Insulin-like growth factor (IGF)-I stimulates IGF-I and type 1 IGF receptor expression in cultured rat granulosa cells: autocrine regulation of the intrafollicular IGF-I system.

A growing body of information documents the existence of a complete rat intrafollicular insulin-like growth factor (IGF)-I system replete with a ligand (IGF-I), a receptor (type 1 IGF receptor) IGF binding proteins (4 and 5), and IGFBP-directed endopeptidases (4 and 5). Previous studies have established the ability of IGF-I to promote the elaboration of granulosa cell-derived IGFBP-5 and to suppress the activity of granulosa cell-derived IGFBP-5-directed endopeptidase. It was the purpose of this article to examine the effects of treatment with IGF-I on the other components of the intrafollicular IGF system, i.e., IGF-I itself and the type 1 IGF-receptor. Granulosa cells, obtained by follicular puncture from 25-d-old estrogen-primed rats were cultured in polystyrene tubes for 72 h under serum-free conditions, in the absence or presence of the indicated agents. At the conclusion of each experiment, media were discarded, and RNA was extracted and subjected to an RNase protection assay. Treatment of cultured rat granulosa cells with IGF-I resulted in a significant 1.8-fold increase in the steady-state levels of IGF-I mRNA. No effect was noted on the total cellular DNA content thereby arguing against the possibility that the relative increase in IGF-I transcripts can be ascribed to a possible treatment-induced increase in cell number in culture. The IGF-I effect was apparent (p < 0.05) at IGF-I doses as low as 1 ng/mL, minimal additional increments being noted thereafter. Treatment with insulin and des (1-3) IGF-I proved equally effective, producing 2.0- and 2.6-fold increases, respectively, thereby suggesting that the IGF-I effect may be mediated via the type 1 IGF receptor. Treatment with IGF-I also resulted in a significant (p < 0.005) increase in type 1 IGF receptor expression (2.3-fold increase), the first significant effect being noted at the 30 ng/mL dose level. Similar results obtained for insulin and des (1-3) IGF-I thereby suggest that the ability of IGF-I to upregulate the expression of its own receptor is probably type 1 IGF receptor-mediated. Taken together, these findings indicate that treatment of estrogen-primed granulosa cells with IGF-I will result in upregulation of the steady-state levels of transcripts corresponding to IGF-I itself and to its type 1 IGF receptor. These observations emphasize the importance of positive autoregulatory phenomena as determinants of the intrafollicular content of IGF-I and its receptor.

Periovulatory and interleukin-1 beta-dependent up-regulation of intraovarian vascular endothelial growth factor (VEGF) in the rat: potential role for VEGF in the promotion of periovulatory angiogenesis and vascular permeability.

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.

Transcriptional and posttranscriptional regulation of intraovarian insulin-like growth factor-binding proteins by interleukin-1beta (IL-1beta): evidence for IL-1beta as an antiatretic principal.

