNA that are generated during apoptotic DNA fragmentation. DNA damage was evident in the 112 c3 5 'Sb £ o -3 -3 113 > S a •o S a 0 © >» O o O © e > o a a E E 2 1 cd a © © C O o CM 8 e | A o o o leuijouqv | 0 % 6D 5 fe .5 114 CCJ X o o tN S 5 3 cu « OH w CCJ l l H-» on H-> co — >> cu o V 2 rs B , co ca 2 O C (U o T3 CU H—I 2 cu c cu cu O T 3 OH ^3 P a > o .5 o 0 £ £ B 1 £ § v 0 N •S OH <U £ 2 .G cu 1 s cu G DO CU cu co -o cu rt « s •rt o -g K V CO DO .SP ja ca fa 3 E fragmented oocytes (Fig. 5.6a) and embryos (Fig. 5.6b). No TUNEL labeling was present in the negative control embryos (Fig. 5.6c; incubated without terminal deoxynucleotide transferase enzyme), indicating no nonspecific labeling, Detection of Bcl-2 and Bax Proteins in Oocytes and Embryos Western analysis revealed the presence of immunoreactive proteins corresponding to both Bcl-2 and Bax in extracts of bovine oocytes and embryos of different qualities. Bcl-2 expression was high in grade I oocytes, good quality 2 to 8-cell embryos and blastocysts but hardly detectable in grade IV oocytes. Expression of Bax was found in all grades of oocytes and stages of embryos with higher expression in grade IV oocytes and degenerate embryos. Neither Bcl-2 nor Bax expression was observed in frozen-thawed blastocysts (Fig. 5.7). DISCUSSION One of the reasons for the limited success rate of in vitro bovine blastocyst production was a lack of reliable methods for predicting the developmental competence of cumulus oocyte complexes (COCs). It has been shown that light microscopical assessment of the COCs was a meaningful tool to discriminate between different categories of COCs based on their morphological appearances. Light microscopical classification of oocytes is a non-invasive technique that can be performed without any extra disturbance of the oocytes while preparing them for IVM, IVF and IVC, and thus, this classification technique has been widely used for selecting COCs and improving in vitro bovine blastocyst production (Lonergan et al., 1992; Madison et al., 1992; Brackett and Zuelke, 1993). The present study provides insight on the molecular mechanism behind this light microscopical classification method. Results suggest that apoptosis is involved in oocyte and embryo degeneration and 117 a. s % «a 03 £.5 o ca T7 *! sj co — — 118 CN 03 a CQ i 1 CO in CU cu causes their reduced developmental competence. The ratio of Bcl-2 to Bax (or anti-apoptotic family members to pro-apoptotic family members) may gauge the sensitivity of oocytes and embryos towards survival or apoptosis. In mammals, the cessation of oogonial mitosis is accompanied by the transformation of oogonia into oocytes. In the very early stages of folliculogenesis, a close association between oocytes and granulosa cells is established through gap junctions to form small nongrowing follicles (primordial follicles) (Szollosi, 1975). Oocytes that do not become enclosed by granulosa cells (denuded, around 50-70% in rats) usually degenerate (Szollosi, 1975; Wassarman and Albertini, 1994). Initiation of follicular growth includes oocyte growth as well as transformation and proliferation of granulosa cells. During follicular development, most follicles become atretic at various stages of folliculogenesis and contain degenerated oocytes. Follicles that do not undergo atresia continue to grow until ovulation, and oocytes complete the first meiosis to become ova just before ovulation. After ovulation, if fertilization does not occur within a certain period of time (mouse, 8 h; human, 6-24 h; rat, 12 h), the ovum loses its capacity to be fertilized and starts to degenerate (Szollosi, 1975; Wassarman and Albertini, 1994; Takase et al., 1995). Although the mechanisms involved in these degeneration processes are not completely understood, it has been thought that a natural pre-programmed cell death exists in the oocytes. In order to improve the production of in vitro embryos, different grades of oocytes, selected on the basis of the morphology of the cumulus layer and the ooplasm, were widely investigated for the possible relationship between morphology and developmental competence (Leibfried and First, 1979; Lonergan et al., 1992; Madison et al., 1992; Brackett and Zuelke, 1993). These studies demonstrated that oocytes with a compact cumulus seemed to originate from healthy follicles or from those with only the initial signs of atresia, whereas oocytes with an incomplete and/or expanded 120 cumulus originated from follicles with more obvious signs of atresia. Blondin and Sirard (1995) reported that denude oocytes had the lowest developmental rate, and differed significantly from oocytes of other classes with compact or incomplete cumulus and homogeneous or granulated ooplasm (Blondin and Sirard, 1995). The possibility exists that oocytes might have been at different levels of degeneration at the time of collection. Our study demonstrated that the percentage of abnormal oocytes was much higher for grade IV than grade I oocytes after in vitro culture. These oocytes had morphological abnormalities, including shrinkage of the ooplasm and cell fragmentation, which are typical characteristics of cells undergoing apoptosis (Kerr et al., 