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 (PGC'S) 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 1995) 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 primordial 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 female's 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 pituitary 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 rat 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- β (TGF-β) 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 suggested 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 day 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 -β2 (TGF-β2) (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-17β (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 coupled 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 embryonic 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 μm (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 hormonal 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. 1996) 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 physiological 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-β (TGF-β) 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 Glister 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 induced estradiol production by murine (Adashi 1995; Erickson et al. 1992) and human (Mason et al. 1998) granulosa cells.

1.4.4.4 Follicular fluid

The follicular fluid plays an important role in ovarian physiology. It provides the mean by which cells of the avascular membrana granulosa can be exposed to a specific environment, which differs from serum and from that in adjacent follicles. Follicular steroidogenesis, oocyte maturation, ovulation and transport of the oocyte to the oviduct as well as preparation of the follicle for subsequent corpus luteum function depend on changes in the physical and endocrine properties of the follicular fluid.

Follicular fluid within a growing follicle becomes readily apparent once the antral stage has been reached. In general, pools of follicular fluid begin to form when the granulosa cells have passed through about 11-12 mitotic cycles and a substantial follicle containing some 2000- 3000 cells has developed (Gosden et al. 1988).

Accumulating between granulosa cells in growing follicles, this ovarian fluid is a mixture of novel secretion, especially of steroids hormones, peptides and glycosaminoglycans (GAGs), and a plasma exudate, especially its proteins, although usually at a lower concentration. The novel secretion is primarily of granulosa cell origin, however, follicular fluid would also contain secretions of the theca interna that have crossed the basement membrane and, molecular products of the oocyte itself.

Although large antral follicles are separated from blood vessels by the inner layers of the follicle, including a relatively permeable basement membrane (Szöllösi et al. 1978), passage of molecules across the follicle wall changes as the time of ovulation approaches. Size and shape will influence the movement of protein molecules into follicular fluid. Small molecules in blood plasma are able to equilibrate rapidly with follicular fluid, and even larger proteins may enter within a few minutes (Edwards 1980). As judged from human samples, most plasma proteins and steroid-binding proteins are found in follicular fluid (Beier-Hellwig and Delbos 1983). Overall, about 80% of protein content in plasma is found in follicular fluid (Lipner 1973). The protein include enzymes, immunoglobulins, transport proteins (e.g. pregnancy-associated plasma protein A (PAPP-A), protease inhibitors) (Baker 1982). An extensive review of steroid hormones in human follicular fluid was presented by Lenton and colleagues(Lenton et al. 1988). The concentration of hormone is varying according to the stage of follicular development. The relative abundance of steroid hormones in follicular fluid changes markedly as ovulation approaches (Baird and Fraser 1975). The antral follicle produces large amounts of steroids. The follicular fluid contains some steroids in concentrations which are 40,000 to 100,000 times higher than those in blood. The major steroids in follicular fluid are progestins, androgens and estrogens (Short 1964; Edwards 1974; McNatty et al. 1975).

Pituitary hormones are present in follicular fluid and their concentrations usually vary with the stage of follicular development. Both FSH and LH concentrations increase in pre-ovulatory follicles, although remaining below values in systemic blood. Prolactin is also present in follicular fluid, and may generally decline in concentration with increasing follicular growth or close to ovulation (McNatty 1978).

1.5 Primate Menstrual Cycle

The term, menstrual cycle refers to the hormonal and reproductive tissue changes that occur in adult female mammals to prepare the reproductive tract for pregnancy. Human beings and the great apes (chimpanzees, gorillas, and orangutans) experience a true menstrual cycle. However, most placental mammals—such as dogs, cats, elephants, and New World Monkeys experience estrus instead. One difference is that animals that have estrous cycles reabsorb the endometrium if conception does not occur during that cycle. Animals that have menstrual cycles shed the endometrium through menstruation instead.