Intraovarian interleukin-1 (IL-1), a putative intermediary in the ovulatory cascade, has recently been implicated as an antiatretic agent. Given the reported antigonadotropic and thus atretogenic potential of granulosa cell-derived insulin-like growth factor-binding proteins (IGFBPs), we evaluated the ability of IL-1beta to regulate ovarian IGFBP-4 and -5, the IGFBP species elaborated by the rat granulosa cell. Treatment of whole ovarian dispersates of immature rat origin with increasing concentrations of IL-1beta for 96 h resulted in substantial and significant time-dependent inhibition of IGFBP-4 and IGFBP-5 transcripts compared with that in untreated controls. The IL-1 effect proved relatively specific in that no significant alterations in IGFBP transcripts were observed in the presence of select ovarian agonists, including transforming growth factor-alpha, tumor necrosis factor-alpha, endothelin-1, hepatocyte growth factor, keratinocyte growth factor, or basic fibroblast growth factor. The inhibitory effect of IL-1beta on ovarian IGFBP-4 and -5 expression was almost completely reversed in the presence of IL-1 receptor antagonist, suggesting mediation via a specific IL-1 receptor. The addition of actinomycin D to IL-1beta-pretreated whole ovarian dispersates produced a pattern of (IGFBP-4 and -5) messenger RNA decay indistinguishable from that noted for the untreated control group. Medium conditioned by IL-1beta-treated (but not untreated) whole ovarian dispersates displayed a marked diminution in the relative content of the IGFBP-4 and IGFBP-5 proteins (24- and 28- to 29-kDa proteins, respectively). Medium conditioned by IL-1beta-treated (but not untreated) whole ovarian dispersates proteolyzed [125I]IGFBP-5 (but not IGFBP-4) into fragments with apparent molecular masses of 18 and 14 kDa, respectively. In conclusion, our present observations demonstrate the ability of IL-1 to 1) inhibit the steady state levels of transcripts corresponding to IGFBP-4 and -5 in a time-dependent, relatively specific, and receptor-mediated fashion; 2) suppress the accumulation of the corresponding IGFBP proteins; and 3) stimulate the activity of the IGFBP-5-directed (but not IGFBP-4) endopeptidase, a posttranscriptional phenomenon. Our findings also suggest, by inference, that the IL-1beta-mediated inhibition of IGFBP-4 and -5 transcripts is due in part to a decrease in the rate of transcription of the corresponding genes and not to a change in the stability of the relevant messenger RNAs. Consequently, the ability of IL-1 to influence ovarian IGFBP economy appears multifaceted, comprising both transcriptional and posttranscriptional effects. To the extent that IGFBP-4 and -5 constitute atretogenic agents, our present findings support the view that IL-1beta may play an antiatretic role in the context of ovarian physiology.

Transforming growth factor-beta1 is a potent inhibitor of interleukin-1beta action in whole ovarian dispersates.

Transforming growth factor beta1 (TGFbeta1) acts as an inhibitor of the actions of interleukin-1beta (IL-1beta) in various organ systems. In order better to understand the inter|P-actions between these polypeptides in the ovary, we evaluated the effect of TGFbeta1 co-treatment on various IL-1beta-mediated actions in cultures of whole ovarian dispersates. Treatment with IL-1beta enhanced media accumulation of nitrites (4.8-fold), prostaglandin E2 (PGE2, 3. 9-fold) and lactate (2.0-fold), and enhanced glucose consumption (2. 1-fold). Treatment with TGFbeta1 alone did not significantly affect any of these parameters. However, the addition of TGFbeta1 inhibited IL-1beta-stimulated nitrite (100%), PGE2 (44%) and lactate (78%) accumulation and inhibited IL-1beta-stimulated glucose consumption (74%) in a dose-dependent manner. The addition of TGFbeta1 also suppressed the steady-state levels of IL-1beta-stimulated IL-1beta, type I IL-1 receptor and IL-1 receptor antagonist transcripts (98, 67 and 83% inhibition respectively). These data suggest that TGFbeta1 is capable of inhibiting several IL-1beta-stimulated endpoints. Since IL-1 has been identified as a possible proinflammatory mediator of ovulation and TGFbeta has been implicated as a promotor of fibrosis and healing, we speculate that IL-1 and TGFbeta might play antagonistic roles in the normal ovulatory sequence.

Characterization and hormonal regulation of a rat ovarian insulin-like growth factor binding protein-5 endopeptidase: an FSH-inducible granulosa cell-derived metalloprotease.

