Stimulatory Effect Of FSH Research Articles

Ghrelin and obestatin can promote human ovarian granulosa cell functions and FSH effects

The aim of the present in-vitro experiments was to examine the direct influence of ghrelin and obestatin on viability, proliferation and progesterone release by human ovarian granulosa cells and their response to FSH administration. Human granulosa cells were cultured in presence of ghrelin or obestatin (both at 0, 1, 10 or 100 ng/ml) alone or in the presence of FSH (10 ng/ml). Cell viability, accumulation of proliferation markers PCNA and cyclin B1 and release of progesterone were analyzed by Trypan blue extrusion test, quantitative immunocytochemistry and ELISA. Ghrelin, obestatin and FSH up-regulated all the measured ovarian cell parameters. Moreover, both ghrelin and obestatin promoted all the stimulatory effects of FSH. The obtained results demonstrate the direct stimulatory action of ghrelin, obestatin and FSH on basic ovarian cell functions, as well as the ability of metabolic hormones to improve FSH action on human ovarian cells.

Fsh stimulates Leydig cell Wnt5a production, enriching zebrafish type A spermatogonia.

Follicle-stimulating hormone (Fsh) modulates vertebrate spermatogenesis by regulating somatic cell functions in the testis. We have found previously that zebrafish Fsh stimulated the differentiating proliferation of type A undifferentiated spermatogonia (Aund) in an androgen-independent manner by regulating the production of growth factors and other signaling molecules in both Sertoli (SCs) and Leydig cells (LCs). For example, Fsh triggered the release of Igf3 that subsequently activated β-catenin signaling to promote the differentiating proliferation of Aund. In the present study, we report that Fsh moreover uses the non-canonical Wnt pathway to promote the proliferation and accumulation of Aund. Initially, we found that the stimulatory effect of Fsh on the proliferation activity of Aund was further strengthened when β-catenin signaling was inhibited, resulting in an accumulation of Aund. We then showed that this Fsh-induced accumulation of Aund was associated with increased transcript levels of the non-canonical Wnt ligand, wnt5a. In situ hybridization of insl3 mRNA, a gene expressed in LCs, combined with Wnt5a immunocytochemistry identified LCs as the cellular source of Wnt5a in the adult zebrafish testis. Addition of an antagonist of Wnt5a to incubations with Fsh decreased both the proliferation activity and the relative section area occupied by Aund, while an agonist of Wnt5a increased these same parameters for Aund. Taken together, our data suggest that Fsh triggered LCs to release Wnt5a, which then promoted the proliferation and accumulation of Aund. Hence, Fsh uses non-canonical Wnt signaling to ensure the production of Aund, while also triggering β-catenin signaling via Igf3 to ensure spermatogonial differentiation.

IGF1R signaling is necessary for FSH-induced activation of AKT and differentiation of human Cumulus granulosa cells.

FSH is routinely administered to in vitro fertilization patients to induce follicle maturation. During this process, granulosa cells differentiate and acquire specific functional characteristics that are required to coordinate ovulation and oocyte maturation. The objective of the study was to gain insight into the molecular mechanisms regulating human granulosa cell differentiation. Design, Setting, Patients, and Interventions: Cumulus and mural granulosa cells were isolated from the follicular aspirates of in vitro fertilization patients and analyzed immediately or cultured in serum-free media in the presence of FSH, IGFs, or an inhibitor of type I IGF receptor (IGF1R) activity. We quantified the mRNA and protein levels of steroidogenic enzymes, components of the IGF system, and gonadotropin receptors; measured 17β-estradiol levels; and examined the activation of intracellular signaling pathways to assess the granulosa cell differentiation as well as the FSH and IGF actions in both cumulus and mural cells. In freshly isolated cells, LH receptor (Lhr) and steroidogenic acute regulator (Star) were expressed at lower levels in cumulus than mural cells, whereas FSH receptor (Fshr) and anti-Müllerian hormone (Amh) were expressed at higher levels in cumulus than mural cells. In vitro, the expression of Igf2, the differentiation markers Lhr, Star, and Cyp19a1 (aromatase) as well as 17β-estradiol production remained low in untreated cumulus cells but increased significantly after FSH treatment. Strikingly, this stimulatory effect of FSH was abolished by the inhibition of IGF1R activity. FSH-induced activation of v-akt murine thymoma viral oncogene homolog 3 (AKT) required IGF1R activity, and overexpression of constitutively active AKT rescued the induction of differentiation markers and 17β-estradiol production by FSH in the presence of the IGF1R inhibitor. The cumulus cell response to FSH resembles the differentiation of preantral to preovulatory granulosa cells. This differentiation program requires IGF1R activity and subsequent AKT activation.

