Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.

Stimulation of rat granulosa cell progesterone production but not other differentiated functions by a splenocyte-derived factor.

Concanavalin A-stimulated splenocytes secrete a factor that stimulates progesterone production in cultured rat granulosa cells. The actions of this progesterone-stimulating factor (PSF) were characterized further by purifying it by sequential chromatographies on heparin-agarose, copper chelating-Sepharose and Mono S columns. Several of its effects on granulosa cells were then compared with those of FSH. Like FSH, PSF induced accumulation of progesterone and 20 alpha-dihydroprogesterone in granulosa cell culture media; however, the time course of progesterone accumulation in response to PSF was much slower than that in response to FSH. In contrast to FSH, PSF induced little accumulation of oestrogen. Induction of other differentiated responses was compared by pretreating cultured granulosa cells with either FSH or PSF, and then challenging with LH, prolactin, epidermal growth factor or the beta-adrenergic agonist, isoproterenol. Pretreatment with FSH induced subsequent responsiveness to all four agents, whereas pretreatment with PSF induced subsequent responsiveness only to isoproterenol. These results indicate that granulosa cell responses to PSF are much more limited than are those to FSH and that these responses are characterized by an increase in progestin production.

Identification of vasopressin mRNA in rat aorta.

We have reported previously that several blood vessels of the rat and cow contain immunoreactive vasopressin and further suggested that this peptide might be produced locally. To provide additional support for this hypothesis, we conducted the present study to determine whether mRNA for arginine vasopressin is also present in blood vessels. Ribonuclease protection analysis of total RNA isolated from rat hypothalamus and aorta revealed the presence of arginine vasopressin message in both tissues but not in RNA isolated from liver, a tissue devoid of vasopressin. Subsequent comparison of the autoradiographic intensities of the signals in these two tissues indicated that vasopressin message was 100- to 1000-fold lower in aorta. Additional studies showed that RNA isolated from endothelium-denuded vessels contained levels of arginine vasopressin message similar to those in intact vessels, indicating that endothelium was not a major source of this message. These data were substantiated by further studies using a vasopressin radioimmunoassay, which showed that vasopressin peptide levels in intact and endothelium-denuded vessels did not differ. Thus, the present study showed that rat aorta contains arginine vasopressin mRNA as well as the vasopressin peptide and that both the message and the peptide are contained in nonendothelial structures. However, the data do not rule out endothelium as a possible source of vasopressin. These studies add further support to the hypothesis that blood vessels are capable of producing vasopressin.

Arginine vasopressin stimulates protein synthesis but not proliferation of cultured vascular endothelial cells.

We previously showed that rat and cow blood vessels contain arginine vasopressin (AVP) of local origin. In the present study, to investigate potential roles for this locally produced peptide, we examined the effects of AVP on growth of cultured vascular endothelial and smooth muscle cells (EC, SMC). Treatment of cultured bovine aortic endothelial (BAE), murine cerebral microvascular endothelial (MME), or murine cerebral microvascular smooth muscle (MMSM) cells with AVP produced dose-dependent increases in total protein content with maximum increases of 11, 46, and 33% over vehicle-treated controls, respectively. AVP also dose-dependently increased new protein synthesis, as reflected by [3H]leucine incorporation into BAE and MME cell protein, with maximal increases of 34 and 24%, respectively. In addition, the AVP-stimulated increase in protein synthesis could be significantly attenuated in both BAE and MME cells by simultaneous addition of a specific AVP V1 receptor antagonist. In contrast, AVP did not affect total cell number or DNA synthesis, as reflected by [3H]thymidine incorporation, in any cell type. Neither was cell size, as measured by forward light scatter with a fluorescence-activated cell sorter (FACS), changed by treatment with AVP. However, the density of individual cells, as measured by orthogonal light scatter with FACS, was increased by AVP. These data demonstrate that AVP stimulates protein synthesis but not proliferation of cultured vascular EC and SMC and suggest that locally produced AVP may function as a growth factor for these cells.

Induction of rat granulosa cell steroidogenic enzyme activities and their messenger ribonucleic acids by a splenocyte-derived factor.

