1 The differential effects of FSH and LH on the human ovary

1 The differential effects of FSH and LH on the human ovary JARON

RABINOVICI

GENERAL CHARACTERISTICS OF GONADOTROPHINS

There are at least two periods during female life when gonadotrophic support of human ovarian function is crucial. The first period is during prenatal gonadal development, when ovarian formation occurs and when absent gonadotrophic support during parts of pregnancy, such as in anencephalic fetuses, leads to a decrease in ovarian follicles and a decrease in ovarian stroma (Rabinovici and Jaffe, 1990). The second period is during the reproductive years, when the cohort of developing primordial follicles depends on gonadotrophic support to escape atresia and to proceed through the process of maturation, ovulation and corpus luteum formation. Absent or inadequate gonadotrophic support during this period results in anovulation and infertility. The neuroendocrine axis is present early during fetal life and recent evidence indicates that at least some of the autocrine/paracrine/endocrine factors that regulate this axis in the adult are already present in the fetus at around 8-10 weeks' gestational age (for review see Rabinovici and Jaffe, 1990). Human fetal pituitaries secrete intact luteinizing hormone (LH) in vitro as early as 5 to 7 weeks' gestational age, and intact follicle-stimulating hormone (FSH) and LH have been detected in fetal pituitaries and in fetal plasma by 10 weeks. In late gestational fetal monkeys, peripheral LH concentrations were pulsatile, presumably representing a pituitary response to pulsatile hypothalamic gonadotrophin-releasing hormone (GnRH) release (Martin et al, 1987). Further, it seems that at least some of the gonadal feedback mechanisms are present and functional during this prenatal period (Rabinovici and Jaffe, 1990; Rabinovici et al, 1991). At the onset of puberty, plasma gonadotrophins rise, their release becomes pulsatile, at first only during sleep. By the end of puberty, pulsatile gonadotrophin secretion is well established and the patterns of pituitary gonadotrophin release are governed by central hypothalamic factors and by gonadal feedback mechanisms that can modulate the gonadotrophin secretion both at the level of the hypothatamus and of the pituitary. The pituitary gonadotrophin hormones, FSH and LH, are glycoproteins composed of two glycosylated, non-covalently linked polypeptide subunits, Baillidre's Clinical Obstetrics and Gynaecology-263 Vol. 7, No. 2, June 1993 Copyright © 1993,by Baillirre Tindall ISBN 0-7020-1754-X All rights of reproduction in any form reserved

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the a- and [3-subunit. The subunits are synthesized independently as precursors encoded by separate genes and are then linked in the endoplasmic reticulum of the gonadot~-ophs before secretion. The [3-subunit confers the receptor specificity of these hormones. The a-subunit is also common to thyroid-stimulating hormone and to chorionic gonadotrophin. Human chorionic gonadotrophin (hCG) is a gonadal trophic glycoprotein hormone produced mainly by the syncytiotrophoblast of the placenta. Because of the structural similarity of the 13-subunits of LH and hCG, both hormones bind to the same receptor and elicit similar effects in vitro and in vivo. The main difference between these two hormones is the more prolonged action of hCG in vivo due to its slower metabolic clearance and its higher affinity to the LH/hCG receptor (see below). Human serum and pituitaries contain low concentrations of hCG [3-subunits as well as intact hCG (Odell and Griffin, 1987; Hoermann et al, 1990), but the physiological role of hCG in non-pregnant women has remained unclear. The biological activity of a hormone on its target organ depends on multiple parameters. These include the circulating concentration of the hormone, the metabolic clearance of the hormone, the number of its receptors on the target tissue, and the activity of a 'postreceptor' signal transduction mechanism. These rules also apply to the actions of FSH and LH on the ovary. Their effects depend on the rate of their synthesis by the pituitary gonadotrophs, their circulating concentrations (which vary throughout life and throughout the menstrual cycle), the relative abundance of the multiple forms of gonadotrophins that have varying biological activity, the presence of their receptors on the different ovarian cell types, the intracellular adenylate cyclase enzyme that causes the production of cyclic AMP (cAMP), and the extra- and intragonadal factors that are able to modulate the effects of gonadotrophins in the ovary. All these multiple variables and their complex interactions need to be taken into account in a review of the ovarian actions of FSH and LH. However, the constraints of this chapter force us to focus mainly on the gonadotrophic-ovarian processes and other important determinants will be discussed only briefly. Different carbohydrate groups are attached at specific locations of the subunits of the gonadotrophins. Therefore, when FSH and LH are extracted from pituitary cells or from serum, based on their different carbohydrate content, they exhibit a charge heterogeneity which can be used to distinguish different FSH and LH isohormones (Ulloa-Aguirre et al, 1988). These isohormones are also distinguishable by their receptor binding, biological activity and plasma half-life. These characteristics influence the action of the hormone at the target organ, and the relative abundance of the different isohormones influences the biological activity of the hormone. Thus, similar circulating concentrations of a hormone, as determined, for example, by radioimmunoassay, can exhibit different biological activities, as determined by in vitro or in vivo bioassays. The ratio of bioactivity to immunoactivity is therefore a determinant of the relative biological activity of the isohormone "mixture" examined. The relative abundance of isohormones can be regulated by GnRH and by sex steroid hormones, so that both the hypothalamus and the gonads can control the bioactivity and bioavailability of