The cAMP analogue, dbcAMP affects release of steroid hormones by cultured rabbit ovarian cells and their response to FSH, IGF-I and ghrelin

The aim of the present study was to examine possible involvement of cAMP-dependent intracellular mechanisms in control of ovarian cell steroidogenesis and its response to hormonal regulators. For this purpose, we examined the influence of administration of dbcAMP, a cAMP analogue (50 µg/animal) in vivo, on release of progesterone, testosterone and estradiol by isolated ovarian fragments, as well their response to hormonal regulators of ovarian steroidogenesis—FSH, IGF-I and ghrelin (all added at doses of 100 ng/ml). It was observed, that administration of dbcAMP resulted reduction in progesterone and testosterone, but not of estradiol release by isolated ovarian fragments. In ovarian tissue isolated from control animals, additions of hormones were able to reduce release of progesterone (FSH, IGF-I and ghrelin) and increase release of testosterone (ghrelin) but did not change estradiol output. Previous administration of dbcAMP modified action of exogenous hormones: it inverted inhibitory action of FSH, IGF-I and ghrelin on progesterone release to stimulatory action and induced stimulatory action of IGF-I on testosterone release and stimulatory effect of FSH on estradiol output. The present observations confirm involvement of peptide hormones FSH, IGF-I and ghrelin in the control of rabbit ovarian steroid hormones release and demonstrate the involvement of cAMP-dependent intracellular mechanisms in down-regulation of rabbit ovarian steroidogenesis and in modification, but not in mediating effect of FSH, IGF-I and ghrelin on ovarian steroid hormones release.

Effects of FSH and LH on Steroid Production by Buffalo (Bubalus bubalis) Granulosa Cells CulturedIn VitroUnder Serum-Free Conditions

The objective of this study was to examine the effects of FSH and LH on oestradiol-17β and progesterone production by buffalo granulosa cells cultured under serum-free conditions. Granulosa cells (3 × 10(5) ) from small (≤ 5 mm diameter) follicles were cultured for up to 4 days in 48-well plates coated with 3.3 μg/cm(2) fibronectin in Dulbecco's modified Eagle's medium (DMEM) : nutrient mixture F-12 Ham (1 : 1 ratio) supplemented with 10(-7) m androstenedione, 5 μg/ml human apo-transferrin and 0.1% bovine serum albumin, in the presence or absence of FSH or LH (0, 1, 2, 4, 8, 16, 32 or 64 ng/ml each). Basal oestradiol-17β production by granulosa cells from small follicles reduced (p < 0.01) from days 1 to 2 of culture and became undetectable by day 3 and basal progesterone production increased (p < 0.05) from day 1 through day 4 of the culture. Although there was no effect of FSH on day 1 of the culture, FSH at 2, 4, 8 and 16 ng/ml increased (p < 0.05) oestradiol-17β production by granulosa cells from small follicles on day 2. Progesterone secretion was increased (p < 0.05) by all doses of FSH on all days of culture. All doses of LH had no effect on oestradiol-17β or progesterone production by granulosa cells from small follicles on any day of the culture. The results of this study demonstrate a serum-free culture system for buffalo granulosa cells and stimulatory effect of FSH but not LH on steroid hormone production by buffalo granulosa cells under these conditions.

Impact of gonadotrophins and steroid hormones on tumour cells derived from borderline ovarian tumours

Conservative surgery is currently proposed for young patients with borderline ovarian tumours (BOT). For those experiencing infertility, the question of medically assisted procreation is raised. We have evaluated in vitro the proliferation of cultured BOT cells in response to FSH or estradiol (E(2)). Primary cell cultures were prepared from BOT. The presence of FSH and E(2) receptors was evaluated by immunochemistry. Cultures in vitro were stimulated with FSH (40 and 200 mUI/ml) or E(2) (300 and 2000 pg/ml) for 96 h and proliferation was evaluated with the WST-1 test. Four primary cultures were obtained that expressed FSH and E(2) receptors to different extents. Growth was generally similar to controls when treated with either FSH or E(2) although 300 pg/ml E(2) caused a significant inhibitory effect on cell proliferation (P = 0.035). No stimulatory effect of FSH or E(2) on cultured BOT cells was found, despite the presence of receptors. Although preliminary, these results suggest that gonadotrophins and E(2) could be used in patients experiencing infertility after conservative surgery.