We have previously identified and purified a splenocyte-derived factor (PSF) that stimulates the accumulation of progesterone and 20 alpha-dihydroprogesterone (20 alpha-OH-P) in rat ovarian granulosa cells independently of FSH. In the present study, time course experiments comparing the response to PSF with that to FSH revealed that PSF-stimulated progesterone accumulation was slower than that of FSH, but PSF-stimulated 20 alpha-OH-P accumulation had a time course similar to that of FSH. To determine the basis for the slower progesterone response to PSF, the effect of these two agents on each step of the steroidogenic pathway was assessed. First, to examine whether PSF-stimulated cholesterol mobilization was limiting, cultured granulosa cells were treated with 22(R)-hydroxycholesterol. While both FSH- and PSF-stimulated progesterone and 20 alpha-OH-P accumulation approximately doubled, the overall time courses did not change indicating that cholesterol availability was not the factor limiting the response to PSF. Next, PSF and FSH induction of steroidogenic enzyme activities and messenger RNAs were compared. While FSH-stimulated cytochrome P450 side chain cleavage enzyme (SCC) activity rapidly increased (peaking at 2 days) and then slowly declined, PSF-stimulated SCC activity gradually increased over 5 days to approximately 35% of the maximal activity stimulated by FSH. PSF also induced slower increases in P450scc mRNA levels than did FSH. In addition, PSF stimulated 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity more slowly than did FSH, but after 3 days of culture, PSF-stimulated activity was significantly higher than that induced by FSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary adenylate cyclase-activating polypeptide stimulates steroidogenesis and adenosine 3',5'-monophosphate accumulation in cultured rat granulosa cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel neuropeptide with considerable homology to vasoactive intestinal peptide (VIP) and GH-releasing hormone (GHRH). Because we have shown previously that VIP and GHRH stimulate steroidogenesis in cultured rat granulosa cells independently of FSH, the present studies evaluated whether PACAP also stimulates steroidogenesis and compared its effects to those of VIP and GHRH. Granulosa cells cultured for 2 days with PACAP-27, VIP, or GHRH (10(-9)-10(-6) M) showed dose-dependent increases in the accumulation of progesterone, 20 alpha-dihydroprogesterone, and estrogen. The rank order of potency for the three peptides was PACAP >> VIP > GHRH. PACAP also increased cAMP accumulation and was again more potent than VIP. In addition, all three peptides synergistically augmented FSH-stimulated progesterone and 20 alpha-dihydroprogesterone accumulation with the same rank order of potency as above; however, only the highest dose of each peptide augmented estrogen accumulation. Further studies examined the effects of various androgens on these responses. PACAP-stimulated progesterone accumulation was minimal in the absence of androstenedione, but increased up to 40-fold in its presence. Other synthetic and naturally occurring androgens produced similar increases with a rank order of potency of R1881 > testosterone = androstenedione > dihydrotestosterone. Analysis of cAMP levels indicated that by 1 h after treatment with PACAP, cAMP levels within the cells increased by 4-fold, and this response was unaltered in the presence of androstenedione; however, by 48 h, cAMP levels had markedly declined, and this response was attenuated by androstenedione. These results demonstrate that PACAP stimulates steroidogenesis and cAMP accumulation in cultured rat granulosa cells, and it is more potent than its two homologs, VIP and GHRH. Because PACAP has been shown to be present within the ovary, these data indicate that this peptide may play a role in modulating ovarian steroidogenic activity.

Characterization of a rapid and sensitive enzyme immunoassay (EIA) for progesterone applied to conditioned cell culture media.

Progesterone accumulation in conditioned media is a frequently employed endpoint for in vitro cell culture of steroidogenic cells. Although radioimmunoassay (RIA) has been the predominant method for measurement of progesterone, a number of nonradiometric immunoassays have been described but they have not been applied to conditioned media. Here, we report the characterization of a microtitre plate enzyme immunoassay (EIA) for determination of progesterone in conditioned media. The EIA has a sensitivity of 0.3 pg per well with intraassay and interassay coefficients of variation of 7.3 and 10.2%, respectively. The specificity of the EIA is no different than that of a comparable RIA showing crossreactivities of less than 0.1% for other steroids except 5 alpha-pregnan-3,20-dione (47%) and 11 alpha-hydroxyprogesterone (18%). Progesterone levels from conditioned cell culture media of either rat or human granulosa cell cultures measured by both EIA and RIA were in close agreement (r = 0.96) and serial dilutions of culture samples in the EIA were parallel to those of the standards. Also, extraction of culture media prior to EIA was found not to be necessary. Thus, this EIA is a highly sensitive and specific assay that provides a rapid, simple, inexpensive, and non-radiometric alternative to radioimmunoassay for measurement of progesterone in conditioned cell culture media.

