Inhibitory actions of a gonadotropin-releasing hormone agonist on ovarian follicle-stimulating hormone receptors and adenylate cyclase in vivo

The regulation of ovarian gonadotropin-sensitive adenylate cyclase and FSH receptors was studied in hypophysectomized diethylstilbestrol-primed rats treated with FSH and/or the potent GnRH agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa). The animals were treated with 7.5 micrograms ovine FSH twice daily for 2 days, either alone or with 10 micrograms GnRHa. FSH-stimulated adenylate cyclase activity was augmented by 2.5- to 3.5-fold in the presence of 5'-guanyl-imidodiphosphate. Adenylate cyclase responses to FSH were almost completely abolished by GnRHa treatment in ovarian homogenates from control animals and rats treated with FSH. This inhibition was receptor specific, since GnRHa did not block adenylate cyclase stimulation by prostaglandin E2 or isoproterenol. No inhibition of 5'-guanyl-imidodiphosphate- or sodium fluoride-stimulated adenylate cyclase activity was noted after any hormone treatment. When GnRHa treatment was initiated at 12, 24, or 36 h during the 2-day period of FSH treatment, inhibition of both FSH- and LH-stimulated adenylate cyclase was observed in ovaries collected at 48 h. Whereas FSH treatment increased the ovarian FSH receptor concentration by more than 100%, concomitant treatment with GnRHa prevented this increase and reduced FSH receptors to 60% of the control level. Treatment with GnRHa alone caused a 65% decrease in FSH receptor levels below the untreated control values. Histological analysis of hormone-treated ovaries indicated that FSH stimulated follicle growth and antrum formation, but caused little luteinization. GnRHa did not completely prevent the effects of FSH on follicle growth, but did induce degeneration and premature cleavage of ova. GnRHa alone suppressed the diethylstilbestrol-stimulated mitotic activity, slightly increased degenerative changes in granulosa cells, and caused oocyte cleavage and premature antrum formation. These findings demonstrate that GnRHa inhibits FSH-dependent adenylate cyclase by a mechanism involving the loss of binding sites for FSH. It is also evident that only short term exposure to GnRHa is necessary for expression of the inhibitory action of the peptide upon FSH- and LH-stimulated adenylate cyclase.     

Human choriogonadotropin-induced desensitization of granulosa-cell adenylate cyclase to gonadotropins and loss of LH/hCG receptor

Immature female rats that had been primed with pregnant-mare serum gonadotropin (PMSG) were injected intravenously with various doses of human choriogonadotropin (hCG) for the investigation of the relationship between adenylate cyclase activities and the concentrations of LH/hCG receptor in luteinizing granulosa cells. Injection of 1 μg of hCG induced a loss of LH and FSH sensitivities of adenylate cyclase within 6 h and a disappearance of free LH/hCG receptors within 24 h. Basal adenylate cyclase activity has a transient maximum at 6 h after hCG injection. After injection of 100μg of hCG the loss of LH sensitivity of adenylate cyclase and free LH/hCG receptors occurred immediately, but the changes in FSH-stimulated and basal activities followed the same time scale as after injection of 1 μg of hCG. When hCG was omitted from the injections the response of the animals to the endogenous gonadotropin surge varied. A complete desensitization of adenylate cyclase to LH and FSH stimulation and a 65% loss of free LH/hCG receptors were found at 24 h if the follicles were ovulated.These results suggest that occupation of a limited number of LH/hCG receptors in granulosa cells induces adenylate cyclase refractory to further stimulation by gonadotropins. The transient elevation of basal adenylate cyclase activity and its desensitization to further stimulation by gonadotropins may have a role in physiological processes leading to ovulation and luteinization.     