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circulating gonadotrophins. Therefore, the bioactivity of gonadotrophins examined in women at various times of their reproductive life is quite variable (Lobo et al, 1983; Veldhuis and Dufau, 1987). Due to their different carbohydrate components, the three gonadotrophins, FSH, LH and hCG, have different metabolic clearance rates despite a similar protein structure. O V A R I A N G O N A D O T R O P H I N RECEPTORS

FSH and LH exert their effects through binding to specific membranebound receptors. Following binding of the ligand, the receptors undergo conformational changes that lead to the activation of an effector system. The major effectors for FSH and LH/hCG are adenylate cyclase which forms cAMP and phospholipases that cause an elevation in cytosolic calcium concentrations (Catt and Dufau, 1991). Following the activation of the G-protein cascade, cAMP phosphorylates a protein kinase that, through phosphorylation of proteins, affects the actions of the gonadotrophins. Gonadal gonadotrophic receptors are acquired during fetal and perinatal development. FSH receptors are already present in the second trimester human fetal testis and in the late gestational rhesus monkey fetal testis and ovary, but are not found in human second trimester ovaries (Huhtaniemi et al, 1987). In a recent study, the activation of the ovarian LH receptor during prenatal life was determined in rats (Sokka et al, 1992). Only truncated versions of LH receptor messenger R N A (mRNA) are present in the developing rat ovary from day 17 of fetal life up to day 7 postpartum. On day 7, LH receptors become functional, as determined by ligand binding and stimulation of cAMP production. Concomitant with the appearance of a functional LH receptor, two larger LH receptor mRNA species (2.6 and 6.7 kb) appear, which are also present in the adult ovary. These findings indicate that the LH receptor gene is probably constitutively expressed in the developing rat ovary, and that the onset of translation of functional LH receptor occurs through a change in the alternative splicing pattern of LH receptor m R N A (Sokka et al, 1992). Until recently we based our knowledge of the ovarian localization and regulation of gonadal gonadotrophin receptors on binding studies of iodinated gonadotrophins (Rajaniemi and Vanha-Perttula, 1972; Channing and Kammerman, 1974; Uilenbroek and Richards, 1979; Solano et al, 1980). In summary, these studies indicated that the granulosa cells of primordial and preantral follicles bind FSH, while theca-interstitial cells bind LH. Granulosa cells of large antral follicles appear to bind both FSH and LH. These granulosa cells acquire the ability to bind LH during folliculogenesis. The detection of the primary structure and the gene function of both gonadotrophin receptors has confirmed these studies and has allowed a better understanding of the genetic regulation of gonadal gonadotrophin receptors. The primary structures of the rat and human FSH receptors were only recently elucidated (Sprengel et al, 1990; Minegish et al, 1991). As

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expected, the human amino acid sequence displays similarity to other Gprotein-coupled receptors. There is a 89% identity in the protein structure of the human and rat FSH receptor, with the putative transmembrane segments showing the highest similarity (95%) (Minegish et al, 1991). Due to very low amounts of FSH receptor mRNA in the ovary, Camp et al (1991) developed a highly sensitive and specific quantitative reverse transcription polymerase chain reaction (PCR) amplification scheme to determine the LH and FSH receptor mRNA levels in ovarian samples. In ovaries of immature rats they found low levels of FSH receptor mRNA in the granulosa cells of small follicles and low levels of LH receptor mRNA in the theca cells of these same follicles (Camp et al, 1991). Ovarian stimulation with pregnant mare serum gonadotrophins (PMSGs) initiated the production of LH receptor mRNA in the granulosa cell layer as well as in the theca cell layer of large follicles. Subsequent addition of ovulatory doses of hCG to these animals decreased the LH receptor mRNA in the granulosa cells to basal values. Similarly, administration of PMSGs increased the FSH receptor mRNA signal, but the degree of change was less marked than that seen for the LH receptor mRNA. In the same study, using in situ hybridization techniques on ovarian sections obtained throughout the oestrous cycle, the signal for LH receptor mRNA was localized predominantly to the theca cells of small follicles on oestrous morning, but appeared in the granulosa cells of growing follicles by dioestrous morning (Camp et al, 1991). LH receptor m R N A was also found in interstitial tissue and corpora lutea throughout much of the oestrous cycle. Some follicles continued to express the LH receptor m R N A only in theca cells on dioestrous morning and the authors speculated that these follicles might represent atretic follicles or follicles doomed for atresia, unable to induce granulosa cell LH receptor mRNA production (Camp et al, 1991). The concentration of the FSH receptor mRNA increased somewhat during the cycle, but FSH receptor mRNA was in lower abundance and not as highly regulated as the LH receptor mRNA (Camp et al, 1991). FSH receptor m R N A was not detected in theca cells, corpora lutea or interstitial tissue, although it cannot be ruled out that the m R N A is expressed in these tissues at levels below the sensitivity of the technique used. Several previous studies that only focused on the LH receptor also found similar results and demonstrated that the LH receptor and its mRNA are localized in theca and interstitial cells and that the expression of both is upand down-regulated during the reproductive cycle (Hu et al, 1990; Nakamura et al, 1990; Segaloff et al, 1990; LaPolt et al, 1991; Peng et al, 1991). Before gonadotrophic stimulation, LH receptor mRNA is usually below the detection level in granulosa cells, cumulus cells and oocytes, while low levels of LH receptor m R N A can be found in theca cells. After gonadotrophic stimulation, expression of LH receptor mRNA is increased in thecainterstitial cells, and a more dramatic increase in receptor m R N A takes place in the granulosa cells of large tertiary follicles. In these follicles, the abundance of LH receptor m R N A varies among different subpopulations of granulosa cells, with mural granulosa cells close to the basement membrane exhibiting higher levels than granulosa cells located closer to the antrum, and cumulus cells and the oocyte lacking a detectable hybridization signal.