Previous studies established the existence of an FSH-inducible rat granulosa cell-derived insulin-like growth factor binding protein (IGFBP)-5 endopeptidase. It was the objective of this communication to characterize this activity in some detail. Exposure of [125I]rhIGFBP-5 substrate to media conditioned by FSH-treated granulosa cells (a cell-free assay) produced two rhIGFBP-5 cleavage products (estimated size 19.5 and 17.5 kDa). The acquisition of IGFBP-5 endopeptidase activity in culture proved FSH (or PMSG) to be dose and time dependent. The addition of oFSH or rhFSH to the cell-free assay in turn, proved without effect on IGFBP-5 endopeptidase activity, thereby arguing against the possibility of an FSH receptor-independent phenomenon or of contaminating pituitary-derived contribution. The ability of FSH to induce IGFBP-5 endopeptidase activity proved relatively specific in that other granulosa cell agonists such as activin-A, IGF-I, GnRH, interleukin-1beta, TNF alpha, TGF beta1, EGF, or endothelin-1 failed to do so. However, the concurrent provision of GnRH, TNF alpha, EGF, or endothelin-1 proved inhibitory to the IGFBP-5 endopeptidase-inducing property of FSH. Activin-A and TGF beta1 in turn further stimulated the FSH effect. Sensitivity to EDTA, 1,10 phenanthroline, and high concentrations (> or = 0.1 mM) of Zn2+ suggested a Zn2+ metalloprotease. Insensitivity to TIMP-1 and TIMP-2 argued against a matrix metalloprotease (MMP). Relative insensitivity to PMSF, AMPSF, aprotinin, TPCK, and benzamidine argued against the possibility of a serine protease. Insensitivity to pepstatin A and E64 argued against aspartic and cysteine proteases, respectively. Insensitivity to plasminogen activator inhibitor-1 (PAI-1) and the presumed lack of free plasminogen in serum-free culture media argued against plasmin. Proteolysis was completely inhibited over the acid pH range but proceeded unencumbered at neutral and basic pH. Competition studies using unlabeled IGFBPs (1-6) as well as cell-free proteolysis assays of [125I]-labeled IGFBP-1, 2, 3, and 6 suggested a significant level of specificity for the FSH-induced/IGFBP-5-directed endopeptidase. Centricon-mediated fractionation of FSH-conditioned media revealed the IGFBP-5 endopeptidase activity in the fraction representing proteins of molecular weight >100K. Taken together, these observations document a secreted, granulosa cell-derived, high molecular weight, FSH-inducible, IGFBP-5-selective, neutral/basic pH-favoring, non-MMP Zn2+ metalloprotease.

The mouse intraovarian insulin-like growth factor I system: departures from the rat paradigm.

Although the rat intraovarian insulin-like growth factor I (IGF-I) system is well documented, the increasing availability of null mouse mutants for components of the IGF system necessitates characterization of the mouse model as well. Therefore, we undertook to define the components of the mouse intraovarian IGF-I system and to examine its operational characteristics. The cellular pattern of ovarian gene expression was comparable in the immature rat and mouse for IGF-I and the type I IGF receptor. In both species, IGF-I messenger RNA (mRNA) is selectively expressed by granulosa cells in growing, healthy appearing follicles. Type I IGF receptor mRNA was also concentrated in granulosa cells, but was uniformly expressed in all follicles large and small, healthy and atretic appearing alike. Cellular patterns of IGF-binding protein (IGFBP) gene expression were similar in mouse and rat, except in the case of IGFBP-2. IGFBP-2 mRNA was localized to the mouse granulosa cell, in contrast to its concentration in the rat thecal-interstitial compartment. This difference in IGFBP expression pattern was also noted in cultured mouse and rat granulosa cells. Although immunoreactive IGFBP-4 (24 and 28 kDa) and IGFBP-5 (29 kDa) were shared by both species, the cultured mouse granulosa cell also featured immunoreactive IGFBP-2 (30 kDa). The mouse paradigm further differed from its rat counterpart in that a maximal dose of FSH, previously shown to suppress the elaboration of rat granulosa cell-derived IGFBPs, was without effect. The addition of IGF-I proved stimulatory to the accumulation of the 28- to 29-kDa IGFBPs, as previously reported for the rat. However, IGF-I proved inhibitory to the accumulation of the 24-kDa IGFBP (presumptive nonglycosylated IGFBP-4); no consistent effect was reported for the rat model. Functional comparisons of mouse and rat ovarian cell cultures revealed qualitatively comparable FSH-stimulated steroidogenesis, disposition of radiolabeled pregnenolone, IGF-I-amplified FSH action, and IGFBP-mediated antigonadotropic activity. These findings indicate that the mouse intrafollicular IGF-I system differs from the rat paradigm in both the makeup and regulation of granulosa cell-derived IGFBPs as well as in the intensity and character of the steroidogenic process. Studies employing the mouse model must take into account these important distinctions relative to the more established rat paradigm.