The cultured rodent follicle as a model for investigations of gonadotrophin surge-attenuating factor (GnSAF) production.

Gonadotrophin surge-attenuating factor (GnSAF) bioactivity (the suppression of GnRH-induced but not basal LH and FSH secretion from pituitary gonadotrophs) is produced by granulosa cells in vitro. Previous studies to investigate this bioactivity used dispersed granulosa cells which lack some cell types and the structural components of the follicle in vivo. The aim of this study, therefore, was to investigate whether intact rodent follicle culture was a suitable model for the study of the production of GnSAF bioactivity, allowing GnSAF to be investigated in a more physiologically realistic environment while still retaining culture conditions from which, as with granulosa cell cultures, extraneous factors can be excluded. Follicles from 16-day-old rats and 21-day-old mice were cultured for 3-6 days in the presence or absence of FSH and/or LH. The follicle-conditioned medium, and matching samples of unconditioned culture medium were added to our established rat pituitary monolayer GnSAF bioassay. Both mouse and rat intact follicles produced GnSAF bioactivity, reducing GnRH-induced LH secretion significantly. GnSAF output from the mouse follicles was highest during days 1-3 of culture, when follicles were at an early antral stage of development, and fell on days 4-6 as the follicles grew to the mid antral stage. While the stimulatory effects of FSH on rat follicle GnSAF secretion was dose-dependent, LH alone did not increase GnSAF production. An antibody against human GnSAF blocked GnSAF bioactivity produced by rat follicles, and recognised proteins within the expected pI and molecular weight range for GnSAF in two-dimensional gels of rat follicle-conditioned medium, showing a good homology between rodent and human GnSAF proteins. In conclusion, the release of GnSAF bioactivity is principally from small follicles stimulated by FSH. Therefore, intact rodent follicle culture systems offer an excellent model for the investigation of factors controlling GnSAF production under relatively physiological conditions.

In vitro effect of FSH and testosterone on Sertoli cell nursing function in wall lizard Hemidactylus flaviviridis (Rüppell)

The present in vitro Sertoli cell-enriched culture study was aimed to understand the precise role of FSH and testosterone either alone or in combination in regulation of Sertoli cell nursing function such as lactate production, which in turn is utilized by the germ cells as an energy substrate. The stimulatory effect of FSH on lactate production by Sertoli cell-enriched culture was significantly enhanced when FSH and dihydrotestosterone were supplemented in the medium either simultaneously or DHT was added 12 h later to FSH (0 h FSH + 12 h DHT). On the contrary, DHT alone or added 12 h prior to FSH (0 h DHT + 12 h FSH) in the incubation medium, significantly reduced the lactate secretion by Sertoli cells as compared to control or FSH alone/FSH + DHT simultaneously/0 h FSH + 12 h DHT, respectively. This provides the conclusive evidence that androgen has additive effect on nursing function of Sertoli cell-enriched clusters only when they are in an activated state in response to gonadotropin.

Glutathione S-transferase alpha expressed in porcine Sertoli cells is under the control of follicle-stimulating hormone and testosterone.

Glutathione S-transferases (GSTs) are a family of detoxification isoenzymes present in different tissues including the testis and that conjugate many toxic substrates to glutathione. Among these substrates are carcinogens, mutagens and products of oxidative processes. In the present report we show that GSTalpha is expressed in somatic testicular Leydig cells and Sertoli cells. GSTalpha expression in Sertoli cells is under the hormonal control of FSH, testosterone, and estradiol. In Leydig cells, immunoreactive GSTalpha was present at the neonatal, pubertal, and adult periods. In Sertoli cells, GSTalpha was predominant in pubertal and adult testes (but not in neonatal testes), suggesting that its expression is controlled by gonadotropins. The regulatory action and the mechanisms of action of FSH and testosterone on GSTalpha mRNA and protein levels were studied by using a model of primary cultures of porcine testicular Sertoli cells. FSH increased GSTalpha mRNA levels in a dose-dependent manner (ED50 = 18.5 nm/ml) with a maximal effect observed after 48 h of exposure (a 3-fold increase; P < 0.001). In addition, FSH increased GSTalpha protein, which was detected as a doublet of 28 kDa. Treatment with testosterone enhanced GSTalpha mRNA levels in a dose-dependent (ED50 = 1.4 ng/ml) and time-dependent manner with a maximal effect delayed at 8 h of exposure (a 2-fold increase; P < 0.001). Similarly, Sertoli cell treatment with testosterone metabolites, dihydrotestosterone (DHT) and estradiol, led to an increase in GSTalpha mRNA levels. Because stimulatory effects of FSH and androgens were also observed on GSTalpha protein, we therefore had to determine whether the different hormones were affecting GSTalpha gene transcriptional activity, or GSTalpha mRNA stability, or both. FSH and 8-Br-cAMP (but not testosterone) increased the stability of GSTalpha mRNA. The effects of FSH and testosterone on GSTalpha protein were additive, confirming that both hormones act through distinct mechanisms on the expression of the enzyme. Taken together, the present observations indicate that Sertoli cell GSTalpha is targeted by FSH, testosterone, and its metabolites, and they reinforce the concept that Sertoli cells exert a protective role and are under endocrine control to ward against toxic agents in the context of Sertoli-germ cell interactions during spermatogenesis.

