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The gonadotrophins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were first isolated in more or less pure forms over half a century ago. Purified pituitary gonadotrophins were used by Fevold (1941) and Greep, Van Dyke & Chow (1942) in their classic experiments which demonstrated unequivocally the need for both FSH and LH to stimulate normal ovarian follicular development and oestrogen secretion in hypophysectomized rats. Human pituitary gonadotrophins of equivalent purity have never been widely available for clinical use, but within the next few years pharmaceutical grades of human recombinant FSH and LH are both likely to become so (e.g. Keene, Matzuk, Otani et al. 1989). Taking account of recent advances in our understanding of gonadotrophin action at the cellular level, it should be possible to use these pure human gonadotrophins to devise improved strategies for stimulating ovarian function in infertile women.
Development-dependent follicular responsiveness to gonadotrophins
FSH receptors are located
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The classic endocrine function of ovarian inhibin is negative feedback control of pituitary FSH secretion (see de Jong, 1988 for a review). There is, however, a long-harboured suspicion that inhibin and related proteins such as activin could have local regulatory roles at or near their sites of formation in the gonads (e.g. Ying, 1988). Here, I survey recent developments in inhibin and activin research which support this likelihood, proposing paracrine functions for both proteins in the regulation of preovulatory follicular development and oestrogen synthesis in human ovaries.
Inhibin, activin and related regulatory proteins
Mature inhibin is a 32 kDa glycoprotein which has been isolated from ovarian follicular fluid as two distinct forms composed of a common α-subunit and one of two β-subunits, βA and βB (Ling, Ying, Ueno et al. 1985; Miyamoto, Hasegawa, Fukuda et al. 1985; Rivier, Spiess, McClintock et al. 1985; Robertson, Foulds, Leversha et al
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ABSTRACT
Catechol oestrogens are formed in the ovary but it is not known if they have a local function. Working with primary granulosa cell cultures prepared from immature rat ovaries, we found that the presence of 2-hydroxyoestradiol in the culture medium (48 h incubation) dramatically enhanced the steroidogenic response (progesterone production) to human FSH (100 ng/ml). The effect of 2-hydroxyoestradiol was dose-dependent and maximal (approximately 40 times the response to FSH alone) at 3.0 μM. The stimulatory action of 1.0 μM 2-hydroxyoestradiol was >10 times more than that of 1.0 μM oestradiol but only half that of 1.0 μM testosterone; other catechol oestrogens (2-hydroxyoestrone, 4-hydroxyoestradiol, 2-methoxyoestradiol and 2-methoxyoestrone) were not stimulatory. The stimulatory actions of 2-hydroxyoestradiol and testosterone were partially additive and each was antagonized in the same way by the presence of a specific antiandrogen (SCH16423). These observations suggest a role for intrafollicular catechol oestradiol in modulating FSH-stimulated granulosa cell steroidogenesis; its mechanism of action may be similar to that of testosterone.
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ABSTRACT
Granulosa-lutein cells from human preovulatory ovarian follicles were cultured for up to 12 days to determine their capacity for production of inhibin in vitro. Using a highly sensitive sheep pituitary cell bioassay we observed time-related changes in basal inhibin production, maximal during the first 4 days of culture (48 ± 15 units/million cells every 2 days, means ± S.E.M.; n = 5 patients) falling to values five times lower by day 12. After 4-6 days of culture in the presence of human LH (hLH) inhibin production was enhanced in proportion to the hLH dose (maximum five fold at 10 ng/ml); hFSH over the same dose-range had no effect. Progesterone production in response to hLH followed a similar pattern to that of inhibin and was also unresponsive to hFSH. In the absence of exogenous aromatase substrate, basal and gonadotrophin-stimulated oestradiol production was negligible after the first 4 days. Addition of testosterone (1 μmol/l) to the culture medium increased oestrogen formation several hundredfold with no effect on progesterone production. Inhibin production was also increased by 50-100% in the presence of testosterone.
These results demonstrate that LH and testosterone stimulate the production of inhibin by granulosa-lutein cells in vitro. It is suggested that inhibin production occurs under hormonal control in the corpus luteum as well as in the preovulatory follicle in the human ovary.
