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Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and permeability factor the role of which in ovarian angiogenesis has been the subject of increasing interest. It was the objective of this communication to explore the possibility that interleukin (IL)-1 may regulate the in vitro expression of rat ovarian VEGF mRNA, as well as to study the in vivo expression of rat ovarian VEGF transcripts during follicular maturation, ovulation, and corpora lutea formation. Taken together, our findings 1) reaffirm the rat ovary as a site of VEGF expression; 2) document an in vivo increase in VEGF transcripts before ovulation; 3) disclose a marked dependence of VEGF on IL-1 beta; 4) reveal the IL-1 beta effect to be receptor mediated and dose and time dependent and to be shared by at least two growth factors--epidermal growth factor and basic fibroblastic growth factor; and 5) demonstrate a lack of VEGF effect on ovarian progesterone biosynthesis as assessed in cultured isolated granulosa cells. It is tempting to speculate that the up-regulatory effect of IL-1 beta on VEGF transcripts may be relevant to the marked angiogenesis and increased vascular permeability displayed by the hyperemic ovarian Graafian follicle during the terminal stages of follicular development. In this context, VEGF may be joined by other IL-1-dependent angiogenesis promoters such as IL-6 or transforming growth factor beta 1. Thus, IL-1-mediated VEGF induction may constitute one of several end points through which IL-1 may coordinate and perhaps amplify the ovulatory cascade.

This study examines the effects of interleukin-1 (IL-1) on plasminogen activator (PA) activity and prostaglandin (PG) E production in pregnant mare serum gonadotropin (PMSG)-primed granulosa cells and the potential involvement of PGE in the regulation of ovarian plasminogen activation.Granulosa cells were obtained from PMSG-primed rat (27-day-old) ovaries and cultured in serum-free conditions for 48 hours in the absence or presence of IL-1 beta (10 ng/mL) with and without transforming growth factor-beta 1 (10 ng/mL). Cellular PA activity was measured through the conversion of plasminogen to plasmin and assay of the plasmin-mediated cleavage of [14C]-labeled globin to acid-soluble products.Exposure of PMSG-primed granulosa cells to IL-1 resulted in a 30% reduction (P.05) in PA activity. Addition of hCG (1 IU/mL) to the granulosa cell cultures resulted in a 2.3-fold increase in PA activity, an effect significantly attenuated by co-administration of IL-1. The IL-1-mediated inhibition occurred concurrent with a 6.6-fold increase in the ability of the corresponding conditioned media to inhibit exogenous urokinase activity. This latter inhibitory capacity was the result of a significant increase in plasminogen activator inhibitor type 1 (PAI-1), given its abolition by a polyclonal anti-rat PAI-1 immunoglobulin G. The IL-1-mediated effects on PA/PAI-1 were accompanied by a sevenfold increase in PGE content of the spent culture medium. This response was dose dependent. The IL-1 effects on plasminogen activation and PG production were abolished by the IL-1 receptor antagonist, suggesting specific IL-1 receptor-mediated responses. Indomethacin, an inhibitor of PG biosynthesis, prevented the IL-1-induced increase in PGE accumulation but failed to affect the response of the PA system. Transforming growth factor-beta 1, a known regulator of IL-1 action, significantly attenuated the IL-1-stimulated PGE production but did not interfere with the ability of IL-1 to affect the PA system.The present observations suggest a pleiotropic response of PMSG-primed granulosa cells to IL-1, characterized by the induction of PAI-1 concurrent with but independent of PG production. These findings corroborate and extend earlier observations suggesting that IL-1 affects PA activity and PGE production in immature rat ovaries. Moreover, these observations support our contention that IL-1 may play a major regulatory role in the cellular events leading to ovulation and early corpus luteum formation.

We have recently documented a marked dependence of ovarian prostaglandin endoperoxide synthase (PGS)-2 transcripts, proteins, and activity on interleukin (IL) 1, a putative intermediary in the ovulatory cascade. The purpose of the present study was to characterize the cellular and molecular mechanisms underlying the ability of IL-1beta to upregulate the steady-state levels of ovarian transcripts corresponding to PGS-2. Results of studies designed to enrich or deplete nitric oxide strongly suggest that the stimulatory effect of IL-1beta on ovarian PGS-2 expression is independent of nitric oxide. Utilization of a series of agents designed to simulate or enhance transduction via the sphingomyelin ceramide cycle suggests that the long-term stimulatory effect of IL-1beta on ovarian PGS-2 gene expression is independent of ceramide. In contrast, inhibition of prostaglandin biosynthesis with a series of distinct inhibitors suggests that the ability of IL-1beta to upregulate ovarian PGS-2 transcripts is due, if only in part, to the generation of endogenous prostaglandin estradiol-17beta (E(2)). Inhibition of protein biosynthesis suggested that the IL-1beta-induced PGS-2 gene expression required de novo protein biosynthesis. Our findings revealed substantial IL-1beta-mediated stabilization of PGS-2 transcripts, as assessed by a threefold increase in the half-life of the message. We have also observed the ability of IL-1beta to upregulate the transcription of PGS-2 promoter constructs subjected to transient transfection into whole-ovarian dispersates (twofold increase as assessed by activation of the luciferase reporter gene). Taken together, these findings suggest that the stimulatory effect of IL-1beta on PGS-2 expression is 1) independent of nitric oxide as well as ceramide, 2) dependent on prostaglandin E(2), 3) contingent on de novo protein biosynthesis, and 4) accounted for by both increased transcription and message stabilization. These observations provide indirect support for the hypothesis that IL-1beta, acting in part through PGS-2 (an obligatory ovulatory principal), may constitute a key intermediary in the ovulatory cascade.

Interleukin (IL)-6, traditionally an IL-1-induced immune regulator, is nevertheless synthesized by a variety of tissues including the ovary. The purposes of this communication were to assess the ovarian expression of IL-6, examine its cyclic variation, and study its regulation by IL-1, a putative intermediary in the ovulatory process. Molecular probing revealed IL-6 transcripts to be most abundant in the thymus, liver, and ovary, which suggests that, in relative terms, untreated immature whole ovarian tissue is a significant site of IL-6 gene expression. Treatment of immature rats with pregnant mare serum gonadotropins resulted in a measurable, statistically significant (P.05) increase in ovarian IL-6 mRNA compared with untreated controls. Isolated and indeed transient increments in the relative abundance of IL-6 transcripts were noted 4 hours after exposure to hCG, a point in time 8 hours from projected follicular rupture, a pattern highly reminiscent of that previously recognized for IL-1 beta transcripts. Treatment of whole ovarian dispersates from immature rats with IL-1 beta for 48 hours resulted in a significant (P.05) increase (11-fold) over control in IL-6 transcripts. The IL-1 effect proved dose dependent; the first significant increase was noted at the 1-ng/mL dose level. Evaluation of the time requirements revealed IL-1 beta to significantly (P.05) up-regulate (4.5-fold) IL-6 transcripts as early as 24 hours into the culture period. Cotreatment with the IL-1 receptor antagonist completely reversed the IL-1 effect, which suggests mediation through a specific IL-1 receptor. Treatment with indomethacin (10 microg/mL), an established inhibitor of prostaglandin biosynthesis, resulted in a significant (P.05) decrease (79%) in the ability of IL-1 beta to up-regulate IL-6 transcripts. Importantly, the addition of prostaglandin E(2) (10 microg/mL) to untreated or indomethacin-treated cells significantly (P.05) augmented the IL-1 beta effect. This suggests a role for eicosanoid signaling in IL-1 beta action. Treatment with aminoguanidine, an established inhibitor of nitric oxide synthase, significantly (P.05) decreased (85%) the IL-1 beta effect. However, the addition of S-nitroso-n-acetyl-penicilamine, an established nitrite generator, failed to reverse the aminoguanidine effect, which suggests that the inhibitor effect of aminoguanidine may be nitrite independent. Treatment with cycloheximide produced dose-dependent inhibition of the ability of IL-1 to up-regulate IL-6 transcripts; the maximal inhibitory effect was 89%. Taken together, these findings (1) reaffirm the rat ovary as a site of IL-6 expression; (2) document an in vivo increase in IL-6 transcripts before ovulation; (3) disclose a marked dependence of IL-6 on IL-1 beta; and (4) reveal the IL-1 beta effect to be dose and time dependent, receptor mediated, contingent upon de novo protein biosynthesis, and eicosanoid dependent but nitric oxide independent. These findings suggest that IL-1 action in the ovary may require the intermediacy of IL-6 in a manner like that encountered in extraovarian sites.