Interleukin (IL)-1beta increases glucose uptake and induces glycolysis in aerobically cultured rat ovarian cells: evidence that IL-1beta may mediate the gonadotropin-induced midcycle metabolic shift.

This communication explores the possibility that interleukin (IL)-1beta, a putative intermediary in the ovulatory process, may take part in the gonadotropin-driven midcycle diversion of ovarian carbohydrate metabolism toward glycolysis. We examined the effect of treatment with IL-1beta on glucose metabolism in aerobically cultured whole ovarian dispersates from immature rats. Treatment with IL-1beta increased cellular glucose consumption/uptake, stimulated extracellular lactate accumulation and media acidification, and decreased extracellular pyruvate accumulation in a receptor-mediated, time-, dose- and cell density-dependent manner. Endogenous IL-1beta-like bioactivity was shown to mediate the ability of gonadotropins to exert these same metabolic effects. The IL-1beta effect was also (1) apparent over a broad range of glucose concentrations, inclusive of the putative physiological window; (2) relatively specific, because tumor necrosis factor-alpha and insulin were inactive; (3) contingent upon cell-cell cooperation (4) and reliant on de novo protein synthesis. Comparison of the molar ratios of lactate accumulation to glucose consumption in IL-1beta-replete vs. IL-1beta-deplete cultures suggests that IL-beta promotes the conversion of all available glucose to lactate but that other substrates for lactate production may also exist. However, no lactate was generated by cells grown under glucose-free conditions. Taken together, our data suggest that IL-1beta may act as a metabolic hormone in the ovary. Subject to the limitations of the in vitro paradigm, our data also suggest that IL-1beta may mediate the gonadotropin-associated midcycle shift in ovarian carbohydrate metabolism. By converting the somatic ovarian cells into a glucose-consuming glycolytic machinery, IL-1beta may establish glycolysis as the main energy source of the relatively hypoxic preovulatory follicle and the resultant cumulus-oocyte complex. The consequent oxygen sparing may conserve the limited supply of oxygen needed for vital biosynthetic processes such as steroidogenesis. This adaptational response may also provide the glycolytically incompetent oocyte with the obligatory tricarboxylic cycle precursors it depends on to meet the increased energy demands imposed upon it by the resumption of meiosis.

Rat ovarian granulosa cell as a site of endothelin reception and action: attenuation of gonadotropin-stimulated steroidogenesis via perturbation of the A-kinase signaling pathway.

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)

IGF-I stimulates granulosa cell-derived insulin-like growth factor binding protein-5: evidence for medication via type I IGF receptors.

Although the precise role of insulin-like growth factor binding proteins (IGFBPs) in ovarian physiology remains a matter of study, existing data suggest a possible antigonadotropic role in the context of follicular atresia. Given the above and the need for improved understanding of the regulation of ovarian IGFBPs, we have set out to explore the ability of IGF-I to modulate IGFBP levels in cultured rat granulosa cells. Specifically, granulosa cells (5 x 10(5) viable cells/dish) from immature (23-25 days old), estrogen-primed rats were cultured under serum-free conditions for 72 h in the absence or presence of IGF-I. At the conclusion of this incubation period, media samples were collected and subjected to Western ligand blotting. Treatment with IGF-I (100 ng/ml) resulted in a substantial (P < 0.05) increase in the accumulation of IGFBP-5, the major 28-29 kDa IGFBP species. Subsequent studies revealed this effect of IGF-I to be both dose- and time-dependent. A similar effect was noted for insulin at dose levels 1-10 micrograms/ml at which cross-reaction with the type I IGF receptor (but not with IGFBPs) has been amply documented. Des (1-3) IGF-I, a type I receptor-selective ligand with markedly reduced avidity for IGFBPs, proved substantially more potent (as a promoter of IGFBP-5 accumulation) than its native counterpart. In contrast, treatment with IGF-II or [Leu27]IGF-II, type II IGF receptor-selective ligands, yielded a more limited effect on IGFBP-5 accumulation in keeping with an overall rank order of potency of des (1-3) IGF-I > IGF-I > IGF-II > or = [Leu27]IGF-II.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat ovarian insulin-like growth factor-binding protein-6: a hormonally regulated theca-interstitial-selective species with limited antigonadotropic activity.