1972). Likewise, Takase et al. (1995) and Matwee et al. (1999) indicated that apoptosis is related to the process of degeneration in mouse and bovine immature oocytes. There are two major protein families involved in the regulation of apoptosis: those that mediate the proteolytic breakdown of the cells, the caspase family; and those that regulate the activity of the caspases, the Bcl-2 family. Therefore, the Bcl-2 family of proteins constitutes a critical intracellular checkpoint of apoptosis within a distal common cell death pathway (Ellis et al., 1991; Oltvai et al. 1993; Cory and Adams, 1998). In our study, the level of Bcl-2 expression was much higher in grade I than in grade IV oocytes, whereas more Bax protein expression was observed in grade IV oocytes. These results suggest that oocytes of grade IV are in a more advanced apoptotic process, which explains why these oocytes have less developmental competence compared with grade I oocytes. In addition, our findings support the concept proposed by Oltvai et al. (1993) that the ratio of Bcl-2 to Bax determines whether a cell lives or dies. The reasons for oocyte apoptotic degeneration were not clear. It may be due to an erratic follicular microenvironment and/or the absence of cumulus cells. The presence of cumulus cells has been shown to be required 121 for the transfer of energy to support oocyte maturation and as the mediator of the luteinizing hormone (LH) effect (Sato et al., 1972). The success rate and quality of preimplantation mammalian embryos obtained via IVM/IVF/IVC are variable. In our in vitro embryo production system, the cleavage rate is 74% and the blastocyst formation rate is only 33%. Unequal sized blastomeres and cellular fragmentation, which are typical signs of apoptosis, were observed in cleaved embryos undergoing degeneration during development. Based on these findings, the possibility that apoptosis could be an underlying mechanism of embryo fragmentation was investigated further. Typical DNA laddering, a biochemical hallmark of apoptosis, was not well demonstrated in our study, possibly due to the limited amount of nuclear chromatin (Jurisicova et al., 1995). The TUNEL technique, which has been used extensively in many cell types to detect apoptosis and can be performed within individual cells in situ, enabled us to do further analysis of embryo fragmentation. In our study, the presence of cells with fluorescent labeling strongly suggested apoptotic cell death. A previous study has shown that when compared to in vrvo-derived embryos, cultured porcine embryos at a similar stage exhibit more DNA fragmentation (Long et al., 1998). These data suggest that the artificial environment for IViWIVF/IVC may be one possible trigger for embryo fragmentation. Recently, evidence has suggested that the generation of reactive oxygen species (ROS) in cells plays a fundamental role in the initiation of apoptotic cell death (Wong et al., 1989). Gardiner and Reed (1994) reported that preimplantation embryos are very sensitive to conditions that cause oxidative stress and that their glutathione levels, a tripeptide which protects cells from reactive xenobiotics and guards against ROS generated as a result of normal oxidative metabolism, change dramatically during development (Gaddiner and Reed, 1994). The localization of abundant Bcl-2 protein to the mitochondria of cells suggests that 122 the mechanism of Bcl-2 may be linked to respiratory metabolism and reduction oxidation reactions (Kane et al., 1993). In this regard, data from in vivo studies indicate a role for Bcl-2 in an antioxidant pathway to prevent free radical-induced cellular damage, including lipid peroxidation. However, purified Bcl-2 protein has never demonstrated peroxidase activity in vitro (Hockenberry et al., 1993; Kane et al., 1993). It is now clear that the activity of Bcl-2 is influenced by coexpression of other members of the Bcl-2 family (Oltvai et al., 1993). To date, the most plausible hypothesis is Bcl-2, through protein-protein interactions, focuses as well as regulates an antioxidant pathway at the selective sites of ROS generation. Our results indirectly support this hypothesis since relative expression of Bcl-2 and Bax was found in embryos of different quality. When Bcl-2 is in excess, the embryo is healthy, but when Bax is in excess, the embryo is fragmented. A very recent study in mice demonstrated similar findings using an immunofluorescent staining method (Exley et al., 1999). To our surprise, we failed to detect expression of either Bcl-2 or Bax in frozen-thawed blastocysts; the reason remains to be elucidated. In conclusion, apoptotic death is responsible for oocyte degeneration, embryo fragmentation and related embryonic loss. The relative expression of Bcl-2 and Bax in oocytes and embryos seems to be associated with different developmental competence. Because all apoptotic pathways appear to terminate in the activation of the caspase family of proteases (Thornberry and Lazebnik, 1998), more studies will be carried out to correlate changes in the expression of Bcl-2 homologues and caspases to the generation of oxygen free radicals during in vitro embryo production, thus contributing to understanding the biochemical mechanisms functioning in the embryo to protect it from oxidants. 