Study undertaken by Corner and Allen (Corner and Allen 2005) established the role for ovarian steroids, estradiol (E2), and progesterone (P4) in regulating the changes across the menstrual cycle. The molecular mechanisms by which sex steroids induce events of menstruation involve complex interactions between the endocrine and immune system (Critchley et al. 2001). The dominant hormones involved in the menstrual cycle are gonadotropin releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), estrogen, and progesterone (Figure 1.7). GnRH is secreted by the hypothalamus, the gonadotropins FSH and LH are secreted by the anterior pituitary gland, and estrogen and progesterone are secreted at the level of the ovary. GnRH stimulates the release of LH and FSH from the anterior pituitary, which in turn stimulate release of estrogen and progesterone at the level of the ovary. The menstrual cycle starts, i.e., menarche occurs, at different ages in different species. Such as, in rhesus monkey, menarche occurs at about 4 years of age and in humans, menarche occurs at about 11-14, but this can vary depending upon heredity, diet, and even, climate. The length of the menstrual cycle differs in different animals. The cycle is usually divided into four phases: menstruation does not occur widely throughout the animal kingdom, but is limited to humans, non-human primates. Follicular phase is the time when the follicles in the ovary are maturing and beginning to secrete estrogen, through the influence of a rise in FSH. In a signal cascade initiated by LH, the follicles secrete estradiol, a steroid that acts to inhibit pituitary secretion of FSH. With diminished FSH supply comes a slowing in growth that eventually leads to follicle death, known as atresia. The largest follicle secretes inhibin that leads to the demise of less competent follicles by further suppressing FSH. This dominant follicle continues growing, forms a bulge near the surface of the ovary, and will ovulate in response to the LH surge. The follicles also secrete estrogens (of which estradiol is the major one). Estrogens initiate the formation of a new layer of endometrium in the uterus, histologically identified as the proliferative endometrium. If fertilized, the embryo will implant itself within the uterus. Finally is the luteal phase, after ovulation, the residual follicle transforms into the corpus luteum (CL) under the support of the pituitary hormones. This CL will produce progesterone in addition to estrogens. Progesterone plays a vital role in converting the proliferative endometrium into a secretory lining receptive for implantation and supportive of the early pregnancy. If fertilization of an egg has occurred, it will travel as an early blastocyst to the uterine cavity and implant itself. In the absence of a pregnancy, the CL demises and inhibin and progesterone levels fall. This will set the stage for the next cycle. Progesterone withdrawal leads to menstrual shedding (progesterone withdrawal bleeding), and falling inhibin levels allow FSH levels to rise to raise a new crop of follicle

1.6 The ovine estrous cycle

Sheep, originating from temperate climates, are seasonally, polyestrous animals (Gordon 1997) i.e. they display estrous cycles that occur only during certain seasons of the year. The estrous cycle of the ewe ranges in length from 14 to 18 days, with an average cycle length of 17.5 days (Marshall 1904), which is highly repeatable (McKinzie and Terrill 1937; Asdell 1946; Hafez 1952). There are some differences in cycle lengths among different breeds of sheep (Asdell 1946) and with age (reproductive performance increases up to the age of 3 or 4 years and then gradually declines (McKinzie and Terrill 1937; Hafez 1952), but these differences are relatively small (≤1 day). The ewe is a spontaneous ovulator (Robertson 1977) and repeated estrous cycles provide the female with repeated opportunities to copulate and become pregnant. O'Shea and colleagues (O'Shea et al. 1986) reported that abnormally long cycles in ewes may be associated with the prolonged lifespan of corpora lutea.

The estrous cycle can be divided into two distinct phases; the follicular phase and the luteal phase (Senger 2003). These two phases can then be further sub-divided.

The follicular phase

The follicular phase of the estrous cycle includes pro-estrus and estrus (Arthur et al. 1989). Pro-estrus is characterised by declining serum concentrations of progesterone as a consequence of luteal regression (Arthur et al. 1989; Senger 2003). There is also an increase in serum estradiol concentrations due to the emergence and growth of the ovulatory follicle (Goodman 1994; Senger 2003) (Figure 1.8). The estrus period immediately follows pro-estrus (Bindon et al. 1979; Quirke et al. 1979; Goodman 1994). Estradiol is the dominant hormone during this period and is the cause of major behavioral changes and the period of sexual receptivity and mating, in the ewe (Robertson 1969). Estrus lasts between 24 to 48 hours, depending on the breed (Land 1970; Land et al.1973). Ovulation in sheep occurs 24 to 30 hours after the onset of estrus behavior (McKinzie and Terrill 1937; Robertson 1969).