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Abstract
In rat ovarian granulosa cells the effects of GnRH are determined by the state of granulosa cell development with mainly inhibitory actions in immature cells and stimulatory actions in differentiated mature cells. These developmentally related effects of GnRH may arise from changes in either one or more of the signal transduction pathways activated by GnRH. The present study therefore measured downstream signalling events associated with the activation of the phospholipase C (PLC) signal transduction pathway in both mature and immature rat ovarian granulosa cells. Results showed that GnRH produced similar total inositol phosphate and intracellular calcium ([Ca2+ ]i) responses in both immature and mature granulosa cells. In contrast to the biphasic GnRH-induced [Ca2+]i response in pituitary gonadotropes, stimulation of the endogenously expressed GnRH receptor in both immature and mature granulosa cells produced a prompt monophasic rise in [Ca2+]i. This calcium transient was abolished by pretreating either cell type with a potent GnRH receptor antagonist or the PLC inhibitor U73122, demonstrating a GnRH receptor-specific activation of PLC. Similarly, pretreatment of cells with the [Ca2+]i antagonists thapsigargin or cyclopiazonic acid abolished the GnRH-induced calcium transient, whereas EGTA and nifedipine, a voltage-operated calcium channel (VOCC) antagonist, had no effect. These results suggest that in either immature or mature granulosa cells GnRH mobilises calcium from thapsigargin/cyclopiazonic acid-sensitive [Ca2+]i stores but does not involve the influx of extracellular calcium through VOCCs. We conclude that GnRH-induced stimulation of the PLC signal transduction pathway is independent of the stage of granulosa cell maturity and that alternative mechanisms account for the opposite effects of GnRH on gonadotrophin-induced steroidogenesis in mature and immature rat granulosa cells in vitro.
Journal of Endocrinology (1996) 149, 449–456
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Abstract
Inhibin is a putative gonadal paracrine factor produced by FSH-stimulated granulosa cells. To assess the paracrine function of inhibin further, preantral follicles (approx. 240 μm) with attached thecal cells were isolated mechanically from immature rat ovaries and cultured individually in vitro for 5 days in medium containing homologous serum and FSH. After 5 days, follicles had grown to preovulatory size (approx. 470 μm) with a commensurate increase in oestradiol secretion but not progesterone. Immunoneutralization of endogenous inhibin resulted in a significant decrease in oestradiol secretion and an increase in progesterone accumulation. When antiserum-treated follicles were supplemented with exogenous inhibin, oestradiol secretion was restored and progesterone accumulation was reduced. Aromatase substrate (androstenedione) levels were too low to measure, regardless of antiserum treatment. However, follicles treated with inhibin antiserum in the presence of exogenous androstenedione also exhibited oestradiol levels similar to untreated controls while progesterone accumulation remained elevated. We interpret these data as evidence that inhibin secreted by FSH-stimulated granulosa cells exerts a physiologically significant paracrine function in the follicle wall. Based on previous observations that inhibin stimulates androgen synthesis by isolated thecal/interstitial cells, it is proposed that granulosa-derived inhibin promotes thecal androgen synthesis and hence oestrogen synthesis during preovulatory follicle development. The antiserum-induced increase in progesterone accumulation is most probably explained by reduced metabolism of C21 steroid substrate to androgen in thecal/interstitial cells deprived of inhibin. It is concluded that inhibin is a physiological modulator of follicular steroidogenesis which exerts its effect at the level of thecal cell androgen synthesis.
Journal of Endocrinology (1994) 140, 437–443
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Abstract
During the luteal phase of the primate ovulatory cycle the predominant inhibin/activin subunit mRNAs produced by the corpus luteum and antral follicles are those for the α- and βB-subunits respectively. The control of expression of these mRNAs and the resultant nature of the endocrine and paracrine signals which they may potentially generate has yet to be elucidated. Inhibin/activin subunit mRNAs may have a role in both the paracrine regulation of follicular and luteal function and modulation of FSH secretion. The aim of this study was to investigate the expression of inhibin/activin subunit mRNAs following luteal regression induced by either withdrawal of LH support (GnRH antagonist treatment), or by a direct inhibitory action (prostaglandin administration). Marmoset monkeys with regular ovulatory cycles were treated on day 8 and 9 of the luteal phase with either GnRH antagonist, prostaglandin or vehicle (n=3 per group). Ovaries were studied 48 h after onset of treatment (on day 10 of the luteal phase) by hybridizing frozen tissue sections with radiolabelled riboprobes specific to the inhibin/activin α-, βA- and βB-subunit mRNAs. After autoradiographic exposure, grain concentrations were quantified by image analysis. In corpora lutea from control marmosets there was high expression of α-mRNA with only marginal expression of βB-mRNA. Corpora lutea in animals treated with GnRH antagonist or prostaglandin had markedly reduced expression of α-mRNA while βB-mRNA was unchanged. In controls, all healthy antral follicles exhibited a high level of expression of βB-mRNA in the granulosa cells and low expression of α-mRNA in theca cells. This was unaffected by either treatment. βA-mRNA was found at a low level in granulosa cells but was not evident at a significant level in the corpora lutea of any of the groups. These results demonstrate (1) the marmoset corpus luteum is a source of high expression of α-subunit mRNA, (2) this α-mRNA is dependent upon LH support, (3) the process of luteal regression takes place without alteration of βB-mRNA. Antral follicle α- and βB-mRNAs are independent of the process of luteal regression or gonadotrophic withdrawal during the period of the luteal-follicular phase transition.