123 REFERENCES Blondin P, Sirard MA. Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes. Mol Reprod Dev 1995; 41: 54-62. Brackett BG, Zuelke KA. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology 1993; 39: 43-64. Cleary ML, Smith SD, Sklar J. Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t (14; 18) translocation. Cell 1986; 47: 19-28. Cory S, Adams JM. Matters of life and death: programmed cell death at cold spring harbor. Biochimica et Biophysica Acta 1998; 1377: R25-R44. Dobrinsky JR, Johnson LA, Rath D. Development of a culture medium (BECM-3) for porcine embryo: Effects of bovine serum albumin and fetal bovine serum on embryo development. Biol Reprod 1996; 55: 1069-1074. Du F, Looney CR, Yang X. Evaluation of bovine embryos produced in vitro vs. in vivo by differential staining of inner cell mass and trophectoderm cells. Theriogenology 1996; 45:211. Ellis RE, Yuan JY, Horvitz HR. Mechanisms and functions of cell death. Annu Rev Cell Biol 1991; 7: 663-698. Exley GE, Tang CY, McElhinny AS, Warner CM. Expression of caspase and Bcl-2 apoptotic family members in mouse preimplantation embryos. Biol Reprod 1999; 61: 231-239. Gaddiner C, Reed DJ. Status of glutathione during oxidant-induced oxidative stress in the preimplantation mouse embryo. Biol Reprod 1994; 51: 1307-1314. 124 Hardy K, Handyside AH, Winston RM. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 1989; 107: 597-604. Hockenberry DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993; 75: 241-251. Jurisicova A, Varmuza S, Casper RF. Involvement of programmed cell death in preimplantation embryo demise. Hum Reprod Update 1995; 1: 558-566. Kane DJ, Sarafian TA, Anton R, Hahn, H, Gralla EB, Valentine JS, Ord T, Bredesen DE. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science 1993; 262: 1274-1277. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239-257. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685. Leibfried L, First NL. Characterization of bovine follicular oocytes and their ability to mature in vitro. J Anim Sci 1979; 48: 76-86. Lonergan P, Fair T, Gordon I. Effect of time of transfer to granulosa cell monolayer and cell-stage at 48 hours post-insemination on bovine oocyte development following IVM/IVF/IVC. Proceedings of the Eighth Conference of the European Embryo Transfer Association 1992: 178. 125 Long CR, Dobrinsky JR, Garrett WM, Johnson LA. Dual labeling of the cytoskeleton and DNA strand breaks in porcine embryos produced in vivo and in vitro. Mol Reprod Dev 1998; 51: 59-65. Madison V, Avery B, Greve T. Selection of immature bovine oocytes for developmental potential in vitro. Anim Reprod Sci 1992; 27: 1-11. Matwee C, Betts DH, King WA. Developmental regulation of apoptosis in the early bovine embryo. Theriogeneology 1999; 51 (1): 185. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vitro with a conserved homology, Bax, that accelerates programmed cell death. Cell 1993; 74: 609-619. Raff MC. Social controls on cell survival and cell death. Nature 1992; 356: 397-400. Sato E, Iritani A, Nishikawa Y. Factors involved in maturation of pig and cattle follicular oocytes cultured in vitro. Jap J An Reprod 1972; 23: 12-18. Szollosi D. Mammalian eggs aging in the fallopian tubes. In: Blandau RJ (ed.), Aging Gametes: Their Biology and Pathology. Karger, Basel; 1975: 98-121. Takase K, Ishikawa M, Hoshiai H. Apoptosis in the degeneration process of unfertilized mouse ova. Tohoko J Exp Med 1995; 175: 69-76. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998; 281: 1312-1316. Wassarman PM, Albertini DF. The mammalian ovum. In: Knobil E, Greenwld GS, Markert CL, Pfaff DW (ed.), The Physiology of Reproduction, vol. 1, 2nd ed. New York: Raven Press; 1994: 79-122. 126 Wong GHW, Elwell JH, Oberly LW, Goeddel DV. Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell 1989; 58: 923-931. Yang J, Liu X, Bhalla K. Prevention of apoptosis by bcl-2: release of cytochrome c from mitochondria blocked. Science 1997; 275: 1129-1136. Yang MY, Rajahamendran R. Involvement of apoptosis in bovine blastocysts produced in vitro. Theriogeneology 1999; 51 (1): 336. 127 CHAPTER 6 GENERAL DISCUSSION The aims of this thesis were to further study the involvement and regulation of apoptosis during bovine follicular atresia and embryonic development. More than 99% of all ovarian follicles in the cow undergo atresia at various stages of follicular development. The earliest and most prominent feature of atresia in antral bovine follicles is the death of granulosa cells (GCs), leading to almost total destruction of the follicle (Rajakoski, 1960). Given that atresia is the consequence of individual cellular components of the follicle dying, one approach to further elucidate the mechanism underlying follicular atresia would be examine the nature of the death in follicle cells. There are essentially two means by which cells die based on morphological and biochemical criteria: necrosis and apoptosis. Cells that are acutely traumatized typically swell and lyse in response to a wide variety of harmful conditions and toxic substances in the form of cell death known as necrosis. On