The luteal phase

The luteal phase of the cycle includes metestrus and diestrus. The first period is metestrus, during which ovulation and the formation of a corpus luteum (CL) occur (Keyes et al. 1983). A structure called the corpus hemorrhagicum forms prior to the CL and is due to the rupture of blood vessels in the follicle wall (Senger 2003). Once the CL is fully functional and secretes high levels of progesterone, this period is referred to as diestrus and is the longest stage of the estrous cycle (Senger 2003). Cyclic activity in the ewe is mainly regulated by the hypothalamic-pituitary-ovarian axis (Goodman 1994).

1.7 Ovulation and follicular rupture

Ovulation is a complex process by which a preovulatory follicle ruptures and releases a fertilizable oocyte into the oviduct where it may be fertilized. Ovarian mechanisms of ovulation have been a subject of investigation for more than a century (Espey 1994) nevertheless, essential regulatory pathways remain uncertain. The physiological mechanisms controlling ovulation in mammals involve a complex exchange of endocrine signals between the pituitary gland and the ovary, and a localized exchange of intraovarian hormones between the oocyte and its adjacent somatic cells. (McNatty et al. 2003). The initiating signal for ovulation is the surge in pituitary secretion of luteinizing hormone (LH) that occurs during the late follicular phase of an estrous or menstrual cycle (Barrell et al. 1992; Fauser and Van Heusden 1997). Many proteolytic enzymes are present in the preovulatory follicle, such as plasminogen activator (PA), plasmin and matrix metalloproteinases (MMPs, e.g collagenase) (Espey 1980; Woessner, Jr. et al. 1989). PA exist in two forms, tissue plasminogen activator (tPA) and urokinase plasminogen activaror (uPA). Both forms are present within the preovulatory follicle and ovulation is associated with a significant increase in PA activity (Tsafriri et al. 1989). Cellular death (apoptosis and inflammatory necrosis) occurs within the formative site of ovulation (stigma) (Murdoch 1994; Murdoch 1995). Outgrowth of a follicle selected to ovulate brings it into close contact with the ovarian surface epithelium (OSE) (Anderson et al. 1976). A role for the OSE in ovulation has been debated for many years. In sheep OSE contiguous with preovulatory ovine follicles secretes a plasminogen activator (PA) (Colgin and Murdoch 1997). Proteases produced from OSE aid in digestion of the collagenous matrices of the tunica albuginea (TA) and follicular theca (Bjersing and Cajander 1975). Tumor necrosis factor (TNF) facilitates follicular dissociation by stimulating collagenase production (Johnson et al. 1999). Collagen breakdown and cellular deletion accompanied by increased permeability of the blood vessels, resulting in leakage of blood cells and edema of follicular tissue (Parr 1975; Abisogun et al. 1988) lead to weakening of the apical ovarian wall, stigma development, and ovarian rupture Immune cells are present in the mature follicle (Chun et al. 1993), as a mediators of inflammation such as vasoactive substances, eicosandoids, interleukins and chemotactic (Espey 1994). Ovarian epithelial cells that overlie the apical aspect of preovulatory follicles become apoptotic and are sloughed with the approach of rupture (Ackerman and Murdoch 1993). Once the oocyte-bearing cumulus mass is expelled from ovary, ovulation is complete (Espey 1999).