Inhibition of FSH action on granulosa cells by low molecular weight components of follicular fluid

Immature female rats were injected with a single dose (10 IU) of pregnant mare serum gonadotropin to induce growth and maturation of ovarian follicles. Using such ovaries as a model, we tested the effects of low molecular weight subfractions of charcoal-absorbed bovine follicular fluid (FF-c) on (a) radioiodinated human FSH (125I-hFSH) binding to ovarian homogenates, (b) ovine FSH-stimulated adenylate cyclase activity in granulosa cell homogenates and (c) cAMP production by intact granulosa cells. The follicular fluid was fractionated by ultrafiltration through membranes of differing pore-sized into molecular weight components of 1000-5000 (passing Amicon H1P-5 hollow fibers but retained by Amicon UM-2 membrane) and 500-1000 (passing Amicon UM-2 membrane but retained by Amicon Um-05 membrane). These low molecular weight fractions inhibited 125I-hFSH binding to ovarian receptors, FSH-stimulated cAMP production by rat granulosa cells and FSH-stimulated, as well as fluoride-ion-stimulated adenylate cyclase activity in granulosa cell homogenates. Inhibition of FSH-stimulated adenylate cyclase activity by the FF subfractions was non-competitive as determined by double reciprocal plot analysis. Our results suggest that modulation of FSH effects on granulosa cells may be mediated by low molecular weight constituents of follicular fluid.     

Increase in catecholamine-stimulated cyclic AMP and progesterone synthesis in rat granulosa cells during culture

Catecholamines stimulated cyclic AMP and progesterone synthesis in rat corpora lutea but not in the pre-ovulatory follicles. We collected granulosa cells from follicles of immature rats treated with pregnant mare's serum gonadotropin (PMSG) and cultured them either in monolayer or in suspension. Freshly collected granulosa cells responded to isoproterenol and epinephrine with 2-fold increases in cyclic AMP accumulation and progesterone synthesis. However, granulosa cells cultured in monolayer for 2 days responded to isoproterenol and epinepherine with a 90-fold and a 6-fold increase in cyclic AMP accumulation and progesterone synthesis, respectively. The accumulation of cyclic AMP in response to Catecholamines gradually increased in cells cultured in suspension, from 2-fold over control after 5 h, to 8-fold after 24 h. Granulosa cells isolated from hypophysectomized and diethylstilbestrol (Hx-DES) treated immature rats (containing pre-antral follicles) also showed an increase in cyclic AMP accumulation in response to Catecholamines during culture. Because these cells are devoid of an LH-responsive adenylate cyclase system, we conclude that luteinization of granulosa cells in culture is not necessarily the process responsible for the increased response to Catecholamines.During culture, the number of β-adrenergic receptors in granulosa cells rose from 6020 ± 400 per 10⁶ cells shortly after isolation to 26400 ± 1800 after 2 days in culture. This increase in receptor density during culture may be responsible for the change in the responsiveness to Catecholamines, although other factors, such as changes in coupling efficiency between the hormone-receptor complex and the adenylate cyclase moiety and/or supersensitivity to Catecholamines, should also be considered.     

Regulation of luteinizing hormone receptor messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells

The induction of LH receptors in granulosa cells is prerequisite for ovarian follicles to ovulate and form corpora lutea. Earlier studies have demonstrated the modulatory role of gonadotropins, growth factors, and GnRH on ovarian LH receptor content. We have now analyzed the influences of gonadotropins (FSH, LH, and PRL), several growth factors, and GnRH on LH receptor mRNA levels in cultured granulosa cells. Cells were obtained from immature estrogen-treated rats and cultured in medium containing FSH with or without growth factors or GnRH for 48 h. Some cells were also treated with FSH for 48 h, followed by treatment with FSH, LH, or PRL for another 2 days. Cellular total RNA was extracted, and blot hybridization with 32P-labeled LH receptor cRNA or 28S ribosomal RNA cDNA probes was performed. Treatment of granulosa cells with FSH increased the levels of five species of LH receptor mRNAs in a dose- and time-dependent manner. In FSH-primed cells, LH receptor mRNA levels were maintained by FSH, LH, and PRL. In contrast, treatment of cells with basic fibroblast growth factor or epidermal growth factor suppressed FSH induction of LH receptor mRNA in a dose-dependent manner, whereas treatment with insulin-like growth factor-I had no effect. In addition, GnRH suppressed FSH-stimulated LH receptor mRNA levels in a dose-dependent manner; the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting mediation by specific GnRH-binding sites. These studies demonstrated that the observed stimulatory effects of gonadotropins (FSH, LH, and PRL) and the inhibitory effects of growth factors (epidermal growth factor and basic fibroblast growth factor) and GnRH on LH receptor content are correlated to their regulation of LH receptor mRNA levels. The granulosa cell culture system should provide a useful model for studying LH receptor gene regulation.     

