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Effects of gonadotropins and insulin-like growth factor-I (IGF-I) and IGF-II on steroid production by… Yang, Ming Yuan 1996

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EFFECTS OF GONADOTROPINS AND INSULIN-LHCE GROWTH FACTOR-I (IGF-I) AND IGF-H ON STEROID PRODUCTION BY BOVINE GRANULOSA CELLS IN VITRO by  MING YUAN YANG B. Sc., Henan Agricultural University, China, 1991 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 1996 © Ming Yuan Yang, 1996  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or by his  or her  representatives.  It  is  understood  that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT  A serum-free culture system for bovine granulosa cells was developed. Effects of follicle-stimulating hormone (FSH), luteinizing hormone (LH), insulin-like growth factor-I (IGF-I), and insulin-like growth factor-II (IGF-II) on steroid production by bovine granulosa cells were determined. Bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (>10 mm) follicles were cultured for 48 h. Concentrations of estradiol-17p (E ) and 2  progesterone (P ) were measured by radioimmunoassay. 4  For granulosa cells from all classes of follicles, the basal E  2  production was high  during day 1 culture. With time of culture, E production decreased dramatically in granulosa 2  cells from all classes of follicles. Estradiol-170 production increased significantly with increasing follicle size (p < 0.01). Basal P4 production increased with the time of culture and with increasing follicle size (p < 0.01). These results indicated that a serum-free culture system can support basal granulosa cell steroidogenesis and can be used to study the effects of FSH, L H , IGF-I, and IGF-II on bovine granulosa cell steroidogenesis. Only a low dose of F S H (1 ng/ml) stimulated E production by granulosa cells from 2  medium and large follicles (p < 0.05); high doses of F S H (10, 100 ng/ml) inhibited E  2  production by granulosa cells from large follicles (p < 0.05). Follicle-stimulating hormone stimulated P production from granulosa cells of all classes of follicles (p < 0.05). Luteinizing 4  hormone inhibited E  2  production in medium and large follicles (p < 0.05). Luteinizing  hormone increased P4 production in medium and large follicles (p < 0.05).  11  A high dose of IGF-I (100 ng/ml) increased E2 production by granulosa cells from medium and large follicles (p < 0.05). Insulin-like growth factor-! increased P4 production from all categories of follicles. Estradiol-170 production from large follicles decreased in response to IGF-II (10, 100, 500 ng/ml; p < 0.05). A dose of IGF-II (500 ng/ml) inhibited E production by granulosa cells from medium follicles. Insulin-like growth factor-II inhibited P  2  4  production by granulosa cells from medium and large follicles (p < 0.05). Progsterone production from small follicles was inhibited only at a high dose of IGF-II (500 ng/ml; p < 0.05). In conclusion, FSH, L H , IGF-I, and IGF-II have significant effects on steroid production by bovine granulosa cells from small, medium, and large follicles in culture. The bovine granulosa cell culture system developed in the present study can be used for the further studying the effects of hormones, growth factors and their interactions on bovine granulosa cells.  iii  TABLE OF CONTENTS  Abstract  ii  List of Figures  vi  Acknowledgments  viii  Chapter 1  General Introduction  1  Chapter 2  Literature Review  4  2. 1 2.2 2. 3 2. 4  Ovarian Follicular Growth and Development 4 Steroidogenesis 17 Regulation of Steroid Hormone Production at the Ovarian Level . . . 20 Insulin-like Growth Factors (IGF) and IGF-Binding Proteins (IGFBP) 24  Chapter 3  Development of a Serum-free Culture System for Bovine Granulosa Cell from Small, Medium, and Large Follicles To Study Effects of Gonadotropins and Growth Factors 35 3. 1 Abstract 35 3. 2 Introduction 36 3.3 Materials and Methods 40 3.4 Results 45 3. 5 Discussion .48  Chapter 4  Effects of Gonadotropins on Steroid Production by Bovine Granulosa CellsfromSmall, Medium, and Large Follicles in vitro 53 4. 1 Abstract 53 4. 2 Introduction 54 4.3 Materials and Methods 56 4.4 Results 57 4.5 Discussion 67  iv  Chapter 5  Effects of Insulin-like Growth Factor I and II on Steroid Production by Bovine Granulosa Cells from Small, Medium, and Large Follicles in vitro 71 5. 1 Abstract 71 5. 2 Introduction 72 5.3 Materials and Methods 75 5.4 Results 76 5. 5 Discussion 86  Chapter 6  General Discussion and Conclusion  References  90  94  LIST OF FIGURES  Fig. 2-1.  Schematic representation for the growth and development of ovarian follicles during the estrous cycle of cattle (Savio et al, 1990). 14  Fig. 3-1.  Morphology of bovine granulosa cellsfrommedium follicles cultured in the absence of serum at 48 h. 46  Fig. 3-2  Basal E secretion by granulosa cells at 24 and 48 h. 2  47 Fig. 3-3  Fig. 4-1  Basal P secretion by granulosa cells at 24 and 48 h. . 4  .49  Effect of FSH on E production (0-24 h). 2  58 Fig. 4-2  Effect of FSH on E production (24-48 h). 2  60 Fig. 4-3  Effect of FSH on P production (0-24 h). 4  61 Fig. 4-4  Effect of FSH on P production (24-48 h). 4  62 Fig. 4-5  Effect of L H on E production (0-24 h). 2  63 Fig. 4-6  Effect of L H on E production (24-48 h). 2  64 Fig. 4-7  Effect of L H on P production (0-24 h). 4  65  vi  Fig. 4-8  Effect of L H on P production (24-48 h). 4  66 Fig. 5-1  Effect of IGF-I on E production (0-24 h). 2  77 Fig. 5-2  Effect of IGF-I on E production (24-48 h). 2  79 Fig. 5-3  Effect of IGF-I on P production (0-24 h). 4  80 Fig. 5-4  Effect of IGF-I on P production (24-48 h). 4  81 Fig. 5-5  Effect of IGF-II on E production (0-24 h). 2  82 Fig. 5-6  Effect of IGF-II on E production (24-48 h). 2  83 Fig. 5-7  Effect of IGF-II on P production (0-24 h). 4  84 Fig. 5-8  Effect of IGF-II on P production (24-48 h). 4  :  vii  85  ACKNOWLEDGMENTS  I want to express my gratitude to my supervisor, Dr. R. Rajamahendran, for his guidance, assistance and financial support during the graduate program. Appreciation is extended to Dr. K. Cheng and Dr. R. M . Beames for serving on my supervisory committee and for contributing to the accomplishment of this goal. Additionally I would like to thank the Department of Animal Science laboratory technicians who gave their time. I appreciate the friendship and support of my fellow graduate students. Finally, I want to express my deepest appreciation to my parents and sister for their dedicated support and encouragement throughout the graduate program.  viii  CHAPTER 1 GENERAL INTRODUCTION Before reproductive senescence, bovine ovaries have a pool of primordial follicles, each consisting of an oocyte arrested in prophase I of meiosis and a single layer of stratified granulosa cells. Once the cohort of primordial follicles has been established, follicles gradually and continually leave the resting pool to begin growth in a wave pattern. Follicular growth development, selection, and dominance is a very selective process. Once a follicle begins to grow, growth seems to be continuous until the follicle meets one of two fates - ovulation or atresia. It is well known that very few follicles that begin growth successfully ovulate; most die before reaching that stage. The granulosa cells have an integral role in the maintenance and control of ovarian function. These cells help to form the ovarian follicle and provide a proper microenvironment and cytoarchitectural support for the developing oocyte. A major functional parameter of granulosa cells is the biosynthesis of estradiol-170 (E ) and progesterone (P ). E and P4play 2  4  2  important roles in the regulation of female reproductive cycle. Therefore, regulation of granulosa cell function influences both local ovarian function and the endocrine status of the female. Information has been obtained regarding the regulation of granulosa cell function from studies with rat granulosa cells (Hsueh et al., 1984). However, due to the small size of the rat ovary the isolation of follicles at various stages of development is difficult. The bovine ovary is of sufficient size to isolate adequate quantities of granulosa cells from follicles at various stages of development. In addition, the bovine is similar to the human ovary in that only one  1  follicle generally ovulates during each estrous cycle. For these reasons, the bovine ovary provides an attractive model system to study the developmental regulation of granulosa cell function. Bovine granulosa cells have been isolated and maintained in serum-supplemented culture (Fortune and Hansel, 1979). These bovine granulosa cell cultures have been useful in elucidating a number of events (Savion et al., 1982; Henderson and Moon, 1979), including luteinization of bovine granulosa cells and formation of the corpus luteum. Since serum contains multiple substances such as unidentified hormones and growth factors that may interfere with granulosa cell functions, developing a serum-free culture system for bovine granulosa cells to optimize analysis of the hormonal regulation of granulosa cell function is necessary. Both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are required for follicular growth and development ( Lostroh and Johnson, 1966; Findlay, 1993). Early in follicular development, L H receptors are located on theca cells and F S H receptors are located on granulosa cells. However, granulosa cells acquire L H receptors during development and the number o f L H receptors increases during the final stage o f follicular maturation (Uilenbroek and Richards, 1979). A recent study (Berndtson et al., 1995) has shown that only low doses of F S H increase E secretion by granulosa cells from bovine preovulatory follicles 2  in vitro. Although previous studies in vivo and in vitro have examined the granulosa cell's P4 and E production response to gonadotropins, these studies have usually focused on a single 2  stage of developmental follicle, but there have been no direct studies on the effects of F S H and L H on the steroidogenesis of bovine granulosa cells from small, medium, and large follicles.  2  Although gonadotropins play a major role in the control of ovarian follicular growth and development, it is now becoming evident that growth factors, which are themselves products of gonadotropin action, may act locally on follicle cells to either amplify or attenuate the timing and direction of granulosa and theca cell differentiation (Erickson et al., 1994; Hillier, 1994). Of these growth factors, insulin-like growth factors (IGFs) have received considerable attention (Howard and Ford, 1992). In vivo studies indicate that follicular fluid concentration of insulin-like growth factor I (IGF-I) increase with increased follicular size in cattle (Spicer and Enright, 1991). In vitro studies have demonstrated that IGF-I and -II stimulate P4 and E production by granulosa cells from large follicles in the sow in serum2  supplemented medium (Xu et al., 1995). No direct studies have been done on the effect of IGFs on bovine granulosa cells from small, medium, and large follicles. Therefore, the main objectives of this study were: 1) to develop a serum-free culture system for bovine granulosa cells to optimize analysis of the hormonal regulation of granulosa cell function; 2) to study the effect of gonadotropins on bovine granulosa cell steroidogenesis from small, medium, and large follicles; and 3) to study the effect of IGFs on bovine granulosa cell steroidogenesis from small, medium, and large follicles. Increasing basic knowledge of the effect of gonadotropins and IGFs on bovine granulosa cell steroidogenesis of different classes of follicles should contribute to an optimization of techniques used for synchronization of estrous, superovulation and aspiration of follicles for in vitro maturation and in vitro fertilization.  3  CHAPTER 2 LITERATURE REVIEW 2.1 Ovarian Follicular Growth and Development 2.1.1 Ovarian Follicular Growth and Development in Embryonic Life The primary sex organs develop from ridges on the ventromedial surface of the mesonephric kidneys. The gonadal ridges of the undifferentiated embryonic gonads contain primordial germ cells and gonadal blastema (Latshaw, 1985). The primordial germ cells arise from endodermal cells of the yolk sack. The primordial germ cells start to migrate to the gonadal ridge around day 26 to 32 in cattle (Richards, 1980). Mitotic division of germ cells (i.e. oogonia) starts when germ cells arrive at the gonadal ridge and onset of meiosis occurs at approximates day 70 of embryonic life in the bovine ovary; however, meiosis is not completed in the embryonic ovary, and germ cells are arrested at diplotene stage of prophase I. In cattle, most primary oocytes have achieved first meiotic arrest by day 170 to 175 of embryonic life and remain in this stage until puberty (Erickson, 1966). The first primordial follicles are observed in the ovary of cattle around day 90 to 100 of embryonic life (Marion and Gier, 1971). At this time a single oocyte is surrounded by squamous, often irregularly shaped epithelial cells. Under appropriate conditions of hormonal stimulation, which normally occur at puberty, the primordial follicles become activated and start to grow. The oocyte enlarges, and the squamous cells that surround it swell and become cubodial. These structural changes herald the transformation of a primordial follicle into a primary follicle. The follicle is termed a secondary follicle once cells surrounding the oocyte  4  divide and form several layers. While these modifications take place, the stroma immediately around the follicle differentiates  to form the theca folliculi. This layer subsequently  differentiates into theca interna and theca externa. As the follicle grows due to an increase in size and number of granulosa cells, accumulation of liquor folliculi (follicular fluid) secreted by granulosa cell appears between the adjacent granulosa cells. Eventually, these spaces fuse, and the follicular fluid becomes contained within a single large cavity called the antrum, these follicles are termed tertiary follicles. In the tertiary follicle, cells of the granulosa layer form a small pedicle of cells, the cumulus oophorus, which contains the oocyte and protrudes towards the interior of the antrum. Granulosa cells that form a layer that surrounds the zona pellucida of the oocyte become elongated and form the corona radiata.  2.1. 2 Gonadotropin Receptors in Ovarian Follicles Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are gonadotropins that belong, together with placental chorionic gonadotropin and thyroid-stimulating hormone (TSH), to a family of glycoprotein hormones formed by two noncovalently linked a and 3 subunits (Fiddes and Talmadge, 1984). The a subunit is responsible for species specificity and the P subunit for hormone specificity. L H and F S H act on target cells through specific receptors. These receptors activate the adenylate cyclase pathway and to some extent phospholipase C and phospholipase A (Misrahi et al., 1993) 2  Development of gonadotropin  receptors on theca interna and granulosa cells is essential to the process of growth and development of rat ovarian follicles (Richards, 1980). Although there is enough evidence to indicate that no gonadotropin support is required to reach the stage of primordial and  5  secondary follicles, antrum formation in most mammals seems to be gonadotropin dependent (Richards, 1980). Hypophysectomy causes atresia of large antral follicles in the rat ovary after a short period of time (Richards et al., 1978). After several days of hypophysectomy, only primordial follicles are observed in the ovaries, supporting the view that gonadotropin support is only required in large antral follicles. Granulosa cells of small antral follicles contain F S H receptors but no L H receptors (Findlay, 1993). It is generally assumed that F S H binding first appears in granulosa cells at or about the primary follicle stage, when the oocyte ceases to grow. Ovarian F S H binding becomes measurable by the end of the first week of life in rats and increases during prepubertal development to a maximum at day 28. In vivo studies using immature, hypophysectomized female rats indicate that F S H increases the content of its own receptors in the granulosa cells. Ovarian F S H receptors in prepubertal rats may be induced by the high serum F S H levels present at this age. In vitro studies using granulosa cells suggest that F S H may also exert a negative effect on its own receptor. Continuous exposure of granulosa cells to F S H in vitro results in 'down regulation" of the F S H receptor and decreased F S H responsiveness. Although estradiol by itself has no effect on the distribution, number, or affinity of affinity of granulosa cell F S H binding sites, estrogens synergize with F S H in vivo to increase the number of F S H receptors per granulosa cell through a process involving both induction and maintenance. F S H stimulation induces L H receptor gene expression in theca interna cells (Segaloff et al., 1990). Ireland and Roche (1982) demonstrated the presence of L H receptors on theca cells of antral follicles. In contrast to the presence of F S H receptors in granulosa cells from  6  follicles of all sizes, L H receptors are found only in granulosa cells of large preovulatory follicles. Granulosa cells from large follicles contain receptors for both F S H and L H in cattle (Ireland and Roche, 1982), humans (Hillier, 1994) and rats (Magoffin and Erickson, 1994). These observations are in keeping with the acquisition of L H receptors under the influence of F S H during ontogeny, as well as with the marked increases in the number of L H receptors in granulosa cells at the time of proestrus. Several in vivo and in vitro studies have clearly demonstrated the ability of F S H to increase L H receptor numbers in whole ovarian tissue, isolated follicular units, or cultured granulosa cells (Richards and Midgley, 1976; Hiller et al., 1978; Casper and Erickson et al., 1981; Erickson et al., 1982). The ability of F S H to induce L H receptors is augmented by the concomitant presence of estrogens in vivo (Richards et al., 1979; Richards, 1980) and in vitro (Rani et al., 1981). Furthermore, progestins (Rani et al., 1981), androgens (Rani et al., 1981) and insulin (May et al., 1980), as well as L H / h C G (Ireland and Richards, 1978) enhance the F S H induction of L H receptor content. In contrast, the F S H action is inhibited by epidermal growth factor (EGF) and gluococorticoids (Mondschein and Schomberg, 1981). Also, in vitro exposure to excessive L H results in down regulation of the L H receptor content (Schwall and Erickson, 1983). Once induced, the L H receptors of the granulosa cells require the continued presence of F S H for their maintenance. Alternatively, these L H receptors can be maintained by prolactin (Jones and Hsueh, 1981). The presence of both L H and F S H receptors in both cell types of antral follicles supports the two cell-two gonadotropin theory (Fortune and Amstrong, 1977, 1978). Thus, L H stimulates the synthesis of A 4 precursors in theca interna cells which are then aromatized to 17p-estradiol in granulosa cells under F S H stimulation.  