1.8 Formation and development of the corpus luteum

The ovarian cycle is characterized by repeated patterns of cellular proliferation, differentiation and transformation that accompany follicular development and the formation and regression of the corpus luteum (CL). The corpus luteum is a transient endocrine organ formed from cells of the follicle following ovulation (Juengel and Niswender 1999). Pituitary derived gonadotropins and growth hormone are the primary regulation of final follicular maturation and CL function. Formation of the CL is initiated by series of morphological and biochemical changes in cells of the theca interna and granulosa of the preovulatory follicle (Juengel and Niswender 1999). The preovulatory LH surge causes differentiation of follicular cells into luteal cells (luteinization). Luteinization is characterized by increased steroid production and a switch from producing oestradiol to progesterone and of enzymes responsible for these changes (Juengel and Niswender 1999). The principal function of the CL is to secrete progesterone (P4) during non-pregnant cycle as well as during pregnancy. The CL is one of the few adult tissues that exhibit regular periods of growth (CL formation), function and luteolysis. In domestic ruminants, the primary luteotropic hormones are LH and growth hormone (GH) (Schams and Berisha 2004). LH is the principal hormone that stimulates progesterone production by the small luteal cells (Niswender and Nett 1988). In cattle it has been demonstrated that the granulosa and theca cells, of the follicular wall, give rise to large and small luteal cells, respectively (Alila and Hansel 1984; Niswender et al. 1985; Meidan et al. 1990). Large luteal cells are responsible for 80% of total P4 production by the CL (Niswender et al. 1985). Most of the LH receptors are located on small luteal cells (Harrison et al. 1987). However, the large luteal cells are unresponsive to LH stimulation (Hoyer and Niswender 1986), suggesting that large luteal cells are not dependent on LH for the production of P4 (Alila and Dowd 1991). Research has shown that small luteal cells may differentiate into large luteal cells when LH is administered to ewes (Farin et al. 1988) and cows (Niswender et al. 1985). In addition to LH, Oxytocin (OT) has intraovarian effects influencing steroidogenesis (Schams 1987). Oxytocin is localized in bovine CL in large and small luteal cells (Kruip et al. 1985). It is appears that luteinization stimulated by the preovulatory LH surge trigger the production of ovarian OT. Progesterone has an effect on the function of the bovine early CL in an autocrine and paracrine fashion (Skarzynski and Okuda 1999). It was indicated that treatment of cultured bovine luteal cells obtained from early CL with progesterone antagonist reduced secretion of P4 (Pate 1988).

1.9 Luteolysis regulation

Luteal regression or luteolysis involves a collapse of the lutein cells, and progressive cell death with consequent fall in the output of progestagens. Luteolysis can be caused by withdrawal or inadequacy of the luteotrophic complex. However in many species, it is not primarily a failure of luteotrophic support, but active production of luteolytic factor that brings about normal luteal regression. The luteolytic factor in ruminants is prostaglandin F2α (PF2α) and is released from the endometrial glands of the uterus (Knickerbocker et al. 1988). PF2α travels to the ovary by way of the uterine venous and lymphatic vessels and ovarian artery (Krzymowski et al. 1989). In the ewe, small luteal cells are insensitive to PF2α, while large luteal cells contain PF2α receptors (Fitz et al. 1982). However, the CL of the ewe is only responsive to PF2α between days 4 and 14 of the estrous cycle (Day 0 = oestrous) (Chamley et al. 1972). Ovarian estradiol, progesterone, and oxytocin are regulators of PF2α secretion, in the ewe. Exposing the uterus to high levels of progesterone for a specific period of time prepares the endometrium for PF2α synthesis (Silvia et al. 1991). Zelinski and colleagues (1982) reported high concentrations of endometrial receptors for progesterone at estrus but then a gradual decline during the luteal phase of the cycle (Zelinski et al. 1982). The exposure to luteal phase progesterone allows the build up of prostaglandin endoperoxidase and arachidonic acid, which are required for PF2α production (Knickerbocker et al. 1988; Silvia et al. 1991). Towards the end of the luteal phase, the formation of endometrial receptors for oxytocin and estradiol increases and is stimulated by follicular estradiol (Roberts et al. 1975; Koligian and Stormshak 1977). Early exposure to progesterone greatly amplifies the effect of estradiol on the recruitment of oxytocin receptors and estradiol amplifies the secretion of PF2α (Ford et al. 1975; Vallet et al. 1990). It is interesting to note that an increase in pulsatile PF2α secretion and an elevation in the number of oxytocin receptors are related to the decrease in circulating progesterone concentrations (Sheldrick and Flint 1985). Immune cells infiltrating the bovine CL play a central role in luteolysis (Pate and Landis 2001). The number of leukocytes (e.g. T lymphocytes, macrophages) increases at the time of luteolysis (Penny et al. 1999; Townson et al. 2002).