Effects of a gonadotropin-releasing hormone agonist on progesterone formation in cultured human granulosa cells

Human granulosa cells from follicles of natural cycles (13 women) in mid- or late follicular phase were cultured in modified Medium-199. Human luteinizing hormone (100 micrograms/l), follicle-stimulating hormone (100 micrograms/l) and gonadotropin-releasing hormone agonist (GnRHa) (10(-12) to 10(-6) mol/l) alone or in combination were added to the culture medium. Medium content of progesterone was analysed. In granulosa cells obtained in the mid-follicular phase, the basal progesterone formation averaged 0.5 pg.cell-1.(48 h)-1. FSH caused a 3-fold stimulation. GnRHa (10(-6) mol/l) had a variable influence on the basal progesterone formation, whereas it consistently inhibited (40-50%) the FSH response. In granulosa cells from late follicular phase basal progesterone formation averaged 5 pg.cell-1.(48 h)-1 and was stimulated 3- to 6-fold by LH. GnRHa (10(-6) mol/l) stimulated the basal progesterone formation and caused a tended to potentiate the LH response on progesterone formation. It is concluded that GnRHa at relatively high concentrations exerts direct effects on gonadotropin-stimulated progesterone formation of human granulosa cells and that these effects are different (inhibitory or stimulatory) dependent on the degree of follicular maturation, and/or type of gonadotropin used.     

GnRH receptors in cultured rat granulosa cells: mediation of the inhibitory and stimulatory actions of GnRH

The regulatory actions of FSH and the GnRH agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa) upon ovarian GnRH receptors were studied in granulosa cells obtained from ovaries of hypophysectomized diethylstilbestrol-treated rats. When granulosa cells were cultured for 48 h in the presence of FSH (5-250 ng/ml) the binding of 125I-GnRHa to granulosa cell receptors was increased in a dose-dependent manner, to a maximum of 3-4 fold above the control value. Addition of FSH (100 ng) also caused a dose-dependent increase of more than 100-fold in the accumulation of cAMP and progesterone in the culture medium. In freshly prepared cells, Scatchard analysis of GnRHa binding data revealed an equilibrium constant (Ka) of 1.1 x 10(10) M-1 and GnRH receptor concentration fo 401 fmoles/mg protein. Granulosa-cell GnRH receptors decreased during culture without FSH, but were maintained in the presence of 100 ng FSH at 580 femoles/mg protein, with Ka of 0.8 x 10(10) M-1. This action of FSH on GnRH receptors was significantly reduced by 10(-8) M GnRHa. Also, GnRHa concentrations of 10(-10) and 10(-8) M caused inhibition of FSH-induced cAMP and progesterone accumulation. In cells cultured with GnRHa alone, there was a slight enhancement of GnRH receptors by GnRHa concentrations up to 10(-8) M, and a decrease below control levels with higher amounts. Also, GnRHa concentrations from 10(-8) to 10(-5) M caused a 3-4-fold increase in cAMP accumulation in the absence of FSH. These results demonstrate that FSH maintains GnRH receptors in cultured granulosa cells, and that GnRHa attenuates this effect, as well as the other actions of FSH on granulosa cell maturation. It is also evident that GnRHa itself can slightly stimulate cAMP production and partially maintain GnRH receptors, but at high concentrations causes loss of the homologous receptor sites. These findings also emphasize the ability of GnRH agonists to exert both positive and negative direct effects on rat granulosa cell function.     