Most but not all of the established insulin-like growth factor-binding proteins (IGFBPs) are expressed in the rat ovary. To continue the process of characterizing these ovarian IGFBPs, a solution hybridization/RNase protection assay was used to study IGFBP-6 gene expression, cellular localization, and hormonal regulation in the immature rat ovary. Total RNA was hybridized with a 458-base long 32P-labeled rat IGFBP-6 cRNA. A single protected fragment (380 bases long) corresponding to IGFBP-6 transcripts was identified in RNA from ovary and lung, but not kidney or liver. The amount of IGFBP-6 transcripts was higher in ovaries from immature rats (25-28 days old) than in ovaries from adult rats and was higher in theca-interstitial than in granulosa cell preparations. Hypophysectomy resulted in a significant (P < 0.05) 2.3 +/- 0.7-fold (mean +/- SD) increase in whole ovarian IGFBP-6 transcripts. This suggests that ovarian IGFBP-6 gene expression is subject to inhibition by one or more pituitary hormones. Therefore, the effect of replacement of FSH, GH, diethylstilbestrol (DES), or combinations thereof was evaluated. Treatment with FSH resulted in a 2.4-fold decrease (P < 0.05) in the abundance of ovarian IGFBP-6 transcripts relative to that in hypophysectomized controls. Provision of DES yielded comparable results. Moreover, the combined provision of FSH and DES resulted in a synergistic decrease (6.0-fold) in ovarian IGFBP-6 transcripts. In contrast, treatment of hypophysectomized rats with GH proved without effect. However, treatment with FSH or DES in addition to GH resulted in a decrease in ovarian IGFBP-6 transcripts (3.9- and 2.7-fold, respectively). To assess the role of ovarian IGFBP-6, its influence on gonadotropin action in primary cultures of rat granulosa cells was also studied. Increasing concentrations (0.01-1 micrograms/ml) of recombinantly expressed human IGFBP-6 did not inhibit the FSH-supported accumulation of either progesterone or estradiol. These findings 1) establish the theca-interstitial compartment of the immature rat ovary as a prominent site of IGFBP-6 gene expression, 2) implicate FSH and DES as inhibitors of IGFBP-6 transcript levels in the whole ovary, 3) and disclose the limited antigonadotropic action of IGBP-6F on the rat granulosa cell.

A kinase-mediated regulation of granulosa cell-derived insulin-like growth factor binding proteins (IGFBPs): disparate response sensitivities of distinct IGFBP species.

Rat granulosa cell-derived insulin-like growth factor (IGF) binding proteins (BPs) have been found subject to biphasic dose-dependent regulation by FSH under in vitro circumstances. Since cAMP may play an intermediary role in FSH hormonal action, we have undertaken to characterize the A kinase-mediated regulation of the elaboration of IGFBPs by cultured rat granulosa cells. Treatment with increasing concentrations of prostaglandin E2 or choleragen, both established cAMP-generating agonists, produced biphasic dose-dependent regulation of the release of the major 28-29 kilodalton (kDa) IGFBP species while promoting the release of their minor 24 (and 19) kDa counterparts. Similar effects were noted for other cAMP-generating agonists including vasoactive intestinal peptide and forskolin (a potent activator of adenylate cyclase). Moreover, concomitant treatment with a functionally inert low dose (10(-7) M) of forskolin, substantially potentiated the FSH (10 ng/ml)-mediated inhibition of the elaboration of the 28-29 kDa IGFBPs. Application of decreasing dilutions of the invasive adenylate cyclase toxin of bordetella pertussis (but not of an inactive mutant strain) yielded monophasic dose-dependent modulation of the release of the 28-29 kDa IGFBPs while effecting biphasic regulation of the 24 kDa moiety. Concurrent treatment with 1-methyl-3-isobutylxanthine (a potent inhibitor of cAMP phosphodiesterase activity) at the 10(-4) M level resulted in profound (P < 0.05) inhibition of the (low dose) FSH (3 ng/ml)-supported accumulation of the major 28-29 kDa IGFBP species, an effect associated with modest (2.5-fold) induction (P < 0.05) of the minor 24 kDa IGFBP moiety. Lastly, provision of increasing concentrations of nondegradable lipophilic analogs of cAMP (i.e. (Bu)2cAMP and 8-bromoadenosine cAMP resulted in biphasic dose-dependent modulation of the release of the major 28-29 kDa IGFBP doublet while producing an increase in the accumulation of the minor 24 kDa IGFBP species. Taken together, these observations suggest that the ability of low dose FSH to stimulate and of high dose FSH to inhibit the elaboration of the 28-29 kDa IGFBP species may entail activation of the A-kinase transduction pathway. Similar conclusions appear to apply for the ability of FSH to regulate (albeit at a lower response sensitivity level) the biphasic elaboration of the 24 kDa IGFBP moiety. As such, these observations point out the disparate response sensitivities of distinct IGFBP species, thereby suggesting a novel potent mechanism through which FSH may determine the relative distribution pattern of granulosa cell-derived IGFBPs and the consequent overall IGF responsiveness of this cell type.