1.10 Apoptosis

Apoptosis is a genetically determined and biologically functional mode of cell death. The studies of the Australian pathologist John Kerr, on liver atrophy, provided the first clue to the existence of apoptosis. Kerr called the apoptosis phenomenon shrinkage necrosis. Over two decades, Kerr and Wyllie have defined apoptosis as cell death in terms of morphology, biochemistry and incidence (Kerr et al. 1972; Wyllie 1993).

Apoptosis is known to serve many critical functions in vertebrate and invertebrate species such as cell deletion during embryonic development, balancing cell numbers in continuously renewing tissues, elimination of damaged, senescent, potentially harmful, or no longer useful cells (Schwartzman and Cidlowski 1993) and many other physiologic processes. Furthermore, numerous pathologically induced conditions such as Alzheimers, autoimmune disease, cancer and AIDS, often show varying levels of apoptosis, with greatest significance lying in whether dysreulation of apoptosis is a primary event in the pathology of these diseases, and many other physiologic processes

The major histological features of apoptosis are condensation of the nuclear chromatin, cell shrinkage, plasma membrane blebbing and the formation of the apoptotic bodies. Stimulation by death ligands or deprivation of survival promoting factors is the main contributor to apoptosis, while other inducers such as stress, drugs, toxicants, oxidative stress and radiation are also known to cause apoptosis. Biochemically, apoptosis is characterized by rapid nuclear DNA cleavage (Wyllie et al. 1980) into 300-400 kilobase pair fragments (Oberhammer et al. 1993) (Figure 1.10).

Upon receiving specific signals instructing the cells to undergo apoptosis a number of distinctive changes occur in the cell. A family of proteins known as caspases is typically activated in the early stages of apoptosis. These proteins breakdown or cleave key cellular components that are required for normal cellular function including structural proteins in the cytoskeleton and nuclear proteins such as DNA repair enzymes. The caspases can also activate other degradative enzymes such as DNases, which begin to cleave the DNA in the nucleus. There are a number of mechanisms through which apoptosis can be induced in cells. The sensitivity of cells to any of these stimuli can vary depending on a number of factors such as the expression of pro- and anti-apoptotic proteins (eg. the Bcl-2 proteins or the Inhibitor of Apoptosis Proteins), the severity of the stimulus and the stage of the cell cycle. In some cases the apoptotic stimuli comprise extrinsic signals such as the binding of death inducing ligands to cell surface receptors called death receptors. These ligands can either be soluble factors or can be expressed on the surface of cells such as cytotoxic T lymphocytes. The latter occurs when T-cells recognize damaged or virus infected cells and initiate apoptosis in order to prevent damaged cells from becoming neoplastic (cancerous) or virus-infected cells from spreading the infection. It might also be a consequence of growth factor deprivation or oxidative stress caused by free radicals. In general intrinsic signals initiate apoptosis via the involvement of the mitochondria. The relative ratios of the various bcl-2 proteins can often determine how much cellular stress is necessary to induce apoptosis.

The standard tool for apoptosis detection is the morphological evaluation. Other techniques include DNA agarose gel electrophoresis with the formation of DNA ladder, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling and in situ end labelling (Wyllie 1993).

Few organs provide such a pattern for apoptosis as the ovary. This is due to the cyclisity of ovarian development. In the ovary, the mechanisms underlying decisions of life and death involve cross interchange between pro-apoptotic and pro-survival molecules. Some of these molecules are involved in the process of atresia such as Bcl-2 family members, Tumor necrosis factor (TNF) and caspases (Van Nassauw et al. 1999; Yoon and Carbon 1999; Fenwick and Hurst 2002). Ovarian cell death is a crucial event in maintaining ovarian homeostasis in mammals. It ensures that every estrus/monstrous cycle only one or very few follicle-enclosed oocytes will reach the stage of a Graafian follicle and will ovulate. Apoptosis is found in ovarian follicles throughout fetal and adult life. During fetal life, apoptosis is localized to the oocytes, whereas in adult life, it is detected in granulose cells of growing follicles. In each stage of the cycle about 50% of the large preantral and antral follicles will be in the process of apoptotic death (Almog et al. 2001). Once a follicle has entered the growth phase, it will continue growth with no stops until it either ovulates or undergoes atresia (Peters et al. 1975). Atresia may occur at any stage of follicular development, although generally the incidence increases with increasing follicular size (Block 1952). The process of atresia involves a series of characteristic changes of the morphology of the components of the follicle, starting with a decrease of granulose cell proliferation and changes of oocyte nuclear morphology, and ending with disintegration of the whole structure of the follicle and replacement of granulose, theca cells and oocyte with interstitial tissue (Byskov 1978). These morphological changes are accompanied by functional changes in follicular responsiveness to gonadotropins and steroidogenic capacity (Westergaard et al. 1986; McNatty et al. 1979). Apoptosis is also responsible for corpus luteum regression through luteolysis (Tilly et al. 1991; Tilly 1996).