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The uneven expression of LH receptor mRNA indicates that different ovarian cells have varying hormonal responsiveness. After an ovulatory dose of LH/hCG, LH receptor mRNA levels decrease dramatically, particularly in the granulosa cells of preovulatory follicles, to reach the lowest levels just before ovulation. During the transformation of ovulated follicles into corpora lutea, the expression of LH receptor mRNA is again increased. These mRNA data agree well with the previously reported changes in ovarian LH binding sites during the menstrual cycle. Thus, the LH receptor gene and protein are regulated in a complex manner during the periovulatory period to achieve cell-specific expression. This regulation occurs mainly in the granulosa cells of developing preovulatory follicles, while the expression of the LH receptor and its mRNA in the theca-interstitial cell layer changes to a lesser degree. Further, the previously reported suppression of LH binding sites following a LH/hCG surge seems to result at least in part from a decline in LH receptor mRNA levels (Segaloff et al, 1990; LaPolt et al, 1991). It remains to be examined whether other mechanisms such as degradation, internalization or modification of the receptor take place during the postovulatory down-regulation of the LH receptor. Following ovulation, the developing corpus luteum contains not only LH receptor mRNA but also active LH binding sites. Binding studies of human ovaries revealed a linear binding plot indicative of a single set of LH/hCG receptors in corpora lutea and no binding sites on corpora albicantia (Yeko et al, 1989). Total (occupied plus unoccupied) receptor concentrations reached maximum levels in the mid-luteal phase and declined thereafter. The authors concluded that in normal corpora lutea total and unoccupied LH/hCG receptor levels parallel progesterone secretion, and that loss of LH/hCG receptors is probably related to luteolysis (Yeko et al, 1989). Despite the declining function of the ovary during the perimenopause and postmenopause, there is clinical evidence for persisting, gonadotrophindependent postmenopausal ovarian steroidogenesis. Administration of a GnRH antagonist to postmenopausal women causes a rapid decline in circulating concentrations of gonadotrophins (Andreyko et al, 1992; Rabinovici et al, 1992a). This decline is accompanied by a parallel decline in testosterone levels (Andreyko et al, 1992; Rabinovici et al, 1992a). A recent study also demonstrated that postmenopausal ovaries contain specific binding sites for FSH and LH in the cells of the cortical stroma (Nakano et al, 1989). These cells are steroidogenically active and exhibit diffuse cytoplasmic immunohistochemical staining for oestrogen and 3[~-hydroxysteroid dehydrogenase activity (Nakano et al, 1989). Thus, it seems that gonadotrophin receptors are present in the ovary from fetal development until senescence, but the localization of these receptors and the effects that they stimulate vary and change throughout development. OVARIAN ACTIONS

The gonadotrophic hormones, FSH and LH/hCG can affect ovarian differentiation and mitogenesis in several ways (see Table 1), and their

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actions depend on the state of ovarian development. This is especially true for the different effects caused by the gonadotrophic hormones during folliculogenesis. As proposed many years ago (Hisaw, 1947), many studies have confirmed that FSH and LH are the primary protein hormones involved in folliculogenesis. FSH and LH play a primary role in the promotion of growth and proliferation of the follicular cells and enhance the capacity of granulosa and theca cells in the recruited and selected follicles to synthesize and secrete steroids. Once a follicle develops adequately, LH and maybe also FSH trigger ovulation and are later essential for the support of corpus luteum function until implantation occurs. The central role of FSH and LH is best illustrated in women with hypothalamic-pituitary hypogonadism. In these women absent pituitary gonadotrophin secretion results in anovulation and ovarian quiescence. Exogenous administration of gonadotrophins to these women leads to the resumption of ovarian folliculogenesis.