Titrating luteinizing hormone surge requirements for ovulatory changes in primate follicles. II. Progesterone receptor expression in luteinizing granulosa cells

The events in granulosa cells that are initiated by the midcycle LH surge during luteinization of the primate follicle are poorly defined. This study was designed 1) to determine whether an ovulatory dose of hCG can induce progesterone receptors (PR) in macaque granulosa cells, and if so, 2) to begin titrating gonadotropin requirements for PR expression and progesterone production by luteinizing granulosa cells. Rhesus monkeys were treated with human FSH and LH for up to 9 days to stimulate the growth of multiple follicles. The next day, animals (n = 4-5/group) received: 1) no ovulatory stimulus; 2) 1000 IU hCG, im; 3) one injection of 100 micrograms GnRH, sc (GnRH-1); 4) three injections of GnRH (GnRH-3) at 3-h intervals (0800, 1100, and 1400 h); or 5) two injections of 50 micrograms GnRH agonist (GnRHa), sc, 8 h apart (0800 and 1700 h). Granulosa cells obtained by follicle aspiration 27 h after the hCG or initial GnRH/GnRHa injection or on days 8 or 10 from animals receiving no ovulatory stimulus were processed for indirect immunocytochemistry using a monoclonal antibody to human PR (JZB39). Specific staining for PR, determined by comparing cells incubated with PR antibody vs. a nonspecific antibody, was undetectable in granulosa cells from monkeys without an ovulatory stimulus. In contrast, the majority (64 +/- 5%) of cells from hCG-treated animals stained intensely for PR. In the GnRH/GnRHa groups, granulosa cells from only one animal (i.e. one GnRH-3 monkey) showed positive staining for PR. During 24-h culture in Ham's F-10 medium containing 10% monkey serum, basal progesterone production by cells from the hCG-treated group (2163 nmol/L.8 x 10(4) cells) was higher than that by cells from the no ovulatory stimulus/GnRH-1/GnRH-3/GnRHa groups (60, 111, 194, and 332 nmol/L, respectively). However, granulosa cells from the hCG-treated group were less responsive to hCG in vitro in terms of enhanced progesterone production (2 times control levels) than cells from the other four groups (up to 30 times control levels). This study provides direct evidence that an ovulatory dose of hCG induces PR expression in granulosa cells of luteinizing follicles during stimulated cycles in rhesus monkeys. However, repeated injections of GnRH/GnRHa that produced surge levels (greater than 100 ng/mL) of endogenous LH for up to 14 h failed to induce PR expression or progesterone production by granulosa cells. Thus, an extended LH surge more typical of that in the normal menstrual cycle (48-50 h) may be necessary for PR expression and luteinization of granulosa cells in primate follicles.     

Adenosine and its analogue (-)-N6-R-phenyl-isopropyladenosine modulate anterior pituitary adenylate cyclase activity and prolactin secretion in the rat

The effect of adenosine and its analogue (-)-N6-R-phenylisopropyladenosine (PIA) on both anterior pituitary adenylate cyclase activity and prolactin secretion was examined in the rat. Adenosine inhibited basal adenylate cyclase activity in a dose-dependent manner and also reduced the stimulation of the enzyme by vasoactive intestinal peptide (VIP). Likewise, in primary cultures of anterior pituitary cells, adenosine decreased prolactin secretion in both basal and VIP-stimulated conditions. In perifusion experiments, adenosine also inhibited prolactin release in both basal and TRH-stimulated conditions. PIA produced a biphasic pattern of response of basal adenylate cyclase activity, being inhibitory at low and stimulatory at high concentrations. In VIP-stimulated conditions, low concentrations of PIA inhibited both adenylate cyclase activity and prolactin release from primary cultures of pituitary cells, while no additive stimulatory effect was seen at high concentrations. Similarly, low concentrations of PIA reduced both basal and TRH-stimulated prolactin release from perifused pituitaries, while increasing PIA concentrations restored prolactin release. These data show that adenosine affects basal and stimulated prolactin secretion from anterior pituitary cells. Adenosine receptors seem to be coupled to the adenylate cyclase system in the anterior pituitary gland, suggesting a possible relationship between the effect of adenosine on adenylate cyclase activity and prolactin secretion.     