1.11 Ovarian surface epithelium

Studies on the ovary have mainly focused on the other cell types such as granulosa and theca cells, which play a critical role in folliculogenesis and steroidogenesis. Ovarian surface epithelium (OSE) was among the least studied part of the ovary due to its inconspicuous histological appearance and apparent lack of significant functions. Interest in the OSE began when it become apparent that OSE cells might be the origin of ovarian cancer (OC) (Herbst 1994). Although the surface epithelium represents only a small fraction of the diverse cell types that comprise the ovary, it accounts for over 90% of all ovarian cancers (Murdoch and McDonnel 2002). Recognition of the principal role of ovarian surface epithelium in malignancy has been credited to Sir Spencer Wells in 1872 (Hamilton 1992). Animal models were not available because, except in aging hens (Fredrickson 1987), ovarian tumours in species other than human do not arise in OSE. In the 1980s, the first tissue culture systems for OSE from different species (Adams and Auersperg 1981), including human (Auersperg et al. 1984), were developed. Subsequently, information about the normal OSE and its relationship to OC expanded rapidly. Epithelial ovarian cancer is the fourth most common cause of cancer death among women and has the highest mortality rate among gynaecologic cancers (Jemal et al. 2005). Worldwide, the total number of cases is approximately 190,000 per year (Gadducci et al. 2004). The etiology of OC remains poorly understood. Recent studies have linked smoking, talcum powder, asbestos and alcohol to an increased incidence of ovarian cancer (Modugno et al. 2002; Green et al. 2001; Chang and Risch 1997; Ness and Cottreau 1999). However, these issues are limited by inconsistent data and or the lack of supportive animal models. Other factors such as family history (Swisher 2003), nulliparity and infertility have been consistently recognized as risk factors of OC, whereas pregnancy, oral contraceptive use, hysterectomy, and tubal ligation have protective effects on OC (Ness et al. 2002).

1.11.1 OSE structure

OSE is the modified pelvic mesothelium that covers the mammalian ovary (Risch 1998). The cells which make up this layer vary morphologically from simple squamous to cuboidal to low pseudostratified columnar epithelial cells (Papadaki and Beilby 1971; Blaustein and Lee 1979). Embryologically, OSE is derived from the mesodermal epithelium of the gonadal ridges and is separated from the underlying stromal compartment of the ovary by a basement membrane and underneath, by a dense collagenous connective tissue layer, the tunica albuginea (TA), which is responsible for the whitish color of the ovary. The TA provides a partial barrier to the diffusion of bioactive agents between the ovarian stroma and the OSE. Intercellular contact and epithelial integrity of OSE are maintained by simple dismosomes, incomplete tight junctions (TJ) (Figure 1.13) (Siemens and Auersperg 1988), several integrins (Kruk et al. 1994; Cruet et al. 1999), and cadherins (Sundfeldt et al. 1997; Davies et al. 1998) (Figure 1.9). Tight junctions play key roles in limiting paracellular permeability of ions and molecules (Johnson 2005). In the human, OSE and granulosa cells are connected by N-cadherins, which characterizes adhesive mechanisms of mesodermally derived tissues (Gulati and Peluso 1997).