Hormone-regulated and glucose-sensitive transport of dehydroascorbic acid in immature rat granulosa cells.

Ascorbic acid is concentrated in granulosa cells of the follicle, and ascorbate deficiency causes follicular atresia. Dehydroascorbic acid (DHAA), the oxidized form of ascorbic acid, serves as an important source for the recycling of ascorbate. As we previously demonstrated endocrine up-regulation of ascorbic acid transport by granulosa cells, we investigated DHAA as an alternate source of ascorbate in the follicle. Granulosa cells were cultured for 24 h, and DHAA uptake was initiated by the addition of 14C-labeled ascorbic acid (300 microM) in the presence of ascorbic acid oxidase (2 U/ml), which catalyzes DHAA production. Almost 90% of accumulated DHAA was present as ascorbic acid within 2 h. Preculture of cells for 24 h with FSH (50 ng/ml) and IGF-I (30 ng/ml) significantly stimulated DHAA uptake compared with the control (158 +/- 16 vs. 43 +/- 8 pmol/10(6) cells, respectively). DHAA uptake by granulosa cells was inhibited by D-glucose (ID50, approximately 2.5 mM) and by the glucose transport inhibitors phloretin (200 microM) and cytochalasin B (10 microM), which reduced uptake to 13 +/- 2% and 8 +/- 3% of the control, respectively. Northern and Western analysis of GLUT1 in granulosa cells following 24 h coincubation with FSH and IGF-I revealed up-regulation of GLUT1 at both the messenger RNA and protein levels (1.6- and 1.3-fold of control, respectively), suggesting that the stimulatory effects of FSH and IGF-I on DHAA transport are mediated by the induction of GLUT1. GLUT4 protein was not detectable by Western analysis. Endocrine-regulated DHAA transport may represent an important mechanism for maintaining adequate antioxidant tone within the developing follicle.

Hormone-Regulated and Glucose-Sensitive Transport of Dehydroascorbic Acid in Immature Rat Granulosa Cells

Ascorbic acid is concentrated in granulosa cells of the follicle, and ascorbate deficiency causes follicular atresia. Dehydroascorbic acid (DHAA), the oxidized form of ascorbic acid, serves as an important source for the recycling of ascorbate. As we previously demonstrated endocrine up-regulation of ascorbic acid transport by granulosa cells, we investigated DHAA as an alternate source of ascorbate in the follicle. Granulosa cells were cultured for 24 h, and DHAA uptake was initiated by the addition of 14C-labeled ascorbic acid (300 μm) in the presence of ascorbic acid oxidase (2 U/ml), which catalyzes DHAA production. Almost 90% of accumulated DHAA was present as ascorbic acid within 2 h. Preculture of cells for 24 h with FSH (50 ng/ml) and IGF-I (30 ng/ml) significantly stimulated DHAA uptake compared with the control (158 ± 16 vs. 43 ± 8 pmol/106 cells, respectively). DHAA uptake by granulosa cells was inhibited by d-glucose (ID50, approximately 2.5 mm) and by the glucose transport inhibitors phloretin (200μ m) and cytochalasin B (10 μm), which reduced uptake to 13 ± 2% and 8 ± 3% of the control, respectively. Northern and Western analysis of GLUT1 in granulosa cells following 24 h coincubation with FSH and IGF-I revealed up-regulation of GLUT1 at both the messenger RNA and protein levels (1.6- and 1.3-fold of control, respectively), suggesting that the stimulatory effects of FSH and IGF-I on DHAA transport are mediated by the induction of GLUT1. GLUT4 protein was not detectable by Western analysis. Endocrine-regulated DHAA transport may represent an important mechanism for maintaining adequate antioxidant tone within the developing follicle.