Interactions of ovarian steroids with pituitary adenylate cyclase-activating polypeptide and GnRH in anterior pituitary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) releases LH and FSH from anterior pituitary cells. Although this effect is relatively weak, it has a strong sensitizing action on GnRH-induced gonadotropin secretion. Here we investigated the possibility that ovarian steroids, which are well-known modulators of LH secretion, interact with PACAP and GnRH in pituitary gonadotrophs. Rat pituitary cells were treated for 48 h with vehicle, 1 nmol/l estradiol, 1 nmol/l estradiol + 100 nmol/l progesterone or 48 h with 1 nmol/l estradiol and 4 h with 100 nmol/l progesterone. The cells were stimulated for 3 h with 1 nmol/l GnRH or 100 nmol/l PACAP. Estradiol treatment alone enhanced basal as well as GnRH- or PACAP-stimulated LH secretion. LH release was facilitated by additional short-term progesterone treatment. Long-term treatment with estradiol and progesterone led to reduced LH responses to GnRH and PACAP. Neither treatment paradigms affected cAMP production. However, estradiol treatment led to enhanced cAMP accumulation in quiescent or GnRH-stimulated cells. PACAP-induced increases of cAMP production were inhibited by estradiol treatment. After 7-h preincubation with 10 nmol/l PACAP, cells responded with enhanced LH secretion to GnRH stimulation. When steroid pretreatment was performed the responsiveness of gonadotrophs to low concentrations of GnRH was still increased. In contrast, at high concentrations of GnRH the sensitizing action of PACAP on agonist-induced LH secretion was lost in steroid-treated cells. There were no significant differences between the steroid treatment paradigms. It is concluded that estradiol but not progesterone acts as a modulator of adenylyl cyclase in gonadotrophs. The stimulatory effect of estradiol is thought to be involved in its sensitizing action on agonist-induced LH secretion. The inhibitory effect of estradiol on PACAP-stimulated adenylyl cyclase activities seems to be responsible for the loss of its action to sensitize LH secretory responses to GnRH.     

Gonadotropin-releasing hormone induces ovulation in hypophysectomized rats: studies on ovarian tissue-type plasminogen activator activity, messenger ribonucleic acid content, and cellular localization

GnRH and its agonists are known to induce ovulation in hypophysectomized rats by acting directly at the ovary. Because tissue-type plasminogen activator (tPA) has been implicated in the gonadotropin induction of ovulation, we examined the effect of an ovulatory dose of GnRH on ovarian tPA activity, mRNA content, and cellular localization. Hypophysectomized immature rats were injected sc with 20 IU PMSG and a single dose of a GnRH agonist (GnRHa; des-Gly10,DLeu6(N alpha Me)Leu7,Pro9NHEt-GnRH) 58 h later. At different times after treatment, ovaries were prepared for morphological analysis. Using a fibrin overlay method, tPA activities were measured in ovarian homogenates and cumulus-oocyte complexes, whereas granulosa cells were cultured for 24 h to estimate tPA secretion. Total ovarian RNA was prepared for hybridization analysis of tPA message levels, and tPA localization was studied by immunohistochemistry of ovarian sections. GnRHa induced ovulation in PMSG-primed hypophysectomized rats 14-16 h after injection in a dose-dependent manner, and the GnRHa action was blocked by concomitant treatment with a GnRH antagonist. GnRHa stimulated the induction of tPA, but not urokinase-type PA, activity in ovarian homogenates and granulosa cell-conditioned medium in a time-dependent manner, reaching a maximum before ovulation. tPA activity in cumulus-oocyte complexes was also increased before ovulation, but this increase was sustained. Hybridization analysis of steady state tPA mRNA levels was performed using a rat cRNA probe. Northern blot analysis of total ovarian RNA demonstrated that GnRHa stimulated tPA mRNA levels 12 h after treatment, with a subsequent decrease 24 h after treatment. Immunohistochemistry indicated substantial increases in tPA staining in granulosa cells and oocytes of preovulatory follicles before ovulation. Thus, GnRHa acts through specific receptors to increase ovarian tPA enzyme activity, mRNA content, as well as immunostaining in granulosa cells and oocytes. Like gonadotropins, GnRH may induce ovulation by directly stimulating tPA levels in the ovary.     