Granulosa cell-derived insulin-like growth factor (IGF) binding proteins are inhibitory to IGF-I hormonal action. Evidence derived from the use of a truncated IGF-I analogue.

An increasing body of information now suggests that insulin-like growth factor (IGF) binding proteins (BPs) may serve as antigonadotropins at the level of the ovary. It is the objective of the present communication to evaluate the functional role of endogenous (granulosa cell-derived) IGFBPs by exploiting the unique properties of des(1-3)IGF-I, a naturally occurring IGF-I analogue characterized as a weak ligand of IGFBPs but not of type I IGF receptors. Given IGFBP-replete circumstances, des(1-3)IGF-I proved more potent (10-fold) than its intact counterpart in promoting the follicle stimulating hormone (FSH)-stimulated accumulation of progesterone by cultured rat granulosa cells. In contrast, des(1-3)IGF-I proved virtually equipotent to the unmodified principle under IGFBP-deplete circumstances. Taken together, these findings are in keeping with the notion and that the apparently enhanced potency of des(1-3)IGF-I (under IGFBP-replete conditions) is due to its diminished affinity for endogenously generated IGFBPs and that rat granulosa cell-derived IGFBPs are inhibitory to IGF (and thus inevitably to gonadotropin) hormonal action. Accordingly, the reported ability of gonadotropins to attenuate IGFBP release by granulosa cells may be designed to enhance the bioavailability of endogenously generated IGFs in the best interest of ovarian steroidogenesis.

Interleukin-1 stimulates ovarian prostaglandin biosynthesis: evidence for heterologous contact-independent cell-cell interaction.

An increasing body of information now suggests the existence of a complete intraovarian IL-1 system replete with ligands, receptor, and receptor antagonist. Since IL-1 is an established mediator of inflammation and since ovulation may constitute an inflammatory-like reaction, consideration may be given to the possibility that IL-1 may play an intermediary role in the ovulatory process. To further assess the above hypothesis, we have set out to determine whether IL-1 is capable of promoting ovarian prostaglandin biosynthesis, an established component of the ovulatory cascade. Cultured whole ovarian dispersates from immature (25 day old) rats constitutively elaborated major prostaglandin species (PGE2 greater than PGF2 alpha) in a cell density-dependent fashion. Treatment with IL-1 produced dose-dependent increments in prostaglandin (PGE2 greater than PGF2 alpha) accumulation as compared with untreated controls. Comparable cellular densities of untreated or IL-1 beta-treated whole ovarian dispersates elaborated substantially more PGE2 as compared with isolated granulosa or theca-interstitial cell preparations suggesting a requirement for cell-cell interaction. Indeed, cell contact-dependent reconstitution experiments involving isolated granulosa and theca-interstitial cells at a projected physiologic ratio of 4:1 revealed synergistic interactions in the elaboration of PGE2 under both basal and IL-1 beta-treated circumstances. Identical results were obtained for cell contact-independent heterologous (but not homologous) coculture experiments. Taken together, our present findings reveal optimal basal and IL-1-stimulated ovarian prostaglandin (PGE2 greater than PGF2 alpha) biosynthesis to require heterologous, contact-independent, presumably humorally-mediated, cell-cell interaction. These observations along with the demonstration of the gonadotropin-dependent preovulatory induction of ovarian IL-1 beta gene expression provide strong support for the view that IL-1 may be the centerpiece of an intraovarian regulatory loop concerned with the promotion of the preovulatory cascade.