Androgens augment vasoactive intestinal peptide- and growth hormone-releasing hormone-stimulated progestin production by rat granulosa cells.

Vasoactive intestinal peptide (VIP) has been shown to stimulate steroid production by cultured rat granulosa cells independently of FSH. In the present study, we have examined the modulatory effects of various steroids on this response. Rat granulosa cells cultured for 2 days with only VIP showed small but significant increases in progesterone and 20 alpha-dihydroprogesterone (20 alpha-OH-P) production. Concomitant treatment with either a synthetic estrogen (diethylstilbestrol), a synthetic progestin (R5020), or cortisol did not augment VIP-stimulated progesterone production; however, the latter two steroids slightly, but significantly, augmented VIP-stimulated 20 alpha-OH-P production. In contrast, concomitant treatment with a synthetic androgen (R1881) dramatically augmented both VIP-stimulated progesterone and 20 alpha-OH-P production. These effects were dose dependent for both VIP and R1881 and could be blocked by the androgen antagonist cyproterone acetate. Time course studies revealed that progesterone content of the culture media rapidly increased over the first 24 h of culture then remained fairly constant for the next 48 h; 20 alpha-OH-P content, on the other hand, was low for the first 12 h of culture and steadily increased thereafter. Dose-response analysis for R1881 revealed an ED50 of approximately 2 x 10(-8) M for the synthetic androgen, and comparison with other naturally occurring androgens provided the rank order of potency R1881 > androstenedione > testosterone = dihydrotestosterone. Additional studies with another member of the VIP peptide family, GH-releasing hormone, showed dose-dependent stimulation of progesterone and 20 alpha-OH-P production by this peptide. These effects were also augmented by R1881 but not by diethylstilbestrol, R5020, or cortisol. These studies demonstrate that androgens, but not estrogens, progestins, or glucocorticoids, augment VIP- and GH-releasing hormone-stimulated progestin production by cultured rat granulosa cells.

Gonadotropin regulation of c-fos and c-jun messenger ribonucleic acids in cultured rat granulosa cells.

c-Fos and c-jun are immediate early proto-oncogenes encoding proteins for the heterodimer AP-1, a DNA binding complex which regulates gene transcription. In order to investigate the presence and potential gonadotropin regulation of mRNAs for these proto-oncogenes in rat granulosa cells, we used Northern blotting of total RNA from cultured cells. Granulosa cells obtained from diethylstilbestrol (DES)-treated weanling rats were challenged with follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), dibutyryl cAMP ((Bu)2cAMP) or tetradecanoyl-13-phorbol acetate (TPA) either 2.5 h after cell isolation (day 0) or following a 2-day pretreatment with FSH (day 2). Freshly isolated cells treated with FSH exhibited 4-fold and 3-fold increases in c-fos and c-jun mRNAs, respectively, within 30 min. Two hours after FSH treatment, both c-fos and c-jun message levels diminished to near control levels. Granulosa cells pretreated for 2 days with FSH, then re-challenged with FSH, showed similar increases in both c-fos and c-jun messages. These effects were dose- and time-dependent on both day 0 and day 2. Likewise, (Bu)2cAMP also increased c-fos and c-jun mRNAs in a time- and dose-dependent manner on both day 0 and day 2. In contrast, LH or hCG minimally increased c-fos and c-jun mRNAs on day 0, but on day 2, both hormones markedly increased message levels in a manner similar to that seen with FSH. Analogous effects were observed with TPA which minimally stimulated c-fos and c-jun mRNAs on day 0, but markedly increased these messages on day 2. These studies demonstrate that c-fos and c-jun mRNAs can be induced in cultured rat granulosa cells by acute gonadotropin, (Bu)2cAMP or phorbol ester treatment and suggest that these immediate early proto-oncogenes may play a role in granulosa cell function.

Characterization of a vasopressin-like peptide in rat and bovine blood vessels.