Folliculogenesis FSH plays a major role in folliculogenesis by stimulating proliferative processes and the synthesis of steroidogenic enzymes and various intragonadal factors. Given that FSH receptors are found in the ovary only on granulosa cells, it can be presumed that FSH primarily affects granutosa cell differentiation and mitogenesis. However, secondary changes in other cells of the follicle may be caused through mediation by intragonadal factors that are synthesized and secreted by the granulosa ceils in response to FSH stimulation (see below). During the follicular phase of the menstrual cycle, a cascade of events takes place which ensures that the proper number of follicles is ready for ovulation. According to Goodman and Hodgen (1983), folliculogenesis includes in primates the following events that ultimately lead to ovulation of the dominant follicle: during recruitment, a gonadotrophin-dependent

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event, a group of follicles responds to FSH stimulation and grow in response to this stimulus; during selection, follicles destined to ovulate are picked out, while all other previously recruited follicles undergo the fate of most ovarian follicles, i.e. atresia; and finally, dominance is the process that allows the follicle that will ovulate to continue to grow despite an environment that is suppressive to the growth of other follicles. Although all these steps are driven by pituitary gonadotrophins, the initial step that makes some of the 'dormant' primordial ovarian follicles responsive to gonadotrophins seems to be independent of pituitary control and probably depends on intraovarian regulation. Following normal prenatal development, the primate ovary harbours at birth probably up to two million primordial follicles (Rabinovici and Jaffe, 1990). A primordial follicle consists of an oocyte, arrested in the diplotene stage of the meiotic prophase, surrounded by the zona pellucida and a single layer of granulosa cells (Peters et al, 1975). Groups of primordial follicles, called cohorts, start their growth cycle throughout life, even during periods when ovarian function does not seem to be regulated by gonadotrophins, i.e. during pregnancy or periods of anovulation (Peters et al, 1975). However, it is not clear why one primordial follicle can resume development and proceed through folliculogenesis while a neighbouring follicle may remain in the resting state for years or decades more. It may be assumedthat some intragonadal regulators govern this characteristic pattern of initiation of folliculogenesis. However, these putative intraovarian factors are not able to maintain the development of the follicle after primary stimulation of its initial growth; if left without FSH stimulation, growing primordial follicles will undergo atresia. Atresia is actually the fate of most growing primordial follicles. Possibly the first role of gonadotrophins, and especially of FSH, in folliculogenesis is to rescue some primordial follicles from atresia by promoting their further growth and differentiation. The primary effect of FSH during folliculogenesis is the promotion of growth and proliferation of primordial follicles. The end of the luteal phase is characterized by a decrease in steroidogenesis which, due to the ovarianpituitary feedback mechanisms, leads to an increase in circulating FSH concentrations. The timely coincidence of this rise of FSH with the initiation of growth of some primordial follicles leads to the rescue of a cohort of primordial follicles from atresia and their recruitment (Goodman and Hodgen, 1983; Mais et al, 1986). At this time FSH induces mitogenesis and proliferation of follicular granulosa cells (Richards et al, 1976). Since primordial follicles start their primary growth at different times of the cycle and at different periods of life, only those follicles that are at an appropriate stage when FSH levels rise at the end of one cycle and the beginning of the next cycle escape atresia (Peters et al, 1975). The second important effect of FSH is the stimulation of the follicular aromatase enzyme. This enzyme promotes the conversion of androgens to oestrogens. Endocrinologically, recruitment is characterized by symmetric oestradiol secretion from both ovaries. This symmetry indicates that small follicles develop in both ovaries, However, due to negative feedback, the