The first large survey on the public perception of infertility and its treatment was conducted in six European countries, the USA and Australia. A representative sample of 8194 adults was polled, using standard validated methodology. The results obtained highlighted the following major aspects: (i) infertility is perceived as a disease by less than half of the people surveyed (38%), in contrast to the accepted medical opinion; (ii) awareness about the definition and incidence of infertility is relatively low, despite the fact that half of the people polled claimed to know someone affected by infertility; (iii) close to 90% of the adults surveyed knew about in-vitro fertilization (IVF), but less than one-quarter of them knew about the chances of success of this assisted reproductive technology; and (iv) when confronted with the knowledge that the cost of three IVF cycles is roughly equivalent to the cost of a hip replacement (a commonly reimbursed procedure), a large majority (70%) of the individuals interviewed agreed that IVF should be reimbursable.

As might be expected, the teleological underpinning of ovarian function draws on the fundamental need to preserve the species. Accordingly, the very existence of the ovary, and for that matter the very existence of the reproductive axis as a whole, is designed to subserve a single central objective, i.e., the generation of a mature fertilizable ovum. In this respect the ovary clearly need not be viewed as playing a secondary role in reproductive biology. Rather, it may be viewed as the “master gland” the very function of which is facilitated by the contribution of the various other components of the hypothalamic-pituitary-ovarian axis. This view differs of course from the more traditional outlook ascribing the role of a “master gland” to the pituitary, the workings of which are highly dependent on hypothalamic principles. However, current information would suggest that the ovary may in fact play an active rather than a passive role in the initiation and maintenance of reproductive cyclicity, the hypothalamus and pituitary being viewed as playing a permissive tonic role in this connection. Indeed, it is the changing tide of ovarian signals that appears to determine to a large extent the nature of the activities of the hypothalamic-pituitary unit. It is for these reasons that the ovary has often been likened to a pelvic clock (“zeitgeber”) dictating in more ways than one the comings and goings of the reproductive process. Stated differently, consideration might be given to the argument that the ovary does, in effect, possess a “mind” of its own, as attested to by a multitude of putative intraovarian regulators, the very action of which is highly reminiscent of events previously viewed as the domain of the central nervous system.

It is hypothesized that the intraovarian interleukin (IL)-1 system plays a prominent role in the regulation of follicular development and ovulation. A central component of the intraovarian IL-1 system is the IL-1 receptor antagonist (IL-1RA), a protein acting as a pure IL-1 receptor antagonist and one for which intracellular (icIL-1RA) and secretory (sIL-1RA) varieties have been described. It was the objective of this study to explore rat ovarian IL-1RA gene expression, to establish the identity and relative abundance of its alternative transcripts, to study its cellular localization, to determine its cyclic variation, and to assess its hormonal regulation. Protected IL-1RA cDNA fragments corresponding to either sIL-1RA or icIL-1RA were barely detectable in untreated whole ovarian tissue of immature rat origin. However, sIL-1RA transcripts reached a maximal value (3.3-fold increase over untreated control values; p0.05) 12 h after hCG administration (time of projected ovulation). In situ hybridization localized IL-1RA to mural, antral, and cumulus granulosa cells. Modestly intense staining was also apparent in oocytes. The basal pattern of sIL-1RA expression by cultured whole ovarian dispersates was characterized by a spontaneous increase to a peak value at 4 h. The early (4 h) sIL-1RA burst proved IL-1-, nitric oxide-, and protein biosynthesis-independent. However, treatment with IL-1beta led to a secondary sIL-1RA peak at 48 h, an effect that was substantially reversed by IL-1RA. This stimulatory effect of IL-1beta on IL-1RA expression proved relatively specific, and nitric oxide independent, but contingent upon de novo protein biosynthesis. The in vitro expression of icIL-1RA was barely detectable. Taken together, these in vivo and in vitro observations 1) document the rat ovary as a site of IL-1RA (sIL-1RAcIL-1RA) expression, 2) localize the relevant transcripts to the granulosa cell, 3) disclose peak expression at the time of ovulation, and 4) establish IL-1 dependence.

Ovulation may constitute a cyclic, inflammatory-like process, wherein interleukin (IL)-1 induction and increased biosynthesis of prostanoids may feature prominently. In excess, glucocorticoids, potent anti-inflammatory agents, may exert an antiovulatory effect. This paper addresses the possibility that the antiovulatory action of glucocorticoids may be partly due to interference with ovarian prostanoid biosynthesis. Specifically, we examined the effect of treatment with dexamethasone, a synthetic glucocorticoid, on the IL-1-induced expression and activity of ovarian prostaglandin endoperoxide synthase (PGS)-2, the inducible variety of the rate-limiting enzyme in the prostaglandin cascade. Treatment of cultured whole ovarian dispersates from immature rats with dexamethasone for 48 h produced a significant decrease (98.9% inhibition) in the IL-1-supported expression of PGS-2 transcripts. Comparably marked inhibition was also noted for the corresponding immunoreactive protein. The dexamethasone effect was not limited to the IL-1-mediated induction of PGS-2 transcripts, comparable suppression being noted for the IL-1-mediated up-regulation of ovarian transcripts corresponding to IL-1beta, the IL-1 receptor antagonist, and the type I IL-1 receptor. The order of potency of the glucocorticoids studied was dexamethasoneprednisolone = cortisol. Dexamethasone proved equally effective in suppressing the induction of PGS-2 transcripts by congeners of the sphingomyelin-ceramide cycle (e.g., C-2 ceramide, sphingomyelinase, and sphingosine). The dexamethasone effect proved glucocorticoid-specific, as synthetic agonists representative of the progestin (R-5020), androgen (R-1881), and estrogen (diethylstilbestrol) steroid series proved to be without effect. Cotreatment with RU-486 resulted in reversal of the ability of dexamethasone to suppress PGS-2 activity or expression. Taken together, these observations suggest that dexamethasone is capable of glucocorticoid receptor-mediated/post-ceramide suppression of IL-1-supported ovarian PGS-2 transcript, protein, and activity. These findings are compatible with the view that the chronic anovulatory state associated with adrenal hyperactivity or glucocorticoid excess may be due in part to inhibition of ovarian prostaglandin biosynthesis.

Ovulation may constitute a cyclic, inflammatory-like process, wherein the increased expression of interleukin (IL)-1 and the biosynthesis of prostaglandins may be established corollaries. In this communication we hypothesize that glucocorticoids, potent anti-inflammatory principles, may exert an antiovulatory effect by interfering with ovarian IL-1-driven prostaglandin biosynthesis. To test this hypothesis, we examined the effect of treatment with dexamethasone on the activity of ovarian phospholipase A2 (PLA2), the event-limiting enzyme in prostaglandin biosynthesis, and on the gene expression pattern of secretory and cytosolic PLA2 (sPLA2 and cPLA2, respectively). Whole ovarian dispersates from immature rats were cultured under serum-free conditions for 48 h in the absence or presence of dexamethasone. At the conclusion of this culture period, PLA2 activity was determined in cell sonicates and conditioned media. Parallel probing for sPLA2 and cPLA2 transcripts was also undertaken using a solution hybridization/RNAse protection assay. Treatment of whole ovarian dispersates with dexamethasone produced a significant (P0.005) decrease in basal cellular and extracellular PLA2 activity to 27 and 40% of controls, respectively. A 5-fold decrease in the basal steady state levels of sPLA2 (but not cPLA2) transcripts was also noted. Co-treatment with dexamethasone produced complete inhibition of IL-1-stimulated cPLA2 transcripts but not of IL-1-supported cellular and extracellular PLA2 activity or sPLA2 transcripts. A glucocorticoid receptor antagonist (RU486), blocked the ability of dexamethasone to inhibit basal sPLA2 transcripts and extracellular PLA2 activity. The inhibitory effect of dexamethasone proved glucocorticoid-specific in that aldosterone and 17beta-estradiol were without effect. Taken together, these observations suggest that dexamethasone is capable of inhibiting basal (but not IL-1-supported) ovarian PLA2 activity, a glucocorticoid receptor-mediated effect due, in part, to a decrease in sPLA2 gene expression. Our findings further suggest that sPLA2 and cPLA2 are differentially regulated and that they may well differ in their relative contribution to ovarian prostaglandin biosynthesis in general and to PLA2 activity in particular. To the extent that IL-1 plays a central role in the ovulatory process, these findings argue against the view that the chronic anovulatory state induced by glucocorticoid excess is due, if only in part, to suppression of ovarian IL-1-dependent PLA2 activity.