The present study examined the possibility that blood vessels of the rat and cow contain arginine vasopressin (AVP) and that the vascular stores of this potent vasoconstrictor are of local, rather than blood-borne, origin. Vessels from rat or cow were homogenized in acid, and the supernatants were assayed for AVP by radioimmunoassay. AVP immunoreactivity was detected in all vessels taken from the rat including aorta, mesenteric artery, renal artery, vena cava, and renal vein, as well as atria of the heart and also in cow aorta. Blood vessels from hypophysectomized and Brattleboro rats also contained AVP at levels similar to those of intact control rats. Further characterization of this immunoreactive material showed that, in both radioimmunoassay and radioreceptor assay, vascular extracts produced competition curves, which were parallel to those of synthetic AVP. Additionally, immunoreactive AVP in aortic extracts comigrated with synthetic AVP and pituitary extract on both high-pressure liquid chromatography and Sephadex G-25 chromatography. Furthermore, intravenous administration of the aortic extract produced pressor responses in conscious rats, and these responses were significantly attenuated by pretreatment with a specific AVP V1-receptor antagonist. These data demonstrate that blood vessels contain an AVP-like peptide that appears indistinguishable from authentic AVP and further suggest that this vascular peptide is of local origin.

Hormonal regulation of rat testicular arginine vasopressin receptors.

Previous studies have implicated arginine vasopressin (AVP) as a negative intratesticular modulator of androgen production. The present studies were undertaken to further define the testicular AVP system by investigating potential influences of pituitary hormones on testicular receptors for AVP. Binding of [3H]AVP to testicular membranes was time, temperature, and pH dependent, with optimum binding at 90 min, 22 C, and pH 8.0. Addition of divalent cations to the incubation mixture significantly increased binding by 4-, 10-, 15-, and 35-fold with Ca2+, Zn2+, Mg2+, and Mn2+, respectively. The influence of pituitary hormones on testicular AVP receptors was initially assessed by comparing binding parameters obtained by Scatchard analysis of saturation binding data between membranes prepared from intact or hypophysectomized rats. Testicular membranes from intact animals bound 66 +/- 2 fmol AVP/mg protein, whereas membranes from animals hypophysectomized 10 days previously showed a significantly lower binding capacity of 40 +/- 3 fmol AVP/mg protein; however, the affinity of the receptors for AVP did not differ between these two groups. A similar decrease was observed when binding was compared between purified Leydig cells isolated from intact and hypophysectomized animals by Metrizamide density gradients. Time-course analysis of this effect showed a continuous decline in total testicular AVP-binding capacity for the first 2 weeks after hypophysectomy and a slight increase at 3 weeks. Treatment of hypophysectomized animals with pituitary hormones beginning on the third day after hypophysectomy and continuing for either 4 or 7 days showed that LH and GH were able to dose-dependently restore testicular AVP-binding capacity to levels found in intact animals, whereas FSH and PRL were ineffective. These data indicate that testicular AVP receptors are under the control of LH and GH from the pituitary and suggest that pituitary hormones may impact on the efficacy of the testicular AVP system by regulating AVP receptor levels.

Central vasopressin raises arterial pressure by sympathetic activation and vasopressin release.

Although central administration of arginine vasopressin (AVP) has been reported to increase arterial pressure mediated by activation of the sympathetic system, we found that peripheral blockade of sympathetic transmission did not attenuate this pressor response. To elucidate the mechanism, rats were pretreated with either phentolamine (3 mg/kg), chlorisondamine (2.5 mg/kg), a vasopressin V1 receptor antagonist d(CH2)5Tyr(Me)AVP (AVP-X) (10 micrograms/kg), or the combinations of phentolamine and AVP-X or chlorisondamine and AVP-X. The pressor response to intracerebroventricular injection of AVP in unrestrained conscious rats was reduced but not significantly altered by intravenous injection of phentolamine or AVP-X; however, combined treatment with these agents abolished the response. To determine that the amount of central AVP leaked to the periphery did not contribute to the pressor effect, tritiated AVP and AVP (100 ng total) were injected intracerebroventricularly. Blood samples collected at 0, 3, and 30 minutes after injection showed that radioactivity in plasma was primarily metabolites and that the amount of intact AVP estimated to leak from the brain was too low to produce a pressor effect. Comparative regional hemodynamic studies between intracerebroventricular and intravenous injection of AVP performed in conscious rats instrumented with Doppler flow probes demonstrated a qualitatively similar pattern of increased resistance in the renal, mesenteric, and hindquarters beds. These data suggest that central pressor action of AVP is mediated by both activation of the sympathetic system and release of AVP.