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increasing circulating oestradiol levels lead to a decrease in pituitary FSH secretion and therefore to a decrease in the occupancy of follicular FSH receptors. A developing follicle requires an oestrogenic microenvironment to proliferate (Richards et al, 1976) and in turn needs FSH stimulation to aromatize androgens and secrete oestrogens (Hillier et al, 1988; Richards and Hedin, 1988). In primates, only the one follicle that contains an adequate number of FSH receptors will continue to proliferate and differentiate during this period. Thus, this phase of folliculogenesis is characterized by the balance between increasing FSH-dependent oestradiol secretion that causes induction of further FSH receptors and decreasing pituitary FSH secretion. Finally, during selection, the follicle that survived this period becomes dominant and becomes the major source of oestradiol. At this time oestradiol levels in ovarian venous affluents are higher at the side of the dominant follicle. In primates, the period of recruitment begins at the end of the luteal phase of the prior cycle and concludes approximately on day 5 of the current cycle (Irianni and Hodgen, 1992). The selection of the dominant follicle takes place on days 5 to 7 of the 28-day cycle. FSH is not the only gonadotrophin involved in early folliculogenesis. The undifferentiated theca cells are not steroidogenically active but they contain LH receptor mRNA and LH binding sites (Erickson et al, 1985; Segaloff et al, 1990; Camp et al, 1991). During folliculogenesis, the theca cells become responsive to LH, and LH stimulates in these cells the activity of enzymes necessary for steroidogenesis: the side-chain cleavage enzyme, 17o~hydroxylase and 17,20-desmolase (Erickson et al, 1985). Immunohistochemical examination of the human ovary throughout all stages of the menstrual cycle shows that aromatase is present in granulosa cells, whereas the 17ot-hydroxylase enzyme is confined to theca cells (Sasano et al, 1989). Further, the side-chain cleavage enzyme is present in theca cells during the follicular phase and in both granulosa and theca lutein cells in the luteal phase (Sasano et al, 1989). The LH-stimulated side-chain cleavage reaction that converts cholesterol to pregnenolone is a rateqimiting step of steroidogenesis. The theca cell enzymes are able to promote the synthesis of progestins and androgens; the aromatase activity of theca cells is very low--about several hundred times lower than that of granulosa cells (Hillier et al, 1981). Thus, theca cells are a good source of de n o v o synthesis of androgens but they are a very poor source of oestrogens. In contrast, the adjacent granulosa cells are incapable of synthesizing steroids de n o v o during early folliculogenesis. The FSH-stimulated acquisition of LH receptors is a characteristic change in the state of differentiation of the granulosa cells during follicular development. Granulosa cells acquire LH/hCG receptors during the transition from small antral follicles to larger preovulatory follicles. The induction of these receptors is FSH and oestradiol dependent (Richards et al, 1976; Nimrod et al, 1977; Erickson et al, 1979). Like most other FSH-stimulated actions, the expression of LH receptors on ovarian granulosa cells is induced by cAMP. This is supported by findings that addition of cAMP or its analogues to cultured granulosa cells increases their LH receptor levels (Knecht et al, 1981; Nimrod, 1981; Knecht and Catt, 1982; Erickson et al, 1982). In the rat

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ovary, oestrogens also induce LH receptors and act synergistically with cAMP (Richards, 1975; Hillier et al, 1989; Segaloff et al, 1990). Both FSH and oestradiol also induce an increase in LH/CG responsive adenylate cyclase activity (Jonassen et al, 1982; Richards and Kersey, 1989), thus furnishing the granulosa cells with the receptor and its second-messenger cascade. The effects of oestradiol in the rat ovary are mediated by specific receptors (Richards, 1975; Hillier et al, 1989). However, it should be noted, that in contrast to the rat ovary, the direct role of oestradiol on folliculogenesis in the human ovary has remained questionable. Primate granulosa cells do not seem to contain oestradiot receptors (Hild-Petito et al, 1988) and administration of the oestradiol agonist stilboestrol (diethylstilbestrol USP) to juvenile monkeys did not affect follicular growth (Koering, 1987). The two-cell theory The question surrounding the relative roles of FSH and LH during the ovulatory process has received wide attention since the original proposal of the 'two-cell theory' and has remained the subject of some controversy (Fevold, 1941; Greep et al, 1942; Falck, 1959; Lohstroh and Johnson, 1966). This theory holds that FSH and LH and their effect on granulosa and theca cells are necessary for ovarian oestrogen synthesis: LH is responsible for theca cell androgen production and FSH stimulates the aromatization of these androgens in granulosa cells. The two-cell theory has been modified since its introduction, but it tries to integrate the findings that gonadotrophin receptors and steroidogenic enzymes are present in different cells during folliculogenesis and that only interaction between the cell layers can enable the follicle to create an oestrogenic microenvironment. Specifically, in the developing follicle FSH receptors are present on granulosa cells and LH receptors are present on theca cells. FSH stimulates in granulosa cells the synthesis of aromatase that is necessary for the conversion of androgens into oestrogens, and the aromatase activity of granulosa cells in vivo is several hundred times that of theca cells (Hillier et al, 1981). It is only after FSH-stimulated induction of LH receptors on granulosa cells (Hillier et al, 1988; Peng et al, 1991) that granulosa cells acquire the ability to produce and secrete progesterone de novo (Sasano et al, 1989). The two-cell theory suggests that during early folliculogenesis C19 steroids (androgens), biosynthesized in LH-stimulated theca cells, traverse the lamina basalis that separates the theca cell layer from the granulosa cell layer. The androgens then enter the granulosa cells and accumulate in antral fluid, and provide the granulosa cell layer with precursors that are transformed by the FSH-stimulated aromatase into oestrogens. Oestrogens, in turn, promote the induction of more FSH receptors on the granulosa cells and enhance the ability of FSH to induce LH receptors on the granulosa cells (Hillier et al, 1988). Some clinical studies in non-human primates (Kenigsberg et al, 1984) and in humans (Couzinet et al, 1988; Hodgen, 1989; Edelstein et al, 1990) seemed to question the validity of the two-cell theory in primate folliculogenesis.