the other hand, cell death occurrs spontaneously, termed apoptosis, in many cases and also could be induced by physiological or noxious agents. Apoptosis has been suggested as a basic physiological process that plays a major role in the regulation of a cell population (Schwartzman and Cidlowski, 1993). Results from Chapter 3 were the first to demonstrate that most degenerating GCs in follicles undergoing atresia display both morphological and biochemical characteristics of apoptotic cell death including different types of apoptotic cells, apoptotic bodies and definitive ladder pattern of oligonucleosomal length DNA fragments in cows. Apoptosis occurred mainly in GCs and in scattered theca cells. No apoptosis was evident in cumulus cells. These results suggest that apoptosis is the most common pathway of GC deletion which leads to the destruction of the GC layer and finally triggers follicular atresia. In contrast to the results from studies in chickens and rats (Tilly et al., 1991), apoptosis was also detected in GCs of some morphologically healthy follicles, indicating apoptosis is detectable before other morphological and biochemical signs of degeneration appear in cows. It is well known that ovarian follicular atresia is regulated by hormones. If apoptosis is the molecular mechanism underlying atresia, how do hormones control follicular apoptosis? To investigate the effects of hormones on apoptosis in follicles at developmental stages [small (< 4 mm), medium (5-8 mm) and large (> 8 mm)], an in vitro GCs culture system was developed. A time-dependent, spontaneous onset of apoptosis occurred in GCs from follicles of all different sizes during culture. The rate of DNA fragmentation in the culture of GCs from small follicles was higher than that from medium and large follicles. Follicle stimulating hormone (FSH) attenuated apoptotic cell death in GCs from medium follicles more effectively than those from small follicles. Insulin-like growth factor-I (IGF-I) also suppressed apoptosis in cultured GC from small follicles. These data suggest FSH and IGF-I are follicle survival factors probably acting by inhibiting GC apoptosis. Although studies in Chapter 3 demonstrated that apoptosis in bovine follicles is regulated by hormones, nothing is known of the molecular pathways used by these signalling factors to control the initiation of follicular atresia. The Bcl-2 family of proteins constitutes a critical intracellular checkpoint of apoptosis within a distal common cell death pathway. Herein we have characterized changes in the expression of the Bcl-2 protein (an inhibitor of apoptosis) and the Bax protein (an inducer of apoptosis) during atresia of dominant follicles (DFs) induced by high concentration of progesterone (P4) during luteal phase of bovine estrous cycle in Chapter 4. An in vivo model in which the phenomena of the 129 luteal phase in the bovine estrous cycle are mimicked by the synthetic progestin (norgestomet) ear implant and exogenous P4 administration during the follicular phase was used. At the time of P4 administration, DFs gradually underwent atresia and exhibited different levels of apoptosis in GCs, confirming our findings in Chapter 3 that apoptosis is responsible for follicular atresia. Importantly, it was demonstrated that with the progression of follicular atresia, there was a shift in the ratio of Bcl-2 (death repressor) to Bax (death inducer) protein expression and that the shift is mediated primarily through alteration in Bax. Analysis of Bcl-2 and Bax protein expression in follicles at different developmental stages obtained from slaughterhouse ovaries demonstrated results similar to those from in vivo study, suggesting that the members of the Bcl-2 protein family are likely important regulators of follicular atresia. In order to clarify whether the effects of P4 on DF atreisa are via negative feedback regulation of luteinizing hormone (LH) or through a direct action at the level of ovary, an in vitro study using the GC culture system developed in Chapter 3 was carried out. Progesterone did not suppress apoptotic cell death in cultured GC, suggesting P4-induced atresia of non-ovulatory bovine DFs is probably via regulation of LH and its inhibitory effect on apoptosis. Involvement and regulation of apoptosis in bovine immature oocytes and embryos were investigated in Chapter 5. Using an in vitro embryo production system, apoptosis was found to be associated with bovine oocyte degeneration and embryo fragmentation, suggesting the existence of a natural preprogrammed cell death mechanism which can respond to external stimuli and/or internal defects in bovine oocytes and embryos. Compared with poor quality bovine oocytes, the number of good quality oocytes undergoing apoptosis after 48 h serum-free culture was lower. In addition, studies found expression of Bcl-2 was 130 high in good quality oocytes and embryos, low in fragmented embryos, and hardly detected in denuded poor quality oocytes. In contrast, the expression of Bax was found in all types of oocytes and embryos with the highest expression in the denuded poor quality oocytes. These data suggest that the ratio of Bcl-2 and Bax protein expression may be an important determinant of developmental competence for both oocytes and embryos. Taken together, this thesis provides important insight into the mechanisms of follicular atresia and early embryonic loss. Evaluation the nature of these events would increase our understanding of the limitations with follicular dynamics and embryo development. Yet, questions remain. Is apoptosis involved in follicular selection and dominance? Are other apoptotic-related genes involved in follicular atresia? What are their effects? How can apoptosis be inhibited to obtain high quality oocytes? How can culture condition be improved to increase the quality of in vitro produced embryos? Clearly more research is required. 