Are estrogens of import to primate/human ovarian folliculogenesis?

The notion that estrogens play a meaningful role in ovarian folliculogenesis stems from a large body of in vitro and in vivo experiments carried out in certain rodent models, (e.g., rats) wherein the stimulatory role of estrogen on granulosa cell growth and differentiation is undisputed. However, evidence derived from these polyovulatory species may not be readily generalizable to the monoovulatory subhuman primates, let alone the human. Only recently, significant observations on the ovarian role(s) of estrogen have been reported for the primate/human. It is thus the objective of this communication to review the evidence for and against a role for estrogens in primate/human ovarian follicular development with an emphasis toward the application of the concepts so developed to contemporary reproductive physiology and to the practice of reproductive medicine. The role(s) of estrogens will be examined not only by analyzing the physiological evidence to the effect that these hormones control ovarian function and follicular growth, but also by summarizing the molecular evidence for the existence and distribution of the cognate receptors.

Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function.

A relatively large body of evidence now appears to support the existence of the essential ingredients for novel intraovarian IGF-driven control mechanisms. Indeed, evidence presented in this communication is in keeping with the possibility that the granulosa cell may be the site of IGF production, reception, and action. Although the relevance of IGFs to ovarian cell types other than the granulosa cell is largely unknown, one cannot at the present time exclude the possibility of nongranulosa cell contributions to intraovarian IGF production, reception, and action. Indeed, preliminary affinity cross-linking studies (Adashi, Resnick, Svoboda, Van Wyk and D'Ercole; unpublished data) suggest the existence of type-I and type-II receptors in nongranulosa cell compartments. The above notwithstanding, IGFs of granulosa (and possibly circulatory) origins may interact with granulosa cell autoreceptors either independently or in synergy with other granulosa cell agonists. According to this view, IGFs may act in the autocrine mode to stimulate granulosa cell replication on the one hand and promote granulosa cell differentiation on the other. Although proliferation and terminal differentiation may prove mutually exclusive under some circumstances, coexistence of the two processes is being increasingly recognized. In this context, some studies of porcine granulosa cells support a dual role for IGFs in granulosa cell ontogeny. As such, the IGFs can be added to a growing list of growth factors known to modulate granulosa cell growth and function, including EGF, PDGF, and FGF. Our findings indicate that Sm-C/IGF-I synergizes with FSH in the induction of rat granulosa cell aromatase activity at nanomolar concentrations compatible with its granulosa cell receptor binding affinity (thus far studied only in porcine cells. A role for Sm-C/IGF-I in the regulation of this key granulosa cell function would be in keeping with the possibility that Sm-C/IGF-I may partake in the assertion and maintenance of dominance by the selected follicle(s) or in promoting juvenile and early follicular development. Moreover, the ability of Sm-C/IGF-I to potentiate this and other FSH-driven ovarian functions may also account, at least in part, for the puberty-promoting effect of growth hormone. This permissive action of growth hormone has been initially suggested by observation in growth hormone-deficient rats, mice (dwarf mutants, and humans (sporadic, hereditary or acquired growth hormone deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

The morphogenic/cytotoxic and prostaglandin-stimulating activities of interleukin-1 beta in the rat ovary are nitric oxide independent.

Nitric oxide (NO) has been implicated as a mediator of physiologic and pathologic cellular injury. Since the cytokine interleukin-1 beta (IL-1 beta) induces nitric oxide synthase (NOS) activity as well as effects morphogenic/cytotoxic changes and increased prostaglandin (PGE2) levels in cultured whole ovarian dispersates, we set out to determine whether these actions are interrelated. Treatment with IL-1 beta resulted in a marked increase in media nitrite and nitrate accumulation, morphological alterations, and increased release of lactate dehydrogenase (LDH) into media. Addition of IL-1 receptor antagonist (RA) eliminated these IL-1 beta effects. In contrast, specific inhibitors of NOS failed to reverse IL-1 beta-induced morphogenic changes or LDH release in spite of complete reduction of media nitrite to control levels. Similarly, treatment with transforming growth factor beta 1, inhibited IL-1 beta-induced nitrite accumulation, but had no effect on the morphologic or cytotoxic endpoints. Moreover, the addition of sodium nitroprusside, an NO generator, resulted in progressive increments in media nitrite content without a corresponding increase in the IL-1 beta-associated morphogenic changes or media LDH content. Furthermore, IL-1-induced PGE2 accumulation remained unaffected by specific NOS inhibition. These observations support the view that NO does not mediate the morphogenic/cytotoxic or inflammatory-like (e.g., PGE2 inducing) properties of IL-1 beta in cultured whole ovarian dispersates. Although the precise role of NO in ovarian physiology remains unknown, it is possible that NO participates in the periovulatory modulation of ovarian blood flow by virtue of its potent vasodilatory activity.

