Effects of Physiological Reproductive Events on Ovary

Effects of Physiological Reproductive Events on Ovary Chapter one Introduction Background The two majors function of the ovary is the differentiation and release of female gametes (oocyte), which can then be successfully fertilised and ensure the survival of species. The ovary is also an endocrine organ that produces steroid hormones that allow the development of female secondary sexual characteristics and supports pregnancy. The mammalian ovary is covered by a single layer of epithelial cells (surface epithelium), which vary in type from simple squamous to cuboidal to low pseudostratified columnar (Anderson et al. 1976). Directly underneath the surface epithelium there is a layer of dense connective tissue known as the tunica albuginea. The mammalian ovary is a heterogeneous organ containing follicles and corpora lutea at various stages of development. The ovarian follicle is the fundamental unit of the ovary, each follicle consists of an oocyte, surrounded by granulosa cells and outer layer of theca cells ( 1.2 Folliculogenesis During embryogenesis, primordial germ cells (PGCS) migrate from the yolk sac through the dorsal mesentery of the hindgut, to the genital ridge. The germ cells then undergo extensive proliferation, and lose their motile characteristics, in addition, somatic cells derived from the mesenchyme of the genital ridge, proliferate as well (Hirshfield 1991). The germ cells cease mitotic division and form association with small numbers of pre-granulosa cells to form primordial follicle (Telfer et al. 1988). The germ cells undergo the first meiotic division and are now called primary oocytes. The oocytes then become arrested in the diplotene stage of prophase I of meiosis until the primordial follicles start to grow and finally reach the ovulatory stage. In the diplotene stage, the oocyte may prepare itself for rapid mitosis and implantation, by producing large amounts of mRNA and ribosomes (Picton et al. 1998). Primordial follicles may be observed from week 22 in the human (Faddy and Gosden 19 95) and week 13 in the cow (van den Hurk and Zhao 2005). The pool of primordial follicles develops during fetal life in some species (e.g primates, ruminants), but in others it develops during the early neonatal period (e.g rodents, rabbits) (Marion et al. 1971). The number of primordial follicles present at birth represents the total population of germ cells available to mammalian females during their entire reproductive life (Kezele et al. 2002), and is believed to serve as the source of developing follicles and oocytes (Eppig 2001). Although recent studies have suggested that postnatal oogenesis may also occur in female mammals (Johnson et al. 2004), they suggested that germline stem cells can repopulate the postnatal ovary and renew the primordial follicle pool. This group subsequently went on to suggest that these cells were derived from bone marrow (Johnson et al. 2005). This has attracted a great deal of attention as well as criticism (Gosden 2004; Byskov et al. 2005; Telfer et al. 2005). This is an ongoing debate but the balance of evidence suggests that renewal is not a major factor in ovarian development (Eggan et al. 2006). In mammals, the number of primordial follicles in the ovaries at birth varies enormously between species, ranging from tens of thousands in mice to millions in humans and domestic species (Gosden and Telfer 1987). These follicles must develop through primordial, primary and secondary stages before reaching the preovulatory stage, and subsequent ovulation (Figure 1.1). Proper follicle development involves maturation of the oocyte, which is surrounded by variable layers of granulosa cells, enveloped by theca cells (Drummond 2005). Granulosa cells provide physical support of the oocyte and mediate signals between the oocyte, outer theca cells and endocrine hormones. Once the pool of primordial follicles has been established, follicles gradually and continually leave the resting pool to begin growth. However, less than 1% of primordi al follicles present at the time of birth of an animal will ever proceed to ovulation (Erickson 1966), with the majority of follicles degenerating by atresia. 