Interleukin (IL)-1beta is a putative regulator of ovulation and perhaps luteal function. In this work, we examine its actions on the steroidogenic cascade of the rat granulosa cell. Whereas treatment of immature granulosa cells with FSH for 72 h produced substantial increments in the accumulation of progesterone, the addition of IL-1beta produced dose-dependent inhibition of this FSH effect. Pulse labeling of cells with [3H]pregnenolone revealed IL-1beta to effect a decrease in the FSH-supported accumulation of [3H]progesterone while enhancing the accumulation of its proximal metabolite, [3H]20alpha-dihydroprogesterone. IL-1beta was without effect on the activity levels of the progesterone-synthesizing enzymes, even though the corresponding transcripts were elevated. The effect of IL-1beta on some progesterone-degrading enzymes was negligible (5alpha-reductase) or modest (3alpha-hydroxysteroid dehydrogenase). In contrast, IL-1beta markedly stimulated both control and FSH-supported 20alpha-hydroxysteroid dehydrogenase activity (4.8- and 3.3-fold, respectively) and transcripts (16.4- and 7.5-fold, respectively). These data demonstrate an IL-1beta-mediated inhibition of gonadotropin-stimulated steroidogenesis via modulation of specific enzymes, and suggest a role for IL-1beta in mediating the observed decline of these bioactive hormones during ovulation and luteolysis.

This laboratory has previously shown that interleukin-1 (IL-1), a putative intermediary in the ovulatory process, is capable of up-regulating PG biosynthesis by cultured whole ovarian dispersates from immature rats. In part, this phenomenon was attributable to the stimulation of ovarian phospholipase A2 activity. In this communication we examine the possibility that the PG-promoting property of IL-1 is also due to the up-regulation of PG endoperoxide synthase (PGS), the rate-limiting step in prostanoid biosynthesis. The in vivo expression of ovarian PGS-2 transcripts in the course of a simulated estrous cycle rose abruptly to a peak (35-fold increase over the control value; P0.05) 8-12 h after hCG administration (i.e. before or during projected ovulation). PGS-1 transcripts, in turn, were not significantly altered during the periovulatory period. Treatment of cultured whole ovarian dispersates with IL-1beta resulted in dose- and time-dependent up-regulation of PGS-2 transcripts (as well as of immunoreactive PGS-2 protein and PGE2 accumulation), characterized by an ED50 of 2 ng/ml and a maximal (72-fold) increase at 10 ng/ml. Although treatment with IL-1beta also led to an increase in PGS-1 transcripts and immunoreactive PGS-1 protein, the relative magnitude of the effect was markedly reduced compared with that of PGS-2. Cotreatment with an IL-1 receptor antagonist completely reversed the IL-1 effects, thereby suggesting mediation via the IL-1 receptor. The ability of IL-1 to up-regulate PGS-2 transcripts proved relatively specific, in that other cellular regulators (insulin-like growth factor I, activin A, endothelin-1, transforming growth factor-alpha, tumor necrosis factor-alpha, vascular endothelial growth factor, leukemia inhibitor factor, hepatocyte growth factor, or keratinocyte growth factor) were not effective. The optimal IL-1 effect required heterologous contact-dependent coculturing of granulosa and thecal-interstitial cells. Taken together, these observations 1) reaffirm (by molecular probing) the granulosa cell as the primary site of ovarian PGS-1 and PGS-2 expression, 2) document an increase in ovarian PGS-2 transcripts before ovulation, and 3) reveal a marked dependence of ovarian PGS (21) transcripts, proteins, and activity on IL-1. The effects of IL-1 proved relatively specific, contingent upon somatic cell-cell cooperation, dose and time dependent, and IL-1 receptor mediated. These results are compatible with the proposition that the PG-promoting property of IL-1 is due, in large measure, to the activation of ovarian PGS transcription and translation. The ability of IL-1 to up-regulate ovarian PGS, an obligatory component of ovulation, is in keeping with the idea that IL-1 may constitute an intermediary in the ovulatory process.

It has been suggested that ovulation may constitute a cyclic inflammatory-like process and that gonadotropin-inducible intraovarian interleukin (IL)-1, an established mediator of inflammation, may play a central role in this regard. In support of this hypothesis, our group has been able to document the ability of IL-1 to potently stimulate prostaglandin biosynthesis by cultured rat ovarian cells. Herein we explore the possibility that the prostaglandin-stimulating action of IL-1 is due, in part, to the enhanced expression of ovarian secretory phospholipase-A2 (sPLA2). A single sPLA2 transcript of 1.4 kilobases was noted in all extraovarian tissues of immature rat origin subjected to Northern blot analysis. However, only a barely detectable signal was apparent in ovarian tissue. In contrast, the more sensitive RNase protection assay revealed the unequivocal presence of ovarian sPLA2 transcripts. Cellular localization studies by way of in situ hybridization documented sPLA2 transcripts primarily in the granulosa cell of the periovulatory ovary. Molecular probing of untreated cultured whole ovarian dispersates disclosed spontaneous elaboration of sPLA2 transcripts as early as 20 h after the introduction of cells into culture. Treatment of cultured whole ovarian dispersates with IL-1beta for 48 h produced a 1.7-fold increase (over the value in untreated controls) in the relative expression of sPLA2 transcripts (p0.01) along with a 1.7-fold increase in media PLA2 activity (p0.01). A more marked increase was documented for IL-1beta-treated cultured isolated granulosa cells (12.5-fold increase, p0.001). Treatment of whole ovarian dispersates with an IL-1 receptor antagonist (IL-1RA) produced a reduction in the basal expression of sPLA2 transcripts (55% at the 5 microg/ml dose level; p0.01) and PLA2 activity (40%; p0.01), thereby suggesting basal endogenous IL-1-like bioactivity. Treatment of cultured whole ovarian dispersates with either hCG or FSH led to 2.6-fold (p = 0.056) and 3-fold (p = 0.029) increases in the abundance of sPLA2 transcripts, respectively, effects blocked by the concurrent presence of IL-1RA. These observations 1) document the immature rat ovary as a site of sPLA2 gene expression, 2) localize the relevant transcripts to the postovulatory granulosa cell, 3) confirm the presence of functional secreted ovarian PLA2 activity, 4) reveal PLA2 expression to be IL-1- and gonadotropin-dependent, and 5) suggest the existence of endogenous PLA2-stimulating IL-1-like activity. These findings also suggest that the ability of hCG or FSH to up-regulate ovarian sPLA2 transcripts may be due, in part, to the endogenous elaboration of IL-like activity.