Human intraovarian interleukin-1 (IL-1) system: highly compartmentalized and hormonally dependent regulation of the genes encoding IL-1, its receptor, and its receptor antagonist.

To delineate the scope of the human intraovarian IL-1 system we used a solution hybridization/RNase protection assay to test for expression of the genes encoding IL-1, its type I receptor (IL-1R), and its receptor antagonist (IL-1RA). IL-1 transcripts were not detected in whole ovarian material from days 4 or 12 of an unstimulated menstrual cycle but transcripts (IL-1 beta much greater than IL-11 alpha) were detected in preovulatory follicular aspirates from gonadotropin-stimulated cycles. Concurrently obtained peripheral monocytes did not contain IL-1 beta transcripts but macrophage-depleted follicular aspirates did, thus implicating the granulosa cells as the site of IL-1 expression. IL-1R transcripts were detected in RNA from whole ovaries and follicular aspirates but not in RNA from peripheral monocytes. IL-1RA transcripts were detected in whole ovarian material as well as in macrophage-free follicular aspirates. Cultured human granulosa and theca cells did not contain mRNA for IL-1 beta or IL-1RA but did contain mRNA for IL-1R. Treatment of cell cultures with forskolin (25 microM) induced IL-1 beta transcripts in granulosa but not theca cells. Forskolin also increased the basal levels of IL-1R transcripts in both granulosa and theca cells but did not induce IL-RA transcripts in either cell type. Taken together, these findings reveal the existence of a complete, highly compartmentalized, hormonally dependent intraovarian IL-1 system replete with ligands, receptor, and receptor antagonist.

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.

The midcycle increase in ovarian glucose uptake is associated with enhanced expression of glucose transporter 3. Possible role for interleukin-1, a putative intermediary in the ovulatory process.

This study characterizes the rat ovary as a site of hormonally dependent glucose transporter (Glut) expression, and explores the potential role of interleukin (IL)-1, a putative intermediary in the ovulatory process, in this regard. Molecular probing throughout a simulated estrous cycle revealed a significant surge in ovarian Glut3 (but not Glut1) expression at the time of ovulation. Treatment of cultured whole ovarian dispersates from immature rats with IL-1beta resulted in upregulation of the relative abundance of the Glut1 (4.5-fold) and Glut3 (3.5-fold) proteins as determined by Western blot analysis. Other members of the Glut family (i.e., Gluts 2, 4, and 5) remained undetectable. The ability of IL-1 to upregulate Glut1 and Glut3 transcripts proved time-, dose-, nitric oxide-, and protein biosynthesis-dependent but glucose independent. Other ovarian agonists (i.e., TNF alpha, IGF-I, interferon-gamma, and insulin) were without effect. Taken together, our findings establish the mammalian ovary as a site of cyclically determined Glut1 and Glut3 expression, and disclose the ability of IL-1 to induce the ovarian expression as well as translation of Glut1 and Glut3 (but not of Gluts 2, 4, or 5). Our observations also establish IL-1 as the first known regulator of Glut3, the most efficient Glut known to date. In so doing, IL-1, a putative component of the ovulatory process, may be acting to meet the increased metabolic demands imposed on the growing follicle and the ovulated cumulus-enclosed oocyte.

Ovulation-selective genes: the generation and characterization of an ovulatory-selective cDNA library.