1.3 Regulation of early folliculogenesis A critical process in ovarian biology is the transition of the developmentally arrested primordial follicle to the developing primary follicle. Follicular growth may begin at any time during the females life. The primordial follicle contains an oocyte arrested in meiosis I surrounded by flattened somatic cells termed the pregranulosa (Kezele et al. 2002). During onset of primordial follicle growth, flattened pregranulosa cells become cuboidal and begin to proliferate. The enclosed oocyte begins to grow at the same time (Anderson et al. 2000; Clark and Eddy 1975). The growths of both the primordial follicles with oocytes characterize the initiation of the growing phase. The oocytes within the primordial follicles remain quiescent for months to years until they receive the appropriate signals to initiate folliculogenesis and primordial to primary follicle transition. So far, little is known about the molecular mechanisms and extracellular signalling factors that regulate this process. These processes directly affect the number of oocytes available to a female throughout her reproductive life. Once the pool of primordial follicles is depleted, ovarian steroidogenesis ends and the series of physiological changes called menopause begins (Richardson et al. 1987). It is unclear whether the signals originate from the oocyte, or/and from surrounding somatic cells, or from outside the ovary. It is also unknown if it is an inhibitory factor preventing resting primordial follicles from leaving the stock or a stimulus acting on the resting primordial follicles store that stimulates some follicle to leave it (Gougeon and Busso 2000). A multitude of factors may act locally to regulate early folliculogenesis by promoting growth (Bennett et al. 1996) or by inhibiting growth (Bukovsky et al. 1995). The initial growth signal appears to be independent of the pitui tary gonadotropins (Peters et al. 1975) (Figure 1.3). Primordial follicles do not possess receptors for FSH hormone (Oktay et al. 1997). Despite some studies suggesting that gonadotropins are involved in the initiation of follicular growth in immature rodents (Lintern-Moore 1977; Neal and Baker 1973), nevertheless, during natural hypopituitary conditions in both animal species and humans, the initiation process is not completely abolished (Howe et al. 1978; Halpin et al. 1986). Although follicles at early stages of development have been shown to express follicle stimulating hormone receptors (FSH-R) (Bao et al. 1998), in the absence of gonadotropins during the early stages of follicle growth, follicles can still develop to the early antral stage (Awotwi et al. 1984; Gong et al. 1996). Knockout mice who are null mutants for either FSH receptor or LH receptor are able to undergo the primordial to primary follicle transition (Zhang et al. 2001; Abel et al. 2000). Several local factors have been found that can regulate the primordial to primary follicle transition. Bone morphogenetic protein-7 (BMP-7) has been shown to promote the primordial to primary follicle transition and to increase granulosa cell proliferation (Lee et al. 2001). Leukemia inhibitory factor (LIF) has also been shown to promote the primordial to primary follicle transition and to up-regulate granulosa cell expression of kit ligand (KL) (Nilsson et al. 2002). Bone morphogenic protein-15 (BMP-15) is a growth factor expressed in the oocytes of developing follicles that plays a role in early follicle progression (Dube et al. 1998) and stimulates granulosa cell proliferation (Otsuka et al. 2000). Growth differentiation factor-9 (GDF-9) has been localized inthe oocytes of mouse (Dong et al. 1996) rat (Jaatinen et al. 1999) and human (Aaltonen et al. 1999) primary follicles. Nilsson and Skinner (2002) have shown that GDF-9 promotesthe development of primary follicles in neonatal r at ovaries,but it has no effect on the growth of primordial follicles (Nilsson and Skinner 2002).Studies by Wang and Roy (2004) have provided the first evidence that GDF-9 can promote the formation of primordialfollicles and their subsequent growth in neonatal hamster ovaries (Wang and Roy 2004) . Anti-Mullerian hormone (AMH) is a member of the transforming growth factor- ÃŽ ² (TGF-ÃŽ ²) is considered as a negative regulator of the early stages of follicular development. AMH is produced by the granulosa cells of developing preantral and small antral follicles which inhibits the primordial to primary follicle transition (Durlinger et al. 1999). AMH is never found in primordial follicles, theca cells or oocytes (Ueno et al. 1989; Hirobe et al. 1994; Baarends et al. 1995). Examination of ovarian follicles