131 SUMMARY OF THESIS RESEARCH FINDINGS 1. Changes in the morphological appearance of GCs during the process of follicular atresia in cattle were found to conform to the characteristic morphological features of apoptotic cell death. 2. The earliest and most prominent feature of atresia in bovine follicles is the death of GCs. Apoptosis was observed mainly in GCs and in scattered theca cells, suggesting apoptosis is the mechanism underlying the follicular atresia. 3. Apoptosis was detected in some morphological healthy bovine follicles, suggesting apoptosis may occur to a certain level during normal follicle growth and development and apoptotic death of GCs may be detectable before other morphological and biochemical signs of degeneration appear in cows. 4. Cultured bovine GCs underwent spontaneous onset of apoptosis. FSH and IGF-I attenuated apoptosis in cultured bovine GC, underscoring their roles as follicle survival factors. 5. Atresia in bovine follicles was found to be linked to a shift in the ratio of antiapoptotic protein (Bcl-2) and proapoptotic protein (Bax) expression and that this shift was mediated primarily through alterations in Bax. 6. Apoptosis was the mechanism underlying the atresia of norgestomet (synthetic progestin)-maintained bovine dominant follicles induced by P4 treatment in vivo. However, P4 did not have direct effect on apoptosis in cultured bovine GCs, suggesting atresia of non-ovulatory bovine dominant follicles that develop during luteal phase of bovine estrous cycle probably via regulation of LH by P4 negative feedback. 7. Apoptotic death was found to be related to oocyte degeneration, embryo fragmentation and related embryonic loss. 8. The relative expression of Bcl-2 and Bax proteins in oocytes and embryos seemed to be connected with their developmental competence. 133 REFERENCES (For General Introduction, Literature Review, and General Discussion) Adams GP, Matted RI, Kastelic JP, Ko, JCH, Ginther, OJ. Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers. J Reprod Fertil 1992; 94: 177-188. Allen RT, Cluck MW, Agrawal DK. Mechanisms controlling cellular suicide: role of Bcl-2 and Caspases. Cell 1998; 54: 427-445. Azzolin GC, Saiduddin S. Effect of androgens on the ovarian morphology of hypophysectomized rat. Proc Soc Exp Biol Med 1983; 172: 70-73. Billig H, Furuta I, Hsueh AJW. Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 1993; 133: 2204-2212. Billig H, Furuta I, Hsueh AJW. Gonadotropin releasing hormone directly induces apoptotic cell death in the rat ovary: biochemical and in situ detection of DNA fragmentation in granulosa cells. Endocrinology 1994; 134: 245-252. Bicsak TA, Tucker EM, Cappel S, Vaughan J, Rivier J, Vale W, Hsueh AJW. Hormonal regulation of granulosa cell inhibin biosynthesis. Endocrinology 1986; 119: 2711-2719. Brackett BG, Zuelke KA. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology 1993; 39: 43-64. Braw RH, BarAmi S, Tsafriri A. Effect of hypophysectomy on atresia of rat preovulatory follicles. Biol Reprod 1981; 25: 989-996. Braw RH, Tsafriri A. Effects of PMSG on follicular atresia in the rat ovary. J Reprod Fertil 1980; 59: 267-272. 134 Buttke TM, Sandstrom PA. Oxidative stress as a mediator of apoptosis. Immuno Today 1994; 15: 7-10. Carson RS, Findlay JK, Clarke IJ, Burger HG. Estradiol, testoterone, and androstenedione in ovine follicular fluid during growth and atresia of ovarian follicles. Biol Reprod 1981; 24: 105-113. Chun SY, Billing H, Tilly, Furuta I, Tsafriri, Hsueh AJW. Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor-I. Endocrinology 1994; 135: 1845-1853. Cohen JJ, Duke RC. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J immuol 1996; 132: 38-42. Cory S, Adams JM. Matters of life and death: programmed cell death at Cold Spring Harbor. Biochimica et Biophysica Acta 1998; 1377: R25-R44. Daud AI, Bumpus FM, Husain A. Evidence for selective expression of angiotensin II receptors on atretic follicles in the rat ovary: an autoradiographic study. Endocrinology 1988; 122: 2727-2734. Dobrinsky JR, Johnson LA, Rath D. Development of a culture medium (BECM-3) for porcine embryo: Effects of bovine serum albumin and fetal bovine serum on embryo development. Biol Reprod 1996; 55: 1069-1074. Don MM, Ablett G, Bishop CJ, Bundesen PG, Donald KJ, Searle J, Kerr JFR. Death of cells by apoptosis following attachment of specifically allergized lymphocytes in vitro. Aust J Exp Biol Med Sci 1977; 55: 407-417. Du F, Looney CR, Yang X. Evaluation of bovine embryos produced in vitro vs. in vivo by differential staining of inner cell mass and trophectoderm cells. Theriogenology 1996; 45: 211. 