1.11.2 OSE function

The OSE transports materials to and from the peritoneal cavity and participates actively in the mechanism of gonadotropin-induced ovulatury follicular rupture (Auersperg et al. 1991). The OSE cells directly over the point of rupture undergo apoptotic cell death before ovulation (Ackerman and Murdoch 1993) and the wound created at the ovulatory site surface is repaired by rapid proliferation of OSE cells from the perimeter of the ruptured follicle (Osterholzer et al. 1985). Studies in rabbits and sheep have shown that OSE release proteolytic enzymes that degrade the basement membrane and the underlying apical follicular wall, weakening the ovarian surface to the point of rupture (Bjersing and Cajander 1975). OSE might contribute to the remodelling, as well as the breakdown, of the ovarian cortex. It is also likely that OSE takes part in the restoration of the ovarian cortex by the synthesis of both epithelial and connective tissue-type components of the extracellular matrix (Auersperg et al. 1994; Kruk and Auersperg 1994). Most OSE functions vary with the reproductive cycle and thus are likely to be hormone dependent (Nicosia et al. 1991).

Recent studies revealed that the proliferation and migration of the OSE are regulated by hormones, growth factors and cytokines. FSH and LH have been involved in OSE proliferation, migration and protection from apoptosis in humans, mice, rats and cows in vivo and in vitro (Stewart et al. 2004; Syed et al. 2001; Choi et al. 2006). Also, steroid hormones such as progesterone, estrogen and androgen modulate the OSE (Risch 1998; Auersperg et al. 2001).

1.11.3 Epithelio-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a complex process, it is suggested to serve as an adaptable mechanism for allowing cellular movement required during tissue regeneration, embryonic development and cancer progression.

Duringpostovulatory repair and in culture, OSE cells undergo an epithelio-mesenchymal transition and gain fibroblast-like characteristics reflecting their developmental relationship to stromal cells (Salamanca et al. 2004). The resulting mesenchymal type cells can be stimulated to differentiate back into the epithelial phenotype (Dyck et al. 1996; Auersperg et al. 1999). This capacity of OSE to undergo epithelio-mesenchymal conversion in response to postovulatory stimuli has been proposed to give advantage to the postovulatory repair of the OSE by altering the motility and proliferative response required for extracellular matrix remodelling (Salamanca et al. 2004). EMT might facilitate the release of trapped OSE cells within the ovary during ovulation into the ovarian stromal fibroblasts (Ahmed et al. 2006). EMT inducers of OSE such as epidermal growth factor (EGF) and collagen are present at the site of ovulatory rupture (Salamanca et al. 2004). Since that EMT is a part of normal OSE physiology, any failure to undergo such process may be one of the reasons of initiation of ovarian cancer.

1.11.4 Role of OSE in ovulation and malignancy

The OSE can release enzymes that contribute to the breakdown of the underlying stroma which is adjacent to the preovulatory follicle and thus has been suggested to be involved in the process of ovulation (Murdoch 1996). After ovulation, the OSE proliferates and covers the site of follicular rupture. The incessant ovulation hypothesis of OC was proposed by (Fathalla 1971). This hypothesis argues that repeated cycles of ovulation without long dormant periods, may causes malignant transformation of epithelium at the site of ovulation. Additionally, it was reported a lack of ovarian carcinomas in species where seasonal ovulation and multiple pregnancies occur (Fathalla 1971). Although, a recent study suggested that inducing ovarian tumours in the OSE not required incessant ovulation; thus, other mechanisms must contribute to ovarian tumorigenesis (Chen et al. 2007). Several investigations indicated the role of ovulation in tumorigenesis. It was reported that ovulation, and therefore ovarian cancer, more common in women than it was in most other species since they are either pregnant or lactating for most of their reproductive lives (Murdoch and McDonnel 2002). According to (Fathalla 1971), successive bouts of apoptosis and regenerative repair of OSE cells at the ovulation site induces genetic instability, which makes this cell layer more susceptible to tumorigenesis. Oxidative DNA damage, expression of P53, and apoptosis occur in the OSE cells within the formative site of ovulation (Murdoch et al. 2001). In agreement with the previous finding about ovulation and tumorigenesis, it has been reported that a low incidence of OC is found in women who have been pregnant or have taken oral contraceptives (Clow et al. 2002). In both groups, pregnant and oral contraceptive usage, there was lack of ovulation for a certain time during the life span (fewer ovulation cycles). Another protective factor of pregnancy from OC could be the exposure of the OSE to high levels of progesterone during pregnancy. It was reported that women with a history of twin pregnancies exhibit a lower risk of developing OC, possibly due to higher levels of progesterone found in maternal circulation during twin pregnancies when compared to singleton pregnancies (Batra et al. 1978; Adami et al. 1994). Another study indicated that pregnancy-equivalent does of progesterone inhibited the growth of normal and malignant human ovarian surface epithelium cells in vitro (Syed and Ho 2003). An increase in ovarian cancer incidence was observed among women with progesterone deficiency (Modan et al. 1998). There is increasing evidence suggesting that it is the hormonal environment of the normal ovarian surface epithelium and ovarian epithelial cancer cells that is associated with the development and progression of ovarian cancer. Repetitive loss of the basement membrane during ovulation has been implicated as an early event in the preneoplastic transformation of OSE (Roland et al. 2003; Capo-Chichi et al. 2002).