in AMH-deficient female mice revealed lower numbers of primordial follicles and more growing follicles compared with wild-type mice, these findings revealed that, in the absence of AMH, ovaries are depleted of their primordial follicles earlier than they are in control mice and these observations led to the propsal that AMH inhibits primordial follicle recruitment (Durlinger et al. 1999). Results were obtained from another in vitro experiments on the bovine ovarian cortex sugges ted that, at least in nonhuman species, the presence of AMH acts as a brake on the activation of primordial follicles and the growth of preantral follicles (Gigli et al. 2005). It has been demonstrated that oocytes from early preantral, late preantral and preovulatory follicles up-regulate AMH mRNA levels in granulosa cells, in a fashion that is dependent upon the developmental stage of the oocyte (Salmon et al. 2004). These findings suggest that oocyte regulation of AMH expression may play a role in intra- and interfollicular coordination of follicle development. ; kit ligand (KL) and basic fibroblast growth factor (bFGF) secreted by pre-granulosa cells and oocyte respectively, have mutual stimulatory effects on oocytes and granulosa cells; they also promote recruitment of theca cells from the surrounding stromal/interstitial cell population. Stromal/interstitial cells and theca cells secrete BMP-4 and BMP-7, which promote follicle activation and survival. GDF-9 and/or BMP-15 secreted by the oocyte of the activated follicle promote granulosa cell proliferation, KL expression and theca formation. Granulosa cells of growing follicles secrete AMH that appears to act as a ‘brake on primordial follicle recruitment (Knight and Glister 2006). Foxo3a (FKHRL1), a member of the FOXO subfamily of forkhead transcription factors, has been implicated in the regulation of follicle activation. It has been indicated that Foxo3a functions at the earliest stages of follicular growth as a suppressor of follicular activation (Castrillon et al. 2003). It was suggested that Foxo3a serves an essential role by suppressing the growth of primordial follicles, thereby preserving them until later in life (John et al. 2007). It was shown that Foxo3a -/-ovaries contained markedly elevated numbers of early growing follicles, and this extensive of follicular growth in Foxo3a -/- females resulted in the progression of increased numbers of follicles to more advanced stages of follicular development and this misregulation of this process can lead to premature ovarian failure (Castrillon et al. 2003). Kit ligand (KL) is produced by the granulosa cells of developing ovaries (Manova et al. 1993; Ismail et al. 1996) and KL receptors (c-kit) are present on oocytes and theca cells (Manova et al. 1990). C-kit is expressed at the surface of mammalian oocytes at all stages of follicular development in postnatal ovaries of the mouse, the rat and humans (Driancourt et al. 2000; Horie et al. 1991; Manova et al. 1990; Orr-Urtreger et al. 1990) and its only known ligand, Kit-ligand (KL), which is also referred to as stem cell factor (SCF). KL acts to recruit theca cells from surrounding ovarian stroma during folliculogenesis (Parrott and Skinner 2000). Therfore, KL is thought to act as a signal from the granulosa cells around primordial follicles to the oocyte and surrounding stroma to promote the events of the primordial to primary follicle transition (Parrott and Skinner 1999). In this study (Parrott and Skinner 1999), treatment of in vitro cultured follicles from postnatal ovaries from 4 da y old rats, with KL dramatically induced the development of primordial follicles, but was completely blocked by the Kit antibody ACK2. 1.4 Follicle development Follicular development is regulated by both endocrine and intraovarin mechanisms which co-ordinate the processes of somatic cell proliferation and differentiation (Moley and Schreiber 1995). The basic functional unit in the ovary is the ovarian follicle that is composed of somatic cells and developing oocyte. The two primary somatic cell types are the theca cells and granulosa cells. These two somatic cell types are the site of action and synthesis of a number of hormone that promote a complex regulation of follicular development. The prolifetation of these two cell types is in part responsible for the development of the antral ovarian follicle. At the same time, the oocyte is undergoing developmental changes necessary to