135 Duvall E, Wyllie AH. Death and the cell. Immunol Today 1986; 7: 115-119. Ellis RE, Yuan JY, Horvitz HR. Mechanisms and functions of cell death. Annu Rev Cell Biol 1991; 7: 663-698. Erickson, BH. Development and radio-response of the preantral bovine ovary. J Reprod Fertil 1966; 11: 97-115. Erickson GF, Li D, Sadrkhanloo R, Liu XJ, Shimasaki S, Ling N. Extrapituitary actions of gonadotropin-releasing hormone: stimulation of insulin-like growth factor-binding protein-4 and atresia. Endocrinology 1994; 134: 1365-1372. Erickson GF, Magoffin DA, Dyer CA, Hofeditz C. The ovarian androgen producing cells: a review of structure/function relationships. Endocrine Rev 1985; 6: 371-399. Exley GE, Tang CY, McElhinny AS, Warner CM. Expression of caspase and Bcl-2 apoptotic family members in mouse preimplantation embryos. Biol Reprod 1999; 61: 231-239. Flaws JA, Kugu K, Trbovich AM, Desanti A, Tilly KI, Hirshfield AN, Tilly JL. Interleukin-1-P-converting enzyme related proteases IRPs and mammalian cell death: dissociation of IRP-induced oligonucleosomal endounclease activity from morphological apoptosis in granulosa cells of the ovarian follicle. Endocrinology 1995; 136: 5042-5053. Giudice LC. Insulin-like growth factors and ovarian follicular development. Endocrine Rev 1992; 13: 641-669. Gorospe WC, Hughes FMJ, Spangelo BL. Interleukin-6: effects on and production by rat granulosa cells in vitro. Endocrinology 1992; 130: 1750-1752. Greenwald GS. Temporal and topographic changes in DNA synthesis after induced follicular atresia. Biol Reprod 1989; 41: 175-181. 136 Gregg GW, Nett TM. Direct effects of estradiol-170 on the number of gonadotropin-releasing hormone receptors in the ovine pitutary. Biol Reprod 1989; 40: 288-293. Gulyas BJ, Hodgen GD, Tullner WW, Ross GT. Effects of fetal and maternal hypophysectomy on endocrine organs and body weight in infant rhesus monkeys: with particular emphasis on oogenesis. Biol Reprod 1977; 16: 216-219. Hammond JM. Peptide regulators in the ovarian follicle. Aust J Biol Sci 1981; 34: 491-504. Hardy K, Handyside AH, Winston RM. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 1989; 107: 597-604. Hay MF, Cran DG, Moor RM. Structural changes occurring during atresia in sheep ovarian follicles. Cell Tissue Res 1976; 169: 515-529. Hengartner MO, Ellis RE, Horvitz HR. Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature 1992; 356: 494-499. Hillier SG, van der Boogaard AMY, Reichert LEJ, Van Hall EV. Alterations in granulosa cell aromatase activity accompanying preovulatory follicular development in the rat ovary with evidence that 5ct-reductase C19 steroid inhibit the aromatase reaction in vitro. J Endocrinol 1980; 84: 409-419. Himelstein-Braw R, Byskov AG, Perters H, Faber M. Follicular atresia in the infant human ovary. J Reprod Fertil 1976; 46: 55-59. Hirshfield AN. Rescue of atretic follicles in vitro and in vivo. Biol Reprod 1989; 40: 181-190. 137 Hsu C, Holmes CD, Hammond J. Ovarian epidermal growth factor-like activity: concentrations in porcine follicular fluid during follicular enlargement. Biochim Biosphys Acta 1987; 147: 242-247. Hubbard CJ, Greenwald GS. Morphological changes in atretic Graffian follicles during induced atresia in hamster. Anat Rec 1985; 212: 353-357. Hughes FM Jr., Gorospe WC. Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Endocrinology 1991; 129: 2415-2422. Hsueh AJW, Billig H, Tsafriri A. Ovarian follicle atresia: A hormonally controlled apoptotic process. EndocrRev 1994; 15: 707-724. Ireland JJ, Roche JF. Effect of progesterone on basal LH and episodic LH and FSH secretion in heifers. J Reprod Fert 1982; 64: 295-302. Ireland JJ, Roche JF. Hypotheses regarding development of dominant follicles during the bovine estrous cycle. In: Roche JF, O'Callagan D (eds) Follicular growth and ovulation rate in farm animals. Martinus Nijhoff Publishers Dordrecht. 1987: pp 1-18. Jolly PD, Smith PR, Heath DA, Hudson NL, Lun S, Still LA, Watts CH, McNatty KP. Morphological evidence of apoptosis and the prevalence of apoptosis versus mitotic cells in the membrana granulosa of ovarian follicles during spontaneous and induced atresia in ewes. Biol Reprod 1997; 56: 837-846. Jacobson M, Raff M. Programmed cell death and BCL-2 protection in very low oxygen. Nature 1995; 374: 814-816. Jurisicova A, Varmuza S, Casper RF. Involvement of programmed cell death in preimplantation embryo demise. Hum Reprod Update 1995; 6: 558-566. 138 Kaipia A, Chun SY, Eisenhauer K, Hsueh AWJ. Tumor necrosis factor-oc and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles. Endocrinology 1996; 137: 4864-4870. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239-257. Kaynard AH, Periman LM, Simard J, Melner MH. Ovarian 3 beta-hydroxysteroid dehydrogenase and sulfated glycoprotein-2 gene expression are differentailly regulated by the induction of ovulation, pseudopregnancy, and luteolysis in the immature rat. Endocrinology. 