Thyroid hormone effects on mouse oocyte maturation and granulosa cell aromatase activity.

In the present study we evaluated the role of T3 on the in vitro processes of mouse cumulus cell-oocyte complex expansion, oocyte meiotic maturation, and granulosa cell aromatase activity. Results obtained from cumuli oophori isolated from immature and adult mice ovaries demonstrated that T3 at all concentrations tested (0.1-100 nM) did not affect basal or FSH-induced cumulus expansion or interfere with oocyte meiotic maturation up to metaphase II stage. On the contrary, T3 inhibited in a time- and dose-dependent manner FSH-induced aromatase activity in cultured granulosa cells obtained from either adult or immature female mice. The half-maximal dose (ED50) of T3 inhibition was 0.87 +/- 0.21 nM, which is in agreement with the reported dissociation constant of T3 nuclear receptor (Kd = 0.4-5 nM) in mammalian granulosa cells. Time-course experiments demonstrated higher sensitivity to T3 of adult granulosa cells with respect to immature granulosa cells in culture. Indeed, in immature granulosa cells T3 inhibition became significantly evident only after 6 days of hormonal treatment, whereas in adult granulosa cells the inhibitory effect was present after only 2 days of treatment. (Bu)2cAMP- or 3-isobutyl-1-methyl-xanthine-stimulated aromatase activity was also significantly decreased by T3, thus suggesting that the inhibition was downstream from cAMP formation. Lastly, analysis of aromatase messenger RNA (mRNA) levels by semiquantitative RT-PCR demonstrated the ability of FSH to increase aromatase mRNA level in cultured granulosa cells by 2.4 +/- 0.5-fold. In agreement with the effect on enzyme activity, the stimulatory effect of FSH on aromatase mRNA level was greatly reduced after T3 cotreatment. In conclusion, T3 inhibition of aromatase activity may be of physiological relevance in the complex multihormonal regulation of mammalian follicle development and may contribute to explaining the alteration in female reproductive functions after thyroid hormone hypo- or hypersecretion.

Regulation of Midkine Messenger Ribonucleic Acid Levels in Cultured Rat Granulosa Cells

To investigate the regulation of ovarian midkine (MK) production at the mRNA level, we evaluated changes in MK mRNA in cultured granulosa cells. The level of MK mRNA increased in a time-dependent manner in the presence of FSH or retinoic acid (RA). FSH increased MK mRNA levels in a dose-dependent manner to a maximum of about 2-fold increase. The stimulatory effects of FSH were mimicked by 8-Br-cAMP. RA increased MK mRNA levels in a dose dependent manner to a maximum of about 2 fold increase at a concentration of 0.3 μM. These results show that granulosa cells produce MK mRNA under the independent control of FSH and RA. The coexistence of FSH and RA in this culture exerted no additive effect on the induction of MK mRNA. Analysis of FSH receptor mRNA induction by both reagents revealed that FSH stimulated expression of the FSH receptor while RA had an inhibitory effect on FSH receptor induction regardless of the presence of FSH. These data show that RA increases MK mRNA, and decreases FSH receptor expression, by a pathway independent of cAMP.

Modulatory role of epidermal growth factor in follicle-stimulating hormone-induced DNA synthesis in cultured rat granulosa cells.

Epidermal growth factor (EGF) modestly increased DNA synthesis by cultured rat granulosa cells. FSH also stimulated DNA synthesis dose-relatedly with the maximal effect occurring at FSH 100 ng/ml. The stimulatory effect of FSH was still greater than that of EGF. However, in the presence of EGF, the stimulatory effect of FSH at any concentration was regulated to the level as high as when EGF alone stimulates. In addition, EGF inhibited DNA synthesis induced by forskolin, but enhanced the action of (Bu)2cAMP additively, which indicates that EGF attenuates DNA synthesis of granulosa cells by suppressing the activity of adenylate cyclase or cAMP production. As it is suggested that cAMP is the most likely intracellular second messenger for FSH, the regulatory effect of EGF on FSH-induced DNA synthesis could be, in part, due to suppression of cAMP production. These results suggest that EGF is involved in granulosa cell proliferation, irrespective of the presence of FSH, in a different pathway from FSH and additionally modulates the FSH growth-promoting effect as a local regulator. The interaction between EGF and FSH may be important in the control of follicular development.