7  Ireland and Roche (1983) characterized changes in growth and in concentrations of steroid in follicular fluid and gonadotropin receptors of individual antral follicles after spontaneous luteolysis in heifers (Ireland et al., 1982) or in nonovulatory follicles after ovulation in herfers (Ireland and Roche, 1983). These studies indicated that development of F S H and L H receptors in estrogen (E ) active (EA) follicles is critical to develop their 2  steroidogenic capacity and give further support to the two cell-two gonadotropin theory (Fortune and Amstrong, 1977, 1978; Fortune and Hansel, 1979).  2.1. 3 Gonadotropic Regulation of Follicular Growth The first signs of follicular growth are the enlargement of the primary oocyte, granulosa cell proliferation and differentiation of theca cells adjacent to the basal lamina. The initial stimulus for these events is unknown; however it would appear to be gonadotropin independent. Granulosa cell proliferation occurs in vitro without gonadotropin stimulation (Baker andNeal, 1973). In addition, treatment of adult mice with anti-gonadotropins does not interrupt early follicular growth, although gonadotrophic stimulation can accelerate the rate of growth. It has been suggested that an oocyte-derived growth factor may stimulate granulosa cell proliferation in primordial follicles. Destruction of primordial germ cells in fetal rats results in development of sterile gonads incapable of steroid synthesis. (Merchant, 1975). As with the initial stimulus for the growth of primordial follicles, the stimulus for the differentiation of interstitial cells in the thecal layers is not clear. It appears that the signal may arise from the primordial follicle itself. Co-culture of dispersed porcine granulosa and thecal  8  cells tend to re-aggregate with the theca cells at the periphery (Stoklosawa et al., 1982). With the acquisition of L H receptors, theca cells became steroidogenically competent. Further growth of follicles from the prenatral to the antral stage and beyond has long been held to require gonadotrophic stimulation, namely that of FSH. With F S H stimulation granulosa cells acquire the enzymes required for the conversion of androgens to estrogens. There is also an increase in mitotic activity, and finally, under the influence of F S H and estradiol-17p, the granulosa cell layer acquires L H receptors (Richards, 1980). Under the influence of both L H and F S H the theca cell layer and the granulosa cell layer of growing antral follicles interact synergistically to synthesize and secret sex steroid hormones. The thecal layer primarily produces progestins and androgens, while the granulosa layer produces estorgens and, to a lesser degree, progestins. Sex steroid hormones produced by growing follicles and the resultant corpus luteum (CL) from ovulatory follicles enter the general circulation and find their way to the hypothalamic-pituitary axis where they can regulate the synthesis and secretion of pituitary gonadotropins. It has long been known that L H and F S H secretion is modulated by a negative feedback loop from factors secreted by gonads. P4 exerts a negative effect on L H secretion by decreasing the L H pulse frequency. The negative effect of P4 is probably primarily at the level of the hypothalamus. The negative effect of P does not appear to extend to FSH. Treatment 4  of ovariectomized wes with P does not decrease circulating F S H (Nett et al., 1981). There is 4  some evidence to suggest that P may also act at the level of the pituitary (Miller et al., 1990). 4  Feedback of estrogen on the hypothalamic-pituitary axis is more complicated than the feedback of P4. Estrogen have been shown to have both negative and positive feedback  9  effects. The inhibition by E is probably due to a direct effect at the pituitary level. Injection of 2  an estradiol bolus in ovariectomized ewes inhibits L H secretion but has no effect on gonadotropins releasing hormone (GnRH) secretion from the hypothalamus (Nett et al., 1984). The positive feedback effect of E2, which generates the ovulatory gonadotropin surges, appears to be the result of E 's effect on the hypothalamus. The L H surge is associated with 2  an increase in GnRH secretion (Miyake et al., 1980). The increase in GnRH leads to the secretion of large amounts of both F S H and L H , producing the ovulatory gonadotropin surges and results in depletion of the pituitary gonadotropin stores (Nett et al., 1990). This is followed by an increase in the pituitary content of gonadotropin subunit mRNAs, presumably to replenish the pituitary (Nett et al., 1990). The positive feedback of E may be enhanced by 2  the actions of E directly on the pituitary. One of the initial effects of E appears to be the 2  2  induction of an increase in pituitary GnRH receptors. Following the gonadotropin surges, the effect of E switches from a positive feedback to a negative one. Most evidence suggests that 2  the long-term effect of E is due to the actions of E on the hypothalamus. GnRH secretion is 2  2  decreased, leading to a decrease in gonadotropin secretion (Karsch et al., 1987).  2.1. 4 Follicular Dynamics During the Bovine Estrous Cycle Before ultrasonic imaging became available in 1984 (Pierson and Ginther, 1984), growth and replacement rates and the fate of bovine ovarian follicles during the estrous cycle were studied extensively by utilizing a variety of microscopic techniques (Rajakoski, 1960; Matton et al., 1981; Dufour and Roy, 1985). Rajakoski (1960) proposed the existence of two waves of follicular growth. One wave culminated on day 12, followed by atresia, whereas the  10  other culminated at estrous. In addition, Rajakoski (1960) concluded: that the first wave dominant follicles that become atretic at day 12 of the estrous cycle remains on the ovary for another 5 days; that both ovaries have a similar number of follicles > 1 mm; that the right ovary has a greater number of follicles > 5 mm, a greater frequency of ovulations, and a greater weight compared to the left ovary. He described morphology of the ovarian follicular system and variations attributed to season, stage of the estrous cycle, asymmetry between right and left ovaries, and corpus luteum development. Furthermore, Dufour et al. (1972) demonstrated that the largest follicle present in the ovary 5 days prior to estrus is not the follicle that ovulates. This provided the first evidence that turnover of the largest follicle in the ovary is quite rapid at the end of the cycle. These studies in cattle provided evidence for growth and replacement rates of large follicles, but no information on small and medium size follicles. Marion and Gier (1971) suggested that growth of an antral follicle to the preovulatory size requires 40 days. Recently Lussier et al. (1987) concluded that a period equivalent to two estrous cycles (42 days) is required for a follicle to grow through the antral phase (0.13 mm) to preovulatory size. In follicles greater than 2.5 mm, they shown that c  follicular growth was the result of antrum development rather than an increase in the number of granulosa cells. Real-time ultrasonic imaging of ovarian structures has provided a noninvasive and nondisruptive technique to image directly daily changes in ovarian follicular dynamics during the estrous cycle in cattle (Pierson and Ginther, 1984; Ginther et al, 1989; Savio et al, 1988; Sirois and fortune, 1988; Rajamahendram and Walton, 1990). Briefly, ultrasound imaging utilizes high-frequency sound waves to produce images of internal tissues and organs. Pulses  11  of electric current produce the vibration of specialized piezoelectric crystals housed in the ultrasound transducer which generate sound waves. These sound waves sample a wide (e.g. 50 mm) but thin (e.g. 2 mm) tissue area which results in a two-dimensional image of the tissue. Thus, sound waves produced by the ultrasound transducer are directed through the tissue of interest by placing the transducer over the tissue and moving and/or varying the angle of the transducer. Ultrasonic characteristics of the tissue dictate the ability of the sound wave to reflect back to the transducer and be converted to electric current which are displayed as an echo on the ultrasound viewing screen. The echoes are displayed as varying shades of gray. Liquids do not reflect sound waves and therefore are displayed as black images (e.g., ovarian follicle and embryonic vesicle, whereas dense tissues reflect a large proportion of the sound waves and hence are displayed on the viewing screen as light gray or white (e.g., pelvic bones). Transducer with three types of frequencies (i.e. 3.5, 5 and 7.5 MHz) are used commonly for transrectal scanning of the reproductive tract of most large animals. Transducers with a higher frequency of transmitted sound waves have a shallower depth of penetration in the tissue and hence have a better resolution of the image. Therefore, a relatively small structure like a 5-15 mm follicle located close to the transducer generally is studied with a 5 or 7.5 M H z transducer. Conversely, transducers with a lower frequency of transmitted sound waves have a lower resolution but a higher degree of penetration, and therefore are more suited for studying large structure located far from the transducer such as the mid- or late-gestation fetus and uterus. Ultrasonic monitoring of ovarian follicles and corpora lutea has been used to study follicular dynamics during the estrous cycle (Pierson and Ginther, 1984; Ginther et al., 1989; savio et al., 1988; Sirois and fortune, 1988) and during  12  the early postpartum (Rajamahendram and Taylor, 1990). Before the utilization of ultrasonic imaging to study folliculogenesis in cattle, there was no agreement on whether antral follicles were in a continuous state of turnover without any distinct pattern of growth and atresia during the estrous cycle (Marsion et al., 1968; Dufour et al., 1972) or if cows had two very distinct periods of turnover during the estrous cycle (Matton et al., 1981; Rajakoski, I960;). Now it is well documented in cattle (Savio et al., 1988; Sirois and fortune, Rajamahendran and Taylor, 1990)  1988;  that one to four waves of follicular growth and  development occur during a single estrous cycle of cattle, and that the preovulatory follicle is derived from the last wave. The processes of recruitment, selection, and dominance were defined by Hodgen (1982) as they relate specifically to the recruitment and selection of the dominant follicle in primates. Recruitment is a process whereby a cohort of follicles begins to mature in a milieu of sufficient pituitary gonadotropic stimulation to permit progress toward ovulation. Selection is the process by which a single follicle is chosen and avoids atresia with the potential competence to achieve ovulation. Dominance is the means by which the selected follicle dominates through inhibition of recruitment  a new cohort of follicles. These  definitions also have been applied to the processes of dominant follicle turnover during the estrous cycle in cattle (Ireland and Roche, 1987). A schematic representation of follicle numbers and size from one ultrasonographic study of follicular dynamics during an esrous cycle is presented in Figure 2-1 (Savio et al., 1990). Early during the estrous cycle a cohort of follicle is recruited out of the pool of smaller antral follicles (2 to 4 mm). The mechanism that controls recruitment of these small follicles and determines which follicles are recruited is unknown, but increased concentrations of F S H in plasma after ovulation, the preovulatory  13  saiomod do u a a w n N  3 < w  to  u  V)  D 0  >• _J O  ¥ I  LU D  cc CO UJ  UJ  1 0  x•  E  LL  a 8  tn ><  z  O  a  1 <  7. >  Q  cr  D  U O <y>«or~<oin*jr->(->j —  • ssmo  t  c ssvio (WH)  U313WVIQ  t  f t 3 SST10  c3  t I SSY10  3iomoj *0  14  F S H surges, and the presence of F S H receptors (Ireland and Roche, 1982) are thought to be responsible for this process. After 2 to 4 days of recruitment (d 2, 3, and 4 of the estrous cycle), several medium-sized follicles (6 to 9 mm) can be detected by ultrasonography. Immediately after recruitment, a selection phase begins in which a single follicle emerges from the pool of recruited follicles and continues to grow, whereas other recruited follicles decrease in size. The mechanism for determining which follicle becomes the selected dominant follicle has not been identified. The prevalent concept is that once recruitment has started due to gonadotropin stimulation, the follicle that gets a head start in the coordinated gene expression of growth factors, steroidogenic enzymes and inhibins, will be the one selected (Erickson et al., 1994). The dominant follicle of the first follicular wave remains active until the middle of the estrous cycle (d 8 to 11; Ginther et al., 1989). When ovaries are examined by ultrasonography during this period of follicular dominance, no new follicles > 5 mm are detected . Based on this evidence, it seems that other follicles are prevented from entering the recruitment phase during the period of dominance. Therefore, a dominant follicle is defined as a large ovarian follicle ( > 10 mm) that is recruited and selected during a follicular wave. In addition, an active dominant follicle is capable of preventing the growth of other follicles on the ovary. In the majority of estrous cycles, the first dominant follicle regress and evolution of a second follicular wave (recruitment, selection, and dominance) results in the presence of a second active dominant follicle. In two-wave cycles, maturation of the second dominant follicle coincides with spontaneous regression of the C L , and this follicle ovulates after luteolysis (Savio et al., 1988; Sirois and Fortune, 1988; Taylor and Rajamahendran, 1991).  15  Alternately, the second dominant follicle may become atretic and, if this occurs, a third follicular wave will be initiated. Second-wave dominant follicles are smaller in mature size than first-wave dominant follicles when there is a third follicular wave during the estrous cycle. In addition, cattle that experience three follicular waves have a longer estrous cycle (Taylor and Rajamahendran, 1991) because estrous is delayed when the second dominant follicle fails to ovulate, with the third dominant follicle requiring additional time to complete development before ovulation. Most cattle have two or three follicular wave during an estrous cycle; however, estrous cycles consisting of one or four waves of follicular growth have been reported (Savio et al., 1988; Sirois and Fortune, 1988). The number of follicles within discrete size classes changes during a follicular wave. Early during the estrous cycle (d 1 to 4), the average number of follicles detected by ultrasonography in Class 1 (3 to 5 mm) decreases, whereas the average number of follicles in Class 2 (6 to 9 mm) increases. The shift apparently occurs because Class 1 follicles are growing into Class 2 and are not being replaced by smaller follicles (< 3 mm in diameter). This movement represents the growth of follicles out of Class 1 into Class 2 during the recruitment phase. On approximately d 4 of the estrous cycle, the average number of Class 3 (10 to 15 mm) follicles increases as an average of one follicle grows into Class 3 as the selected follicle. At the same time, the average number of follicles in Class 2 decreases because follicles that did not become dominant decrease in size and become atretic. This results in the detection of fewer Class 2 follicles and more Class 1 follicles after d 7 of the estrous cycle. Later during the first follicular wave (d 7 to 9 of the estrous cycle) the average number of Class 4 follicles increases because Class 3 follicles (dominant follicles) grow to a diameter > 15 mm and are  16  detected by ultrasonography as Class 4 follicles. The sustained suppression of Class 2 follicles (d 9 to 11 of the estrous cycle) is probably due to effects of dominance exerted by the firstwave dominant follicles.  