Granulosa cell fusion allows heterologous receptor stimulation of adenylate cyclase and progesterone accumulation

The hallmark of the preovulatory follicles in the rat ovary appears to be the presence of receptors for both LH and FSH on follicular granulosa cells. We have tested the possibility that both gonadotropin receptors could share a common adenylate cyclase system utilising cell fusion techniques. Adenylate cyclase (and concomitant stimulation of progesterone synthesis) was inactivated in granulosa cells possessing both FSH and LH receptors by incubation of the cells with N-ethylmaleimide. This treatment did not affect hormone binding to the cells. Subsequent fusion, using polyethylene glycol, of the cyclase-inactivated cells with granulosa cells possessing FSH receptors only and an active cyclase restored LH stimulation of cAMP and progesterone production. These findings support the hypothesis that the LH and FSH receptors on granulosa cells of preovulatory follicles share a common adenylate cyclase system.     

Paracrine mechanisms involved in granulosa cell differentiation

Since the heterogeneous development of individual follicles in a given ovary cannot be accounted for by changes in circulating gonadotropin levels, local modulatory factors play an important role in the paracrine control of follicular development. The important paracrine role of ovarian steroids has been well established. Oestrogen is important in the augmentation of gonadotropin action. High local concentration of oestrogens enhances the gonadotropin stimulation of aromatase activity, resulting in further increases in oestrogen production. The elevated local oestrogens in the follicular fluid are also capable of enhancing the FSH induction of LH receptors. Similar to oestrogens, local high concentrations of progesterone may enhance the gonadotropin stimulation of progesterone biosynthesis in granulosa and luteal cells. This positive autofeedback mechanism is believed to be important for the autonomy of luteal cell steroidogenesis. Ovarian actions of androgens are diverse. In the absence of FSH, androgens exert mainly negative effects at the follicular level by causing atresia and granulosa cell death, whereas in the presence of FSH, androgens augment FSH stimulation of progesterone and oestrogen biosynthesis. Since androgen and oestrogen appear to antagonize each other's actions, the ratio of these two steroids is important in determining the fate of an individual follicle. In contrast to ovarian steroids, the role of ovarian peptides as paracrine signals is less clear. In vitro studies clearly demonstrated that GnRH exerts both stimulatory and inhibitory actions on follicular functions, while IGF-I and VIP stimulate ovarian steroidogenesis. The actions of these peptides are presumably mediated through specific granulosa cell receptors that have been tentatively identified. It is presumed that GnRH and IGF-I may be produced by ovarian cells, while VIP may be derived from ovarian nerves. It is anticipated that new methodologies will be developed to study individual follicles as independent units, capable of synthesizing hormones, releasing them, and exerting local paracrine functions.     

Regulation of gonadotropin receptors by luteinizing hormone in granulosa cells.

In association with luteinization, LH induces a decrease in the content of receptors for FSH and LH and an increase in that for PRL. To elucidate if the mechanism by which LH regulates its own receptors involved occupancy of sites and/or loss of receptors the effects of a luteinizing dose of LH were examined in the preovulatory follicles of immature hypophysectomized rats primed with estradiol and FSH. The measurable LH receptor content declined by 82% 24 h after LH administration. Serum concentration of the hormone declined by 24 h to 1.4% of the concentration measured 2 h after LH administration. Administration of iodinated LH to demonstrate occupancy of sites in vivo, resulted in a decline in the amount of hormone bound in vivo, over a period of time. This decline in occupancy paralleled the decrease in the number of available sites as measured in vitro. Furthermore, a large dose of highly purified hFSH administered in lieu of LH induced luteinization and an associated loss of gonadotropin receptors. These results indicate that luteinizing doses of LH and FSH induce a loss in gonadotropin receptors by mechanisms other than occupancy.     

Follicle luteinization in hyperandrogenic follicles of polycystic ovary syndrome patients undergoing gonadotropin therapy for in vitro fertilization