1.11.5 OSE inclusion cysts

The OSE is separated from the underneath stroma by the collagenous tunica albuginia layer and a basement membrane. Ovulation and aging related trapping of OSE fragments result in surface invaginations (cleft) and inclusion cysts (IC) in the ovarian cortex () (Murdoch 1994).

Some investigators have suggested that the formation of inclusion cysts is related to the repair process of the ovarian surface after ovulation (Godwin et al. 1993). However, it has been indicated that the first step in tumorigenesis of the surface epithelium is the formation of epithelial inclusion cysts derived from crypts or invaginations of the ovarian surface epithelium (Cramer and Welch 1983). The formation of the cysts would result in the disruption of the underlying connective tissue, and as the cysts come into contact with stromal cells, a microenvironment which may under certain conditions be conducive to proliferation (figure 1.14). Normal OSE cells have the ability to undergo epithelio-mesenchymal conversion, which may function as a homeostatic mechanism to accommodate OSE cells that become trapped in the ovary at ovulation (Auersperg et al. 2001). It has been proposed that failure to undergo EMT under such conditions may favour the aggregation of epithelial cells within the stroma resulting in the formation of inclusion cysts which are a common site of ovarian cancer initiation (Auersperg et al. 2001). However, Scully (Scully 1995) reported that inclusion cysts are more numerous in ovaries of multiparous women than in nulliparous women who ovulate more frequently and the cysts are particularly numerous in women with polycystic ovarian disease, a condition that is characterized by anovulation or infrequent ovulation. He proposed an alternative that inclusion cysts arise through inflammatory adhesions of surface OSE which becomes apposed at sites of surface invaginations, combined with localized stromal proliferation. Ness and Cottreau (1999) have suggested that it is an inflammatory microenvironment such as cell damage, oxidative stress, and elevations of cytokines and prostaglandins, rather than the trapping of the OSE into the stroma that could mediate the mutagenesis induced by ovarian ovulation (Ness and Cottreau 1999). However, whether the surface and /or cyst epithelial cells directly progress to malignant neoplasm is still not clear.

1.12 Aims of the project

The OSE plays an important role in normal ovarian physiology, specifically in the cyclic ovulatory ruptures and repair. Thus the mitogenic activity of OSE cells in the reparative process may favour the survival of OSE with accumulated mutations leading to OSE cell transformation and malignancy.

The overall aim of this thesis is to define how the physiological reproductive events affect the ovarian surface epithelium activity specifically:

  • To examine the proliferative activity of the OSE through the regular ovarian cycle and pregnancy by monitoring proliferation in vivo using immunohistochemistry (IHC) for cell cycle markers (PCNA and Ki-67) (sheep and cow) and BrdU incorporation (marmoset).
  • Using primary culture system of OSE (sheep and cow) to examine the effect of steroids and gonadotropin on OSE proliferation and apoptotic cell death.
  • Study the expression and localization of P53 tumour suppressor gene within OSE cells in consideration of its role as a genomic guardian gene (cows).

Different species were used as models to examine the physiological differences among OSE from different animals.

For in vivo studies, it appears that bovine OSE is the best model for study, because of the relative similarity between the reproductive physiology between human and bovine. Marmoset monkey is a suitable model to study the effects of multiple ovulations on OSE activity in non-human primates as marmosets is only the non-human primates which ovulate 2-4 ooctyes every cycle.