2. 2 Steroidogenesis Growing follicles and the corpus luteum synthesize and secrete most of the body's sex steroids. Ovarian theca cells (TCs) and granulosa cells (GCs) work in concert to produce progestins, androgens and estrogens by what is known as the two cell-two gonadotropins theory of steroidogenesis. Falk's (1959) results were the first to show critical experimental evidence that follicular estradiol production is dependent on an interplay between theca interna and granulosa cells. Further support for the two cells model for E production comes 2  from experiments done by Fortune and Armstrong with rats (1977) and with bovine proestrus follicles (Fortune, 1986). All three types of steroids, progestins, androgens and estrogens, are secreted and enter the circulation to be delivered to target tissues, including cells in the hypothalamic-pituitary axis where they modulate gonadotropin biosynthesis and secretion.  2. 2.1 Progestin Biosynthesis Cholesterol, the common steroid precursor for the biosynthesis of progestins, androgens and estrogens, is newly synthesized from acetate or mobilized from its esterfied derivative stores in the cytoplasmic lipid droplets. In mitochondria, cholesterol is converted into pregnenolone (P5) by cytochrome P450 cholesterol side-chain cleavage enzyme (P450 SCC). This is the first and rate-limiting step in the synthesis of steroids hormones in ovarian  17  follicles. The conversion step is done by three successive monooxygenations on cholesterol (hydroxylations at C-22, followed by C-20, and finally cleavage of the C-20, 22 bond). Immunoflourescent staining has been used to localize cytochrome P450 SCC to inner mitochondria membranes in theca and granulosa cells (Farkash et al., 1986). P450 SCC is expressed in the theca interna cells; its expression in granulosa cells depends on stage of follicle development (Waterman and Simpson, 1985). F S H stimulates small increases in cytochrome P450 SCC mRNA in rat granulosa cells, and this effect is potentiated by E . 2  Stimulation of granulosa and theca cells by L H leads to dramatic increases in cytochrome P450 SCC transcripts. Induction of cytochrome P450 SCC by gonadotropins is via the cAMP-adenylate cyclase signal transduction system. Forskolin, and other agonists which increase intracellular cAMP, stimulate increases in cytochrome P450 SCC mRNA and increase progestin production (Richards and Hedin, 1988).  2. 2. 2 Androgen Biosynthesis Progestins produced in theca and granulosa cells can be further metabolized in theca cells to yield androgens. Pregnenolone  is converted  subsequently  pregnenolone and dehydroepiandrosterone (DHEA) through the A  5  into  17-hydroxy-  pathway, or into  progesterone (P4) by cytochrome P450 3p-hydroxysteroid dehydrogenase/A -A -isomerase 5  (P450  33-HSD)  and  subsequently  converted  to  4  17-hydroxy-progesterone  and  androstenedione A 4 through the A pathway. The A pathway is the predominant pathway for 4  5  the production of A4 in cattle and humans, while the A4 pathway is the predominant pathway for the production of A4 in rats, mares, and sows (Lacroix et al., 1974). Both of these  18  reactions are catalysed by the enzyme cytochrome P450 17a hydroxylase (P450i ) which has 7a  been localized in theca but not granulosa cells (Richards et al., 1986). This single chain polypeptide has both hydroxylase and 17-20 lyase activity to convert C-21 progestins to C-19 androgens. In rats, small amounts of L H or human chorionic gonadotropin (hCG) stimulate androgen biosynthesis by cultured theca cells (Carson et al., 1981). The increase in androgen is associated with an increase in theca cell cytochrome P450i  7a  protein and mRNA.  Conversely, with large amounts of L H , as seen during the ovulatory surge, there is a decrease in  theca  cell  androgen  biosynthesis,  P450n  a  activity,  protein,  and  mRNA.  Dehydroepiandrosterone can be acted upon by 30 H S D to produce androstenedione. Androstenedione can then be converted to testosterone by 170 hydroxysteroid dehydrogenase (170 HSD). The conversion of androstenedione to testosterone occurs in both theca and granulosa cells, although the majority of the activity appears to be associated with granulosa cells. (Bogovich and Richards, 1984).  2. 2. 3 Estrogen Biosynthesis Estradiol and estrone are the major estrogens synthesized by the follicle. The conversion of androgens to estrogens in the ovary occurs primarily, if not exclusively, in granulosa cells. The reaction is catalysed by the microsomal enzyme aromatase cytochrome P450. Aromatase has been shown to be a single polypeptide capable of catalysing both the aromatization of the A ring and the NADPH-cytochrome reductase reaction. Aromatase is capable of converting androstenedione to estrone and testerone directly to estradiol-170. Estrone is converted to estradiol-170 by 170 HSD, though there is evidence to suggest that  19  this is a separate isozyme from the 17B H S D that converts androstenedione to testerone (Mendelson et al., 1990). It is generally accepted that theca and granulosa cells act synergistically in estrogen production. The so-called 'two-cell theory" was first proposed by Flack (1959) and later modified a documented by studies in rats (Fortune and Armstrong 1977) and sheep (Moor 1977). This theory asserts that the theca interna cells, under the influence of L H , produce androgens that are transported to granulosa cells where they are converted to estrogen by aromatizing enzymes induced by FSH. Thus granulosa cells are the source of follicular estrogen in these species. However, in primate an porcine follicles the theca interna has been shown to provide an additional source of estrogen. Also granulosa cells can produce androgens, indicating that steroidogenesis is not rigidly compartmentalized in all species and throughout follicular differentiation (Ryan, 1979). Aromatase is induced by F S H stimulation, as seen by a dramatic increase in both mRNA and protein (Chan and Tan, 1987). Luteinizing hormone has been shown to be capable of maintaining the F S H induced aromatase activity, as has E , testerone and dihydrotesterone 2  (Richards et al., 1987). In addition, a number of growth factors synergize with F S H to further increase aromatase activity, including IGF-I (Adashi et al., 1985) and TGFfj (Bendell and Dorrington, 1990). Conversely, E G F and TNFoc (Mondschein and Schomberg, 1981) inhibit FSH-stimulated aromatase activity.  2. 3 Regulation of Steroid Hormones Production at the Ovarian level Steroid hormone output at the ovarian level depends on the rate of steroid biosynthesis and catabolism (Hanukoglu, 1992). The pathway for steroid hormone biosynthesis starts with  20  cholesterol derived primarily from circulating lipoproteins (i.e. high-density lipoprotein [HDL] and low-density lipoprotein [LDL]) rather than from de novo synthesis from acetate (Hinshelwood et al., 1994). In cattle and sheep, the addition of either H D L or L D L increases P production in cultured luteal cells, and supports the role of H D L and L D L as a substrate 4  source for steroid hormone biosynthesis  at the ovarian level (Carrol et al., 1992).  Gonadotropin hormones activate a series of reactions that lead to the hydrolysis of cholesterol and transport of cholesterol into the mitochondria. Conversion of cholesterol to pregnenolone in mitochondria by P450 SCC is the first rate-limiting and hormonally regulated step in the synthesis of all steroid hormones (Hanukoglu, 1992; Miller, 1988). Subsequent biosynthetic steps proceed with the flow of substrates through enzymes located in the endoplasmic reticulum (Miller, 1988). Furthermore, steroids hormones are hydrophobic molecules that can penetrate biological membranes and can flow into the blood stream after being synthesized without being stored in intracellular vesicles. Steroid hormone production is regulated by at least four different mechanisms (Hanukoglu, 1992): 1) mRNA levels of steroidogenic enzyme as determined by transcription, stability and translation of mRNAs encoding the enzymes; 2) steroidogenic enzyme activity as , determined by postranslational modifications of the enzymes, by cofactor availability and by the conditions of the intracellular milieu; 3) substrate availability, and 4) tissue growth as determined by cell division multiplication (i.e., growth of the dominant follicle and the corpus luteum). Gonadotropin stimulation of granulosa and theca cells in culture generates an increase of mRNA for steroidogenic enzymes within a few hours, with this reaching a maximum value at 6-12 hrs post stimulation (Steinkampf et al, 1987). The 'two cell, two  21  gonadotropin" theory for E  2  synthesis in ovarian follicles states that L H stimulates the  synthesis of A 4 precursors in theca interna cells whereas F S H stimulates aromatization of A 4 in granulosa cells (Hillier, 1994; Magoffin and Erickson, 1994). The interaction of F S H or L H with the granulosa and theca interna cell receptors activates adenylate cyclase and phospholipase, causing a transient increase in intracellular levels of second messages such as cAMP, cGMP, inositol triphosphate, C a  2+  and diacylglycerol (Leung and Steelt, 1992).  Thereafter, cAMP activates protein kinase (PKA), and C a  2+  and diacylglycerol activiates  protein kinase C (PKC); both kinases phosphorylate protein and hence affect their function. The list of FSH-responsive genes is extensive and include P450 SCC, P450 3p-HSD, P450 17oc-HYD, L H receptors, IGF-I, IGFBPs and inhibin (Richards et al., 1987; Magoffin and Erickson, 1994; Segaloff et al., 1990;). The list of L H responsive genes is less extensive compared to FSH. L H positively regulates P450 17a-HYD and IGF-I receptors in theca cells. Dramatic evidence that IGF-I and gonadotropins act synergistically to control follicle cytodifferentiation has been reported. In general, IGF-I itself has little or no effect on the differentiated function of either granulosa or theca cells. However, when gonadotropins are present, activities of theca and granulosa cells are dramatically enhanced by IGF-I. IGF-I and FSH, both which act synergistically to control progestin and aromatase steroidogenesis and L H receptors (Adashi et al., 1985), whereas IGF-I and L H act synergistically to control androgen biosynthesis (Erickson et al., 1985). The dominant follicle of the first follicle wave is recruited, selected, becomes dominant, and then undergoes atresia within the first 12 days of the estrous cycle. During this period, the physiological function of the dominant follicle changes as it grows and develops.  22  For example, the concentrations of estradiol and inhibin in plasma increase during the development of a preovulatory dominant follicle. Secretion of estradiol and inhibin by the dominant follicle continues until it eventually becomes atretic or is ovulated. During the same period, other follicles on the ovary (second-largest or subordinate follicles) do not become dominant and do not continue to grow after selection. The failure of these second-largest follicles to continue to grow suggests that they are functionally different from dominant follicles (Badinga et al., 1991). Badinga et al. (1991) hypothesized that the steroidogenic function (aromatase enzyme activity and steroids in follicular fluid) of dominant and subordinate follicles would differ during the initial stages of selection (d 5 of the estrous cycle), during the dominance phase (d 8 of the estrous cycle), and finally during the initial stages of atresia for the dominant follicle of the first follicular wave (d 12 of the estrous cycle). Furthermore, Lucy et al. (1992) concluded that major differences in the amounts of aromatase activity in follicle walls, as well as estradiol in follicular fluid, exist between dominant and subordinate follicles. The differences in aromatase activity in follicle walls, and estradiol in follicular fluid, were detected as early as d 5 of the estrous cycle when dominant and subordinate follicles were diverging in size (12 mm vs 7 mm). Progesterone, which is low at all times in the follicular fluid of dominant follicles, increases from d 5 to 12 in the follicular fluid of subordinate follicles. Dominant follicles collected on d 5 and d 8 had similar aromatase activity in the follicular wall but that the d-8 dominant follicles has less estradiol in follicular fluid than did the d-5 dominant follicles. Therefore, relative to the d-5 dominant follicle, the d-8 dominant follicle may have insufficient concentrations of androgens (substrate for aromatase) for the level of aromatase activity that is found in the walls of these follicles. One  23  hypothesis implied by this is that the atresia of the first-wave dominant follicle is initiated by a limited concentration of androgen substrate for estrogen synthesis. Because L H stimulates androgen biosynthesis, a reduction in androgen availability might occur when L H pulse frequency declines toward the middle of the estrous cycle in the cow. This may reduce the flow of androgens through the steroidogenic pathway, causing a decline in aromatizable substrate (and estradiol in follicular fluid) and thus initiate atresia.  2. 4 Insulin-like Growth Factors (IGFs) and IGF-Binding Proteins (IGFBPs) 2. 4.1 Ovarian IGFs Granulosa cells provide the cytoarchitectural support for the developing oocyte and participate in follicular antrum formation, whereas theca cells surround granulosa cells and form the exterior wall of the follicle. Both cell types must undergo extensive proliferation and functional differentiation to develop from preantral to antral follicles. Once the follicle is selected and becomes dominant, further growth is required to achieve ovulatory size. Conversely, the cohort of follicles that does not become selected and/or the nonovulatory dominant follicle become atretic and cell growth is arrested. Therefore, the regulation of cell proliferation in ovarian follicles requires both stimulatory and inhibitory mechanisms. Follicular development is primarily regulated by L H and F S H (Richards, 1980; Lucy et al., 1992), but ample evidence is emerging that growth factors, which are themselves products of gonadotropin action, may act locally on follicle cells to either amplify or attenuate the timing and direction of granulosa and theca cell differentiation (Erickson et al., 1994; Hillier, 1994). Of these growth factors, insulin-like growth factors are receiving more and more attention.  24  Over 20 years after the determination of the primary amino acid sequence of insulin in 1955, a role for insulin as a regulator of ovarian function was suggested. It took an additional 10 years to determine the mechanism of insulin action on ovarian cell steroidogenesis as well as to determine the presence of insulin receptors in ovarian cells (Poretsky et al., 1987). In general, insulin's effect on ovarian cells is positive, stimulating granulosa cell proliferation and the production of P , and enhancing luteal cell steroidogenesis, regardless of species. The 4  insulin-like growth factor-I (IGF-I) and factor-II (IGF-II) molecules were first purified from human plasma and sequenced by Rinderkneckt and Humbel 18 years ago (Rinderkneckt and Humbel, 1978ab). Both IGFs are low molecular weight single chain polypeptides that are structurally related to proinsulin and promote cellular mitosis and differentiation in a variety of tissues and systems. Furthermore, IGF-I is formed of 70 amino acids, whereas IGF-II is formed by only 67 amino acids. Approximately 45% of the amino acids comprising IGF-I, IGF-II and insulin are identical; The presence of insulin and IGFs in the ovary was first reported by Hammond (Hammond et al., 1985) in the pig. Since that time, the presence of IGFs in the follicular fluid of several other species has been reported, including cattle, sheep, horses, and humans (Hammond et al., 1985). In general, concentrations of insulin (0.5 to 10 ng/ml), IGF-I (100 to 500 ng/ml) and IGF-II (20 to 1,000 ng/ml) in follicular fluid are equal to or lower than concentrations in plasma (Samaras et al., 1994). However, after a 2-day fast in heifers, follicular fluid concentrations of IGF-I were found to be higher than plasma concentrations (Spicer et al., 1992). Similarly, in fasted women, follicular fluid concentrations of insulin are higher than plasma concentrations (Diamond et al., 1984). Thus, it appears that  25  nutritional status influences the relationship between ovarian and systemic levels of insulin and IGFs. IGF-I is produced by granulosa cells under control of F S H and E (Hammond et al., 2  1985) and have been shown to have a synergistic effect with F S H to stimulate granulosa cells in vitro (Adashi et al., 1991; Giudice 1992). Studies have reported the that presence of IGF-I and/or IGF-II mRNA in ovarian tissue of humans, rats, cattle, pigs, and sheep including granulosa, theca, stromal, and luteal cells (Echternkamp et al., 1991; Spicer et al., 1993, 1995; Barreca et al., 1993; Samaras et al., 1994). Rat granulosa cells appear to contain exclusively IGF-I mRNA, whereas rat theca cells contain exclusively IGF-II mRNA (Hernandez et al., 1989; Oliver et al., 1989) Both granulosa and theca cells of bovine follicles contain IGF-I mRNA (Spicer et al., 1993). Recent studies indicate that IGF-II mRNA exists in porcine ovaries (Samaras et al., 1994) as well as in ovine follicular walls (Spicer, 1995). In addition, porcine and bovine granulosa cells secrete IGF-I (Spicer et al., 1993; Hsu and Hammond, 1987), whereas human granulosa cells predominantly secrete IGF-II (Ramasharma et al., 1987), exclusively localize IGF-II, and contain IGF-II mRNA (Barreca et al., 1993). Some studies have indicated that somatotropin (ST) and gonadotropin can stimulate IGF-I and(or) IGF-II production in porcine (Hsu and Hammond., 1987) and human granulosa cells, respectively. In contrast, the production of IGF-I by bovine granulosa cells in vitro was unaltered by treatment with ST and FSH, but was inhibited by treatment with insulin. In vivo, 20- to 40-d treatment of pST in prepubertal gilts was thought to increase serum and follicular fluid IGF-I concentrations but decrease or have no effect on serum and(or) follicular fluid IGF-II concentrations (Samaras et al., 1994).  