allow the resumption of meiosis after the preovulatory surge of gonadotrophins (Montgomery et al. 2001). This regulation occurs according to endocrine principles, involving hormones such as pituitary gonadotropins , ovarian steroids and locally produced factors that act either on the cell that produces them (autocrine) or on neighbouring cells (paracrine) (Salha et al. 1998). Ovarian follicular development is a long process which can take around 6 months from the initiation of growth of primordial follicles until development of a preovulatory follicle in humans, cattle and sheep (Lussier et al. 1987; Cahill and Mauleon 1980) and around 4 months in pigs (Morbeck et al. 1992). The majority of this time is spent in the pre-antral stages of development. Already at this stage, a considerable proportion of growing follicles fail to survive and they degenerate through a process termed follicular atresia. Observations in humans and in animals suggest that apoptosis is the mechanism of follicular atresia (Tilly 1996; Kaipia and Hsueh 1997). In women, the dominant follicle orginates from primordial follicle that was recruited to grow almost 1 year earlier Folliculogenesis can be divided into two stages: the gonadotropin-independent (preantral) and gonadotropin-dependent (antral or Graafian) periods (Erickson and Shimasaki 2000). Locally produced growth factors are critically involved in controlling preantral follicle development during the gonadotropin-independent period. After antraum formation, the follicle becomes dependent on FSH stimulation for continued growth and development. Interestingly, it was discovered that the process of folliculogenesis is controled by growth factors secreted by the oocyte (Matzuk 2000). Five growth factors have been identified in mamalian oocytes: growth differentiation factor-9 (GDF-9) (McGrath et al. 1995), bone morphogenetic protein -15 (BMP-15) (Dube et al. 1998; Laitinen et al. 1998), bone morphogenetic protein -6 (BMP-6) (Knight and Glister 2006), transforming growth factor -ÃŽ ²2 ( TGF-ÃŽ ²2) (Schmid et al. 1994), and fibroblast growth factor-8 (FGF-8) (Valve et al. 1997). Experiments with knockout mice have demonstrated that in the absence of GDF-9, folliculogenesis is blocked at the primary to preantral stage (Dong et al. 1996). Consequently, there are no Graafian follicles, no ovulations, and no pregnancies. 1.4.1 Follicular cell types and follicle classification The two primary somatic cell types in the ovarian follicle are the theca cells and granulosa cells. These two somatic cell types are the site of action and synthesis of a number of hormones that are involved in the complete regulation of follicular development. 1.4.1.1 Granulosa cells Granulosa cells are the primary somatic cell type in the ovary that provides a physical support of the oocyte and mediated signals between the oocyte, outer theca cells and endocrine hormone. Regulation of granulosa cell differentiation during folliculogenesis requires the actions of a number of hormones and growth factors. Specific receptors have been demonstrated on granulosa cells for gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) (Richards and Midgley, Jr. 1976). In addition, receptors have been found for factors such as insulin-like growth factor (IGF) (Adashi 1998) epidermal growth factor (EGF) (Vlodavsky et al. 1978) and anti-Mullerian hormone (AMH) (Josso et al. 2001). Actions of these hormones and growth factors on granulosa cells vary with the stage of differentiation. Follicular growth and steroidogenesis are dependent on the coordinated actions of FSH and LH with their receptors on granulosa cells and thecal cells of ovarian follicles. Both granulosal and thecal cells are involved in production of estradiol-17ÃŽ ² (two cell/two gonadotropin model which is well accepted for many species). 