Effects of interleukins 1, 2 and 3 on follicle-stimulating hormone-induced differentiation of rat granulosa cells.

A growing body of evidence indicates that substances released by activated immune cells can directly influence the functions of various endocrine cells. In the present study, the direct in vitro effects of interleukins (IL) 1, 2, and 3 on follicle-stimulating hormone (FSH)-stimulated steroidogenesis and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor induction in granulosa cells were examined. In the absence of FSH, none of the interleukins stimulated steroid production or LH/hCG receptor induction during a 2-day culture period. However, in the presence of FSH, both IL-1 alpha and IL-1 beta inhibited, in a dose-dependent manner, progesterone and estrogen production as well as LH/hCG receptor induction in response to FSH. In contrast, both agents augmented 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) production stimulated by FSH. In all cases, less IL-1 beta than IL-1 alpha was required to produce a comparable effect. IL-2 slightly, but significantly, enhanced both FSH-stimulated progesterone and 20 alpha-OH-P production but had no effect on FSH-stimulated estrogen production or LH/hCG receptor induction. IL-3 potentiated the 20 alpha-OH-P response to FSH by up to 65% but had no effect on FSH-stimulated progesterone or estrogen production or LH/hCG receptor induction. These data suggest that the interleukins, which are key mediators of immune responses, may affect mechanisms crucial for the maturation and differentiation of granulosa cells and thus may also play a regulatory role in reproductive function.

Gamma-interferon inhibits rat granulosa cell differentiation in culture.

A growing body of evidence indicates that factors secreted by cells of the immune system can directly affect a variety of endocrine phenomena. In the present study we examined the direct effects of the cytokine, interferon (IFN), on FSH-stimulated steroidogenesis and LH/hCG receptor induction in rat granulosa cells. We show that gamma-IFN, but not alpha-IFN, inhibits FSH-stimulated progesterone, 20 alpha-hydroxypregn-4-en-3-one and estrogen production as well as gonadotropin-induced LH/hCG receptor formation in a dose-dependent manner. The results suggest that gamma-IFN may play a role in the maturation and differentiation of granulosa cells and thus may serve as a regulatory link between the immune and reproductive endocrine systems.

Lymphokines from concanavalin-A-stimulated lymphocytes regulate rat granulosa cell steroidogenesis in vitro.

Animals with impaired immune function show numerous reproductive disorders. To determine whether immune factors might play a direct role in the regulation of ovarian function, we examined the effects of Concanavalin-A-stimulated lymphocyte supernates (CAS) on steroidogenesis by cultured rat granulosa cells. Granulosa cells from immature estrogen-primed rats were incubated for 48 h with increasing doses of CAS in the presence or absence of FSH. In the absence of FSH, CAS produced a dose-dependent increase in progestin (progesterone and 20 alpha-hydroxyprogesterone) production to a maximum of 190-fold greater than untreated controls, but did not stimulate estrogen production. In the presence of FSH, the stimulatory effect of CAS on progestin production was additive with that of FSH. In contrast, CAS inhibited FSH-stimulated estrogen production in a dose-dependent manner. The maximum inhibition was greater than 90%. Addition of the phosphodiesterase inhibitor isobutylmethylxanthine to CAS-containing cultures significantly enhanced the stimulatory effect of CAS on progesterone production, suggesting that this action may be exerted through a cAMP-mediated pathway. The stimulatory component of CAS was heat labile, acid stable, and required a trypsin-sensitive cell surface recognition site, whereas the inhibitory component was both heat and acid stable. Neither the stimulatory nor the inhibitory actions of CAS were mimicked by treating granulosa cells with supernates from Concanavalin-A-stimulated neonatal cardiac or hepatic cell cultures. Thus, the present studies demonstrate that secretory products of lymphocytes (collectively termed lymphokines) can affect steroidogenesis in cultured rat granulosa cells. These data imply that immune cell factors may play a significant role in the differentiation and maturation of granulosa cells.

Insulin-like growth factor-I augments gonadotropin-stimulated androgen biosynthesis by cultured rat testicular cells.