26  In summary, it appears that, regardless of species, ovarian IGF-I production and IGFII production are under different control mechanisms and that concentrations of IGF-I increase as follicles develop. However, species differences may exist in terms of the specific cell layer(s) within the follicle that may produce IGF-I and IGF-II and in terms of the hormones that regulate their production.  2. 4. 2 Characterization of IGF-I and IGF-II Receptors The IGFs exert their physiological actions by interacting with specific cell surface membrane receptors, described as type I and type II 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 very similar three dimensional structures, leading to cross reactivity between the IGFs , insulin and their respective receptors (Giudice, 1992). Specific receptors for insulin and IGFs and their mRNA exist in various types of ovarian cells, including granulosa cells, theca cells, luteal cells, and ovarian stromal cells (Davoren et al., 1986; EIRoeiy et al., 1993). Whether numbers of insulin and IGF receptors change as follicles develop is unclear. In swine, granulosa cells from small follicles have fewer insulin receptors than do granulosa cells from medium or large follicles (Otani et al., 1985). Similarly, in.women, granulosa and theca cells from dominant follicles localize more insulin receptor mRNA than do cells from small antral follicles (EI-Roeiy et al., 1993).In comparison to insulin receptors, the number of granulosa cell IGF-I receptors is similar among small, medium, and large porcine follicles (Hylka et al., 1989). In contrast, granulosa cells from large bovine follicles, cultured in vitro,  27  have a 15-fold higher number of IGF-I receptors than do cells form small follicles (Spicer et al., 1994). A recent report showed that specific IGF-I binding increased as follicles enlarged from preantral to antral size, concomitant with increases in F S H and L H binding. Atretic follicles from these ovaries contained a significantly lower number of specific IGF-I binding sites than did nonatretic follicles (Wandji et al., 1992). A similar study using in situ hybridization histochemistry in rat ovaries indicated that IGF-I mRNA is lost whereas IGF-I receptor mRNA is increased as the granulosa cells differentiate and luteinize, whereas IGF-I receptor mRNA disappears from the granulosa cells of atretic follicles. In further support of the notion that the number of IGF-I receptors in granulosa cells is higher in large than is small follicles, Shaw et al. (1993) has shown that steroid production by granulosa cells from large follicles is dramatically increases by IGF-I whereas IGF-I has little or no effect on steroid production by granulosa cells from small follicles. Recent studies using the cellular localization of IGF-I receptor mRNA in human ovaries indicate that IGF-I receptors are present in granulosa cells of primary and antral follicles of infant ovaries and graffian follicles of adult ovaries (EI-Roeiy et al., 1994). Collectively, IGF-I receptors are present in granulosa cells throughout ovarian follicular development and increase as small antral follicles develop into graffian follicles (i.e., as granulosa cells differentiate), and decrease during atresia. The numbers of IGF-I receptors in granulosa cells of small bovine follicles are increased by E G F , estradiol, and F S H but are unaffected by L H and progesterone. In contrast, numbers of IGF-I receptors in granulosa cells of large bovine follicles were unaffected by E G F , estradiol, FSH, or L H in the experiment of Spicer et al (1994). The stimulatory effect of EGF, estradiol and  28  F S H on IGF-I receptors in granulosa cells of small follicles may explain why these hormones synergize with IGF-I to enhance granulosa cell mitogenesis and(or) differentiation. In contrast to IGF-I receptors, IGF-II receptors mRNA and protein are localized within both granulosa and theca cells of human antral follicles (EI-Roeiy et al., 1994). Receptors for IGF-II have been localized in rat ovaries. In sheep, IGF-II receptors appear to be primarily present in granulosa cells of atretic follicles and in theca cells of healthy follicles, although specific [ I] IGF-II binding was also detected in oocytes and stroma (Teissier et al., 125  1994). Determination of whether follicles of pigs and cattle contain IGF-II receptors awaits further study. Experimental evidence indicates that expression of IGF receptors is under gonadotropin control, because hypophysectomy decreases the biosynthesis of IGF receptors, and treatment with F S H and L H restores IGFs receptors to levels similar to those prior to hypophysectomy (Spicer et al., 1992). Changes in plasma and FF concentration of IGF-I in cattle have been reported in several studies. In vivo studies indicate that concentrations of IGF-I increase with increased follicular size in cattle (Spicer and Enright, 1991) and pigs (Bryan et al., 1989; Spicer et al., 1992). In vivo data also reveal a positive correlation between follicular fluid IGF-I and progesterone concentrations in postpartum anestrous and cyclic cows. (Spicer & Enright, 1991). Echternkamp et al. (1994) reported that FF IGF-I concentrations increase with increasing concentrations of E in FF in preovulatory follicles. The IGFs have several effects 2  on ovarian cells. One of the more important is the mitogenic effect of IGF-I, which stimulates D N A synthesis and cell proliferation in granulosa cells (Baranao and Hammond, 1984; Olsson et al., 1990). IGF-I also increases progestin production in porcine and bovine granulosa cells  .29  cultured in vitro, probably by acting in synergy with FSH to increase  P450  side-chain cleavage  activity, gene expression and lipoprotein metabolism (Veldhuis et al., 1985;  Gong et al.,  1994). In rat granulosa cells IGF-I increases FSH-stimulated, but not basal, P and E 4  2  production. It also increase the FSH-stimulated induction of granulosa cell L H receptors, FSH-stimulated basal inhibin synthesis (Adashi et al., 1986), suggesting IGF-I plays an important role in follicle selection, maturation and atresia. Ovarian follicular fluid contains considerable amounts of IGF-II (Giudice, 1992). Less appears to be known of the actions of IGF-II than of IGF-I, although it would appear that it does stimulate P production by granulosa cells collected from mature pigs (Veldhuis et al., 4  1985) and that IGF-II can enhance progesterone biosynthesis by facilitating sterol delivery and increasing levels of cholesterol side-chain cleavage enzyme mRNA in pig (Garmey et al., 1993). In humans, the concentrations of IGF-I, IGF-II and insulin in ovarian and peripheral venous blood samples obtained from women undergoing abdominal hysterectomy have revealed a decreased ovarian gradient for IGF-II but not for IGF-I or insulin, suggesting that IGF-II but not IGF-I may be locally regulated by the ovary (Jesionowaska et al., 1990). Indeed, human granulosa cells contain IGF-II mRNA but not IGF-I mRNA, and IGF-II is secreted by proliferating granulosa cell cultures from human ovaries (Ramasharma and Li, 1987). Based on immunohistochemical studies, the cells in the ovary producing the IGF-I peptide may be the theca-interstitial cells. El-Roeiy and coworkers (1993) found by studying the expression of the genes encoding the IGFs, their receptors and the localization of their gene products in specific compartments of the human ovary, that IGF-II is synthesized in theca cells in small antral follicles and in granulosa cells in dominant follicles. Because IGF-II  30  mRNA is detectable in human granulosa cells before ovulation, this peptide probable plays an autocrine-paracrine role in follicle maturation (Geisthovel et al., 1989). While apparently not produced by human granulosa cells, IGF-I stimulates DNA synthesis in human granulosaluteal cells from both natural and stimulated cycles (Olesson et al., 1990). This suggests a role for IGF-I in the regulation of human granulosa cell proliferation. All of these indicate that both endocrine and paracrine actions of IGF-I are likely to modulate granulosa cell function.  2. 4. 3 Ovarian Insulin-like Growth Factor Binding Proteins (IGFBPs)  Follicle development is primarily regulated by F S H and L H . Recently, evidence has become available to indicate that IGFs themselves are the products of gonadotropin action, and that they have a very active role in folliculogenesis by acting locally to modulate the timing and direction of granulosa and theca cell differentiation and gene expression. Furthermore, an increasing body of evidence suggests that in the ovary, the biological responses of the IGF-I and IGF-II under normal F S H stimulation also may be regulated by IGF-binding proteins (IGFBPs) (Ui et al., 1989). These IGFBPs may bind to IGF-I and IGF-II present in the FF and therefore selectively negate their tropic activity (Ui et a l , 1989). The IGFBPs are high-affinity carrier proteins that function as transport proteins for the IGFs in the systemic circulation. The IGFBPs major functions are: reservoir of IGFs molecules, increasing the half-life, regulating endocrine effects of IGFs in peripheral circulation, and modulating paracrine and autocrine effects of IGFs in specific tissues (Giudice, 1992). To date, six unique species of IGFBPs have been identified: IGFBP-1, -2, -3, -4, -5, and -6. In general, there is about a 50% amino acid homology among the IGFBPs within species and about an 80%  31  amino acid homology for each IGFBP across species. The liver produces IGFBPs along with numerous other tissues, including the ovary. Since 1989, when the structure of an IGFBP and its mRNA in ovarian tissue were first reported (Ui et al., 1989), research has implicated the IGFBPs as potential regulators of follicular development. In particular, the IGFBPs in the ovary appear to play an important role in regulating the biologic activity of IGF-I (Hammond et al., 1991; Giudice et al., 1992; U i et al., 1989). For example, IGFBP-2, -3, -4, and -5 inhibit FSH-induced estradiol synthesis in cultured rat and human granulosa cells by sequestering IGF-I (Ui et al., 1989). Consistent with the idea that IGFBPs inhibit follicular steroidogenesis, levels of follicular fluid progesterone are negatively correlated (r = -0.70) with follicular wall IGFBP-2 mRNA (Samas et al., 1993). Also, follicular fluid estradiol is negatively correlated with follicular fluid IGFBP-2 in ovine follicles (r = -0.74) (Spicer et al., 1995) and bovine follicles (r = -0.57). Follicular fluid levels of IGFBP-2 as well as other lower molecular weight IGFBPs (-4, -5, but not IGFBP-3), assessed by ligand blotting, decrease as follicles enlarge in sheep (Monget et al., 1993), pigs (Mondschein et al., 1991) , and cattle (Echternkamp et al., 1994). In pigs, IGFBP-2, IGFBP-4 and IGFBP-5 are present predominantly in atretic and in small immature follicles. Similar result have been described in cattle, where concentration of FF IGFBP-2 and two unidentified IGFBP forms (29-27 kDa and a 22 kDa) were related inversely to FF E concentrations. There is at least one report in cattle that provides evidence 2  to indicate that IGFBPs levels are higher in FF from bovine follicles that contained oocytes with reduced developmental potential and in follicles that contained oocyte cumulus complexes that failed to develop to the blastocyst stage (Hazeleger et al., 1993). Collectively, ligand blot analyses of FF from human and rat follicles indicate that IGFBP-3 is predominant  32  IGFBP in FF and that amounts of IGFBP-2, -4 and -5 are higher in atretic follicles. In addition, studies in porcine indicate that low amounts of IGFBP-2, -4 and -5 are present in preovulatory follicles, and that IGFBP-3 is abundant in FF. In cattle, one study partially characterized the presence of IGFBPs in FF during the periovulatory period, but did not include atretic dominant follicles. Studies on comparing IGFBPs content in dominant and atretic follicles in cattle FF are lacking. The identification of factors that regulate the production of ovarian IGFBPs has been an active area of research in recent yeas. Of the hormones evaluated, F S H (200 ng/ml) is one of the most potent inhibitors of IGFBP-2 and -3 activity produced by porcine granulosa cells (Mondschin et al., 1990). In cultured rat granulosa cells, total IGFBP activity in spent media was also reduced after F S H (> 30 ng/ml) treatment (Adashi et al., 1991). The administration of exogenous F S H to cyclic cows decreased the amounts of IGFBP-2 and lower molecular weight IGFBPs in large (> 8 mm) estrogen-active follicles, but had no effect on these IGFBPs in large estrogen-inactive follicles or on IGFBP-3 in either type of large follicle (Echternkamp et al., 1993). In contrast to FSH, IGF-I and insulin have been shown to stimulate IGFBP-2 and -3 activity in cultured porcine granulosa cells, whereas estradiol, E G F , and transfroming growth factor-3 had no significant effect on IGFBP-2 or -3 activity when corrected for changes in cell numbers (Mondschein et al., 1990). There are at least two FSH-dependent mechanisms that are thought to regulate the FF amounts of IGFBP-2, -4 and -5 in rat follicles. The first potential mechanism to regulate FF amount of IGFBPs involves inhibition of gene expression of IGFBP-2, -4 and -5. Therefore, the F S H signal transduction mechanism may act either to inhibit gene transcription or to  33  increase degradation of mRNAs for these low molecular IGFBPs. The second potential mechanism to regulate FF amounts of IGFBPs involves stimulation of protease(s) that cleave IGFBP-2, -4 and -5 into lower molecular mass fragments that do not bind IGFs in FF (Liu et al., 1993). In summary, IGFBP activity in the follicular fluid of pigs, sheep, and cattle decreases as follicles develop and become estrogen active. As follicles undergo atresia and become estrogen inactive, IGFBP activity increases. These changes in IGFBP activity are due to changes in IGFBP-2 and other lower molecular weight IGFBPs and not to changes in IGFBP3. The hormones that regulate these changes in IGFBP activity are not well characterized for sheep and cattle but, on the basis of studies in rats and pigs, may involve FSH, insulin, and IGF-I. The decrease in IGFBP activity, together with an increase in IGF-I concentrations as follicles develop, likely provides a coordinated sequence of events that facilitates IGF-I and -II bioavailability for the support of follicular growth and increased steroidogenesis by granulosa cells.  34  CHAPTER 3 DEVELOPMENT OF A SERUM-FREE CULTURE SYSTEM FOR BOVINE GRANULOSA CELLS FROM SMALL, MEDIUM, AND LARGE FOLLICLES TO STUDY EFFECTS OF GONADOTROPINS AND GROWTH FACTORS  3.1 Abstract This study developed a serum-free culture system for bovine granulosa cells (GCs). GCs from healthy small (< 5 mm), medium (6-10 mm), or large (> 10 mm) follicles were cultured at a concentration of 5 x 10  5  Progesterone (P4) and estradiol-170  cells/well in a serum-free medium for 48 h.  (E ) content in the medium were determined by 2  radioimmunoassay. E production from all classes of follicles was higher at 24 h compared to 2  production at 48 h. At both time points E production increased significantly (p < 0.01) with 2  increasing follicle size.With the time of culture, P production from all categories increased 4  significantly. P production at both time points increased significantly (P < 0.01) with the 4  increasing follicle size. In conclusion, the serum-free culture system can mimic bovine G C function in vivo. The system can be used to study direct effects of hormones and growth factors on bovine G C function.  35  3. 2 Introduction Ovarian follicles have been shown to be the basic functional unit of the ovary and consist of an outer layer of the theca interna cells which encircle an inner layer of granulosa cells. Granulosa cells, in turn, surround the innermost oocyte-cumulus cell complex. Various immunocytochemical, histochemical and autoradiographic studies have clearly demonstrated that the granulosa cells inside a given follicle are arranged in a stratified manner and that the states of their differentiation are not uniform. At least three populations of granulosa cells can be distinguished. The mural granulosa cells, i.e. those found near the basement lamina, are usually the most active in various functional parameters (Zoller and Weisz, 1979). The antral granulosa cells are closer to the antral cavity, while the cumulus cells are those granulosa cells surrounding the oocyte. The mural cells are probably most active in steroid hormone production, as these cells possess high levels of 3(3-hydroxysteroid dehydrogenase and cytochrome P450 activities (Zoller and Weisz, 1979). The maturation of ovarian follicles, follicle atresia, and follicle transformation into corpora lutea is regulated by a specific set of hormones and has been a major research area for ovarian physiologists for long time. In response to cyclic pituitary gonadotropin secretion, various follicular compartments interact in a highly integrated manner to secrete sex steroids (estrogens and progestins) and to produce a fertilizable ovum. Although pituitary gonadotropins ( L H and FSH) perfusing the ovary are the major regulators of follicular development, not all follicles in a estrous cycle, indeed, only a limited number of selected follicles ovulate during the life span of the female while most of the follicles become atretic. After ovulation, the granulosa cells undergo profound changes in their hormonal responsiveness and in their capacity to produce steroids.  