1992; 130: 2192-2200. Kim JM, Boone DL, Auyeung A, Tsang BK. Granulosa cell apoptosis induced at the penultimate stage of follicular development is associated with increased levels of Fas and Fas Ligand in the rat ovary. Biol Reprod 1998; 58: 1170-1176. Kruip THAM, den Daas JHG. In vitro produced and cloned embryos: Effects on pregnancy, parturitin and offspring. Theriogenology 1997; 47: 43-52. Leibfried L, First NL. Characterization of bovine follicular oocytes and their ability to mature in vitro. J An Sci 1979; 48: 76-86. Leung PC, Steele GL. Intracellular signaling in the gonads. Endocr Rev 1992; 13: 476-498. Ling N, Ying SY, Ueno N, Shimasaki S, Esch F, Hotta M, Guillemin R. Pituitary FSH is released by a heterodimer of the beta-subunits from the two forms of inhibin. Nature 1986; 321:779-782. Liston O, Roy N, Tamai K, Lefebvre C, Baird S, Cherton-Horvat G, Farahani R, McLean M, Ikeda JE, MacKenzie A, Korneluk RG. Suppression of apoptosis in mammalian cells by NAIP and a related family. Nature 1996; 379: 349-353. 139 Lonergan P, Fair T, Gordon I. Effect of time of transfer to granulosa cell monolayer and cell-stage at 48 hours post-insemination on bovine oocyte development following IVM/IVF/IVC. Proceedings of the Eighth Conference of the European Embryo Transfer Association 1992: 178. Long CR, Dobrinsky JR, Garrett WM, Johnson LA. Dual labeling of the cytoskeleton and DNA strand breaks in porcine embryos produced in vivo and in vitro. Mol Reprod Dev 1998; 51: 59-65. Loos de F, van Vliet C, van Maurik, P Kruip. Morphology of immature of bovine oocytes. Gamete Res 1989; 24: 197-204. Madison V, Avery B, Greve T. Selection of immature bovine oocytes for developmental potential in vitro. Anim Reprod Sci 1992; 27: 1-11. Manikkam M, Rajahendran R. Progesterone-induced atresia of the proestrous dominant follicle in the bovine ovary: changes in diameter, insulin-like growth factor system, aromatase activity, steroid hormones, and apoptotic index. Biol Reprod 1997; 57: 580-587. Marion GB, Gier HT. Ovarian and uterine embryogenesis and morphology of the non-pregnant female mammal. J Anim Sci 1971; 32 (Suppl. 1): 24-47. Matter A. Microcinematographic and electron microscopic analysis of target cell lysis induced by cytotoxic T lymphocytes. Immunology 1989; 36: 179-190. Matwee C, Betts DH, King WA. Developmental regulation of apoptosis in the early bovine embryo. Theriogeneology 1999; 51 (1): 185. Miyashita T, Reed JC. Bcl-2 oncoprotein blocks chemotherapy induced apoptosis in a human leukemia cell line. Blood 1993; 81: 151-157. Nagata S. Apoptosis by death factor. Cell 1997; 88: 355-365. 140 Nett TM, Crowder ME, Moss GE, Duello TM. GnRH receptor interaction. V. Down regulation of pituitary receptors for GnRH in ovariectomized ewes by infusion of homologous hormone. Biol Reprod 1981; 24: 1145-1155. Odell WD, Moyer DL. Sperm and ovum transport. In: Physiology of Reproduction, The C.V. Mosby company, Saomt Louis, 1971; pp. 107-122. Ohno S, Smith JB. Role of fetal follicular cells in meiosis of mammalian oocyte. Cytogenetics 1964; 3: 324-333. Olsson JH, Carlsson B, Hillensjo T. Effect of insulin-like growth factor-I on deoxyribonucleic acid synthesis in cultured human granulosa cells. Fertil Steril 1990; 54: 1052-1057. Oppenheim RW, Prevette D, Tytell M, Homma S. Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and RNA synthesis: evidence for the role of cell death genes. Dev Biol 1990; 138: 104-113. O'shea JD, Hay MF, Cran DG. Ultrastructural changes in the theca interna during follicular atresia in sheep. J Reprod Fertil 1978; 54: 183-187. Payne RW, Hellbaum AA. The effect of estrogens on the ovary of hypophysectomized rat. Endocrinology 1955; 57: 193-197. Peluso JJ, Pappalardo A. Progesterone and cell adhension interact to regulate granulosa cell apoptosis. Biochem Cell Biol 1994; 72: 547-551. Peluso JJ, Pappalardo A. Progesterone maintains large rat granulosa cell viability indirectly by stimulating small granulosa cells to synthesize basic fibroblast growth factor. Biol Reprod 1999; 60: 290-296. Raff MC. Social controls on cell survival and cell death. Nature 1992; 356: 397-400. 141 Rajakoski E. The ovarian follicular system in mature heifers with special reference to seasonal, cyclical and left-right variations. Acta Endocrinologica Suppl. 1960: 52: 7-68. Ratts VS, Flaws JA, Kolp R, Sorenson CM, Tilly JL. Albation of Bcl-2 expression decreases the numbers of oocytes and primordial follicles established in the post natal female mouse gonad. Endocrinology 1995; 136: 3665-3668. Reed JC, Meister L, Tanaka S, Cuddy M, Yum S, Geyer C, Pleasure D. Differential expression of Bcl-2 protooncogene in neuroblastoma and other human tumor cell lines of neural origin. Cancer Res 1991; 52: 6529-6538. Roberson MS, Wolfe MW, Stumpf TT, Kittok RJ, Kinder JE. Luteinizing hormone secretion and corpus luteum function in cows receiving two levels of progesterone. Biol Reprod 1989; 41: 997-1003. Roche JF. Control and regulation of folliculogenesis - a symposium in perspective. Rev Reprod 1996; 1: 19-27. Roche JF, Foster DL, Karsch FJ, Cook B, Dziuk PJ. Levels of luteinizing hormone in sera and pituitaries of ewes during the estrous cycle and anestrus. Endocrinology 1970; 86: 568-572. Russell DL, Doughton BW, Tsonis CG, Findlay JK. Pituitary and ovarian function in ewes immunized against the amino-terminal peptide ocN of the inhibin a 4 3 subunit. J Reprod Fertil 1994; 100: 115-122. Salveson GS, Dixit VM. Caspases: Intracellular singaling by proteloysis. Cell 1997; 91: 443-446. Savio JD, Boland MP, Hynes N, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle in heifers. J Reprod Fertil 1988; 83: 663-671. 