CONTEXT: Polycystic ovary syndrome (PCOS) is a reproductive disorder of ovarian hyperandrogenism and insulin resistance characterized by abnormal luteinization of small follicles. After exposure to GnRH analog/FSH stimulation for in vitro fertilization (IVF), however, it is unclear whether such PCOS follicles remain abnormally luteinized during the resumption of oocyte maturation in vivo. OBJECTIVE: The aim of this study was to determine whether PCOS follicles exposed to GnRH analog/FSH stimulation for IVF show abnormal luteinization. DESIGN: This study was a prospective cohort. SETTING: The setting was an institutional practice. Patients: Eleven PCOS and 30 normoandrogenic ovulatory women were included. INTERVENTION(S): All subjects received GnRH analog/FSH therapy after basal serum hormone determinations. MAIN OUTCOME MEASURE(S): Follicle fluid aspirated at oocyte retrieval from the first follicle of each ovary was assayed for gonadotropins, steroids, insulin, and glucose. LH receptor mRNA expression was determined in granulosa cells of the same follicle. RESULTS: In PCOS patients with basal hyperandrogenemia and hyperinsulinemia, total oocyte number was increased and follicle diameter was decreased, despite normal maximal serum estradiol levels. Within PCOS follicles, progesterone levels were reduced (P < 0.01), despite comparable bioactive LH and insulin levels and granulosa cell LH receptor mRNA expression; estradiol levels were normal, despite diminished FSH availability (P < 0.004). Elevated androstenedione (P < 0.01), testosterone (P < 0.001), and glucose (P < 0.01) levels also occurred. In PCOS follicles containing mature oocytes, however, elevated androgen levels were accompanied by both normal progesterone concentrations and a normal inverse relationship between glucose depletion and lactate accumulation. CONCLUSION: Hyperandrogenic follicles with mature oocytes from PCOS women receiving GnRH analog/recombinant human FSH therapy for IVF show sufficient glucose utilization for normal luteinization.     

Characterization of gonadotropin-sensitive adenylate cyclase activity in human testis: uncoupling of the receptor-cyclase complex by specific hormonal antagonist

Basal and gonadotropin stimulated adenylate cyclase activity was assessed in testicular tissues obtained from men (20-80 years). A disparity was observed in the gonadotropin responsiveness of the human testicular adenylate cyclase system to hFSH and hCG stimulation. Of the tissues analyzed, 61% were FSH responsive and 22% showed low response to hCG. Forskolin, a diterpene which activates adenylate cyclase by a receptor independent mechanism, stimulated adenylate cyclase activity in the gonadotropin unresponsive tissues. This suggests that the tissue unresponsiveness is due to an uncoupling of the catalytic subunit of the adenylate cyclase. Several functional properties of the FSH responsive human testicular adenylate cyclase were investigated. hFSH and oFSH stimulated the enzyme activity in a concentration dependent manner. However, the hormone (DG-oFSH) in which 80% of the carbohydrate residues had been removed was inactive, despite its good binding ability to the FSH receptor. hFSH stimulated adenylate cyclase activity was inhibited by DG-oFSH but not by DG-hCG (deglycosylated hCG). The data demonstrates the existence of specific FSH and LH(hCG) receptors in human testicular membranes. The FSH receptors in some tissues are coupled to adenylate cyclase. The link between the FSH receptor and adenylate cyclase may be uncoupled in the presence of the deglycosylated form of oFSH resulting in a loss of hormone response.     

Regulation of prostaglandin E, progesterone, and cyclic adenosine monophosphate production in ovarian granulosa cells by luteinizing hormone and gonadotropin-releasing hormone agonist: comparative studies

The early direct effects of GnRH on the ovary were investigated using cultured granulosa cells from preovulatory rat follicles, and compared to the known stimulatory effects of LH. Stimulation of ovarian functions by a GnRH agonist include a rapid receptor-mediated phosphatidylinositol turnover (approximately 5 min). On the other hand, LH action on granulosa cells is initiated by increased cAMP production (approximately 10-15 min), consisting of an indomethacin-resistant and indomethacin-sensitive pools (40% and 60%, respectively). The GnRH agonist [D-Ala6] des-Gly10 N-ethylamide (GnRHa) at concentrations of 10(-12)-10(-8) M had no effect on basal or LH-stimulated cAMP production during a 4-h incubation test. Both LH and GnRHa increase progesterone formation (30 and 120 min, respectively) with ED50 values of 2.5 ng/ml and 10(-9) M, respectively and the stimulatory effect is not blocked by indomethacin. LH and GnRHa increase also prostaglandin E (PGE) formation (180 and 120 min, respectively) and the ED50 values were 0.1 microgram/ml and 10(-9) M, respectively. No inhibitory effect of GnRHa on LH actions was observed during 4 h of incubation. It is concluded that: 1) GnRH mimicks LH stimulation of ovarian PGE and progesterone production; 2) cAMP does not play a role in mediating the direct stimulatory effects of GnRH agonists on ovarian PGE and progesterone production; 3) PGE is not involved in mediating GnRH and LH stimulation of progesterone formation. 4) LH-induced cAMP production consists of indomethacin-sensitive and indomethacin-resistant pools.     