36  These luteinized granulosa cells constitute a major component of the corpus luteum and are the main source of ovarian progestins. The granulosa cell has an integral role in the maintenance and control of ovarian function. This cell not only helps form the ovarian follicle but also provides the proper microenvironment and cytoarchitectural support for the developing oocyte. A major functional parameter of granulosa cells is the biosynthesis of estrogen and progesterone. Therefore, regulation of granulosa cell function influence both local ovarian function and the endocrine status of the female. To understand the basis for the disparate maturation of ovarian follicles and the luteinization of maturing granulosa cells, it is necessary to correlate functional and morphological changes with the intraovarian hormonal profile. Follicular granulosa cells are separated from blood vessels and theca cells by a basement membrane lining the follicle. Although Smith (1949) observed that rabbit cumulus cells survived during culture, Bjersing and Carstensen (1964) first isolated granulosa cells from porcine follicles and performed a short term incubation of these cells to demonstrate their steroidogenic capacity in vitro. Granulosa cells from many animal species were subsequently cultured for a prolonged period in serum-containing medium (Channing,  1970) and were shown to maintain progestin  biosynthetic capacity and to undergo luteinization in vitro (Richards and Midgley, 1976; Dorrington and Armstrong, 1979). Since serum contains multiple substances that may interfere with granulosa cell function, further attempts were made to culture these cells in serum-free conditions (Erickson and Hsueh, 1978). There are several potential benefits from the use of serum-free media for studying developmental granulosa cell function: 1) serum-free  37  media provide an opportunity to study effects of circulating hormones and various serum factors; 2) primary culture of granulosa cells in a serum-free medium retain hormonal responsiveness and physiological functions resembling the in vivo conditions; 3) serum-free media improve reproducibility between cultures, and avoidance of batch-to-batch variations of sera; 4) serum-free medium avoid serum cytotoxicity; and 5) there is less protein interference in bioassays. Extensive information has been obtained regarding the regulation of granulosa cell function with the use of serum-free cultures of rat granulosa cells (Hsueh et al., 1984). However, due to the small size of the rat ovary, the isolation of follicles at different stages of development is difficult. The bovine ovary is of sufficient size to isolate adequate quantities of purified granulosa cells from follicles at various stages of development. In addition, the bovine is similar to the human in that only one follicle is generally ovulated during each estrous cycle. For these reasons, the bovine ovary provides an attractive model system to study the developmental regulation of granulosa cell function. Bovine granulosa cells have been isolated and maintained in serum-supplemented culture (Fortune and Hansel, 1979). This bovine cultures have been useful in elucidating a number a events (Henderson and Moon, 1979), including the steroid interactions between thecal and granulosa cells (Fortune, 1986). However, in these studies, it was difficult to determine estradiol concentration in medium. It is likely due to methodological differences (i.e.: tissue source, culture conditions) for each species. In particular, it is recognised that inclusion of serum in the culture medium may have a profound influence on the function of the cells in vitro, not only by affecting cell plating efficiency, but also by modifying secretory responses to various exogenous hormones and  38  other factors (Henderson and Franchimont, 1981). Information obtained from rat indicated that granulosa cells in serum-free culture system retain remarkable hormonal responsiveness. The development of a serum-free culture system for bovine granulosa cells to optimize analysis of the normal regulation of cellular function is urgently needed. The ovarian follicle has several functionally distinct developmental stages during the processes of folliculogenesis.  These include development of the primordial follicles to  preantral, antral, Graffian, and ovulatory follicles. In addition, follicles may undergo atresia at any stage of development, and ovulated follicles luteinize to form the corpus luteum. The present  study was restricted to an analysis of the developmental stages of small antral,  medium antral, and Graffian follicles. Granulosa cells within the small and medium antral follicles undergo rapid cell proliferation as the follicle enlarges and develops an increased capacity to produce estrogen. Granulosa cells initiate the formation of the antrum and produce many components of follicular fluid. Granulosa cells in the Graffian follicle form a large antrum,  produce  large  amounts  of  follicular fluid  components,  and  provide  the  cytoarchitectural support for the developing oocyte. Cells in these follicles maintain a high level of estrogen production while developing an increased ability to produce progesterone. This study was designed: 1) to develop a serum-free culture system for bovine granulosa cells from small, medium, and large follicles; 2) to determine whether major differences of steroid production exist in bovine granulosa cells obtained from various stages of preovulatory follicle development, i.e. granulosa cells from small, medium, and large follicles.  39  3. 3 Materials and Methods 3. 3.1 Preparation of Experimental Reagents Medium is Dulbecco's Modified Eagles medium (DMEM) and Ham's F12 in a 1:1 ratio obtained from Stemcell Technologies Inc. (Vancouver, B.C., Canada). The medium was supplemented with penicillin (100 ug/ml)/streptomycin (100 ug/ml), insulin (5 pg/mg) /transferrin  (5  pg/ml)/sodium  selenite  (5  ng/ml),  and  androstenedione  (1  uM).  Androstenedione was initially dissolved in absolute ethanol, followed by subsequent dilution in the culture medium. The final ethanol concentration in the cell cultures was less than 0.1% (vol./vol.). All the reagents, unless otherwise specified, were obtained from Sigma Chemical Co. (St. Louis, M O , USA).  3. 3. 2 Preparation of Granulosa Cells Ovaries were obtained at nearby commercial abattoirs from beef and dairy cows and heifers less than 5 min after slaughter. Ovaries were transported to the laboratory (within 120 min) on ice in a buffered salt solution supplemented with 1% (w/v) bovine serum albumin and 1% (v/v) penicillin/streptomycin. Ovaries were washed three times in saline (0.15 M NaCl) then immersed in 70% ethanol for 30 s, and washed again three times with saline. The ovaries were kept in saline on ice until the granulosa cells were collected. The follicles were classified into three types according to their diameters: small (< 5 mm), medium (5-10 mm) and large (> 10 mm). Granulosa cells were harvested only from healthy follicles. Healthy follicles were punctured with an 18 gauge needle and follicular fluid was aspirated. The inner wall of the punctured follicle was scraped gently to remove granulosa cells, leaving the basement  40  membrane and theca cells intact. Then, a glass pipet was used to aspirate serum-free medium and rinse the follicular wall ten times. Media obtained from different classes of follicles were placed separately in 15 ml culture tubes immersed in ice. The granulosa cells were harvested by centrifuging media at 400 x g for 10 min. The cells were washed three times with serumfree medium and cell concentrations were estimated using a hemacytometer. Viability of the cells was assessed by trypan blue exclusion (cell suspensions were mixed 1:1 with 0.4% trypan blue solution and incubated for 10-15 min, dead cells are stained blue but live cells do not take up colour) and ranged from approximately 40-60% (Campbell, 1979). The cell suspension was then diluted with media to obtain a concentration of approximately 5 x 10 cells/50 pi cell 6  suspension.  3. 3. 3 Selection of Healthy Follicles Only granulosa cells recovered from healthy follicles were used to culture. Two methods were used. First, healthy and atretic follicles were classified according to previously established morphological criteria (Metcalf, 1982). Follicles were classified as non-atretic if they had a uniformly bright appearance; extensive and very fine theca blood capillaries when observed at lOOx magnification; a regular granulosa layer, and no free-floating particles in the follicular fluid. Follicles with some loss of translucency and a slightly greyish appearance or with some very small free floating particles in the follicular cavity were classified as light-atretic. Follicles were classified as atretic if they had a dull, grey appearance, blood vessels either irregularly filled with clotted blood of partial detachment of the membrane granulosa and many large  41  free-floating globules in the antral cavity. Follicles with a dark, often spotted appearance and a very dark cumulus were classified as heavy-atretic. Second, bovine follicles were confirmed as non-atretic or atretic according to their viable cell numbers in each follicles (McNatty et al., 1979). In this procedure, the maximum number of granulosa cells in follicles ranging from 1 mm to 18 mm in diameter was determined. A follicle was judged to be healthy if it contained > 75% of the maximum number of granulosa cells for the diameter it had reached: these follicles were shown to have high levels of F S H and estradiol-17p in their antral fluid and, in most cases, a healthy oocyte; most follicles which contained between 51 and 75% of their maximum number of granulosa cells, also contained a healthy oocyte but their levels of follicular fluid estradiol-170 were low. Although these follicles were undergoing early degenerative changes, their granulosa cells were still capable of aromatizing androgen to estrogen (McNatty et al., 1979), therefore these follicles could still be considered to have the potential for further development. Follicles with less than 50% of their maximum number of cells were judged to be atretic. The granulosa cells present in atretic follicles were unable to aromatize androgen to estrogen even in the presence of FSH. In bovine follicles ranging in diameter from 1, 4, 6, 8, 10, 12, 14, 16 or 18 mm the respective maximum numbers of granulosa cells that have been recovered were 1, 4, 7, 10, 14, 18, 24, 36 and 40 million.  42  3. 3. 4 Cell Culture Conditions 3. 3. 4.1 Coating of plates To facilitate cell adhesion, one day before collecting the ovaries, each well of 24-well plastic tissue culture plates was coated with 500 pi fetal bovine serum. Animal cell surfaces and the traditional glass and plastic culture surfaces are negatively charged, so for cell attachment to occur, cross-linking with glycoproteins which are positively charged and/or divalent cations (Ca , M g ) is required. Glycoproteins are present in serum and other 2+  2+  physiological fluid. The plates were then incubated overnight at 37°C in an atmosphere of 5% C O 2 and 95% air. C O 2 (5%) was used to maintain the correct pH in media.  3. 3. 4. 2 Plating out Fetal bovine serum in the precoated plates were decanted and the plates were washed three times with serum-free medium. Aliquots of granulosa cell suspension (50 pi, 5 x 10  6  cells) were added to 950 pi medium in wells of plates. For each experiment, six wells were plated for each dose at beginning. The cells were cultured at 37°C in a humidified atmosphere of 5% C O 2 and 95% air. Media were withdrawn and replaced completely with fresh media every 24 h for 2 d. At 24 h, after media withdrawn from six wells, viable cell number of three wells were determined and the remaining three wells were used for the second day's culture. Media were always pre-warmed to the operating temperature (37°C) and pH stablized before changing. Culture media were collected  and stored at -20°C until assayed for progesterone  (P ) and estradiol-170 (E ). 4  2  43  3. 3. 5 Hormone Assays Concentrations of  estradiol and progesterone at 24 and 48 h in media were  determined using a solid phase radio-immunoassay kit (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA). This kit had been previously validated in our laboratory for measurement of P4 and E in bovine follicular fluid (Manikkam and Rajamahendran, 1995). 2  Cultured media or reference standard (0.1 ml) was added to tubes coated with a specific antibody. Reference standards contained buffered  125  I P4 or  125  I E (1.0 ml) was added to all 2  tubes, mixed, and left to incubate for 3 h. Tubes were aspirated after the incubation period. Tubes were counted for 1 min in a gamma counter (Packard Auto-Gamma 500, Packad Instruments, Downers Grove, IE). The sensitivity of the assay for E and P was 8 pg/ml and 2  4  0.03 ng/ml, respectively.  3. 3. 6 Statistical Analysis Hormone concentrations  were expressed  as nanograms of estradiol-170  and  progesterone per 10 viable cells per 24 h. Viable cell numbers at 24 and 48 h which were 6  determined by trypan blue exclusion method, were used for this calculation. Experimental data are presented as the least squares means ± SE of measurements from triplicate culture wells. Each experiment was performed three times with different pools of granulosa cells from small, medium, and large follicles. Treatment effects were assessed using G L M procedures of SAS (1988). Data from each day of culture were subjected to one-way analysis of variance (ANOVA). Duncan's Multiple Range Test was used to make comparisons among the means  44  when the A N O V A showed a significant size effect. Statistical differences between different day of culture were analysed by t-test.  3. 4 Results Granulosa cells in serum-free culture had the characteristic morphology, shown in Fig. 3-1. From this morphological analysis, and considering the cell isolation procedure, we concluded that the granulosa cell cultures contained no major cell contaminant. Granulosa cells obtained from small and large follicles demonstrated similar serum-free culture characteristics. Cells isolated from large follicles had a slightly lower plating efficiency and a greater loss in cell number with duration of culture than the small or medium follicles. To examine specific functions of granulosa cells, the steroidogenic capacities of cells isolated from small, medium, and large follicles were examined. The ability of granulosa cells to aromatize androstenedione to produce estrogen was determined by measuring estrogen concentration in media. Granulosa cells from small, medium, and large follicles were cultured, and at designated time points (24 and 48 h) the cell cultures were terminated and assayed for estradiol concentration. Estradiol concentration was the higher at 24 h than at 48 h. Similar results were obtained with granulosa cells isolated from different follicles, i.e. from small, medium, and large follicles. Comparison of estradiol concentration by granulosa cells obtained from small, medium, and large follicles is shown for 24 h and 48 h of culture (Fig.3-2). Estradiol concentration in granulosa cells increased significantly with the size of the follicle. The ability of granulosa cells to produce P was examined. Progesterone concentration 4  in media increased with the time of culture. The time course of progesterone concentration by  45  Fig. 3-1. Morphology of bovine granulosa cells cultured in serum-free culture system at 48 h. Granulosa cells were isolated from medium follicles (6-10 mm in diameter) Photographed at lOOx magnification.  46  Fig. 3-2. Estradiol-17 P ( E 2 ) secretion by granulosa cells isolated from bovine small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 2 d in serum-free medium. Values are least square means ± SE of triplicate cultures from three separate experiments. Data were subjected to A N O V A . significant differences relative to small follicles are indicated: ** p <  0.01. 47  granulosa cells isolated from small, medium, and large follicles were similar. Comparison of progesterone concentration at 24 h and 48 h from cells isolated from small, medium, and large follicles was shown in Fig. 3-3. Progesterone concentration in granulosa cells increased significantly with the size of the follicle (p < 0.01).  3. 