Ovulation-selective/specific genes, that is, genes preferentially or exclusively expressed during the ovulatory process, have been the subject of growing interest. We report herein studies on the use of suppression subtractive hybridization (SSH) to construct a 'forward' ovulation-selective/specific cDNA library. In toto, 485 clones were sequenced and analyzed for homology to known genes with the basic local alignment tool (BLAST). Of those, 252 were determined to be nonredundant. Of these 252 nonredundant clones, 98 were analyzed by probing mouse preovulatory and postovulatory ovarian cDNA. Twenty-five clones (26%) failed to show any signal, and 43 cDNAs tested thus far display a true ovulation-selective/specific expression pattern. In this communication, we focus on one such ovulation-selective gene, the fatty acid elongase 1 (FAE-1) homolog, found to be localized to the inner periantral granulosa and to the cumulus granulosa cells of antral follicles. The FAE-1 gene is a beta-ketoacyl-CoA synthase belonging to the fatty acid elongase (ELO) family, which catalyzes the initial step of very long-chain fatty acid synthesis. All in all, the present study accomplished systematic identification of those hormonally regulated genes that are expressed in the ovary in an ovulation-selective/specific manner. These ovulation-selective/specific genes may have significant implications for the understanding of ovarian function in molecular terms and for the development of innovative strategies for both the promotion of fertility and its control.

Monocyte chemotactic protein-1 expression is increased in human gestational tissues during term and preterm labor.

OBJECTIVE: Microarray analysis was used to characterize the labor-selective transcriptome of the human myometrium during labor. One highly up-regulated transcript, monocyte chemotactic protein-1 (MCP-1), was further characterized. METHODS: Expression of MCP-1 was evaluated in the myometrium, the placenta, the gestational membranes (GM) and the amniotic fluid (AF) by real time RT-PCR, Northern blot analysis and ELISA. The level of immunoreactive (IR) MCP-1 content of primary myometrial cultures treated with inflammatory cytokines was quantified by ELISA. RESULTS: Up-regulation of the myometrial MCP-1 transcript in term laboring patients was demonstrated by microarray and confirmed by real time (RT)-PCR and Northern blot analysis. Increased MCP-1 transcripts were demonstrated in GM during term labor. The IR content of myometrial MCP-1 was increased during term labor and in the AF from patients experiencing preterm delivery. Levels of IR MCP-1 increased in myometrial cultures in response to interleukin 1-beta. CONCLUSION: The expression of myometrial MCP-1 was significantly increased during term labor and was similarly increased in vitro in response to interleukin 1-beta, a pro-inflammatory substance known to play a role in preterm birth. The increased IR content of MCP-1 within the AF preceding preterm delivery may render this protein a useful predictor of preterm birth.

Intraovarian regulation: peptidergic signaling systems.

Intraovarian regulation, an evolving field, is now at a crossroad. Although a number of putative intraovarian regulators appear to be of import to ovarian physiology, none has thus far been demonstrated to be indispensable to in vivo ovarian function. That notwithstanding, it is already clear that optimal gonadotropin hormonal action is highly contingent upon the input of tissue-based regulatory principles. It is with a strong sense of excitement that future work in this evolving area is anticipated.

Insulin-like growth factor receptor gene expression in the rat ovary: divergent regulation of distinct receptor species.

The intraovarian insulin-like growth factor (IGF) system constitutes a triad composed of ligands, receptors, and binding proteins. Although conventional radioligand receptor assays have documented the presence of specific receptors for insulin and insulin-like peptides in some rat somatic ovarian cell types, the exact cellular localization and hormonal regulation of the receptors in question remain matters of inquiry. To reevaluate the very presence, cellular localization, and hormonal regulation of the IGF receptor gene family in the rat ovary, solution hybridization/RNase protection assays were used wherein ovarian total RNA (20 micrograms) from immature (21-23 days old) rats was hybridized with 32P-labeled type I IGF receptor, type II IGF/mannose-6-phosphate receptor, and insulin receptor riboprobes. Single protected fragments 261 (type I IGF receptor), 500 (type II IGF/mannose-6-phosphate receptor), and 478 (insulin receptor) bases long were evident in whole ovary, granulosa, and theca-interstitial cells. Hypophysectomy of immature rats led to significant (P less than 0.05) albeit variable decrements in the relative (densitometrically quantified) ovarian abundance of transcripts corresponding to the type I IGF (but not insulin or type II IGF/mannose-6-phosphate) receptor. Treatment of immature hypophysectomized rats with FSH (10 micrograms/rat.day x 2.5 days) resulted in a significant (P less than 0.05) increase (4-fold) in transcripts corresponding to the type I IGF receptor in both whole ovarian material and freshly isolated granulosa cells. Similar (3.7-fold) increments (P less than 0.05) were noted after treatment with a diethylstilbestrol-containing sc silastic implant applied for a total of 5 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonistic interactions of transforming growth factors in the regulation of granulosa cell differentiation.