A body of information now supports the existence of an ovarian intrafollicular insulin-like growth factor (IGF)-I system concerned with the amplification of FSH action at the level of the rat granulosa cell. In this study we examined the ability of IGF-I to modulate the basal and FSH-supported activity and expression of key steroidogenic enzymes concerned with progesterone generation and metabolism in cultured granulosa cells from immature rats. The provision of IGF-I stimulated FSH-supported (20 ng/ml) accumulation of progesterone in a dose-dependent manner, reaching a plateau at an IGF-I dose of 50 ng/ml. This dose of IGF-I substantially enhanced FSH action over a broad range of FSH concentrations, reaching a maximum at an FSH dose of 20 ng/ml. Pulse labeling of FSH-pretreated cells with [3H]pregnenolone revealed relatively rapid (5 h) transformation to [3H]progesterone and other distal products that was accelerated by the concurrent addition of IGF-I. These changes in progesterone metabolism were associated with IGF-I-mediated enhancement of the activities and expression of key steroidogenic enzymes. Specifically, treatment with IGF-I produced significant augmentation of the FSH-stimulated activities of cholesterol side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase/ isomerase (3 beta-HSD) enzymes (2.4- and 1.8-fold, respectively). Similarly, P450scc and type I 3 beta-HSD transcripts were elevated by FSH in a dose-dependent manner, the concurrent addition of IGF-I further increasing expression (up to an additional 3-fold) in the range of 1-5 ng/ml (but not at the maximally stimulating dose of 20 ng/ml FSH). The addition of IGF-I also increased basal levels of type I 3 beta-HSD transcripts (3.8-fold). IGF-I enhanced FSH-stimulated 20 alpha-HSD activity and transcripts (2.3-fold and 1.8-fold, respectively) and increased the basal levels of 20 alpha-HSD transcripts (3-fold). Basal levels of 5 alpha-reductase were slightly elevated (1.3-fold) by IGF-I, but the FSH-attenuated activity was unchanged. Taken together, these findings suggest that IGF-I enhances the FSH-supported accumulation of progesterone in cultured granulosa cells through up-regulation of the expression and activity of key enzymes in the steroidogenic pathway. The acceleration of progesterone accumulation reflects a newly established steady state, favoring the activities of progesterone-forming over progesterone-metabolizing enzymes.

Given the suggestion that intraovarian insulin-like growth factor (IGF)-binding proteins (IGFBPs) may constitute markers of follicular atresia, we investigated the possibility that activin, a putative antiatretic principle, may modulate granulosa cell-derived IGFBPs. Untreated granulosa cells cultured for 72 h exhibited a progressive increase in the steady-state levels of transcripts corresponding to IGFBP-4 and IGFBP-5 (1.5-fold and 12-fold, respectively). Transcript levels corresponding to IGFBP-5 were consistently higher than their IGFBP-4 counterparts. Treatment with activin-A (50 ng/ml) for 72 h produced significant (p0.05) decrements in the steady-state levels of IGFBP-4 and IGFBP-5 transcripts (46% and 79%, respectively) as compared to controls. Thus, treatment with activin-A appears to be capable of blocking the spontaneous increase in IGFBP-4 and IGFBP-5 transcripts exhibited by untreated cultured granulosa cells. Consistent activin-A-induced decrements were also observed in the accumulation of the IGFBP-5 (but not the IGFBP-4) protein. Dose-response analysis revealed monophasic dose dependence (half maximal inhibitory doses of 16.2 and 7.8 ng/ml for IGFBP-5 and IGFBP-4 transcripts, respectively). The addition of increasing concentrations of the putative activin-binding protein, follistatin, produced dose-dependent reversal of the activin-A effect on IGFBP transcripts (IGFBP-5IGFBP-4). Activin-B was as effective as activin-A in reducing IGFBP-4 transcripts (31% decrement, p0.05) whereas it had little or no effect on IGFBP-5 transcripts (21% decrement, p0.1). No apparent effect was observed for the corresponding proteins. Activin-A action was specific in that treatment with transforming growth factor (TGF)-beta1, inhibin-A, or Müllerian-inhibiting substance (MIS)--all related peptides--failed to produce statistically significant alterations in the steady-state levels of IGFBP-4 and IGFBP-5 transcripts. Taken together, these observations reveal that activin-A exerts a substantial, relatively rapid, follistatin-neutralizable, dose- and time-dependent inhibitory effect on granulosa cell-derived IGFBP transcripts (IGFBP-5IGFBP-4). Other members of the TGFbeta superfamily (e.g., inhibin-A, TGFbeta1, and MIS) were without significant effect on the expression of IGFBP-4 and IGFBP-5. To the extent that the inhibition of IGFBP-4 and IGFBP-5 expression is associated with, and possibly causally related to, the promotion of follicular health, the present observations are in keeping with the proposition that activin may play an antiatretic role in the dynamic process of follicular selection.

Interleukin (IL)-1 beta has been shown to stimulate ovarian prostaglandin biosynthesis. We hypothesized that this effect entails the induction of phospholipase A2 (PLA2). Treatment of cultured whole ovarian dispersates of immature rat origin with IL-1 beta produced significant increases in [3H]arachidonic acid (AA) release and [3H]prostanoid accumulation as well as increases in cellular PLA2 activity and in secretory PLA2 and cytosolic PLA2 transcripts. Cotreatment with IL-1 receptor antagonist reversed IL-mediated (and basal) release of [3H]labeled AA and prostaglandin products, as well as cellular PLA2 activity. Treatment with IL-1 beta also promoted a significant decrease in the cellular content of [3H]phospholipids (apparently phosphatidylethanolamine but not phosphatidylcholine). These observations establish the ovary as a site of IL-1-dependent sPLA2 and cPLA2 gene expression, document the presence of a possible phosphatidylethanolamine-dependent PLA2 activity in cultured whole ovarian dispersates, reveal the up-regulatory, receptor-mediated action of IL-1 beta in this regard and suggest the existence of endogenous PLA2-stimulating/ IL-1-like bioactivity.

We have previously demonstrated that interleukin-1 beta (IL-1 beta), a putative intermediary in the ovulatory process, is a potent stimulator of ovarian PG biosynthesis. In this communication, we examine the possibility that this IL-1 effect reflects in part the induction of arachidonic acid mobilization by phospholipase A2 (PLA2). Molecular probing of whole ovarian material revealed the immature rat ovary to be a site of modest secretory PLA2 (sPLA2) gene expression. However, no change in ovarian sPLA2 gene expression was noted during the periovulatory period. Comparable probing for cytosolic PLA2 (cPLA2) failed to disclose a quantifiable signal. However, in situ hybridization localized both sPLA2 and cPLA2 (sPLA2cPLA2) transcripts to the granulosa cell layer of the ovarian follicle. Treatment of cultured whole ovarian dispersates with IL-1 beta produced significant (P0.01) increments in the steady state levels of transcripts corresponding to both sPLA2 (1.7-fold increase) and cPLA2 (5-fold increase), an effect reversed by an IL-1 receptor antagonist, suggesting mediation via a specific IL-1 receptor. Treatment with cycloheximide, a protein synthesis inhibitor, resulted in significant attenuation of the ability of IL-1 beta to up-regulate sPLA2 and cPLA2 gene expression as well as medium PLA2 activity. Treatment with aminoguanidine, an inhibitor of inducible nitric oxide synthase, led to augmentation of the ability of IL-1 beta to up-regulate sPLA2 and cPLA2 gene expression as well as medium PLA2 activity. Total cellular PLA2 activity proved time, cell density, and calcium dependent, with an optimal pH of 8.0-9.0 and K(m) values in the low micromolar range (2-5 microM). Our observations 1) establish the rat ovary as a site of sPLA2 and cPLA2 gene expression, 2) localize the corresponding transcripts to the granulosa cell layer, and 3) establish IL-1 beta as an up-regulatory agent for ovarian sPLA2 and cPLA2 gene expression as well as for ovarian PLA2 activity. These findings also indicate that the IL-1 effect is 1) receptor mediated, 2) contingent in part upon de novo protein biosynthesis, and 3) inhibited by nitric oxide. These observations support the proposition that PLA2 may be a key component in the IL-1-stimulated biosynthesis of ovarian PGs.

To study the expression, localization, and in vivo hormonal regulation of type I and type II interleukin-1 (IL-1) receptors in the rat ovary.Segments of the cDNAs for rat type I and type II IL-1 receptors were cloned and used as probes in RNase protection assays and in situ hybridization. Tissues obtained from immature rats and hormonally treated rat ovaries were examined.Type I IL-1 receptor (IL-1R(1)) was ubiquitously expressed in rat tissues, including granulosa cells prepared from immature ovaries, whereas type II IL-1 receptor (IL-1R(2)) expression was restricted to macrophages, thymus, and lung. Hypophysectomy and subsequent treatment with FSH and/or diethylstilbestrol did not alter significantly the abundance of IL-1R(1) transcripts in the whole ovary. However, the relative amount of ovarian IL-1R(1) transcripts increased 7.3-fold 6 hours after the administration of hCG to pregnant mare serum gonadotropin-primed immature rats. During this time, IL-1R(1) mRNA was localized primarily in the granulosa cells. The increased expression of IL-1R(1) persisted 24 hours after hCG administration but declined to baseline by 48 hours. Ovarian expression of IL-1R(2) mRNA was observed only before ovulation in amounts that were approximately 70-fold lower than IL-1R(1).The increased intraovarian expression of IL-1R(1) in granulosa cells during the periovulatory period implies that this cell type has a heightened receptivity to IL-1 and provides further indirect evidence that this cytokine is involved in the ovulatory process.