5 Discussion The bovine ovary is of sufficient size to allow the isolation of adequent number of purified granulosa cells from follicles at various stages of development. Therefore, the current study has focused on the use of developing small antral, medium antral, and Graffian follicles to study developmental and hormonal regulation of granulosa cell function. As previously described (Kruip and Dieleman, 1982), these follicles are referred to as small, medium, and large developing follicles. A comparison of functional parameters of granulosa cells isolated from small, medium, and large follicles provides information on the developmental regulation (Ryan, 1979). Therefore, the tissue in the current study was selected for developing preovulatory follicles, but may contain some early stage atretic tissue. This is a technical limitation that will need to be considered in any data interpretation. Primary cultures of bovines granulosa cells have previously been described and used to examine a number of events (Fortune and Hansel, 1979). The predominate functional parameter previously examined is the steroidogenic capacity of the granulosa cell (Fortune, 1986) and its ability to influence theca cell steroidogenesis. Bovine granulosa cell cultures have also been used to examine the production of individual protein products, such as inhibin (Henderson  and  Franchimont,  1981).  In  48  the  majority  of  these  studies,  bovine  200  Fig. 3-3. Progesterone (P4) secretion by granulosa cells isolated from bovine small (< 5 mm), medium (6-10 mm), and large (> 10 mm)follicles. Cells were cultured for 2 d in serum-free medium. Values are least square means ± SE of triplicate cultures from three separate experiments. Data were subjected to A N O V A . significant differences relative to small follicles are indicated: ** p < 0.01.  49  granulosa cells were cultured in the presence of serum. Serum contains many unknown substance that may interfere with granulosa cell function. Serum is also known to mask the effects of regulatory agents. Langhout et al (1991) demonstrated stimulation of bovine granulosa cell steroidogenesis with serum alone. Information obtained from rats indicated that granulosa cells retain remarkable hormonal responsiveness in serum-free medium. For these reasons a serum-free culture system was necessary in the current study to optimize analysis of the developmental and normal regulation of bovine granulosa cell function. Under the serum-free culture conditions used in this study, bovine granulosa cells from small, medium, and large follicles retained the functional characteristics of non-luteinized cells and were able to secrete estradiol and progesterone. Although the rate of progesterone secretion increased significantly during the culture period, the overall increase was relatively small and was comparable to that observed in a study involving serum-supplemented culture of bovine granulosa cells (Langhout et al., 1991). This small rise in progesterone output was less than the about 20-fold increase reported by Luck et al. (1990) who found that bovine granulosa cells from large follicles underwent rapid, spontaneous luteinization (characterized by loss of granulosa cell aromatase activity and marked increase in secretion of progesterone) even when serum was omitted from culture medium. A major function of granulosa cells is the aromatization of testosterone to estrogen. Estradiol production was dramatically reduced after the first day of granulosa cell culture. Although morphologically viable granulosa cell could be maintained for several days in culture, estradiol production was only high on the first day of culture then decreased with the time of culture. This result is in agreement with the findings of studies in which spontaneous  50  luteinization was seen with bovine granulosa cells cultured in serum-supplemented medium (Henderson and Franchimont, 1981; Shinner and Osteen, 1988). However, compared to results from serum-supplemented culture, estradiol secretion under the serum-free culture conditions used in the present study is higher. These observations suggested that the capacity of biosynthesis of steroid by granulosa cells in serum-free culture system increased or that breakdown is decreased. These data also imply that the first day of bovine granulosa cell culture may be more representative of in vivo granulosa cell function at the time of isolation than longer periods of cell culture. This rapid reduction in estradiol production indicates the possible absence of regulatory agents required to maintain estrogen production. A comparison of estradiol production in granulosa cell from small, medium, and large follicles indicates an increase in estradiol production with the size of the follicle. This correlates with the increase in steroidogenic capacity of the granulosa cell during the follicle development. Before ovulation, granulosa cells require L H receptors and develop the capacity to produce progesterone. After ovulation, progesterone production becomes the predominant steroidogenic activity of the granulosa cell. Therefore, estrogen and progesterone are primarily produced at different stages of follicular development. Progesterone concentration by cultured bovine granulosa cells increased with the time of culture, and comparison of granulosa cells from small, medium, and large follicles demonstrated an increase in progesterone concentration with the size of the follicle in the present study. This supports previous observations of an increase in progesterone concentration with time in serumsupplemented bovine granulosa cell cultures (Fortune and Hansel, 1979; Henderson and Moon, 1979) even though the progesterone concentration is lower in this study. Although  51  only little fetal bovine serum was used for pre-coating in the present study, cells would inevitably have come into contact with low concentrations of various fetal bovine serum constituents (including growth factors, mitogens ect). It is thought to be these very constituents which are at least partially responsible for the spontaneous luteinization of granulosa cells from small, medium, and large follicles.. The demonstration of low level of progesterone content and high level of estradiol content support that granulosa cells differentiate in vitro to change functional parameters from an estrogen-producing cell to a progesterone-producing cell. Whether this in vitro alteration in cellular differentiation is due to promotion of a differentiation process or occurs as a result of a spontaneous dedifferentiation (i:e. regression) remains to be investigated. However, data suggest that these two steroidogenic capacities are predominant at different stages of follicle development. Present observations also supported in vivo studies that granulosa cells in vivo develop in response to F S H to produce a high level of estrogen while acquiring L H receptors. Granulosa cells then respond to L H and luteinize, which reduces estrogen production and allows high levels of progesterone to be made. Information obtained in the current study indicates that a this serum-free bovine granulosa cell culture system can mimic in vivo conditions and will be useful to examine developmental hormonal regulation of granulosa cell function. The research also shows that bovine granulosa cells are developmentally regulated. The limitation of this cell culture system is that the culture of isolated granulosa cells in vitro does not take into account major interactions between the theca and granulosa cells.  52  CHAPTER 4 EFFECTS OF GONADOTROPINS ON STEROID PRODUCTION BY BOVINE GRANULOSA CELLS FROM SMALL, MEDIUM, AND LARGE FOLLICLES IN VITRO  4.1 Abstract This study examined the gonadotropic regulation of bovine granulosa cell (GC) function in small, medium and large antral follicles. GCs from small (< 5 mm), medium (6-10 mm), or large (> 10 mm) follicles were cultured at a concentration of 5 x 10 cells/well in a 5  serum-free medium for 48 h with varying doses (0, 1, 10 and 100 ng/ml) of FSH or L H . Progesterone (P4) and Estradiol (E ) concentrations in the medium were determined by 2  radioimmunoassay. At 24 h, F S H had no significant effect on E production in all categories 2  of follicles. F S H (10, 100 ng/ml) increased P production by GCs from medium and large 4  follicles ( p < 0.05). At 48 h, FSH (1 ng/ml) increased E production in medium and large 2  follicles (p < 0.05), whereas the high dose (100 ng/ml) inhibited E production in large 2  follicles. F S H (100 ng/ml) increased P production of GCs from all classes of follicles. At 24 h, 4  L H (100 ng/ml) inhibited E production in medium and large follicles (p < 0.05) but had no 2  effect on P  4  production. At 48 h, L H did not affect E . L H (100 ng/ml) increased P 2  4  production by GCs from medium and large follicles (p < 0.05). In summary, F S H and L H significantly influence steroidogenic activity of bovine GCs from small, medium and large antral follicles.  53  4. 2 Introduction Steroidogenesis in bovine preovulatory follicles requires uptake or de novo synthesis of cholesterol to pregnenolone by cytochrome P450 cholesterol side-chain cleavage enzyme (P450 SCC; Miller, 1988). Pregnenolone is further metabolized to either 17-hydroxy pregnenolone and dehydroepiandrosterone (DHEA) by cytochrome P450 17a-hydroxylase/C17, C-20 lyase (P450 17a-HYD) through the A pathway or to progesterone (P ) by 5  4  cytochrome Fortune,  P450  3P-hydroxysteroid dehydrogenase/A -A -isomerase 5  1986; Fortune and Quirk,  4  (P450  3P-HSD;  1988). Dehydroepiandrosterone is converted to  androstenedione (AO by P450 30-HSD in theca interna cells (Fortune, 1986, Fortune and Quirk, 1988). Subsequently, in granulosa cells, A t undergoes aromatization by cytochrome P450 aromatase (P450 ARO) to estradiol-170 (E ), supporting the concept of the 'two cells" 2  theory (Flack, 1959; Lacroix et al., 1974; Fortune, 1986) in that both theca and granulosa cells are essential for follicular E production. In contrast to rat preovulatory follicles where 2  the A pathway is the major pathway for A 4 production, in bovine preovulatory follicles most 4  A 4 production occurs via the A pathway (Fortune and Quirk, 1988). 5  Development of gonadotropins receptors on theca interna and granulosa cells is essential to the process of growth and development of ovarian follicles (Richards, 1980). Classic studies of Greep et al. (1942) and Lostroh and Johnson (1966) indicate that both L H and F S H are required for follicular growth and development, even though there is enough evidence to indicate that no gonadotropin support is required to reach the stage of primordial and secondary follicles. Granulosa cells of small antral follicles contain F S H receptors but no L H receptors (Findlay, 1993; Hillier, 1994; Richards, 1980). F S H stimulation induces L H  54  receptor gene expression in theca interna cells (Segaloff et al., 1990). Ireland and Roche (1982) demonstrated the presence of L H receptors on theca cells of antral follicles. Granulosa cells from larger follicles contain receptors for both F S H and L H in cattle (Ireland and Roche, 1982), humans (Hillier, 1994) and rats (Magoffin and Erickson, 1994; Richards, 1980). Therefore, the presence of both L H and F S H receptors in both cell types of antral follicles supports the 'two cell-two gonadotropin" theory (Fortune and Armstrong, 1977; Fortune, 1986). Thus, L H stimulates the synthesis of A» precursors in theca interna cells which are then aromatized to estradiol-17(3 in granulosa cells under F S H stimulation. In rats and humans, granulosa cell aromatization is induced or increased only by F S H during early follicular development and by F S H and L H during late follicular development (Yong et al., 1992). In contrast, similar concentrations of gonadotropins do not stimulate aromatization of androgen by granulosa cells from proestrous follicles of domestic species (Henderson and Swanston, 1978; Fortune and Quirk, 1988). However, studies in vivo with cattle and sheep have provided indirect evidence that normal plasma concentrations of F S H and L H are essential for the differentiation of preovulatory follicles and for the preovulatory rise in circulating estradiol-173 (Schallenberger et al., 1984). The failure of gonadotropins to stimulate the aromatizing capacity of granulosa cells from domestic species is puzzling and has impeded research into the regulation of estradiol170 and progesterone secretion by these species. Although previous studies (Moor, 1977; Fortune and Quirk, 1988; Sirois et al., 1991) have examined granulosa cells steroid production in response to gonadotropins, these studies have usually focused on a single stage of follicle. There are few direct studies on bovine granulosa cell steroidogenesis. Recently,  55  Berndtson (1995) reported that very low doses of F S H increased estradiol-173 secretion by bovine granulosa cells. Therefore, we set out to determine the effects of F S H and L H on estradiol-170 and progesterone production by bovine granulosa cells from small, medium, and large follicles under serum-free culture condition.  4. 3 Materials and Methods 4. 3.1 Preparation of Experimental Reagents Medium is Dulbecco's Modified Eagles medium (DMEM) and Ham's F12 in a 1:1 ratio obtained from Stemcell Technologies Inc. (Vancouver, B C , Canada). The medium was supplemented with penicillin (100 ug/ml)/streptomycin (100 ug/ml), insulin (5 ug/mg) /transferrin (5 ug/ml)/sodium selenite (5 ng/ml) and androstenedione (1 uM); bFSH and b L H obtained from the U S D A National Hormone and Pituitary Program (Bethesda, M D , USA). All the reagents, unless otherwise specified, were obtained from Sigma Chemical Co. (St. Louis, M O , USA); Androstenedione was initially dissolved in absolute ethanol, followed by subsequent dilution in the culture medium. The final ethanol concentration in the cell cultures was less than 0.1% (vol./vol.). L H and F S H were added to the supplemented medium at a concentration of 100 ng/ml and a serial dilution was performed to yield different treatment media: L H (1, 10, 100 ng/ml) and F S H (1, 10, 100 ng/ml). Aliquots of granulosa cell suspension (50 ul, 5 x 10 cells) were 6  added to 950 ul treatment medium in wells of plates.  56  4. 3. 2 Preparation of Granulosa Cells 4. 3. 3 Selection of Healthy Follicles 4. 3. 4 Cell Culture Conditions 4. 3. 5 Hormone Assay The methods for preparation cells, selection of follicles, cell culture conditions, hormone assay and statistical analysis have been described in chapter 3.  4. 3. 6 Statistical Analysis Hormone concentrations were  expressed  as nanograms of estradiol- 17B and  progesterone per 10 viable cells per 24 h. Viable cell numbers at 24 and 48 h which were 6  determined by trypan blue exclusion method, were used for this calculation. Experimental data are presented as the least squares means ± SE of measurements from triplicate culture wells. Each experiment was performed three times with different pools of granulosa cells from small, medium, and large follicles. Treatment effects were assessed using G L M procedures of SAS (1988). Main effects were treatment and experiment.  4. 4 Results Effect of F S H on E and P secretion 2  4  During the first 24 h of culture F S H had no effect on E secretion by granulosa cells 2  from small, medium, and large follicles (Fig. 4-1). However, during the second days of culture, the lowest dose (1 ng/ml) increased (p < 0.05) E secretion by granulosa cells from 2  medium (71%) and large (22%) follicles, whereas higher concentrations of F S H (10 and 100  57  Fig. 4-1. Dose-response of FSH on E production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for Id in the serum-free culture system treated with various doses of FSH. Values are least square means ± SE of triplicate culture wellsfromthree separate experiments. Data were subjected to ANOVA. 2  58  ng/ml) inhibited (p < 0.05) E accumulation by granulosa cells from large follicles by 13.8% 2  and 19.3%, respectively. All concentrations of F S H had no effect on E secretion by granulosa 2  cells from small follicles (Fig. 4-2). In contrast, P 4 secretion at both 24 and 48 h was unaffected by the doses of F S H (1 ng/ml) that stimulated E  2  secretion, whereas the highest doses of F S H (100 ng/ml)  consistently stimulated P 4 accumulation above control values by granulosa cells from both medium and large follicles by 76% and 30%, respectively (Fig. 4-3 and Fig. 4-4). F S H (100 ng/ml) increased P 4 secretion (p < 0.01) by granulosa cells from small follicles by 44% during the second day of culture.  Effect of L H on E and P4 secretion 2  During the first 24 hours of culture, L H (100 ng/ml) inhibited (p < 0.05) E secretion 2  by granulosa cells from medium and large follicles by 36% and 21%, respectively (Fig. 4-5). The inhibitory effects of higher doses of L H on E secretion were similar to F S H treatments 2  observed at 48 h (Fig. 4-6). During the second day of culture, there was a trend that L H inhibited (p < 0.1) E secretion from medium and large follicles. L H had no effect on E 2  2  secretion by granulosa cells from small follicles during both day 1 and day 2 of culture. During the first 24 hours of culture, L H had no effect on P secretion by granulosa 4  cells (Fig. 4-7). During the second day of culture, high dose of L H (100 ng/ml) enhanced (p < 0.05) P secretion by granulosa cells from medium and large follicles by 45% and 94%, 4  respectively (Fig. 4-8).  59  Fig. 4-2. Dose-response of FSH on E production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 2 d in the serum-free culture system treated with various doses of FSH. Values are least square means ± SE of triplicate culture wellsfromthree separate experiments. Data were subjected to ANOVA; significant differences relative to control (0 ng/ml) are indicated: * p < 0.05. 2  60  70 -  i_ O _a. w  CM  o o CD o T— Ol  Q.  60 50 40 30 20 10 -  o  L  0  1  10  100  F S H (ng/ml) Small  22!  