1.4.1.2 Theca cells Another important cell type in the ovary is the ovarian theca cell. These are differentiated stromal cells that surround the follicle and have also been termed theca interstitial cells (Erickson and Case 1983). The thecal cells can be distinguished as two distinct layers, the inner layer of cells, the theca interna has a basement membrane separating it from the outmost layer of granulosa cells. The theca interna is a highly vascular layer. One of the major functions of theca cells in species such as the cow, human and rodent is the secretion of androgens (Fortune and Armstrong 1977). At the primordial stage, no theca cells are present; however during the transition to the primary stage, theca cells at this stage of development are gonadotropin and steroid independent and non-steroidogenic (Braw-Tal and Roth 2005). Theca externa which is less vascularized layer and merges into the stromal tissue without clear boundaries. The ‘two-gonadotrophin, two-cell model of follicular estradiol biosynthesis According to the two-cell-two-gonadotrophin theory, the ovary has two cellular compartments that are driven independently by LH and FSH to produce ovarian steroids. Androgen production by theca cells is a function of LH, whereas aromatization of these androgens to oestradiol by granulosa cells is controlled by FSH (Gougeon 1996) (Figure 1.5). Androgen synthesis occurs in the theca interna regulated by LH, by expressing P450c17, the rate-limiting steroidogenic enzyme in androgen synthesis (Sasano et al. 1989). Theca interna are capable of synthesizing all the steroids from cholesterol to testosterone and are the major source of follicular androstenedione. In contrast, granulosa cells are the major source of follicular estradiol. Granulosa cells are intrafollicular sites of androgen metabolism (Ghersevich et al. 1994) and express aromatase P450arom (Whitelaw et al. 1992) this enzyme converts androgens to estrogens. FSH also induces granulosa cell LH receptors that are functionally coup led to aromatase. Thus, uniquely in the preovulatory follicle, both the synthesis of androgen (in theca cells) and its aromatization to estradiol (in granulosa cells) are directly regulated by LH (Fortune and Armstrong 1977). . In the theca, under the influence of LH, cholesterol is converted to pregnenolone and metabolised through a series of substrates ending in androgen production. The two-cell, two-gonadotrophin model comes into play with androgens produced by the theca cells transported to the granulosa cells where they are aromatised to oestrogens (Drummond 2006). 1.4.2 Classification of follicle stages In sheep, the stages of follicular development have been classified on the basis of the number of granulosa cells in the largest cross-section of follicles (McNatty et al. 1999), 1.4.3 Follicular development in primates 1.4.3.1 Oocyte growth and maturation Once follicles have been initiated to grow, the granulosa cells proliferate to form the different stages of follicular development (Telfer et al. 2000). During follicular activation and early development in mice, for example, the oocyte growth occurs rapidly with an approximatly 300-fold increase in volume during the 2-3 week growth phase (Wassarman and Albertini 1994), which is also accompanied by a 300-fold increase in RNA content (Sternlicht and Schultz 1981) and a 38-fold increase in absolute rate of protein synthesis (Schultz et al. 1979). These events are indicative of a period of cell growth with high metabolic activity (Wassarman and Albertini 1994). Oocytes complete most of their growth phase before the formation of a follicular antrum (Wassarman and Albertini 1994), and the increase in oocyte diameter and volume during antral follicular growth is relatively small (Eppig 2001; Wassarman and Albertini 1994). If the oocyte is to be capable of fertilization and subsequent embry onic development, it must acquire the ability to resume meiosis. Oocytes from immature follicles are unable to resume meiosis (Iwamatsu and Yanagimachi 1975) however, by the time the follicles have reached the antral stages the oocytes of most species have acquired the ability to resume meiosis (Mattioli and Barboni 2000; Telfer 1998). 1.4.3.2 Follicular growth Early in oocyte growth, a homogenous glycoprotein layer called the zona pellucida (ZP) is secreted shortly after initiation of follicular growth (Epifano and Dean 1994). It forms a translucent acellular layer separating the oocyte from the surrounding granulosa cells. However, contact between granulosa cells and the oocyte is maintained via cytoplasmic processes, which penetrate the zona and form gap junction at the oocyte surface. Progressively, follicles become secondary follicles. In addition to oocyte growth and granulosa cell proliferation, the preantral follicle also increases in size through formation and growth of ovarian stromal cells on the outer membrane of the follicle forming the theca layers of the follicle. The thecal cells can be distinguished as two distinct layers: highly vascular theca interna, surrounded by a