142 Schwartzman RA, Cidlowski JA. Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocrine Rev 1993; 14: 133-151. Shimasaki S, Ling N. Identification and molecular characterization of insulun-like growth factor binding proteins (IGFBP-1, -2, -3, -4, -5, and -6). Prog Growth Factor Res 1991; 3: 243-266. Siracusa G, De Felici M, Salustri A. The proliferative and meiotic history of mammalian female germ cells. In: Metz CB, Monroy A (eds) Biology of Fertilization. Academic press, Orlanda, 1999: pp253-297. Sirois J, Fortune JE. Ovarian follicular dynamics during the estrous cycle monitored by real time ultrasonography. Biol Reprod 1988; 39: 308-317. Spicer LJ, Alpizar A, Echternkamp SE. Effects of insulin, insulin-like growth factor-I, and gonadotropins on bovine granulisa cell proliferation, progesterone production, estradiol production, and (or) insulin-like growth factor-I production in vitro. J Anim Sci 1993; 71: 1232-1241. Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 1993; 75: 1169-1178. Taylor C, Rajamahendran R. Follicular dynamics, corpus luteum growth and regression in lacating dairy cattle. Can J Anim Sci 1991; 71: 61-68. Terranova PF. Steroidogenesis in experimentally induced atretic follicles of the hamster: a shift from estradiol to progesterone synthesis. Endocrinology 1981; 108: 1885-1890. Thompson CB, Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267: 1456-1462. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998; 281: 1312-1316. 143 Tilly JL, Billing H, Kowalski KI, Hsueh AJW. Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol Endocrinol 1992; 6: 1642-1650. Tilly JL, Kowalski KI, Schomberg DW, Hsueh AJW. Apoptosis in atretic ovarian follicles is associated with selective decreases in messenger ribonucleic acid transcripts for gonadotropin receptors and cytochrome P450 aromatase. Endocrinology 1992; 131: 1670-1676. Tilly JL, Kowalski KI, Johnson AL, Hsueh AJW. Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 1991; 129: 2799-2801. Tilly JL, Tilly KI, Kenton ML, Johnson AL. Expression of members of the Bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased Bax and constitutive Bcl-2 and Bcl-X long messenger ribonucleic acid levels. Endocrinology 1995; 136: 232-241. Tsafriri A, Braw RH. Experimental approaches to atresia in mammals. Oxford Rev Reprod Biol 1984; 6: 226-265. Turzillo AM, Fortune JE. Suppression of secondary FSH surge with bovine follicular fluid is associated with delayed ovarian folliuclar development in heifers. J Reprod Fertil 1990; 89: 643-653. Uilenbroek JT, Woutersen PJ, van der Schoot P. Atresia of preovulatory follicles: gonadotropin binding and steroidogenic activity. Biol Reprod 1979; 23: 219-229. Vale W, Rivier J, Vaughan J, McClintock R, Corrigan A, Woo W, Karr D, Spiess J. Purification of characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature 1986; 321: 776-779. 144 Vaux DL, Weissman IL, Kim, SK. Preventition of programmed cell death in Caenorhabditis elegans by human Bcl-2. Science 1992; 258: 1955-1956. Veldhuis JD, Furlanetto RW, Juchter D, Garmey, J, Veldhuis P. Trophic actions of human somatomedian-C/insulin-like growth factor I on ovarian cells: in vitro studies with swine granulosa cells. Endocrinology 1985; 116: 1235-1242. Vinatier D, Dufour PH, Subtil D. Apoptosis: a programmed cell death involved in ovarian and uterine physiology. European J Obs & Gyn Reprod Biol 1996; 67: 85-102. Walker NI, Harmon BV, Gobe GC, Kerr JFR. Patterns of cell death. Methods Achiev Exp Pathol 1988; 13: 18-54. Wiesen JF, Midgley ARJ. Expression of connexin 43 gap junction messenger ribonucleic acid protein during follicular atresia. Biol Reprod 1994; 50: 336-348. Whitelaw PF, Edine KA, Seller R, Smyth CD, Hillier SG. Gonadotropin-releasing hormone receptor messenger ribonucleic acid expression in rat ovary. Endocrinology 1995; 136: 172-179. Williams PC. Effects of stilbestrol on ovaries of hypophysectomized rats. Nature 1940; 145: 388-389. Woodruff TK, Lyon RJ, Hansen SE, Rice GC, Mather JP. Inhibin and activin locally regulate rat ovarian folliculogenesis. Endocrinology 1990; 127: 3196-3205. Wyllie AH. Cell death: a new classification separating apoptosis from necrosis. In: Browen ID, Lockshin RA (eds) Cell Death in Biology and Pathology. Chapman & Hall, London pp 9-34. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1980; 284: 555-556. 145 Xiao CW, Kim JM, Boone DL, Li J, Carnegie JA, Yoon YD, Tsang BK. The role of laps, Fas/Fas ligand and p53 in follicular development and atresia. Frontiers in Endocrinology 1999; 21: 195-202. Younis AI, Brackett BG, Fayrer-Hosken RA. Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro. Gamete Res 1989; 23: 189-201. Zeleznik AJ, Ihrig LL, Bassett SG. Developmental expression of Ca2+/Mg2+-dependent endonuclease activity in rat granulosa and luteal cells. Endocrinology 1989; 125: 2218-2220. 146 

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