Because of the potential importance of insulin-like growth factor binding protein-4 (IGFBP-4) to ovarian physiology and the obvious limitations imposed by in vivo-exclusive experimental paradigms, we set out to delineate the characteristics and hormonal regulation of granulosa cell-derived IGFBP-4 under in vitro circumstances.Granulosa cells obtained by follicular puncture of the ovaries from diethylstilbestrol-primed intact immature rats were subjected to culture for up to 72 hours. Insulin-like growth factor binding protein-4 mRNA extracted from culture was subjected to Northern blot hybridization. Data normalization was assured by reprobing with the hamster Chinese hamster ovary B (CHOB) cDNA, and the IGFBP-4/CHOB ratio was calculated. Conditioned culture media were subjected to Western ligand blot before and after immunoprecipitation with a rat IGFBP-4-directed polyclonal antiserum (alpha-B104).Immunoprecipitation studies revealed granulosa cell-derived IGFBP-4 to be composed of a major 24-kDa species as well as a relatively minor 27-kDa moiety. Given cultures of untreated granulosa cells from immature estrogen-treated rats, transcripts corresponding to IGFBP-4 displayed an initial temporary decline culminating in a 6-hour nadir (a decrease of 67%; P.05) followed by relatively prompt recovery (within 24 hours) to levels comparable to those noted at the outset of the culture (time 0). However, additional (albeit statistically insignificant) increments were noted at the 48-hour (but not 72-hour) time point. Treatment of granulosa cells with increasing concentration of FSH resulted in decrements of up to 30% (P.05) in the steady-state levels of IGFBP-4 transcripts. A modest, biphasic, time-dependent response was noted for IGFBP-4 transcripts after treatment with high-dose FSH (100 ng/mL), an effect characterized by 24- and 48-hour increments (51% [P.05] and 26% [P = .052] over untrated controls, respectively) and a 72-hour decrement (25%; P = .16). The concurrent provision of the C19 aromatase substrate androstenedione (10(-7) mol/L) to the culture medium from 72 hours enhanced the inhibitory effect of FSH (100 ng/mL) for a maximal decrement in IGFBP-4 transcripts of 49% (P.05). Treatment with insulin-like growth factor (IGF)-I produced limited inhibition (up to 26%) of the steady-state levels of IGFBP-4 transcripts (P.05).Findings indicate the existence of heterogeneously-sized IGFBP-4 species, of which the 27-kDa (as distinct from the 24-kDa) IGFBP-4 moiety constitutes a relatively minor component. The steady-state levels of granulosa cell-derived IGFBP-4 transcripts display relatively limited regulation in response to treatment with either FSH or IGF-I.

To review the English-language literature as it relates to the rat intraovarian insulin-like growth factor (IGF) system, special emphasis being placed on the hypotheses under study.A thorough literature review of the English-language literature.Current data support the view that intraovarian IGF-I may act as an amplifier of gonadotropin hormonal action. The role of IGF-I as a determinant of follicular fate remains a matter of study.The intraovarian IGF-I system may play a central role in ovarian physiology by amplifying gonadotropin hormonal action and, possibly, by playing a role in determining follicular fate.

Despite the potential importance of insulin-like growth factor binding protein-5 (IGFBP-5) to follicular development, the hormonal regulation of this antigonadotropic IGFBP has not been investigated. Therefore, it was the objective of this study to eludicate the role of gonadotropins and estrogen in the in vivo regulation of IGFBP-5 mRNA expression.Two models of follicular development in immature rats were used. Specifically, rats were hypophysectomized and treated with FSH and/or diethylstilbestrol (DES). Alternatively, terminal follicular development was induced in intact immature rats by pregnant mare serum gonadotropin (PMSG) and hCG. The IGFBP-5 mRNA in whole ovarian RNA was assayed by Northern blot hybridization. Localization of expression in PMSG and hCG-stimulated ovaries was further assessed by in situ hybridization.Expression of IGFBP-5 mRNA was increased in ovaries from hypophysectomized rats. Treatment with FSH and/or DES did not alter the abundance of this mRNA. Treatment with PMSG induced a transient increase in IGFBP-5 expression that was localized in a subset of alpha-inhibin-negative follicles. At later times after PMSG, IGFBP-5 expression persisted in the surface epithelium but was not detected in large preovulatory follicles. In vitro studies affirmed the antigonadotropic action of IGFBP-5.In vivo expression of IGFBP-5 in the rat ovary is moderated by hormonal treatment both in terms of total expression and follicular localization.

The understanding of the pathogenesis of polycystic ovary syndrome has advanced significantly, and improvements have been made in the treatment of the commonly associated symptoms. New data continue to implicate a derangement in insulin secretion and metabolism as a cause of the disease. Therapeutic regimens for those patients unsuccessfully treated by traditional approaches have been further validated, especially for the treatment of infertility. Future therapeutic strategies involving the retrieval of unstimulated, immature oocytes that are fertilized and matured in vitro are a possibility.

During studies on the regulation of rat ovarian steroidogenic enzymes by interleukin-1 beta (IL-1 beta), we observed substantial metabolism of 25-hydroxycholesterol to two unusual polar products. This unexpected effect was observed both in isolated granulosa cells and in whole ovarian dispersates and was also induced by tumor necrosis factor alpha, but not by insulin-like growth factor I or follicle-stimulating hormone. The effect was dependent on time and the dose of IL-1 beta and was blocked by and IL-1 receptor antagonist. The formation of the polar metabolites was inhibited by ketoconazole and trilostane, but not by aminoglutethimide. Subsequent purification of these novel metabolites and analysis by gas chromatography/ mass spectrometry, NMR, and high performance liquid chromatography revealed them to be related 7 alpha-hydroxylated hydroxycholesterols (cholest-4-ene-7 alpha,25-diol-3-one and cholest-5-ene-3 beta,7 alpha,25-triol). IL-1 beta-stimulated ovarian 7 alpha-hydroxylase activity (3-10 pmol/min/mg of cellular protein) was nearly 4-fold that of control levels using 25-hydroxycholesterol as substrate. Activities at or below control levels were observed when IL-1 beta-treated cell sonicates were boiled or assayed in the presence of NADH (rather than NADPH), indicating that involvement of a nonenzymatic process was unlikely. IL-1 beta-stimulated 7 alpha-hydroxylase activity was inhibited to basal levels by a 10-fold excess of unlabeled 25- or 27-hydroxycholesterol, but not by cholesterol, pregnenolone, progesterone, testosterone, or dehydroepiandrosterone, suggesting that ovarian 7 alpha-hydroxylase is specific for hydroxycholesterols. Furthermore, when IL-1 beta-treated ovarian cultures were incubated with radiolabeled cholesterol or testosterone, no 7 alpha-hydroxylated products were observed. We were also unable to detect any mRNA transcripts for liver cholesterol 7 alpha-hydroxylase in IL-1 beta-stimulated ovarian cultures. This study describes an ovarian hydroxycholesterol 7 alpha-hydroxylase that differs from liver cholesterol 7 alpha-hydroxylase and from other nonhepatic progestin/ androgen 7 alpha-hydroxylases. The novel finding of the regulation of a 7 alpha-hydroxylase by IL-1 beta (and tumor necrosis factor alpha) suggests a unique role for cytokines in the regulation of cholesterol metabolism in the ovary and possibly other tissues.