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These studies described that 'pure' FSH preparations were able to stimulate normal folliculogenesis after suppression of pituitary gonadotrophic function by G n R H analogues (Kenigsberg et al, 1984; Edelstein et al, 1990), and that both folliculogenesis and oestradiol production occur in women with gonadotrophin deficiency when treated with 'pure' FSH (Couzinet et al, 1988). However, the results of these studies were probably influenced by at least two factors: firstly, suppression of pituitary gonadotrophin secretion by G n R H analogues is not complete (Loumaye, 1990), and therefore minimal amounts of bioactive LH could be secreted in concentrations sufficient to support theca cell steroidogenesis; and secondly, 'pure' FSH preparations contain albeit small amounts of LH and the administration of pharmacological amounts of 'pure' FSH may again suffice to maintain theca cell steroidogenesis. These reservations are supported by recent comparisons of ovarian reactivity to stimulation with human menopausal gonadotrophin (hMG) and 'pure' FSH in women with hypogonadotrophic hypogonadism. In some, but not all, women with hypogonadotrophic hypogonadism 'pure' FSH preparations caused the development of ovarian follicle-like structures without a concomitant elevation in serum oestradiol levels (Shoham et al, 1991 a). Further, only three out of nine patients treated with 'pure' FSH versus all patients treated by hMG showed an adequate mid-luteal elevation of circulating progesterone concentrations (Shoham et al, 1991a). In a similar study, we were able to observe ovarian follicular development during administration of 'pure' FSH while serum oestradiol levels remained very low. Administration of hCG caused a rapid rise in serum oestradiol levels and luteinization, as judged by elevated serum progesterone levels (J. Rabinovici et al, unpublished data). The recent use of genetically derived FSH preparations that are virtually free of any LH contamination has allowed a re-examination of the two-cell theory (Mannaerts et al, 1991). In rats recombinant FSH was able to induce ovarian follicular growth without increasing plasma oestradiol levels under LH-free conditions (Mannaerts et al, 1991). Histological examination of the ovaries of the placebo-treated animals demonstrated the absence of antral follicles. In contrast, in recombinant FSH-treated animals the ovaries contained large antral follicles. Thus, these studies further confirmed the growth-promoting effect of FSH on developing follicles. Supplementation of FSH with small doses of hCG led to a further rise in ovarian weight, aromatase activity and plasma oestradiol levels (Mannaerts et al, 1991). The FSH threshold

According to current concepts of preovulatory folliculogenesis in primate ovaries, each growing follicle has a 'threshold' requirement for stimulation by FSH which must be met if it is to enter the oestrogen-secretory phase of preovulatory development. This concept is partly based on a clinical study that showed that by stepwise increases of the doses of exogenously administered gonadotrophins, one was able to arrive at a dose--the threshold-that caused the development of only one ovarian follicle (Brown, 1978). Theoretically, this threshold is determined by the number of FSH receptors

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present on the follicular granulosa cells. During the natural cycle, the dominant follicle is the one with most FSH receptors that therefore succeeds in maintaining an oestrogenic microenvironment. The concept of a FSH threshold has resulted in a re-evaluation of treatment with exogenous gonadotrophins, especially in women with polycystic ovary syndrome (PCOS) (Meldrum, 1991; Shoham et al, 1991b). During ovulation induction with exogenously administered gonadotrophins the features of normal cycles cannot be exactly reproduced. Selection and dominance of one follicle does not occur spontaneously and, depending on dosage and the sensitivity of the ovarian follicles, multifollicular development and ovulation can take place. Especially in women with PCOS, the main problem during treatment with hMG/FSH is the high rate of multifollicular development, which leads to high rates of multiple pregnancies and of ovarian hyperstimulation, a condition that carries a serious risk and can even be fatal (Wang and Gemzell, 1980). Several recent studies indicated that by using a low dose protocol and stepwise increases of 'pure' FSH it was possible to reduce the rate of multifollicular development and ovarian hyperstimulation (Meldrum, 1991; Shoham et al, 1991b). Currently conducted studies will determine whether this approach will also result in an adequate pregnancy rate.

The periovulatory period Following the formation of the dominant follicle, its steroidogenic output is crucial for the triggering of the LH surge. An oestradiol pulse of sufficient potency and lasting for an adequately long duration alone is sufficient to trigger off the LH surge. The hypothalamic-pituitaryaxis requires oestradiol priming by approximately 200 pg/ml for at least 36 h to release an LH surge that is sufficient to cause ovulation (Knobil, 1980; Irianni and Hodgen, 1992). Thus, ovulation occurs secondary to an LH surge triggered by increasing levels of oestradiol acting in a positive feedback loop on the pituitary. The LH surge stimulates three major events in the dominant follicle: maturation of the oocyte and resumption of meiosis, luteinization, and extrusion of the oocyte and its cumulus. To date it is not clear whether the accompanying midcycle surge of FSH is of any physiological importance. After fetal gonadal development oocytes remain in the prophase of the first meiotic division. The LH surge signals the oocyte to resume meiosis (Tsafriri et al, 1972). Since oocytes do not seem to contain LH receptors (Lawrence et al, 1980), this process is probably mediated through local factors that are released from the surrounding follicular cells following LH stimulation. During the periovulatory period, LH stimulates the luteinization of the theca and granulosa cells. At this time the follicle contains high levels of progestins and oestrogens and the secretion of these steroids affects the secretion pattern of pituitary gonadotrophins. It is not clear whether this gonadotrophin-mediated increased steroidogenesis of the preovulatory follicle also plays an important intrafollicular role. Several studies suggested a positive association between the concentrations of steroids in follicular