In addition to the well-known growth stimulating effects of insulin-like growth factors (IGFs), recent studies suggest that these peptides may also modulate the differentiated functions of endocrine cells. Thus, in the present studies, we have investigated the actions of IGFs on androgen biosynthesis by cultured testicular cells. Treatment of cells obtained from adult hypophysectomized rats with LH (1 ng/ml) stimulated testosterone production 60-fold over basal levels. In contrast, treatment with either synthetic human IGF-I or IGF-II failed to stimulate androgen production. However, concomitant treatment of the LH-containing cultures with increasing doses of IGF-I (10-500 ng/ml) augmented testosterone production up to 70% over that seen with LH alone (ED50 = 67 ng/ml). Similar effects were obtained with IGF-II but this peptide was about 10-fold less potent than IGF-I. In addition, these peptides also stimulated the accumulation of pregnenolone and progesterone in the culture medium. Additional studies demonstrated specific binding of [125I]iodo-IGF-I to testicular cells. This binding was competed by IGF-related peptides with the potency order IGF-II = IGF-I greater than insulin whereas unrelated peptides did not compete. The cellular localization of these receptors was examined in testicular cells separated on a metrizamide density gradient. IGF-I receptors were evenly distributed between two cell peaks containing subpopulations of Leydig cells whereas much less binding was found in other testicular cell types. Coupled with recent findings indicating testicular production of IGF-I, the present results suggest that this peptide may act as a positive intratesticular modulator of Leydig cell differentiation.

Vasoactive intestinal peptide stimulates plasminogen activator activity by cultured rat granulosa cells and cumulus-oocyte complexes.

Vasoactive Intestinal Peptide (VIP), originally considered to be a gut hormone, has recently been found to increase estrogen and progesterone production by ovarian granulosa and luteal cells. Because several studies indicate that granulosa cells and oocytes are capable of producing plasminogen activators, we have studied the effects of VIP on plasminogen activator activity in cultured granulosa cells and cumulus-oocyte complexes collected from the ovaries of hypophysectomized, estrogen-treated immature rats. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a fibrin overlay technique to assess plasminogen activator activity, we observed that treatment with VIP stimulated the secretion of tissue-type plasminogen activator (tPA), but not urinary-type plasminogen activator (uPA), in a dose-dependent manner by cultured granulosa cells as well as by cumulus-oocyte complexes, but not by denuded oocytes. However, preparation of cumulus-free oocytes from cumulus-oocyte complexes which had previously been treated with VIP indicated substantial increases in tPA activity within the oocyte. The actions of VIP on tPA activity in granulosa cells were specific, because other closely related peptides (PHM-27 and glucagon) were ineffective. These effects of VIP, in addition to the previously observed effects on steroidogenesis, suggest that VIP may be an important regulator of ovarian function.

Specific insulin-like growth factor (IGF) I- and II-binding sites on rat granulosa cells: relation to IGF action.

Although insulin-like growth factor-I (IGF-I) and insulin have been shown to augment rat granulosa cell differentiation, their mechanism(s) of action has not yet been elucidated. In the present study, we have examined granulosa cells obtained from immature hypophysectomized estrogen-treated rats for specific IGF-binding sites that might mediate the effects of the insulin-like peptides. Using synthetic [125I]iodo-IGF-I, we have found specific high affinity, low capacity (Kd = 1.36 +/- 0.131 nM; 3250 +/- 662 sites/cell) IGF-I-binding sites that have lower affinities for the related peptides IGF-II and insulin (potency ratio, 1:9:700 for IGF-I, IGF-II, and insulin). We have also found specific binding sites for [125I]iodo-IGF-II, a newly available synthetic peptide. The IGF-II-preferring sites were of a single class (Kd = 1.54 +/- 0.32 nM; 4728 sites/cell) and exhibited a rank competition order of IGF-II greater than IGF-I much greater than insulin. To study the functional correlates of these binding activities, granulosa cells were cultured for 2 days in serum-free medium in the presence of FSH, with or without increasing concentrations of IGF-I, IGF-II, or insulin. Medium steroids were then determined by specific RIA, and cellular LH/hCG receptors were measured by specific [125I]iodo-hCG binding. Treatment with FSH increased estrogen and progestin production and induced the formation of LH/hCG receptors. Concomitant treatment with the three peptides dose-dependently enhanced both FSH-stimulated steroidogenesis and LH/hCG receptor induction, with a rank order of potency of IGF-I greater than IGF-II greater than insulin (potency ratio, 1:8:36). This rank order of potency of the insulin-like peptides was more closely correlated with their ability to compete for IGF-I binding rather than IGF-II binding, suggesting the preferential involvement of IGF-I receptors in the ovarian actions of the IGFs, although the involvement of IGF-II and insulin receptors cannot be dismissed. Our results demonstrate, for the first time, a biological action of synthetic IGF-II in granulosa cells and further show a novel insulin effect, enhancement of LH/hCG receptor induction. These findings also indicate that rat granulosa cells possess specific IGF-I and IGF-II-binding sites that may mediate the gonadotropin-enhancing actions of the insulin-like peptides. Since IGF-I appears to be the most biologically potent peptide, it is likely to be the most important insulin-like peptide involved in granulosa cell differentiation in vivo.