The role of transforming growth factors (TGFs) in the acquisition of granulosa cell aromatase activity was investigated in vitro in a primary culture of granulosa cells harvested from immature, diethylstilbestrol-treated rats. Basal aromatase activity, as assessed by the generation of radioimmunoassayable estrogen, was negligible, remaining unaffected by treatment with either TGF alpha or TGF beta applied by themselves at the 10 ng/ml dose level. Whereas treatment with FSH produced a substantial increase in the extent of aromatization, concurrent treatment with TGF beta (0.01-10 ng/ml) resulted in dose-dependent augmentation of the FSH effect with an apparent median effective dose of 224 +/- (SE) 32 pg/ml (ca. 9 pM), and a maximal effect 3.6-fold greater than that induced by FSH alone. In contrast, concomitant treatment with TGF alpha (0.01-10 ng/ml) resulted in dose-dependent attenuation of FSH action with an apparent median inhibitory dose of 330 +/- (SE) 40 pg/ml (ca. 60 pM), and a maximal inhibitory effect of 91 +/- (SE) 2%. However, combined treatment with identical (10 ng/ml) maximally effective doses of both TGFs had little or no effect on the FSH-stimulated accumulation of estrogen, suggesting mutual neutralization by the opposing actions of these peptides. Further evaluation of the antagonistic interaction of the TGFs revealed it to be dose-dependent in that maximally effective doses of TGF alpha (10 ng/ml) partially overcame the stimulation of aromatase activity brought about by relatively low (less than 0.3 ng/ml) but not higher (greater than 1 ng/ml) concentrations of TGF beta, thereby shifting the TGF beta dose-response curve to the right. Treatment with either TGF had no significant effect on granulosa cell DNA content or synthesis, plating efficiency or viability. Taken together, these findings suggest that picomolar concentrations of exogenously provided TGF alpha TGF beta exert potent but diametrically opposed effects on the acquisition of granulosa cell aromatase activity and that the interaction between these two peptides is antagonistic in nature. Our findings further suggest that these direct cytodifferentiative effects of the TGFs may represent intrinsic novel properties of these peptides distinct from their well-established role in the regulation of cellular growth.

Direct inhibition of testicular androgen biosynthesis by arginine-vasopressin: mediation through pressor-selective testicular recognition sites.

We have observed that arginine vasopressin (AVP) and related neurohypophysial hormones exert direct inhibition of testicular androgen biosynthesis in vitro. In this report, the functional identity of the putative testicular recognition sites mediating the antigonadal activity of the neurohypophysial hormones was investigated. Pressor-selective (but not antidiuretic- or oxytocic-selective) agonists of neurohypophysial hormones exerted a dose-dependent inhibition of the hCG-stimulated accumulation of testosterone by cultured rat testicular cells. In addition, potent pressor (but not oxytocic) antagonists brought about a dose-dependent blockage of the AVP-induced inhibition of testicular androgen biosynthesis. Thus, the antigonadal activity of neurohypophysial hormones is mediated by specific testicular recognition sites similar to those mediating the pressor actions of these neuropeptides but distinct from those involved in their antidiuretic or oxytocic effects.

Stimulation of beta 2-adrenergic responsiveness by follicle-stimulating hormone in rat granulosa cells in vitro and in vivo.

The development of adrenergic responsiveness in ovarian granulosa cells cultured in the presence of androstenedione was investigated. Progesterone production by granulosa cells obtained from immature hypophysectomized diethylstilbestrol-treated rats was stimulated by FSH, but was only minimally affected by various adrenergic agents. In contrast, FSH treatment for 2 days in vivo or in vitro resulted in an increase in the adrenergic responsiveness of granulosa cells. Subsequent treatment with (-)epinephrine, (-)norepinephrine, or (-)isoproterenol (a potent beta-adrenergic agonist) resulted in time- and dose-dependent increases in progesterone production. The adrenergic agents enhanced the effects of hCG and PRL with respect to progesterone production, but were without effect on estrogen production or the maintenance of LH/hCG receptors in FSH-primed granulosa cells. A selective beta 2-adrenergic agonist (terbutaline) stimulated progesterone production in FSH-treated granulosa cells, whereas a beta 1-agonist (dobutamine) was 10,000-fold less effective. Furthermore, progesterone production induced by (-)epinephrine was blocked by a selective beta 2-antagonist (IPS 339), but the beta 1-antagonist (practolol) was 7,000-fold less effective. These results suggest that FSH treatment in vivo and in vitro increases beta 2-adrenergic responsiveness in ovarian granulosa cells and that this functional responsiveness is coupled to progesterone, but not to estrogen, biosynthesis.