fibrous capsule, the theca externa. The granulosa cells continue to proliferate, resulting in a further increase in follicular size. The formation of the follicular antrum marks the beginning of the antral phase of development. The appearance of an antral cavity starts with the development of small fluid-filled cavities that aggregate to form the antrum. As the follicular antrum grows, the oocyte, surrounded by a dense mass of granulosa cells called the cumulus oophorus which become suspended in fluid. It is connected to the rim of peripheral granulosa cells only by a thin stalk of cells. Attached to the zona pellucida, which surrounds the oocyte, is a small ring of granulosa cells called the corona radiata, these cells will be expelled with the oocyte during ovulation. In humans and monkeys, follicles pass from the preantral to the early antral stage at a follicular diameter between 180 and 250 ÃŽ ¼m (Koering 1983; Bomsel-Helmreich et al. 1979). Further, growth of the follicle is under the influence of follicle stimulating hormone (Gougeon 1996). The follicle is then termed a Graafian follicle. When the proper ho rmonal balance is present, normally one Graafian follicle in mono-ovulatory mammals (e.g. primates, ruminants, equine) and several ones in poly-ovulatory animals (e.g. rodent, porcine) fully develop and the oocyte matures and ovulate (Hafez 1993). 1.4.4 Regulation of follicular development 1.4.4.1 Oocytes somatic cell communication Oocyte growth is dependent on signals, growth factors and nutrients from granulosa cells; at the same time oocytes play an important role in the proliferation and differentiation of granulosa cells. Communication between oocytes and somatic cells have a crucial role in ovarian follicular development (Albertini and Barrett 2003; Eppig 2001). Several studies have shown the importance of oocyte-derived factors such as growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) in female reproduction. GDF-9 and BMP-15 promote the proliferation of granulosa cells from small antral follicles (Hayashi et al. 1999; Vitt et al. 2000; Otsuka et al. 2000). Mutation of the GDF-9 gene in mice prevented the development of somatic cells beyond the primary follicle stages (Dong et al. 1996). C-kit receptor or (Kit) is expressed at the surface of mammalian oocytes at all stages of follicular development in postnatal ovaries of the mouse, the rat and humans (Driancourt et al . 2000b; Horie et al. 1991; Manova et al. 1993). An in vivo study by (Yoshida et al. 1997) has indicated the requirement for C-kit in development of the ovarian follicle in mice. In this study, postnatal mice were injected with Kit-blocking antibody ACK2, and the first wave of follicular development was studied. The blockade of Kit signalling was found to disturb the onset of primordial follicle development, primary follicle growth and follicular fluid formation of preantral follicles (Yoshida et al. 1997). On the other hand, primordial follicle formation, ovulation and luteinization of the ovulated follicle were not affected by ACK2 (Yoshida et al. 1997). 1.4.4.2 Gonadotropins Before the onset of puberty, the normal fate of growing follicles is atresia. After puberty, stimulation by cyclic gonadotropins allows the survival and continued growth with only a limited number of antral follicles that will reach the preovulatory stage. Activation and maintenance of normal follicular function is dependent on gonadotropins secreted by the pituitary. Follicle stimulating hormone (FSH) and Luteinizing hormone (LH) are glycoprotein, secreted by gonadotrophs in the anterior pituitary under the influence of hypothalamic GnRH (Gonadotrophin releasing hormone) neurones, which regulate the synthesis and secretion of those hormones). Gonadotropins are probably not involved in the initiation of follicle growth (Wandji et al. 1992; Fortune et al. 2000; McNatty et al. 1999). Whilst there may not be an absolute requirement for FSH at these early stages the presence of FSH receptor (FSHr) in granulosa cells of immature follicles in cow (Wandji et al. 1992), human (Zheng et al. 1 996) and sheep (Eckery et al. 1997) suggests an involvement. A role for LH in the early stages of development has not been described, although expression of LH receptor (LHr) mRNA is first detected when the theca interna forms around the granulosa cells (Bao et al. 1998; Bao and Garverick 1998). FSH and LH are involved in endocrine control of follicle