Pituitary gonadotropin-releasing hormone receptors during gonadotropin surges in ovariectomized-estradiol-treated rats

In intact cycling rats, the number of pituitary GnRH receptors varies markedly during the estrous cycle. Concentrations are maximal on diestrus and early proestrus, before falling rapidly for a brief period immediately before the preovulatory gonadotropin surge. In this study we investigated whether dynamic changes in ovarian steroids, pituitary hormones, and GnRH itself, all of which are changing at the time of the surge, play a role in the acute transient down-regulation of the pituitary GnRH receptors. We used the ovariectomized-estradiol-treated female rat as a model, as these animals exhibit daily gonadotropin surges at a predictable time of the day and also allow studies in a situation where concentrations of ovarian steroids are stable. The pituitary GnRH binding capacity (GnRH-BC) was measured using the analog D-Ala6des Gly10-GnRH ethylamide as ligand. GnRH-BC was stable between 0900-1530 h [range, 288 +/- 29 to 262 +/- 33 fmol protein (mean +/- SE)] and fell abruptly to 123 +/- 17 fmol/mg at 1630 h, before returning to the initial level by 1730 h. This abrupt fall in GnRH-BC preceded the afternoon gonadotropin surge and was similar in timing, magnitude, and duration to that observed in intact cycling rats. Serum PRL decreased from peak levels at 1630 h, coincident with the fall in GnRH-BC, before rebounding at 1730 h. Pentobarbital given at 1400 h abolished both the gonadotropin surge and the acute fall in GnRH-BC, but did not change serum PRL levels, suggesting that PRL is not causally related to the fall in GnRH-BC. The stable morning levels of GnRH-BC were not reduced after iv injections of LH, FSH, or both hormones despite elevations in serum gonadotropins to concentrations greater than those seen during the afternoon surge. Additionally, multiple iv injections of GnRH at 30- or 10-min intervals did not decrease the stable morning levels of GnRH-BC, although serum LH and FSH were markedly elevated. The data suggest that dynamic fluctuations in ovarian steroids, gonadotropins, PRL, and GnRH are not causally related to the acute transient reduction of pituitary GnRH receptors before the afternoon gonadotropin surge. These results also suggest that another hypothalamic or pituitary factor(s) is involved in the acute regulation of GnRH receptors, and the ovariectomized-estradiol-treated rat appears to be a good model for the elucidation of the factor(s) involved.     

Properties of detergent-solubilized adenylate cyclase and gonadotropin receptors of testis and ovary.

The relationship between solubilized hormone-binding sites and adenylate cyclase was examined in detergent extracts of particulate testis and ovarian fractions. Both basal and fluoride-stimulated activities of the particulate enzyme were markedly increased in the presence of detergents, and about 60% of the enzyme activity was recovered as the soluble form in the 300,000 g supernatant. Enhancement of adenylate cyclase activity was more marked with Lubrol PX and WX than with Triton X-100, and the highest recovery and activation of adenylate cyclase were obtained with 0.5% Lubrol PX. The particulate and solubilized testicular enzymes were more active in the presence of Mn2+, and the detergent-extracted soluble ovarian cyclase showed a small and inconstant response to gonadotropin. Fractionation of Lubrol-solubilized testis and ovarian preparations on Sepharose 6B showed two peaks of free gonadotropin receptors. The binding activity eluted with Kav of 0.32 corresponded to the receptor sites previously characterized in detergent-solubilized gonadal particles, and was coincident with the elution profile of adenylate cyclase activity. An additional peak of binding activity with Kav of 0.56 was not accompanied by detectable adenylate cyclase activity. These observations suggest that the peak of larger molecular size could represent dissociated receptors or binding sites which were not coupled to adenylate cyclase.