Forskolin-induced differentiation of cultured rat granulosa cells: new evidence for an intermediary role of adenosine 3',5'-monophosphate in the mechanism of action of follicle-stimulating hormone.

The intermediary role of cAMP in the mechanism of action of FSH was reinvestigated in vitro using forskolin, a highly specific adenylate cyclase probe. Granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were cultured for 3 days in the absence or presence of forskolin. Treatment with increasing concentrations (10(-7)-10(-4) M) of forskolin led to dose-dependent increments in the accumulation of extracellular cAMP, with an apparent median effective dose of 1.6 +/- 0.5 X 10(-5) M. Concomitant blockade of cAMP phosphodiesterase activity further enhanced the forskolin effect. Treatment with forskolin also brought about dose- and time-dependent increments in progesterone and estrogen accumulation. Granulosa cells not pretreated with forskolin displayed negligible LH/hCG binding and remained unresponsive to luteotropic (LH/hCG), beta 2-adrenergic (terbutaline), or lactogenic (PRL) stimulation. In contrast, forskolin (10(-5) M)-pretreated granulosa cells displayed significant increases over controls in LH/hCG binding (46-fold) as well as in progesterone accumulation stimulated by hCG (3.3-fold), terbutaline (1.9-fold), and PRL (1.8-fold). Furthermore, concomitant treatment with a functionally inert low dose (10(-7) M) of forskolin, substantially potentiated the FSH-stimulated accumulation of extracellular cAMP, progesterone, and estrogen as well as the FSH-mediated increase in LH/hCG binding. Taken together, our findings indicate that forskolin, like FSH, is capable of inducing the differentiation of cultured rat granulosa cells by itself, and that a functionally inert low dose of forskolin can potentiate FSH hormonal action. Inasmuch as forskolin-simulated and forskolin-potentiated hormonal action are acceptable as novel criteria of cAMP dependence, our findings provide new evidence in support of the notion that cAMP may be an intracellular second messenger of FSH.

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)

Insulin-like growth factor-I (IGF-I) and IGF-II hormonal action in cultured rat granulosa cells: mediation via type I but not type II IGF receptors.

Both insulin-like growth factor-I (IGF-I) and IGF-II have been shown to promote granulosa cell differentiation and proliferation. While both type I and type II IGF receptors have been observed in rat granulosa cells, the identity of the IGF receptor type(s) mediating IGF hormonal action remains uncertain. Whereas the role of the rat type I IGF receptor cannot be completely evaluated at this time due to the lack of specific reagents, the availability of antibodies specific for the rat type II IGF receptor (R-II-PAB1) has made studies of this receptor type possible. To validate the utility of the R-II-PAB1 antiserum at the level of the rat granulosa cell, its ability to immunoneutralize the granulosa cell type II IGF receptor was examined. Significantly, R-II-PAB1 (10-100 micrograms/ml) proved a potent inhibitor of [125I]IGF-II (but not [125I]IGF-I) binding to granulosa cell membrane preparations. Substantial, albeit finite, R-II-PAB1-mediated inhibition of the cross-linking of [125I]IGF-II was also observed. Moreover, R-II-PAB1 proved highly potent in immunoprecipitating the rat granulosa cell type II IGF receptor. In light of these observations, we have proceeded to use R-II-PAB1 to assess the functional role of the rat granulosa cell type II IGF receptor in IGF-I and IGF-II hormonal action. To this end, FSH (20 ng/ml)-primed granulosa cells were cultured for 72 h in the absence or presence of IGF-I or IGF-II (50 ng/ml) with or without increasing (receptor-active) concentrations of R-II-PAB1 (10-100 micrograms/ml). Control incubations were carried out with an ammonium sulfate precipitate of nonimmune rabbit serum dialyzed against PBS. Significantly, both R-II-PAB1 and nonimmune rabbit serum were without effect on the cytodifferentiative action of either IGF-I or IGF-II. Subject to limitations inherent to the immunoneutralizing potency of R-II-PAB1, these findings are in keeping with the notion that (inasmuch as the conventional cytodifferentiative process is concerned) the granulosa cell type II IGF receptor does not appear to participate in transmembrane IGF signalling. By inference, these findings also suggest that IGF-I and IGF-II hormonal action at the level of the granulosa cell may be exerted largely, if not exclusively, via the type I IGF receptor. Thus, the potential relevance and the functional role(s), if any, of the granulosa cell type II IGF receptor remain to be determined.