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fluid and successful fertilization (Carson et al, 1982; McNatty et al, 1979; Botero-Ruiz et al, 1984). However, a woman with enzymatic defects of steroidogenesis that blocked the synthesis of normal amounts of androgens and oestrogens was still able to produce oocytes that underwent normal fertilization in vitro (Rabinovici et al, 1989; Pariente et al, 1990). The biochemical changes that occur in the preovulatory follicle following the LH surge include progressive depolymerization of the mucoproteins of follicular fluid with an increase in osmotic pressure, leading to further fluid accumulation and follicular enlargement (Catt and Dufau, 1991). Increased proteolytic activity and collagenase-like enzyme activity lead to degradation of the connective tissue of the follicular wall and to gradual follicular rupture (Espey, 1974; Catt and Dufau, 1991). Further, LH increases the content of plasminogen activator, which also acts as a proteolytic agent in follicular fluid and granulosa cells (Reich et al, 1985). In response to all these changes, the follicular wall is weakened and latent collagenase is released (Yoshimura and Wallach, 1987). The latent collagenase is then activated, and it appears that leukotrienes and prostaglandins, as well as plasmin, may be involved in this process (LeMaire, 1989). The direct or indirect activation of these processes by LH results in a dissociation of the follicular wall, the formation of the stigma and follicular rupture. Ovulation and luteinization are accompanied by histological inflammation-like changes in the ovarian tissue surrounding the follicle and corpus luteum that are probably also gonadotrophin dependent (Espey et al, 1982,1989; Tanaka et al, 1989). Factors such as interleukins (Rivier and Vale, 1989), platelet-activating factor (PAF) (Abisogun et al, 1989; Espey et al, 1989; Rabinovici and Angle, 1991) and plasminogen activator (Reich et al, 1985), that are released by macrophages during inflammatory processes, can cause changes in the differentiation and proliferation of follicular ovarian cells. Specifically, homogenates of periovulatory ovaries obtained from gonadotrophin-primed immature rats contained detectable PAF levels. Four hours after administration of hCG, ovarian PAF levels decreased significantly by up to 50% (Espey et al, 1989). In addition, intraovarian administration of a PAF-specific antagonist, BN52021, inhibited follicular rupture in rats stimulated to ovulate with hCG and decreased the hCG-stimulated increase in ovarian collagenolysis and vascular permeability (Abisogun et al, 1989). Further, PAF at physiological concentrations and in a dose-dependent manner promoted progesterone secretion by luteinized granulosa cells and also induced morphological changes in human luteinized granulosa cells in vitro (Rabinovici and Angle, 1991). Thus, there seems to be an interrelationship between the inflammatory-like processes that accompany ovulation that are directly or indirectly triggered by gonadotrophins and a modulatory effect of these processes on the other ovarian actions of the gonadotrophins.

Corpus luteum The role of gonadotrophins in folliculogenesis does not end with ovulation. Normal corpus luteum development and function depends on adequate

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gonadotrophic support both before and after ovulation. Adequate corpus luteum function, i.e. adequate steroidogenesis for a defined period of time, ensures that the embryo can implant in the endometrium and secrete hCG that will then ensure the prolongation of ovarian corpus luteum function. As discussed previously, corpora lutea contain LH receptor mRNA and active LH receptor binding sites, and loss of LH/hCG receptors is probably related to luteolysis (Yeko et al, 1989). The strong association between LH and luteal steroidogenesis is also demonstrated by the fact that progesterone secretion by the corpus luteum is pulsatile and these pulses correspond to the pulses of pituitary LH release (Filicori et al, 1984). In addition, a strong correlation between inadequate gonadotrophin secretion and inadequate corpus luteum function exists. Suppression of pituitary secretion of LH by a GnRH antagonist during the luteal phase decreases luteal steroidogenesis and shortens significantly the duration of the luteal phase (Mais et al, 1986; Fluker et al, 1991). The importance of FSH for adequate corpus luteum function has remained unclear. Suppression of FSH during the preovulatory period will result in lower preovulatory oestradiol levels, decreased mid-luteal progesterone concentrations and decreased corpus luteum mass (Smith et al, 1985). Whether FSH is necessary during the luteal phase is doubtful. Corpus luteum function and pregnancies can be achieved in women with hypothalamic/pituitary hypogonadism following administration of hMG for ovarian hyperstimulation and only hCG for the induction of ovulation and maintenance. The circulating FSH levels in these women during the luteal phase are very low and corpus luteum function depends mainly on hCG stimulation. Further, FSH receptor mRNA was not detected in the corpora lutea of rat ovaries, although it cannot be ruled out that the mRNA in these tissues is at levels below the sensitivity of the technique used (Camp et al, 1991). GROWTH FACTORS AND FSH/LH