Medium  Large  Fig. 4-3. Dose-response of FSH on P production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 1 d in the seriim-free culture system treated with various doses of FSH. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA; significant differences relative to control (0 ng/ml) are indicated: * p < 0.05, ** p < 0.01. 4  61  Fig. 4-4. Dose-response of FSH on P production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 2 d in the serum-free culture system treated with various doses of FSH. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA; significant differences relative to control (0 ng/ml) are indicated: ** p < 0.01. 4  62  Fig. 4-5. Dose-response of LH on E production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 1 d in the serum-free culture system treated with various doses of LH. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA; significant differences relative to control (0 ng/ml) are indicated: * p < 0.05. 2  63  9 r  Fig. 4-6. Dose-response of LH on E 2 production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 2 d in the serum-free culture system treated with various doses of LH. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA; significant differences relative to control (0 ng/ml) are indicated: * p < 0.05.  64  Fig. 4-7. Dose-response of LH on P4 production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 1 d in the serum-free culture system treated with various doses of LH. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA.  65  Fig. 4-8. Dose-response of LH on P4 production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 2 d in the serum-free culture system treated with various doses of LH. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA; significant differences relative to control (0 ng/ml) are indicated: * p < 0.05.  66  Effect of follicle size on steroid secretion When data were combined from all the control treatments, during both first and second day of culture, there was a significant difference in E and P secretion by granulosa 2  4  cells obtained from small, medium, and large follicles (small vs medium; small vs large; medium vs large: p < 0.05).  4. 5 Discussion The bovine ovary is of sufficient size to allow the isolation of adequate numbers of granulosa cells from follicles at various stage of development. Therefore, the bovine ovary is quite useful for analysis of the developmental and hormonal regulation of granulosa cell function. A comparison of steroid production by granulosa cells isolated from small, medium, and large follicles provides information on the developmental and hormonal regulation of granulosa cell function. The effects of factors observed in the current study could not be accounted for by their effects on cell proliferation, as steroid secretion was corrected for cell number. In this study, only a low dose of F S H (1 ng/ml) stimulated E secretion by granulosa 2  cells from medium and large follicles above levels in control cultures after the first day of culture. This may explain the reason for high concentrations of F S H not stimulating aromatization of androgen by granulosa cells from proestrous follicles of cows (Turzillo and Fortune, 1993) and ewes (Goodman et al., 1981). Low F S H levels (1 ng/ml) did not affect small follicle E secretion. This possibly means that a lower F S H concentration is needed for 2  small follicles. Even in the presence of effective doses of FSH, E secretion decreased with the 2  67  time in culture. The range of stimulatory doses of F S H was very narrow. Furthermore, higher doses of F S H (10 and 100 ng/ml) were inhibitory to E production. This biphasic effect of 2  F S H on E  2  secretion has also been observed with granulosa cells from immature calves  (Saumande et al., 1991) in vitro culture. Like FSH, high doses of L H were inhibitory to estradiol secretion. The lack of a stimulatory effect on E secretion might suggest that L H does not stimulate E production 2  2  during follicular development in vivo. This is unlikely, since granulosa cells from larger follicles in cattle contain receptors for both F S H and L H (Ireland and Roche, 1982), and during the last few days of preovulatory follicular growth in cattle, granulosa cells acquire more L H receptors (Henderson et al., 1984; Kawate et al., 1989)  and plasma L H  concentrations increase, whereas plasma F S H concentrations decrease (Bulter et al., 1983). These observations suggest that L H is responsible for the rise in plasma E through the effects 2  on both theca and granulosa cells. This is in agreement with the one previous in vitro study (Fortune, 1986). Fortune suggested that bovine theca interna cells use the A pathway to make 5  androstenedione, that pregenenolone made by the granulosa cell compartment enhances the production of androstenedione by the theca and that testosterone increases the capacity of granulosa cells to make pregnenolone. His model thus postulates that two interactions between theca and granulosa cells, in addition to the conversion of theca androgen to E by 2  granulosa cells, serve to increase the capacity of the follicle to make E . 2  Before ovulation, granulosa cells acquire L H receptors and develop the capacity to produce P . After ovulation P production become the predominant steroidogenic activity of 4  4  the granulosa cell. Therefore, E and P are primarily produced at different stages of follicle 2  4  68  development. In domestic species, both theca and granulosa cells from preovulatory follicles undergo what has been termed 'spontaneous luteinization" in vitro and secrete P (Geenen et 4  al., 1985; Denning-kendall and Wathes, 1994). Since P is the primary biologically active 4  progestin metabolite, other metabolites were not examined in the current study. P production 4  by cultured bovine granulosa cells increased with time in culture. Comparison of granulosa cells from small, medium, and large follicles demonstrated an increase in P production with 4  the size of the follicle. F S H stimulate P production by modulating the activities of various 4  steroidogenic enzymes. The major regulatory site of F S H is probably at the cholesterol sidechain cleavage  step.  In cultured porcine granulosa cells,  F S H treatment  increases  mitochondrial cytochrome P450 levels and cholesterol side-chain cleavage activity. Zeleznik et al. (1987) have shown that F S H treatment increases 3P-hydroxysteroid dehydrogenase activity in the granulosa cells.  L H stimulate progesterone secretion and the major site of  action of L H on progesterone biosynthesis is the conversion of cholesterol to pregnenolone. The low levels of P production on day 1 of granulosa cell culture are inversely related 4  to the data obtained on E secretion. When E secretion is high and responsive to L H and 2  2  FSH, P production is low. These observations support that granulosa cells differentiate in 4  vitro to change functional parameters from an E -producing cell to a P -producing cell similar 2  4  as granulosa cells in vivo develop in response to F S H to produce high levels of estrogen while acquiring L H receptors. Granulosa cells then respond to L H and luteinize, which reduces estrogen production and allows high levels of progesterone to be made. Information obtained in the current study regarding the developmental regulation of granulosa cell function indicates: 1) during development from a small to a large follicle,  69  steroidogenic capacity is increased; 2) only a very low dose of F S H is needed to stimulate E  2  production typical of the follicular phase whereas higher doses accentuate the natural tendency of granulosa cell to 'luteinize" in vitro; and 3) under the influence of L H , bovine theca and granulosa cells interact to promote E production. In conclusions, F S H and L H 2  significantly influence the steroidogenic activity of bovine granulosa cells under serum-free medium culture conditions.  70  CHAPTER 5 EFFECTS OF INSULIN-LIKE GROWTH FACTOR-I (IGF-I) AND IGF-H ON STEROID PRODUCTION BY BOVINE GRANULOSA CELLS FROM SMALL, MEDDJM, AND LARGE FOLLICLES IN VITRO  5.1 Abstract This study examined regulation of bovine granulosa cell (GCs) function in the small, medium and large antral follicles by insulin-like growth factor I and II (IGF-I, IGF-II). GCs from small (< 5 mm), medium (6-10 mm), or large (> 10 mm) follicles were cultured at a concentration of 5 x 10 cells/well in a serum-free medium for 48 h with varying doses of IGF5  I or IGF-II. Progesterone (P ) and Estradiol (E ) content in the media were determined by 4  2  radioimmunoassay. At 24 h, in response to high dose IGF-I (100 ng/ml), E  2  production by  GCs from large follicles increased (p < 0.05). IGF-I (10, 100 ng/ml) increased P production 4  by GCs from all categories of follicles (p < 0.05). At 48 h, IGF-I (100 ng/ml) increased E and 2  P production in medium and large follicles (p < 0.05). At 24 h, E production from large 4  2  follicles decreased in response to all doses of IGF-II (p < 0.05). At high dose (500 ng/ml), IGF-II inhibited P production by GCs from medium and large follicles. At 48 h, IGF-II had 4  no effect on E production from all categories of follicles and inhibited P production by GCs 2  4  from medium and large follicles. P production from small follicles was inhibited only at high 4  dose (500 ng/ml) (p < 0.05). In summary, IGF-I and IGF-II influence steroidogenic activity of bovine GCs from small, medium and large antral follicles.  71  5. 2 Introduction Ovarian physiology requires rapid and continuous regulation of growth associated with folliculogenesis. Granulosa cells provide the cytoarchitectural support for the developing oocyte and participate in follicular antrum formation, whereas theca cells surround granulosa cells and form the exterior wall to the follicle. Both cell types interact to produce steroid hormones and must undergo extensive proliferation and functional differentiation to develop from preantral to antral follicles. Once a follicle is selected and becomes dominant, further growth is required to achieve ovulatory size. Conversely, the cohort of follicles that do not become selected and/or the non-ovulatory dominant follicle all become atretic and cell growth is arrested. Folliculogenesis in mammalian species is a highly selective process. Only a very small proportion of total follicles ( « 0.1%) survive atresia and give rise to dominant follicles (Aria, 1920). Therefore, probably the most important question in folliculogenesis concerns the mechanisms by which a single follicle within a cohort is selected to become dominant or undergo programmed cell death by atresia (Erickson et al., 1994). Follicular development is thought to be primarily regulated by L H and F S H (Richards, 1980; Ireland and Roche, 1987; Lucy et al., 1992), but why under the same circumstance do follicles have such different fates? What are the signals that choose some follicles others and that then assure maturation of one or more follicles. These questions have led to the postulation of several factors acting within the ovary or between ovaries. Ample evidence is emerging that growth factors which are themselves products of gonadotropins action, may act locally on follicle cells to either amplify or attenuate the timing and direction of granulosa and theca cell differentiation (Erickson et  72  al., 1994; Hillier, 1994). Of these growth factors, insulin-like growth factors (IGFs), IGF-I and IGF-II, have received considerable attention (Howard and Ford., 1992). IGF-I and IGF-II are low molecular weight single chain polypeptides that are structurally related to proinsulin and promote cellular mitosis and differentiation in a variety of tissues and systems. Both IGF molecules have 40% structural homology with insulin and 60% of structural homology between each other (Giudice, 1992). Expression of the IGF-I gene has been identified in granulosa cells of rats (Hernandez et al., 1989; Oliver et al, 1989), pigs (Hammond et al, 1985), and cattle (Echternkamp and Spicer, 1991; Spicer et al, 1993). Receptors for IGF-I have been localized in rat (Adashi et al, 1988), porcine (Baranao and Hammond, 1984), and cattle (Davoren et al, 1986) granulosa cells and rat theca cells (Hernandez et al, 1989). The intraovarian IGF system is complete with ligands, receptors and binding proteins and it is possible that these are indispensable to ovarian function (Adashi et a l , 1991). The actions of IGFs are mediated by three types of receptors in the ovary: Type I IGF receptor, insulin receptor and /or the type II IGF/mannose-6-phosphate binding site. These are induced by gonadotropins and gonadal steroid and hence the latter represents an important control locus. Insulin-like growth factor binding proteins (IGFBPs) are a complex family of proteins that bind IGF-I and IGF-II in body and tissues. An increasing body of evidence suggests that in the ovary, the biological responses of IGF-I and IGF-II under normal F S H stimulation also may be regulated by IGFBPs (Ui et al, 1989). These IGFBPs may bind to IGF-I and IGF-II present in the follicular fluid (FF) and therefore selectively negate their tropic activity (Ui et al, 1989; Magoffin and Erickson, 1994).  73  Several  studies in  recent  vivo  have  demonstrated  that  recombinant bovine  somatotrophin (bST) treatment increases peripheral concentrations of IGF-I and insulin significantly, without altering circulating concentrations of F S H and L H or numbers of ovarian FSH- and LH-binding sites (Gong et al., 1994). These results support the concept that IGF-I and insulin may be involved in the regulation of ovarian function (Adashi et a l , 1985; Hsu and Hammond, 1987; Adashi and Rohan, 1992). Evidence from other studies for rats, pigs, cattle, and humans also indicates that IGF-I and/or IGF-II can be produced by granulosa cells and thus may act as a local regulator of ovarian function (Hammond et al., 1991; Spicer et al., 1993). In vivo studies indicate that concentrations of IGF-I increase with increased follicular size in cattle (Spicer et.al., 1988; Spicer and Enright, 1991) and pigs (Spicer et al., 1992). In vivo data also reveal a positive correlation between FF IGF-I and progesterone concentrations in postpartum anestrous and cyclic cows (Spicer et al., 1988; Echternkamp et al., 1990). Positive correlations between FF IGF-I and estradiol concentrations were found in two studies where follicles > 4 mm were collected during the follicular phase (Spicer et al., 1988), whereas negative correlations between FF IGF-I and estradiol were found where follicles > 8 mm were collected (Spicer et al., 1988). IGF-I also increases progestin production by porcine (Veldhuis et al., 1985) and bovine (Gong et al., 1994) granulosa cells in vitro under serumsupplemented and serum-free conditions, respectively. In rat, IGF-I increases FSH-stimulated, but not basal, P4 and E production (Adashi et al., 1986). Reports on direct effects of IGF-I 2  on steroid production of bovine ovarian cells from small, medium and large follicles are meager.  74  Although the granulosa cell is well recognized as a site of IGF-I action and receptor expression (Hernandez et al., 1989), the specific modulatory effects of IGF-II in the ovary have not been defined in similar detail. Thus far, studies have shown that rat granulosa cells are a locus of IGF-I and IGF-II gene expression (Levy et al., 1992). Porcine granulosa cells express the gene for IGF-II peptide (Samaras et al., 1991), and human granulosa cell also express the gene for IGF-II peptide (rather than IGF-I) as well as both IGF-I and IGF-II receptors (Hernandez et al., 1992). IGF-II is capable of augmenting granulosa cell differention in the rat (Di Blasio et al., 1994) and the pig (Xu et al., 1995). Very little or no information about the effect of IGF-II on bovine granulosa cell steroidogenesis has been reported. Therefore, the objective of this experiment was to determine the effects of IGF-I and IGF-II on estradiol and progesterone production by bovine granulosa cells from small, medium, and large antral follicles under serum-free culture conditions.  5. 3 Materials and Methods 5. 3.1 Preparation of Experimental Reagents The medium used was Dulbecco's Modified Eagles medium (DMEM) and Ham's F12 in a 1:1 ratio obtained from Stemcell Technologies Inc. (Vancouver, B C , Canada). The medium was supplemented with penicillin (100 ug/ml)/streptomycin (100 ug/ml), insulin (5 ug/mg)/transferrin (5  ug/ml)/sodium selenite (5 ng/ml) and androstenedione  (1 uM);  Recombinant human IGF-I and IGF-II. All the reagents, unless otherwise specified, were obtained from Sigma Chemical Co. (St. Louis, M O , USA); Androstenedione was initially  75  dissolved in absolute ethanol, followed by subsequent dilution in the culture medium. The final ethanol concentration in the cell cultures was less than 0.1% (vol./vol.). Recombinant human IGF-I and IGF-II were dissolved in 0.1 N acetic acid; Aliquots were stored frozen at -70 °C and used within 6 months. When used, these solutions were thawed, IGF-I and IGF-II were added to the medium at a concentration of 100 ng/ml and 500 ng/ml, respectively, a serial dilution was then performed to yield different treatment media: IGF-I (1, 10, 100 ng/ml) and IGF-II (10, 100, 500 ng/ml). Aliquots of granulosa cell suspension (50 pi, 5 x 10 cells) were added to 950 pi treatment medium in wells of plates. 6  5. 3. 2 Preparation of Granulosa Cells 5. 3. 3 Selection of Healthy Follicles 5. 3. 4 Cell Culture Conditions 5. 3. 5 Hormone Assay 5. 3. 6 Statistical Analysis The methods for preparation cells, selection of follicles, cell culture conditions, plating out, hormone assay and statistical analysis have been described in chapter 4.  