Insulin-like growth factors (IGFs) block FSH-induced proteolysis of IGF-binding protein-5 (BP-5) in cultured rat granulosa cells.

Rat granulosa cells (GC), in vitro, express IGFBPs under the influence of both FSH and IGF-I. The major IGFBP produced by GC is a 28-29 K IGFBP, presumed to be IGFBP-5. When GC-conditioned medium (GC-CM) was assessed by Western Ligand Blotting (WLB), FSH appeared to decrease IGFBP-5, whereas IGF-I appeared to increase IGFBP-5 and to partially block the effects of FSH treatment. When GC-CM from FSH-treated cells was incubated with pure IGFBP-4 and IGFBP-5, the amount of IGFBP-5 (measurable by WLB) was decreased. Similarly, when GC-CM from FSH-treated cells was incubated with iodinated IGFBP-4 and IGFBP-5, IGFBP-5 (but not IGFBP-4) was proteolyzed into fragments of approximately 18 and 14 K. The ability of FSH-treated GC-CM to proteolyze IGFBP-5 was reduced by the addition of IGF-I to the reaction mixture. When the IGFBPs in GC-CM were evaluated by affinity crosslinking, GC-CM from control cultures contained one band with an apparent M(r) of approximately 34 K, whereas GC-CM from FSH-treated cultures displayed a decrease in the intensity of the 34 K band, as well as a new band of approximately 24 K. These data suggest that rat GC cells produce an FSH-inducible IGFBP-5 protease activity, and reveal that the ability of this protease to cleave IGFBP-5 is blocked by IGFs.

Rat ovarian insulin-like growth factor I (IGF-I) gene expression is granulosa cell-selective: 5'-untranslated mRNA variant representation and hormonal regulation.

We, and others, have recently reported that the ovary is a site of insulin-like growth factor (IGF)I gene expression. It was the objective of the present studies to assess the relative ovarian abundance of IGF-I transcripts with alternative 5'-untranslated (UT) regions, their cellular localization, and hormonal regulation. To this end, a solution hybridization/RNase protection assay was employed wherein total rat ovarian RNA was hybridized with a 404-base 32P-labelled rat IGF-I riboprobe corresponding to the Class A 5'UT variant. As in liver, three protected bands [322 (Class A), 297 (Class B), and 242 (Class C) bases long] were noted, in keeping with established alternative 5' UT transcripts. The ovarian (as the hepatic) Class C variant proved the most abundant. The ovarian Class B variant was barely detectable. Cellular localization studies revealed these ovarian IGF-I transcripts to be primarily, if not exclusively, of granulosa but not theca-interstitial cell origin. Treatment of immature (21-23 days old) hypophysectomized rats with a diethylstilbestrol (DES)-containing subcutaneous silastic implant for a total of 5 days resulted in a 2-fold increase in the (densitometrically quantified) abundance of ovarian IGF-I transcripts, a diametrically-opposed effect (2.6-fold decrease) being noted at the level of the liver. Whereas treatment of hypophysectomized rats with oGH by itself (150 micrograms, qd, sc x5 days) resulted in a 5-fold increase in hepatic IGF-I gene expression, a limited, albeit distinct inhibitory effect was observed on the steady-state levels of ovarian IGF-I mRNA. In contrast, combined treatment with oGH and DES yielded a 3-fold increase in the abundance of ovarian IGF-I transcripts, there being no net alteration in hepatic IGF-I gene expression. Taken together, these findings reveal ovarian expression of the 3 known 5'-UT IGF-I mRNA variants, document the granulosa cell as the main somatic ovarian cell of IGF-I mRNA generation, and indicate that hepatic and ovarian IGF-I gene expression are differentially regulated in diametrically opposed directions.

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.