Insulin-like growth factors: the ovarian connection.

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.

Cytokine-mediated regulation of ovarian function: interleukin-1 inhibits gonadotropin-induced androgen biosynthesis.

Resident ovarian macrophages have long been recognized as potential in situ regulators of ovarian function, presumably through local paracrine secretion of regulatory molecules (i.e. cytokines). One such macrophage product, interleukin-1 (IL-1) has recently been shown to exert profound regulatory effects at the level of the ovarian granulosa cell. In this report, we examine the possibility that the adjacent theca-interstitial (androgen-producing) cell may also be a site of IL-1 reception and action. The basal accumulation of androsterone, the major androgenic steroid synthesized by whole ovarian dispersates from immature rats, in the presence of insulin (1 microgram/ml), increased 8- to 9-fold after treatment with human CG (1 ng/ml). Although IL-1 alpha or IL-1 beta (10 ng/ml) by themselves were without effect on basal androsterone accumulation, both cytokines (IL-1 beta greater than IL-1 alpha) inhibited human CG hormonal action (in the presence of insulin) in a dose-dependent manner, the maximal inhibitory effect being 75%. Similar results were obtained when using highly purified theca-interstitial cells derived from the same animal model suggesting that IL-1-attenuated androgen biosynthesis is due, at least in part, to IL-1 acting directly at the level of the theca-interstitial cells. The IL-1 effect proved relatively specific since all other known interleukins (IL-1, IL-3, IL-4, IL-5, and IL-6) were without effect. Moreover, IL-1 beta action was effectively immunoneutralized when concurrently applied with anti-IL-1 beta (but not nonimmune) sera. Significantly, the antigonadotropic action of IL-1 could not be accounted for by a decrease in the viable cell mass. Tracer studies with radiolabeled steroid substrates suggested that IL-1-attenuated ovarian androsterone accumulation is due, if only in part, to inhibition of transformations catalyzed by (theca-interstitial) 17 alpha-hydroxylase/17:20 lyase, stimulation of theca-interstitial (or granulosa 20 alpha-hydroxysteroid dehydrogenase-mediated conversions, or both. Taken together, these findings indicate that relatively low concentrations of IL-1, possibly originating from somatic ovarian cells or resident ovarian macrophages, are capable of exerting an inhibitory effect upon gonadotropin-supported androgen production. As such, these and previous observations suggest that intraovarian IL-1 may play a dual regulatory role in the developing ovarian follicle by targeting both the granulosa and theca-interstitial cells as its sites of action.

In vivo regulation of granulosa cell type I insulin-like growth factor receptors: evidence for an inhibitory role for the putative endogenous ligand(s) of the ovarian gonadotropin-releasing hormone receptor.