5. 4 Results Effects of IGF-I on E and P4 secretion 2  During thefirst24 hours of culture, IGF-I stimulated (p < 0.05) the secretion of E by 2  granulosa cells from large follicles by 18% (Fig. 5-1). During the second day of culture, IGF-I  76  Fig. 5-1. Dose-response of IGF-I on E 2 production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for Id in the serum-free culture system treated with various doses of IGF-I. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA. significant difference relative to control (0 ng/ml) are indicated: * p < 0.05.  77  enhanced (p < 0.05) E secretion by granulosa cells from both medium and large follicles by 2  54% and 40%, respectively. (Fig. 5-2). IGF-I (100 ng/ml) stimulated (p < 0.05) the secretion of P by granulosa cells from all 4  three size classes of follicles during the first 24 h of culture (Fig. 5-3). There were no differences in granulosa cell responsiveness between cells from different size categories of follicles. During the second day of culture, IGF-I (100 ng/ml) increased (p < 0.05) P  4  secretion by granulosa cells from medium and large follicles (Fig. 5-4).  Effect of IGF-II on E and P secretion 2  4  During the fist 24 h of culture, IGF-II had no effect on E secretion by all three size 2  classes of follicles (Fig. 5-5). However, during the second day of culture, IGF-II (10-500 ng/ml) treatment inhibited (p < 0.05) the secretion of E by granulosa cells from large follicles 2  in a dose-dependent manner (Fig. 5-6). IGF-II (500 ng/ml) also inhibited (p < 0.01) the secretion of E by granulosa cells from medium follicles (Fig. 5-4). 2  During the first 24 h of culture, IGF-II (100 ng/ml) stimulated P  4  secretion by  granulosa cells from large follicles; however, IGF-II (500 ng/ml) inhibited (p < 0.05) the P  4  secretion by granulosa cells from medium and large follicles (Fig. 5-7). During the second day of culture, IGF-II (500 ng/ml) inhibited (p < 0.05 or p < 0.01, respectively) secretion of P by 4  granulosa cells from all three size classes of follicles (Fig. 5-8).  78  Fig. 5-2. Dose-response of IGF-I on E production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 2 d in the serum-free culture system treated with various doses of IGF-I. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA; significant differences relative to control (0 ng/ml) are indicated: * p < 0.05. 2  79  70 CM  \— CD  Q. W 0)  o  o  60 50 " 40 30 -  CO  c  20 10 o  L  0  1  10  100  IGF-I (ng/ml) Small  d Medium  Large  Fig. 5-3. Dose-response of IGF-I on P production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 1 d in the serum-free culture system treated with various doses of IGF-I. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA; significant differences relative to control (0 ng/ml) are indicated: * p < 0.05, ** p < 0.01. 4  80  Fig. 5-4. Dose-response of IGF-I on P production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 2 d in the serum-free culture system treated with various doses of IGF-I. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA; significant differences relative to control (0 ng/ml) are indicated: * p < 0.05. 4  81  Fig. 5-5. Dose-response of IGF-II on E production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 1 d in the serum-free culture system treated with various doses of IGF-II. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA. 2  82  Fig. 5-6. Dose-response of IGF-II on E production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 2 d in the serum-free culture system treated with various doses of IGF-LL Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA; significant differences relative to control (0 ng/ml) are indicated: * p < 0.05, ** p < 0.01. 2  83  Fig. 5-7. Dose-response of IGF-II on P4 production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 1 d in the serum-free culture system treated with various doses of IGF-II. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA. significant differences relative to control (0 ng/ml) are indicated: * p < 0.05; ** p < 0.01.  84  Fig. 5-8. Dose-response of IGF-II on P production by bovine granulosa cells from small (< 5 mm), medium (6-10 mm), and large (> 10 mm) follicles. Cells were cultured for 2 d in the serum-free culture system treated with various doses of IGF-II. Values are least square means ± SE of triplicate culture wells from three separate experiments. Data were subjected to ANOVA; sigriificant differences relative to control (0 ng/ml) are indicated: * p < 0.05; ** p < 0.01. 4  85  Effect of follicle size on steroid secretion When data were combined from all the control treatments, during both the first and the second day of culture, There was a significant difference in E and P4 secretion by granulosa 2  cells obtained from small, medium, and large follicles (small vs medium; small vs large; medium vs large: p < 0.05).  5. 5 Discussion The bovine ovary is of sufficient size to allow the isolation of adequate numbers of granulosa cells from follicles at various stage of development. Therefore, the bovine ovary is quite useful in an analysis of the developmental and hormonal regulation of granulosa cell function. Comparison of steroid production of granulosa cells isolated from small, medium, and large follicles provides information on the developmental and hormonal regulation of granulosa cells functions. The effects of factors observed in the current study could not be accounted for by their effects on cell proliferation, as steroid secretion was corrected for cell number. In agreement with the results of this study, a number of previous reports have shown that IGF-I and insulin can enhance P4 production by granulosa cells in vitro, both in cattle (Savion et al., 1981, Skinner and Osteen, 1988; Langhout et al., 1991; Saumande, 1991), sheep (Manniaux and Pisselet, 1992), pigs (Maruo et al., 1988), and rats (Jia et al., 1986). However, there is little information regarding E production by granulosa cells in vitro, 2  especially for the large animal species. This is probably due to difficulties in maintaining aromatase activity of granulosa cells in culture, especially when serum-containing media is  86  used, as cells can undergo spontaneous luteinization and lose their ability to synthesize estrogen (May and Schomberg, 1981). In our serum-free culture system, IGF-I significantly stimulated the secretion of E  2  by bovine granulosa cells from large follicles. This is in  agreement with the results of Saumande (1991) and Spicer et al. (1993). But, one study (Spicer et al., 1993) have shown that IGF-I (200 ng/ml) inhibited E secretion in the serum 2  supplemented culture system. These observations indicated that IGF-I could stimulate E  2  production by bovine granulosa cells, depending on the concentration, size of follicles, and methods (i.e., presence of serum factors). IGF-I was more effective at stimulating E  2  production by granulosa cells from large vs small follicles. Thus, differences in hormone concentrations, size of follicle, and (or) presence of gonadotropins in a medium could explain why studies evaluating the regulation of E production in cultured granulosa cells show 2  variable results. Although the granulosa cell is well recognized as a site of IGF-I action and receptor expression (Hernandez et al., 1989), the specific modulatory effects of IGF-II in the ovary are not defined in equivalent detail, especially in cattle. In the present study, IGF-II inhibited E  2  production. This is in contrast to the previous studies in pig (Xu et al., 1995) and humans (Barreca et al., 1993) where IGF-II can stimulate steroidogenesis by facilitating sterol delivery and increasing levels of cholesterol side-chain cleavage enzyme mRNA. Thus far, studies have shown that rat ganulosa cells are a locus of IGF-I and IGF-II gene expression. Porcine granulosa cells express the gene for IGF-II, and human granulosa cells express the gene for IGF-II peptide rather than IGF-I (Garmey et al., 1993). The gene expression and the presence of receptors for IGF-II in bovine granulosa cells has yet to be confirmed. Thus, there may be  87  species differences. Results of the current study also indicate that IGFs may act as insulin antagonists at the level of the ovary. Insulin can enhance P and E production by granulosa 4  2  cells in vivo and in vitro (Langhout et al., 1991; Spicer et al, 1993). Insulin was more effective at stimulating E production by granulosa cells from small vs. large follicles. Both 2  IGF-I and IGF-II cross-react by 2-5% with the insulin receptor (Krett et al, 1987). A recent study has shown nearly a 50% cross-reactivity of IGF-II with insulin receptors (Kenner and Heidenreich, 1991). Another recent study in sheep has suggested that IGF-II may act as an IGF-I antagonist (Koea et al, 1992). Thus, antagonistic actions of IGFs are possible and not without precedence. The physiological role of IGFs acting as antagonists on steroidogenesis at the level of the ovary, as well as the hormone or specificity of this effect, remains to be elucidated. The source of ovarian IGFs, such as IGF-I in cattle, is uncertain but may include blood transudate, thecal cells, granulosa cells, ovarian stromal cells, and/or luteal cells. Previous studies have shown that: 1) blood and follicular fluid IGF-I concentrations are positively correlated (Echternkamp et al, 1990); 2) rat thecal cells (Hernandez et al, 1990), human granulosa-luteal cells (Geisthovel et al, 1990) and porcine granulosa cells(Garmey et al, 1993) contain IGF-II mRNA; 3) human ovarian stromal cells (Hernandez et a l , 1992), rat (Hernandez et a l , 1989) and bovine granulosa cells (Spicer et al, 1993) contain IGF-I mRNA, and porcine (Hsu and Hammond, 1987) and bovine (Spicer et al, 1993) granulosa cells secrete IGF-I in vitro. Thus, IGFs may act as autocrine or paracrine regulators of ovarian follicular function without originating from the systemic circulation.  88  Information obtained in the current study regarding the developmental regulation of granulosa cell function indicates: 1) During development from a small to a large follicle, steroidogenic  capacity is increased; 2) IGF-I and IGF-II significantly  influence  the  steroidogenic activity of bovine granulosa cells in serum-free conditions, depending on the dose and the size of follicle; and 3) IGFs may act as insulin antagonists on E production by 2  granulosa cells in antral follicles in cattle and this may provide a mechanism whereby IGFs inhibit  granulosa  cell  differentiation  by  smaller  differentiation of future ovulatory follicles.  89  follicles,  thus  avoiding  premature  CHAPTER 6 GENERAL DISCUSSION AND CONCLUSION  In cattle, follicles develop in a wave-like pattern. This is a highly selective process including recruitment, selection and dominance (Erickson et al., 1994). Only a very small proportion (<0.1%) of the total follicles survive atresia and become dominant follicles. Hence, one of the important questions in research on folliculogenesis  concerns the  mechanisms by which a cohort follicles is either selected to become a dominant ovulatory follicle or destined for programmed cell death (e.g. atresia; Erickson et al., 1994). Growth and development of the follicle is characterized morphologically by the number of granulosa cell layers, the extent of the thecal layer, antrum formation, the size of the oocyte and signs of atresia. The granulosa cells have an integral role in the maintenance and control of ovarian function. A major functional parameter of granulosa cells is the biosynthesis of E and P4. 2  Therefore, regulation of granulosa cell function influences both local ovarian function and the endocrine status of the female. Hormonal treatment affecting the mechanisms regulating folliculogenesis and receptors for FSH, L H , IGF-I have been found in bovine granulosa cells. With the recent introduction of cell biology methodologies into endocrine research, direct hormonal control of granulosa cells differentiation under defined conditions (usually in a serum-supplemented cell culture system) have been studied. Since serum contains multiple substances that may interfere with granulosa cell function, direct study on hormonal control of granulosa cells in serum-free culture is needed.  90  In chapter 3, a serum-free culture system for bovine granulosa cells was developed. This serum-free culture system: 1) provides an opportunity to study effects of circulating hormones and various growth factors; 2) improves reproducibility between cultures, and avoidance of batch-to-batch variations of sera; 3) avoids serum cytotoxicity; and 4) reduce protein interference in bioassays. Granulosa cells cultured under serum-free conditions retain remarkable hormonal responsiveness compared to cells under serum-supplemented conditions. Estradiol secretion by granulosa cells was stimulated whereas spontaneous luteinization of granulosa cells decreased. On the first day of culture, E secretion was high whereas P4 2  secretion was low. With time of culture, E secretion increased. The capability of E  2  2  secretion was dramatically reduced and P  4  and P4 secretion increased with the size of follicles.  These data is in agreement with the data from in vivo studies. Thus, the bovine granulosa cell serum-free culture system developed in this study can mimic bovine granulosa cell development conditions in vivo. Also, this culture system can be used to elucidate the direct hormonal control of granulosa cell differentiation. Effects of F S H and L H on steroid production by bovine GCs from small, medium, and large follicles were studied in chapter 4. Both F S H and L H stimulated P production. The 4  major regulatory site of action of gonadotropins is probably at the cholesterol side-chain cleavage step. Only a low dose of F S H stimulated E secretion, whereas high doses of F S H 2  were inhibitory to E secretion. This provides an explanation for the high concentrations of 2  F S H not stimulating aromatization of androgen by granulosa cells from proestrous follicles of cows (Turzillo and Fortune, 1993) and ewe (Goodman et al., 1981). The biphasic effect of F S H on E secretion is in agreement with previous studies (Saumande, 1991). In the present 2  91  study, results show that high doses of L H were inhibitory to E secretion. This was 2  unexpected, since granulosa cellsfromlarger follicles contain receptors for both FSH and L H in cattle (Ireland and Roche, 1982) and L H is responsible for the rise in plasma E through 2  effects on both theca and granulosa cells (Fortune, 1986). This indicated the importance of interaction between granulosa and theca cells. Follicular development is primarily regulated by gonadotropins (Lucy et al., 1992), but ample evidence is emerging that IGFs may have both a paracrine and an autocrine effect on follicle cells to either amplify or attenuate the timing and direction of granulosa and theca cell differentiation (Hillier, 1994). Effects of IGF-I and IGF-II on steroid production by granulosa cells from small, medium, and large follicles were studied. In this study, IGF-I stimulated E secretion by granulosa cells from large follicles. This indicated that IGF-I could stimulate E  2  2  production by granulosa cells, depending on the dose and on the size of follicles. This is in agreement with the study that receptors for IGF-I increase with the size of follicles. IGF-I increased P production from all classes of follicles. Studies from sheep (Manniaux and 4  Pisselet, 1992), pigs (Maruo et al., 1988) and rats (Jia et al., 1986) have also indicated that IGF-I can stimulate P and E production. IGF-II inhibited E production by granulosa cells 4  2  2  from large follicles. P production was inhibited by granulosa cells from medium and large 4  follicles in response to IGF-II. Only high doses of IGF-II inhibited P production from 4  granulosa cells from small follicles. These results are in contrast to the previous studies in pigs (Xu et al., 1995) and humans (Mason et al., 1994) where IGF-II stimulated P and E 4  2  production. Porcine and human granulosa cells express the gene for IGF-II (Garmey et al., 1993). The gene expression and the presence of receptors for IGF-II in bovine granulosa cells  92  is yet to be confirmed. Studies also indicate that IGFs may act as insulin antagonists at the level of the ovary. A recent study has shown nearly a 50% cross-reactivity of IGF-II with insulin receptors (Krett et al., 1987). Another study in sheep has suggested that IGF-II may act as an IGF-I antagonist (Koea et al., 1992). Because both IGF have 40% structural homology with insulin and 60% of structural homology between each other (Guidice, 1992), thus, a antagonistic action of IGFs is possible. Information obtained in the current studies regarding the developmental regulation of granulosa cell function indicates: 1) this bovine granulosa cell culture system is useful for further examining developmental hormonal regulation of granulosa cell function, 2) during development from a small to a large follicle, steroidogenic capacity is increased, 3) only a low dose of F S H is needed to stimulate E production whereas higher doses of F S H accentuate the 2  natural tendency of granulosa cells to 'luteinize" in yitro, 4) under the influence of L H , bovine theca and granulosa cells interact to promote E production, 5) IGF-I and IGF-II influenced 2  the E and P production depending on the dose and on the size of follicle, 6) IGFs may act as 2  4  insulin antagonists on steroid production. 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