To evaluate the putative benefits of combined continuous hormone replacement therapy for postmenopausal women.A Medline search was performed for relevant English-language studies published during 1981-1995.Forty-two studies were identified, all using a continuous daily regimen of an estrogen and a progestin given to postmenopausal women with intact uteri.Each study was reviewed for the design, number of subjects enrolled, duration of protocol, and type and dosage of medications used. Data were extracted from texts, tables, figures, or personal communications regarding the effects of treatment on patient compliance (ie, drop-out rates), the occurrence of vasomotor symptoms, uterine bleeding patterns, endometrial histology, and lipid and bone density measurements. These data were then arranged in tabular form for the purpose of comparing and identifying trends. The lipid data from six randomized, double-blind studies that compared sequential and combined continuous regimens of conjugated equine estrogen and medroxyprogesterone acetate were further analyzed by meta-analysis. Findings revealed compliance rates of approximately 80% (range 35-100). Vasomotor symptoms improved almost universally. Irregular uterine bleeding was noted to be a common problem in the first 6 months of treatment; thereafter, most studies reported rates of amenorrhea of 75% or greater. In patients undergoing endometrial biopsy, rates of atrophic endometrium were noted to be 90-100%, and rates of endometrial hyperplasia were less than 1%. Adenocarcinoma of the endometrium was documented in two patients with a history of atypical endometrial hyperplasia and bleeding after established amenorrhea. The effects of treatment on lipid levels varied from study to study, but a meta-analysis revealed the combined continuous and sequential regimens to produce a treatment-associated decline in total and low-density lipoprotein cholesterol and an increase in high-density lipoprotein cholesterol. Studies examining bone density documented either no change or an increase with treatment.Combined continuous hormone replacement is well accepted by patients in clinical trials, effective in relieving vasomotor symptoms, and produces amenorrhea (though often after an initial period of irregular bleeding), an atrophic endometrium, and favorable changes in circulating lipids as well as maintaining bone density. Data on the impact of this regimen on long-term patient compliance, cardiovascular disease risk, and urogenital atrophy are lacking.

Endothelins (ETs) are a family of vasoactive peptides that may be involved in granulosa cell (GC) luteinization or follicular maturation. However, the precise role of ET in ovarian physiology remains unknown. We have investigated whether the rat GC is a site of ET reception and have characterized the antigonadotropic effect of ET in cultured GC from immature rats. Two major ET binding species (52 and 30 kDa) were observed after cross-linking of GC membranes with radiolabeled ET-1, although the smaller protein may represent a degradative product. Unlabeled ET-1, ET-2, or ET-3 were equipotent in displacing radiolabeled ET-1 from these putative ET receptors, with EC50s of 0.3-0.7 x 10(-9) M. Similarly, ET-1, ET-2, and ET-3 were equipotent (EC50s of about 10(-10) to 10(-9) M) in inhibiting the FSH-supported accumulation of progesterone. ET-1 (10(-7) M) also inhibited (90%) FSH-supported estrogen accumulation. Maximum progesterone inhibition (90%) by ET-1 (10(-7) M) was achieved throughout the range of FSH does and cell densities tested and by 48 h or 72 h of culture. ET-1 was not cytotoxic in the dose range tested. Forskolin-stimulated progesterone accumulation was similarly inhibited by ET-1, suggesting that ET-1 inhibits cAMP-mediated (e.g., FSH or forskolin-stimulated) progesterone accumulation. ET-1 inhibited (74%) the FSH-stimulated accumulation of cAMP, suggesting that it acts at sites related to cAMP generation or degradation. In addition, ET-1 inhibited 8-bromo-cAMP-generated progesterone accumulation (60%), suggesting that it also acts at sites distal to cAMP generation.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to elucidate the role of 5 alpha-reductase in the ovarian pubertal transition from 5 alpha-reduced to non-5 alpha-reduced steroids, we examined the characteristics and regulation of granulosa cell (GC) 5 alpha-reductase activity. Maximum activity was observed at 37 degrees C and at a pH of 6.5-8.0. Synthetic 4-aza-3-oxosteroids proved to be potent inhibitors (76% inhibition at 0.1 microM) of ovarian 5 alpha-reductase activity, and 20 alpha-DHP was a better substrate than either progesterone or testosterone (4- or 7-fold higher affinity constants, respectively). The Km (20 alpha-DHP) of the enzyme was 0.50 +/- 0.03 microM and 0.75 +/- 0.20 microM in homogenates of whole ovaries and GC, respectively. 17 beta-Estradiol was a non-competitive inhibitor (KI = 6.97 microM). 5 alpha-Reductase activity was 22-fold (immature) to 68-fold (mature) higher in liver than ovary and 4-fold higher in theca-interstitial shells than in isolated GC. Ovarian 5 alpha-reductase activity decreased markedly with age (greater than 60% inhibition in mature, randomly cycling rats as compared to immature rats). In vivo administration of follicle-stimulating hormone (FSH) to immature rats produced a dose-dependent decrease in GC 5 alpha-reductase activity (36 +/- 1.1% and 46 +/- 5.9% inhibition following 12 micrograms and 24 micrograms FSH, respectively). Similarly, the in vitro provision of FSH (100 ng/ml) to cultured GC from immature rats resulted in (36-59%) inhibition in 5 alpha-reduced steroids. Inasmuch as FSH promotes GC development and the advancement of puberty, its ability to "switch-off" ovarian 5 alpha-reductase activity may enhance the formation of biologically potent (i.e. non-5 alpha-reduced) progestins as well as the availability of aromatizable androgens, in the best interests of pubertal steroidogenesis.

The occurrence of pregnancy in the face of untreated Cushing syndrome is rare because of the high incidence of ovulatory disturbances experienced by patients with the disorder. A total of 58 patients with 65 pregnancies has been reported in the literature to date. Although pituitary-dependent adrenal hyperplasia is the most common etiology of Cushing syndrome in the general population, adrenal adenoma is more common in the pregnant population. Significant maternal morbidity is attributable to hypertension, congestive heart failure, and poor tissue healing. Prematurity and intrauterine growth retardation account for most of the perinatal morbidity; perinatal mortality is substantial. Treatment directed at relieving hypercortisolism has been instituted during pregnancy: Pituitary or adrenal surgery, chemotherapy, and pituitary irradiation have all been reported, with variable results. Information is lacking on any alteration of maternal morbidity after treatment. The impact of therapy on perinatal outcome appears limited to a reduction in the prematurity rate, but overall numbers are small and such a conclusion should be viewed with caution. No significant maternal or perinatal complications secondary to treatment itself were reported.

We, and others have recently demonstrated the ovary to be a site of interleukin-1 (IL-1) reception and action. Since IL-1 is an established mediator of inflammation and since ovulation may constitute an inflammatory-like reaction, consideration was given to the possibility that IL-1 may play an intermediary role in the ovulatory process. To begin to evaluate the above hypothesis, we have set out to evaluate rat ovarian IL-1 beta gene expression, to determine its cellular localization, and to study its modulation by key endocrine and autocrine regulatory signals. To this end, use was made of a solution hybridization/RNase protection assay in which rat ovarian total RNA (20 micrograms) was hybridized with a [32P]-labeled 272 base rat IL-1 beta antisense riboprobe. To assess rat ovarian IL-1 beta gene expression under in vivo circumstances, use was made of an established experimental model capable of simulating naturally-occurring follicular maturation, ovulation, and corpus luteum formation. Specifically, a single subcutaneous injection of PMSG (15IU/rat) was followed (48h) later by an ovulatory dose (15IU) of human chorionic gonadotropin (hCG). A faint protected fragment 222 bases long corresponding to the IL-1 beta message was detectable in whole ovarian material prior to gonadotropic stimulation. Treatment with PMSG for 48h resulted in a modest, albeit measurable increase in the densitometrically-quantified steady state levels of the ovarian IL-1 beta message. Most striking, however, were the increments noted in the relative abundance of ovarian IL-1 beta transcripts following a 6h exposure to hCG producing a 4 to 5-fold increase (P less than 0.05) over the untreated state at a time point approximately 6h prior to projected follicular rupture. Subsequent evaluation of ovarian IL-1 beta transcripts, 24 and 48h following hCG administration, revealed significant (P less than 0.05) decrements (relative to the 6h peak) to a level comparable to that seen at the conclusion of 48h of treatment with PMSG. Cellular localization studies revealed the gonadotropin-dependent IL-1 beta mRNA to be theca-interstitial cell-exclusive. To assess rat ovarian IL-1 beta gene expression under in vitro circumstances, we have set out to determine whether IL-1 itself may influence the relative level of its own message. Treatment of whole ovarian dispersates with rhIL-1 beta (10ng/ml) for 4 and 24h resulted in a marked (P less than 0.05) time-dependent increase (up to 12-fold) in the relative abundance of IL-1 beta transcripts when compared with untreated controls.(ABSTRACT TRUNCATED AT 400 WORDS)

A large body of information now supports the existence of a complete intraovarian insulin-like growth factor (IGF) system replete with ligands, receptors, and binding proteins. Although much remains to be learned, the emerging consensus would suggest that the intraovarian IGF system is concerned largely with the amplification of gonadotrophin hormonal action for the facilitation of follicular growth and development. Future studies are likely to address the central issue of indispensability and the documentation of a meaningful in vivo role for this and related putative intraovarian regulators.