The putative endogenous occupant(s) of the ovarian GnRH receptor may play a role as an intraovarian regulator. In this communication we explore the possibility that in vivo activation of the ovarian GnRH receptor may prove heteroregulatory to the murine granulosa type I insulin-like growth factor (IGF) receptor complement. To eliminate potentially confounding pituitary involvement, exclusive use was made of hypophysectomized rats, thereby allowing study of the direct ovarian effect(s) of GnRH receptor ligands. Treatment of immature hypophysectomized rats, thereby allowing study of the direct ovarian effect(s) of GnRH receptor ligands. Treatment of immature hypophysectomized diethylstilbestrol-primed rats with a GnRH agonist (25 micrograms/rat, twice daily) for 2.5 days resulted in a 2.1-fold decrease in FSH (10 micrograms/rat, twice daily)-inducible (but not basal) specific [125I]IGF-I binding to isolated granulosa cells. Scatchard analysis of the binding data revealed this effect to be due in large measure to decreased binding capacity (46% inhibition) rather than affinity (2.5 x 10(-9) M). Although in vivo treatment with a GnRH antagonist (25 micrograms/rat, twice daily) by itself proved without significant effect on the specific binding of IGF-I to isolated granulosa cells, the concurrent provision of a minimally effective dose of FSH (1 microgram/rat, twice daily) resulted in a 2.8-fold amplification of the FSH effect consequent to enhanced binding capacity (110%), but not affinity (2.2 x 10(-9) M). Significantly, this level of binding proved comparable to that induced by a maximally effective dose of FSH when used by itself. Combined pretreatment with identical doses of both peptide analogs had little or no effect on granulosa cell IGF-I binding, suggesting stereospecificity of action and mutual neutralization by the opposing actions of the GnRH receptor ligands employed. The ability of ligands of the GnRH receptor to alter the granulosa cell type I IGF receptor complement proved functionally significant, as assessed by corresponding alterations in IGF-I hormonal action. Indeed, pretreatment with a GnRH antagonist resulted in a 2.1-fold enhancement of IGF-I (50 ng/ml)-supported proteoglycan biosynthesis, with the agonistic analog producing a diametrically opposite effect. Moreover, the ovarian effects of GnRH receptor ligands were not limited to type I IGF receptor binding; comparable effects were noted on basal and hCG-stimulated accumulation of progesterone by cultured granulosa cells, ovarian weight, ovarian protein content, as well as size and number of antral follicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytokine-mediated regulation of ovarian function. Tumor necrosis factor alpha inhibits gonadotropin-supported ovarian androgen biosynthesis.

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.

Ovarian granulosa cell-derived insulin-like growth factor binding proteins: modulatory role of follicle-stimulating hormone.

Recent observations disclosed the multiplicity of granulosa cell-derived high affinity insulin-like growth factor binding proteins (IGFBPs) and revealed the striking ability of high doses of FSH to suppress their constitutive release under both in vitro and in vivo circumstances. It is the objective of this communication to characterize in greater detail the modulatory effect(s) exerted by FSH on the elaboration of IGFBPs by granulosa cells from immature, estrogen-primed rats. The ability of FSH to regulate the elaboration of granulosa cell-derived IGFBPs proved dose-dependent but biphasic in nature. Specifically, FSH concentrations in the range of 1-3 ng/ml applied for 72 h produced a significant (P less than 0.05) increase in polyethylene glycol-precipitable [125I]IGF-I binding activity corresponding to all IGFBP species detectable by ligand blotting. In contrast, treatment with higher concentrations (greater than or equal to 10 ng/ml) of FSH resulted in progressive dose-dependent inhibition of the constitutive release of IGF-I binding activity (80% inhibition at the 100 ng/ml dose level) and the virtual elimination of all detectable IGFBP species. Time-course studies disclosed a significant (P less than 0.05) initial (apparent at the 24-h time point) stimulatory effect of a high dose of FSH (100 ng/ml) corresponding mostly (but not solely) to the single minor (23K) IGFBP band. In contrast, more prolonged exposure to FSH (greater than or equal to 48 h) produced progressive time-dependent decrements in IGF-I binding activity, an effect associated with a decrease in the relative representation of the major band doublet (28-29K), the 23K species being all but eliminated under these experimental circumstances. Hypophysectomy produced significant (P less than 0.05) inhibition of the subsequent in vitro release of granulosa cell-derived precipitable IGF-I binding activity strongly suggesting that (presumptively stimulatory) pituitary principles other than FSH are likely involved in the regulation of granulosa cell IGFBP release and that the trophic influence of these putative agents appears to outweight the potential disinhibition of FSH hormonal action. Taken together, these findings indicate that the pituitary regulation of granulosa cell-derived IGFBPs is complex and is not FSH-exclusive. Our findings further indicate that the ability of FSH to regulate granulosa cell-derived IGFBPs is dose- and time-dependent but biphasic in nature, an effect characterized by an early low-dose stimulatory effect and a late high-dose inhibitory effect.(ABSTRACT TRUNCATED AT 400 WORDS)