development, FSH stimulates granulosa cell division and the formation of glycosaminoglycans that are essential components of antral fluid (Hillier 1991). FSH is vital for the formation of the antral cavity (Nayudu and Osborn 1992) in mouse ovarian follicles cultured in vitro. Granulosa cells are the only cells in the female body possess FSH receptors, and binding of FSH to its receptor on the cell surface altered expression of multiple genes crucial to cytoproliferation and differentiation (Richards 1994). Granulosa cell genes that are responsive to FSH include: aromatase (P450arom) the steroidogenic cytochrome P450 crucial to estrogen synthesis (Simpson et al. 1994); cholesterol side-chain cleavage (P450scc) (Richards 1994) and LH receptors (Segaloff and Ascoli 1993). FSH and LH are important factors for the proliferation and survival of follicular somatic cells and the cyclic recruitment of antral follicles. Suppression of serum gonadotropins after hypophysectomy leads to atresia and apoptosis of developing follicles (Nahum et al. 1996). Whereas FSH treatment of cultured early antral follicles prevents the spontaneous onset of follicular apoptosis (Chun et al. 1996). Although FSH is the central regulator of dominant follicle survival and development, LH signalling pathways play fundamental physiological roles. LH-dependent signal pathways in the theca interstitial cells induce changes in gene expression that are critical for estrogen production (Erickson et al. 1985). Activation of the LH receptors in theca cells leads directly to the stimulation of high levels of androstenedione production. The major physiolog ical significance of this LH response is to provide aromatase substrate to the granulosa cells where it is metabolized by P450 aromatase to E2. Additionally, the preovulatory surge of LH is responsible for ovulation and corpus luteum formation. Also, LH is essential for P4 and E2 production by the CL during the early and midluteal phase of the menstrual cycle. The hypothalamus produces and secretes luteinizing hormone-releasing hormone (LHRH) into a system of blood vessels that link the hypothalamus and the pituitary gland. LHRH stimulates the pituitary gland by attaching to specific molecules (i.e., receptors). After the coupling of LHRH with these receptors, a cascade of biochemical events causes the pituitary gland to produce and secrete two hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH are two of a class of hormones commonly known as gonadotropins. They are secreted into the general circulation and attach to receptors on the ovary, where they trigger ovulation and stimulate ovarian production of the hormones estrogen and progesterone. Adapted from (Kanis 1994) 1.4.4.3 Growth factors A number of locally produced growth factors are known to be important for follicle development, they exert paracrine communication within follicles. During preantral follicle development, growth factors such as epidermal growth factor (EGF), transforming growth factors (TGF) may influence folliculogenesis (van den and Zhao 2005). Vascular endothelial growth factor (VEGF) (Danforth et al. 2003) and mullerian inhibitory substance (MIS) (McGee et al. 2001) have been reported to stimulate preantral follicle growth. It has been demonstrated that bone morphogenetic proteins (BMP) can alter bovine granulosa cell steroidogenesis and proliferation in vitro (Glister et al. 2004). Transforming growth factor-ÃŽ ² (TGF-ÃŽ ²) superfamily contains a range of proteins, including inhibins and activins. The precise roles of these factors are not known, but it is likely that they are involved in follicular differentiation by enhancing the action of gonadotropins (Campbell and Baird 2001; Knight and Gli ster 2001; Montgomery et al. 2001). Insulin growth factors (IGF) and their receptors play important role in follicle growth and development (Poretsky et al. 1999). The IGF family comprises of IGF peptides, IGF receptors which are family of proteins called insulin-like growth factor binding proteins (IGFBP) that regulate the availability of the IGF to their target cells, and IGFBP proteases (Giudice 1992). The IGFs affect biosynthetic processes in granulosa and theca cells and have an influence on mitotic activity in the granulosa cells (Giudice 1992; Poretsky et al. 1999). Additionally, a functional link between the IGF system and FSH action has been demonstrated by the finding that IGFBP-4 is a potent inhibitor of FSH