It has become apparent in recent years that regulation of the ovulatory cycle cannot be accomplished solely through the mediation of the two pituitary gonadotrophins, FSH and LH. Within the last decade, discoveries of additional regulatory mechanisms involved in the neuroendocrine control of the events of the ovulatory cycle have provided new insight into the control of this complex biological phenomenon. The cells of the ovarian follicle contain a plethora of receptors for factors that are able to modulate their functions. These factors include endocrine hormones, i.e. substances secreted in distant organs, as well as substances that are synthesized and secreted by adjacent ovarian and follicular cells (paracrine hormones) and those that are secreted by the target cells themselves and thus act as autocrine hormones. The last decade has seen the list of these intragonadal paracrine/autocrine factors growing quickly, and it is beyond the scope of this chapter to try to describe all of them. However, the synthesis, secretion and ovarian cellular receptors of some of these factors are modulated by

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FSH and LH, and some of these factors modulate the ovarian actions of gonadotrophins (Findlay and Risbridger, 1987; Eden et al, 1989; Tonetta and diZerega, 1989; Cara et al, 1990; Rabinovici et al, 1990, 1992b; Risbridger et al, 1990; Woodruff and Mayo, 1990; Ad ashi et al, 1991 ). Thus, we are faced with complex gonadal regulatory processes and many of these processes are not yet completely elucidated. Activins and inhibins serve as good examples for these intraovarian mechanisms. Activin and inhibin are structurally related dimeric ovarian glycoproteins that were initially characterized by their ability to alter FSH secretion from the pituitary (Vale et al, 1988). However, later studies have shown that both activin and inhibin modulate intragonadal processes as well as functions in other organ systems (Dye et al, 1992). In the human ovary, activin-A decreases basal and gonadotrophin-stimutated steroidogenesis and promotes mitogenesis of luteinized follicular cells in vitro (Rabinovici et al, 1990, 1992). Furthermore, immunoreactive activin-A dimer is present in follicular granulosa cells and corpus luteum cells of human ovaries, and its presence in cultured human follicular cells is regulated by cAMP, FSH and LH (Rabinovici et al, 1992). Activin-A also has binding sites on rat granulosa cells, and modulates both steroidogenesis and LH receptor accumulation in this species (Hasegawa et al, 1988; Sugino et al, 1988a,b). Thus, activins affect the action of gonadotrophin on three levels: (a) they are able to modulate pituitary FSH secretion; (b) they modulate the ovarian action of gonadotrophins; and (c) their ovarian synthesis and secretion can be regulated by gonadotrophins. Observations suggesting similar complex control mechanisms have been made for other factors, such as insulin-like growth factor I, interleukin-l, epidermal growth factor/transforming growth factor-a and transforming growth factor-t3. Our increasing knowledge about the effects of these factors on gonadotrophic actions will help us better understand ovarian physiology and provide us with better clinical tools to overcome ovulatory dysfunction in the future. SUMMARY

The basic foundation for normal puberty and adult reproductive function is established during fetal life with the adequate development of the hypothalamus, pituitary and gonads. Further maturation and differentiation of the hypothalamic-pituitary-gonadal axis continues throughout childhood, puberty, adult life and senescence. Pituitary FSH and LH play a central role in the cascade of events in the hypothalamic-pituitary-gonadal axis by mediating between the brain and hypothalamus on one hand and the endorgan, the ovary, on the other. Absent or low pituitary secretion of FSH and LH, as occurs in hypothalamic/pituitary hypogonadism, leads in women to anovulation, amenorrhoea and absent ovarian follicular development. The ability of gonadotrophins to modulate ovarian function depends on their rate of synthesis by the pituitary gonadotrophs, on their circulating concentrations (which vary throughout life and throughout the menstrual cycle), on the relative abundance of the multiple forms of gonadotrophins that have

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varying biological activity, on the presence of their receptors on the different cell types of the ovary, on the intracellular adenylate cyclase enzyme that causes the production of cAMP, and on the extra- and intragonadal factors that are able to modulate the effects of gonadotrophins in the ovary. Recent clinical and basic research with recombinant gonadotrophins, molecular biological studies on the localization, function and regulation of the long sought after gonadotrophin receptors, as well as research on the interaction between gonadotrophins and local intragonadal factors have widened our knowledge about the function and role of FSH and LH in the ovary and have provided new insights into previously unanswered questions of ovarian physiology and pathophysiology and will provide the basis for the design of new treatment strategies to overcome ovulatory gonadotrophin-dependent dysfunction in the future.

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