To compare the circulating levels of bromocriptine after oral and vaginal administration of the drug.ExperimentalSeven ovulatory female volunteers and one hyperprolactinemic patient.Ovulatory volunteers were randomized to receive either oral or vaginal bromocriptine (2.5 mg). In a second session, the subjects were crossed-over to bromocriptine by the alternate route. An additional hyperprolactinemic patient received vaginal bromocriptine only.Serum bromocriptine and prolactin (PRL) levels were measured hourly for 12 hours in the normal volunteers and for 10 hours in the hyperprolactinemic patient.Circulating bromocriptine levels were significantly higher after vaginal bromocriptine after the 7th hour (P less than 0.05). The reduction in serum PRL was significantly greater after oral administration between 2 and 6 hours.Vaginally administered bromocriptine may result in a reduction in the overall dose required, thereby improving compliance without compromising therapeutic efficacy.

Resident ovarian macrophages have been implicated in the regulation of ovarian function, presumably through local paracrine secretion of regulatory molecules (i.e. cytokines). One such macrophage product, tumor necrosis factor (TNF) alpha, has been shown to attenuate the gonadotropin-dependent differentiation of the somatic ovarian (estrogen-producing) granulosa cell. This study examines the possibility that TNF alpha may also regulate the adjacent androgen-producing theca interstitial cell. The basal accumulation of androsterone (the major androgenic steroid), synthesized by whole ovarian dispersates from immature rats, remained unchanged following treatment with TNF alpha (30 ng/ml) alone. In contrast, concurrent treatment with increasing concentrations of TNF alpha (0.03-30 ng/ml), yielded dose-dependent inhibition of the human chorionic gonadotropin (1 ng/ml)-stimulated accumulation of androsterone. This reversible and immunoneutralizable effect of TNF alpha was characterized by a minimal effective dose of 0.1 ng/ml, a median inhibitory dose of 0.9 ng/ml, a maximal inhibitory effect of 90%, and a minimal time requirement of less than or equal to 48 h. Comparable results were obtained when using highly purified theca interstitial cells, thereby indicating that TNF alpha is capable of exerting a direct inhibitory effect at the level of the ovarian androgen-producing cell. TNF alpha action was not accounted for by alterations in the plated viable cell mass. Instead, treatment with TNF alpha resulted in significant inhibition of the human chorionic gonadotropin-supported accumulation of cAMP, the putative second messenger of gonadotropin hormonal action. TNF alpha action at sites distal to cAMP generation was associated with profound inhibition of the conversion of the [3H]pregnanolone (3 alpha-hydroxy,5 alpha-pregnane-20-one) and [3H]17 alpha-hydroxypregnanolone (3 alpha, 17 alpha-dihydroxy,5 alpha-pregnane-20-one) substrates to androsterone, suggesting stimulation of 20 alpha-hydroxysteroid dehydrogenase activity, inhibition of 17 alpha-hydroxylase/17:20 lyase activity, or both. Taken together, these findings indicate that TNF alpha, acting at relatively low concentrations, is capable of inhibiting gonadotropin-supported ovarian androgen biosynthesis by selectively modulating the activity of relevant key steroidogenic enzymes. As such, these observations suggest that the theca interstitial cell is a site of TNF alpha reception and action and that TNF alpha, possibly of resident ovarian macrophage origin, may partake in the regulation of ovarian androgen production, an effect due in part to inhibition of the activity of the key steroidogenic enzymes 17 alpha-hydroxylase/17:20 lyase.

To determine if ovarian hyperandrogenism represents enhanced gonadotropic stimulation, augmented ovarian sensitivity to gonadotropins, or both, we have undertaken to evaluate (1) the 24-hour integrated concentrations of serum total testosterone (T) and luteinizing hormone (LH) and (2) the ovarian response of T to exogenous gonadotropic stimulation. To this end, two groups of women, hyperandrogenic anovulatory (n = 4) and early follicular phase (n = 4) normally-cycling controls, were subjected to continuous blood withdrawal over 24 hours with a portable Cormed pump (Cormed Inc., Middleport, NY) and to exogenous stimulation with human chorionic gonadotropin. Our current observations support the notion that ovarian hyperandrogenism represents the combined impact of an overall increase in gonadotropic support coupled with augmented ovarian sensitivity to gonadotropic stimulation.

Summary. The present review summarizes our studies on the hormonal control of the differentiation of granulosa cells in vitro. Studies on the hormonal regulation of granulosa cell oestrogen and progestagen biosynthesis by various regulatory agents facilitate our understanding of the control processes involved in follicular maturation and luteinization in vivo. This multiple hormonal control mechanism also provides an interesting model for future studies on the diverse mode of hormone action.

A 37-year-old woman with a symptomatic 18-mm prolactin-secreting pituitary adenoma could not be managed with oral bromocriptine mesylate because of unacceptable gastrointestinal side effects. However, when given the medication intravaginally, the patient was successfully treated as assessed by evident tumor shrinkage and diminished secretory activity. Serum bromocriptine levels were approximately six to eight times higher than those reported after oral administration. The vaginal route may help reduce some of the adverse effects of bromocriptine mesylate experienced during oral administration and may possibly allow lowering of the overall effective dose by avoiding first liver passage.

The ovarian granulosa cell has recently been shown to be the site of Somatomedin C (Sm-C) production, reception, and action. To further elucidate the relevance of Sm-C to granulosa cell physiology, we have undertaken to study the regulation of its receptor under in vitro conditions using a primary culture of rat granulosa cells. Granulosa cells cultured without treatment for 72 h displayed limited, albeit measurable, specific Sm-C binding. However, continuous treatment with increasing concentrations of follicle-stimulating hormone (FSH) for the duration of the 72-h incubation period resulted in dose-dependent increments in Sm-C binding (1.7-, 2.9-, 3.9-, and 3.6-fold increases over untreated controls for 50, 100, 180, and 330 ng/ml of FSH, respectively). This apparent up regulatory action of FSH proved time-dependent, with a minimal time requirement of 24-48 h. Granulosa cell Sm-C binding was similarly enhanced following elevation of the intracellular cAMP content by a series of cAMP-generating agonists, inhibition of cAMP-phosphodiesterase activity, or the provision of nondegradable cAMP analogs. Our findings further indicate that high dose forskolin, like FSH, is capable of augmenting Sm-C binding by itself, that a relatively inactive low dose of forskolin synergizes with FSH in this regard, but that combined treatment with maximal stimulatory doses of both agonists does not prove additive. Taken together, these observations indicate that FSH is capable of exerting a stimulatory effect on granulosa cell Sm-C binding and that cAMP, its purported intracellular second messenger, may play an intermediary role in this regard.