Vasoactive intestinal peptide stimulates androgen biosynthesis by cultured neonatal testicular cells.

Vasoactive intestinal peptide (VIP) was originally isolated from porcine duodenum and considered to be a gut hormone. Recent evidence indicates that it may also be involved in reproductive functions. In this study, a possible action of VIP on steroidogenesis by cultured testicular cells was investigated. Neonatal testicular cells were treated in vitro with hormones for 3 days and medium steroid or cAMP content was measured by radioimmunoassay. Treatment of cultured cells with VIP (10(-9) to 10(-6) M) increased the production of testosterone, progesterone, and pregnenolone in a dose-dependent fashion. Testosterone production in response to 10(-6) M VIP was about 5-10% of that maximally induced by LH. Addition of methyl-isobutyl-xanthine, a phosphodiesterase inhibitor, to the VIP-containing cultures significantly enhanced production of testosterone by 13-fold, of progesterone by 9-fold, and of pregnenolone by 2.5-fold as compared to treatment with VIP alone. Additional experiments also showed a dose-dependent stimulation of cAMP production by VIP. The VIP-related hormones PHM-27, secretin, and glucagon also stimulated progesterone and testosterone production with a potency order (PHM-27 greater than secretin greater than glucagon) consistent with that observed for other VIP receptor-mediated actions. A direct stimulatory effect of VIP on Leydig cells was indicated in studies on steroidogenesis by testicular cells separated on a metrizamide density gradient. In these studies, VIP stimulated androgen production in an LH-responsive subpopulation of testis cells but failed to affect steroid production in non-LH-responsive cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct biphasic modulation of gonadotropin-stimulated testicular androgen biosynthesis by prolactin.

Prolactin (PRL) exerts both stimulatory and inhibitory effects upon testicular steroidogenesis in vivo. The direct effects of PRL on biosynthesis of testicular androgen were studied in primary cultures of testicular cells obtained from adult, hypophysectomized or neonatal, intact rats. In cells from adult animals, treatment with human chorionic gonadotropin (hCG) (10 ng/ml) significantly increased testosterone and progesterone production relative to their respective controls. In contrast, neither steroid was increased by treatment with rat PRL (rPRL) or ovine PRL (oPRL) alone. Upon addition of 0.1-3 ng/ml of either rPRL or oPRL to the hCG-treated cultures, testosterone production was progressively increased up to a maximum of 70% greater than with hCG alone. However, when PRL exceeded 3 ng/ml, the testosterone response began to decline and was 39 or 24% less than from cells treated with hCG alone at 300 ng/ml of rPRL or oPRL, respectively. A similar biphasic response pattern was observed in cells from neonatal animals. In contrast to the biphasic effect of PRL on production of androgen, PRL treatment enhanced hCG-stimulated production of progesterone in a dose-related manner without exerting an inhibitory effect. At 3 and 300 ng/ml, rPRL augmented hCG action by 2.5- and 8-fold, respectively. Similarly, in the presence of inhibitors of pregnenolone metabolism, rPRL also enhanced hCG-stimulated production of pregnenolone. Quantitation of steroid intermediates in the testosterone biosynthetic pathway revealed that the stimulatory effect of 3 ng/ml rPRL on testosterone production was associated with 1.3- and 2.8-fold increases in accumulation of androstenedione and 17 alpha-hydroxyprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)