Journal of Endocrinology (1995) 144, 201–208
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Peritoneal surface epithelial (PSE) cells participate in adhesion formation following inflammatory injury yet adjacent ovarian SE (OSE) cells regenerate without scarification after ovulation. OSE cells show inflammation-associated expression of 11β hydroxysteroid dehydrogenase type 1 (11βHSD1) enzyme, enabling intracrine generation of anti-inflammatory cortisol to minimise tissue damage. We asked if human PSE cells show an 11βHSD1 response to pro-/anti-inflammatory stimulation and if so, how the 11-oxoreductase activity generated compares with OSE. PSE collected from premenopausal women undergoing surgery for benign gynaecological conditions were used to establish primary PSE cell cultures that were treated for 48 h with interleukin-1α (IL-1α) with/without anti-inflammatory steroid (cortisol or progesterone). mRNA levels corresponding to the genes of interest (11βHSD1, 11βHSD2, cyclooxygenase-2, COX-2) were measured by quantitative RT-PCR. IL-1α (0.5 ng/ml) stimulated 11βHSD1 and COX-2 mRNA levels in PSE cells but 11βHSD2 was unaffected. Cortisol (1 μM), not progesterone (1 μM), increased 11βHSD1 mRNA and synergistically enhanced IL-1α action. Cortisol suppressed IL-1α-stimulated COX-2 more effectively than progesterone. PSE cells had a significantly lower basal 11-oxoreductase enzyme activity than OSE cells; IL-1α did not significantly increase the 11-oxoreductase activity in PSE cells but did so in OSE cells. We conclude that PSE cells respond to IL-1α and anti-inflammatory steroids in qualitatively similar ways as OSE. However, the enzymatic activity of 11βHSD1 is lower in PSE and less responsive to IL-1α. This could help explain why peritoneal healing often leads to adhesion formation, whereas postovulatory ovarian healing is scar-free.
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ABSTRACT
The bioactivity of a synthetic peptide fragment which mimics the N-terminal sequence of the 134-amino-acid porcine Inhibin α-subunit (pl- α1-26-Gly27Tyr28-OH) was tested and compared with the bioactivity of GnRH in rat granulosa cell cultures. Granulosa cells from immature female rat ovaries were cultured with hFSH and testosterone to stimulate the production of cyclic AMP, progesterone and oestradiol. Addition of pl- α1-26-Gly27Tyr28-OH to the culture medium caused a dose-dependent suppression of all three parameters (ID50 700-1,000 nmol/l). GnRH caused similar but higher-potency inhibition (ID50 2-4 nmol/l). Suppression of granulosa cell function by both peptides was fully reversible by a synthetic GnRH antagonist. Moreover, specific binding of the porcine inhibin fragment to ovarian GnRH receptors was demonstrated by radioreceptor assay. This is evidence that the porcine inhibin α-subunit fragment suppresses FSH-induced rat granulosa cell function via a mechanism of action similar to that of GnRH.
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ABSTRACT
A primary monolayer cell culture system was developed to investigate human corpus luteum (CL) function in vitro. Steroidogenic cells were isolated by collagenase dispersal and Percoll density-gradient fractionation from CLs enucleated at progressive stages of the luteal phase (tubal surgery patients). 'Pure' granulosa-lutein cells were aspirated from ovulatory follicles at mid-cycle (in-vitro fertilization patients). The steroidogenic capacity (progesterone/20α-dihydroprogesterone biosynthesis and aromatase activity) of isolated luteal cells was assessed in relation to CL development. Basal luteal cell steroidogenesis was maximal at around the expected time of ovulation and declined with CL age during the luteal phase. Conversely, human chorionic gonadotrophin (hCG)-responsive steroidogenesis was initially undetectable but developed as the luteal phase progressed. These results show that luteal cell steroidogenesis becomes increasingly dependent upon gonadotrophic support with CL age. This is evidence that functional luteolysis in human ovaries (1) is pre-programmed to occur at the cellular level, (2) is initiated automatically at the time of ovulation and (3) is reversed at the time of CL 'rescue' in early pregnancy by the direct action of trophoblastic hCG on steroidogenic luteal cells. The culture system described should be of value in further defining the control of human CL form and function at the cellular level.
Journal of Endocrinology (1989) 122, 303–311