To characterize and clarify the time-related pattern of steroidogenesis in primary testicular cultures from adult hypophysectomized rats, we have determined the pattern of C19 and C21 steroids using novel enzymatic assay techniques that rely on highly specific bacterial hydroxysteroid dehydrogenases. Steroids contained in culture media were separated in a standardized high performance liquid chromatography system and the 17 beta-hydroxy- and 17-oxosteroids were quantified by a transydrogenase assay. The individual 3 alpha-, 3 beta-, 17 beta-, and 20 alpha-hydroxysteroids were in turn measured by enzymatic oxidation. Presumptive steroid identities were confirmed by enzymatic oxidation or reduction to products that were rechromatographed and identified by co-elution with standards. Although human chorionic gonadotropin stimulated an increase in the "adult" hormones, testosterone and 4-androstene-3, 17-dione, on both Days 1 and 11 of culture, the majority of the steroids found, even on Day 1, were 3 alpha-hydroxy-5 alpha-dihydrosteroids rather than delta 4-3-oxosteroids. A specific 5 alpha-reduced, C21 steroid: 5 alpha-pregnane-3 alpha, 20 alpha-diol, increased over time and became the most abundant gonodotropin-stimulated steroid (about 5-fold in excess of testosterone) by Day 11. In contrast, testosterone was the identifying steroid of nondispersed testes from both intact and hypophysectomized rats. Studies with tracer quantities of [3H]pregnenolone in culture confirmed the initial (Day 1) preponderance of 3 alpha-hydroxy-5 alpha-dihydrosteroids, as well as the accumulation with time of 20 alpha-hydroxysteroids. These findings suggest that contrary to expectation, cultured testicular cells from young adult hypophysectomized rats display a relatively atypical steroidogenic pattern. Although the cellular mechanisms underlying the time-dependent accumulation of C21 steroids remain uncertain, these patterns suggest either regressive changes in the original parent cells or the emergence of a population of latent cells. Although of limited utility as a model for examining adult testicular physiology, primary cultures of dispersed whole testes should prove useful in studies of culture-induced phenotypic regression and the attendant alteration at the level of gene expression.

Recent studies have demonstrated the ability of somatomedin-C (Sm-C) to synergize with follicle-stimulating hormone (FSH) in the activation of cultured rat granulosa cell progesterone biosynthesis as well as the induction of luteinizing hormone (LH) receptors. Neither effect could be attributed to Sm-C-enhanced granulosa cell survival or replication, but could be accounted for, in part, by increased adenosine 3',5'-cyclic monophosphate (cAMP) generation. The present study was undertaken to determine if the synergistic property of Sm-C is FSH-selective and hence limited in relevance to follicular maturation, as well as to clarify further the role of cAMP in Sm-C-amplified agonist action. To this end, the ability of Sm-C to modulate the hormonal action of a series of physiologic as well as pharmacologic granulosa cell agonists was examined in vitro using cultured granulosa cells from immature, hypophysectomized, diethylstilbestrol-treated rats. Concurrent treatment with highly purified Sm-C (50 ng/ml) resulted in marked increases over controls in the LH-stimulated [1 ng human chorionic gonadotropin (hCG)]-and beta 2-adrenergic-stimulated (10(-6) M terbutaline) accumulation of cAMP (3.8- and 2.6-fold, respectively and progesterone (3.2- and 7.4-fold, respectively). Similarly, concurrent treatment with Sm-C also augmented the vasoactive intestinal peptidergic stimulation of granulosa cell cAMP generation (4.1-fold) and progesterone biosynthesis (2.1-fold). In contrast, Sm-C was incapable of enhancing progesterone accumulation in response to stimulation with rat prolactin, a cAMP-independent granulosa cell agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Since pituitary gonadotropins are essential for normal gonadal functions and since hypothalamic GnRH was believed to act solely on the pituitary gland, treatment with high doses of GnRH or its agonists had been predicted to be a potential means for enhancing fertility. However, early studies using GnRH as an ovulation-inducing agent in anovulatory women have been only partially successful. Similarly, the early use of GnRH and GnRH agonists in the therapy of hypogonadotropic hypogonadism in men resulted in only limited success, and actual decreases in testicular steroidogenesis were reported.

Administration of an intravenous bolus (1.0 mg) or a constant rate infusion (250 microgram/h for 4 h) of 2-hydroxyestradiol (20H-E2) to hypogonadal women, resulted in no discernible alteration in the circulating levels of LH and FSH. In contrast, progressive increments in the release of prolactin were unequivocally detected at 4.5 h after receipt of the bolus and 2.5 h following the initiation of the infusion.

It has been suggested that resident ovarian macrophages may play a role in the regulation of ovarian function through local paracrine secretion of regulatory molecule(s). It is the objective of the in vitro studies reported herein to evaluate the potential ovarian relevance of one such macrophage product, tumor necrosis factor alpha (TNF-alpha). To this end, use was made of a primary culture system of rat ovarian granulosa cells, the functional status of which was monitored by the acquisition of estrogen, progestin, and proteoglycan biosynthetic capacity. Whereas treatment with the gonadotropin follicle-stimulating hormone (FSH), a potent functional regulator, resulted in a substantial increase in the extent of aromatization (conversion of androgenic steroid precursors to estrogens), concomitant exposure to TNF-alpha (10 ng/ml) produced significant (p less than 0.05), yet reversible inhibition (71 +/- 7%) of this FSH effect. This specific activity of TNF-alpha was characterized by a projected minimal effective dose of less than 0.1 ng/ml, an apparent median inhibitory dose of 0.56 +/- 0.14 ng/ml, and a minimal time requirement of 48 h. Significantly, the direct effect of TNF-alpha could not be accounted for by a decrease in cellular viability or plating efficiency, nor by a decrease in the number of cells or their DNA content. Instead, TNF-alpha inhibited FSH hormonal action at the level of stimulatable adenylate cyclase activity, exerting no apparent effect either at the level of the FSH receptor or at site(s) of action distal to cAMP generation. The effect of TNF-alpha was not limited to the attenuation of estrogen biosynthesis, exerting qualitatively similar effects on FSH-supported progestin and proteoglycan biosynthetic capacity. As such, these findings are in keeping with the notion that subnanomolar concentrations of TNF-alpha, possibly of ovarian macrophage origin, may comprise the signal of a paracrine loop designed to attenuate gonadotropin action thereby playing a potential role in the development and/or demise of the ovarian follicle.

Methods to induce ovulation in anovulatory women have blossomed over the last three decades. The introduction of clomiphene citrate in 1960 allowed us for the first time to provoke follicle development in patients with normo or hyperestrogenic forms of anovulation. The development of human menopausal gonadotropins in the early 1960s gave us a much more powerful tool with which to influence ovulation in all forms of ovulatory disturbances. Elucidation of the pulsatile secretion of gonadotropin-releasing hormone together with its isolation and synthesis has allowed us to streamline our methods of inducing ovulation in hypothalamic amenorrheic patients by using endogenous control mechanisms to maximize both safety and effectiveness. However, there are problems yet to solve. Polycystic ovarian disease has long eluded our efforts to resolve its pathophysiology as well as to devise a consistently effective and safe means of treatment. Methods to restore ovulation in patients with polycystic ovarian disease refractory to clomiphene citrate is the quest of future investigations.

Chronic and transient hyperprolactinemia have been associated with luteal phase dysfunction in both spontaneously cycling women and those subjected to ovarian hyperstimulation by exogenous gonadotropins. It is the objective of the in vitro and in vivo studies reported herein to examine the possibility that prolactin (PRL), a known constituent of ovarian follicular fluid, may account, at least in part, for the luteinization inhibitory (LI) activity exerted by follicular fluid (FF) under both physiologic and pathophysiologic circumstances. In vitro treatment with human PRL (100 ng/ml) reduced by 57% the specific binding of luteinizing hormone (LH) to follicle-stimulating hormone(FSH)-primed cultured rat granulosa cells, an effect associated with a 2.6-fold diminution of human chorionic gonadotropin (hCG; 10 ng/ml)-stimulated progesterone biosynthesis. Importantly, the ability of PRL to exert a direct inhibitory effect on FSH-supported LH binding was fully reproduced under in vivo conditions. Inasmuch as the acquisition of LH receptors and progesterone (P) biosynthetic capacity are acceptable as criteria of granulosa cell luteinization, the authors' findings indicate that PRL, acting at concentrations achievable in vivo within the ovarian FF, may account, if only in part, for physiologically encountered LI-like activity. Moreover, subject to limitations imposed by the rat model employed, consideration also must be given to the possibility that hyperprolactinemia-associated luteal phase dysfunction may not only reflect the suppressive effect(s) of PRL at the central level of the reproductive axis, but may also be due, at least in part, to the consequences of inappropriately elevated PRL at the level of the follicular microenvironment.