Production of estradiol by the fetal rat testis.

Testes from 17- to 20-d-old rat fetuses were cultured in vitro for various time intervals in Medium 199 alone or with added gonadotrophins. Estradiol released into the culture media was determined by radioimmunoassay. A basal estradiol secretion rate was inexistant or undetectable at all stages studied. In early stages (17 and 18 d) there was no difference in the stimulatory effect of FSH or LH at the same concentration. At 19 d, the prevalence of FSH became apparent. At 20 d, a significant action of FSH was noted after only 3 h culture time. 1-Methyl-1, 4-androstadiene-3,17-dione, an aromatase inhibitor, markedly depressed FSH-stimulated estradiol secretion. In the 20-d old testis, stimulation of estradiol production by FSH was more rapid and reached a higher level than by (Bu)2cAMP. It is suggested that the difference in the action of LH and FSH reflects the difference in the time of appearance of the corresponding receptors in the developing fetal testis.

The antagonistic effect of exogenous LH pulses on FSH-stimulated preovulatory follicle growth in ewes chronically treated with a gonadotrophin-releasing hormone agonist

The hypogonadotrophism model induced by the chronic administration of gonadotrophin-releasing hormone (GnRH) agonist was used to investigate the effects of different concentrations of FSH with or without LH pulses on the stimulation of follicular development in the ewe. Continuous administration of an agonist (buserelin) by osmotic minipump to thirty-six Welsh Mountain ewes from the early luteal phase for 5 weeks resulted in a sustained suppression of the plasma concentration of FSH and inhibited the pulsatile release of LH. The inhibition of gonadotrophin secretion was due to the desensitization and/or down-regulation of pituitary gonadotroph function, since the agonist-treated animals showed no response to a challenge of 1 microgram GnRH. During week 6 of agonist treatment, ewes were infused with either 4-hourly pulses of ovine LH (9 micrograms/pulse), low concentrations of ovine FSH (3 micrograms/h) or high concentrations of FSH (9 micrograms/h) alone or with 4-hourly pulses of LH. After 5 days of gonadotrophin infusion, there was no difference between the mean number of follicles per ewe from the animals treated with LH alone, low concentrations of FSH with or without LH pulses or the high concentration of FSH alone compared with the mean number of follicles from control ewes on day 8 of the luteal phase. Infusion of the high concentration of FSH alone stimulated the development of an increased number of large oestrogenic follicles (follicles greater than 2.5 mm in diameter and secreting greater than 3.7 nmol oestradiol/h in vitro) compared with control ewes. The addition of high-amplitude LH pulses to the infusion of the high concentration of FSH prevented follicles developing beyond 2.5 mm in diameter, but doubled the number of small follicles (less than or equal to 2.5 mm) present in the ovaries. These results show that normal follicular development can be induced by physiological concentrations of FSH alone in the absence of pulsatile LH release. The addition of high-amplitude LH pulses antagonized this stimulatory effect of FSH on follicle growth in the ewe.

The Effects of Insulin and Insulin-Like Growth Factors-I and -II on Estradiol Production by Granulosa Cells of Polycystic Ovaries*

The objective of this work was to examine the effects of insulin-like growth factors (IGFs) on estradiol (E2) production by granulosa cells obtained from ovaries of patients with polycystic ovary disease (PCO). Granulosa cells, isolated from ovaries of three PCO patients, were cultured in serum-free medium containing either androstenedione alone (10(-7) M) or androstenedione plus graded doses of FSH, IGF-I, IGF-II, and/or insulin. At the end of the culture period (2, 4, or 6 days) E2 levels in the medium were measured by RIA. The results from each patient were similar, and therefore, the data were pooled. In the 6-day time-course experiments, the control (untreated) cells produced relatively high levels of E2 at 2 days; however, none was detected thereafter. Treatment with FSH (30 ng/mL) stimulated E2 production 4-fold at 2 days, but the stimulatory effects of FSH were not sustained during culture. IGF-I at 30 ng/mL mimicked the effects of FSH. Concomitant treatment with FSH and IGF-I caused synergistic increases in E2 production (3-, 13-, and 33-fold at 2, 4, and 6 days, respectively). Dose-response studies revealed that FSH and IGF-I stimulated E2 production in a dose-dependent fashion (ED50 of FSH and IGF-I, were 1.1 +/- 0.3 and 7.6 +/- 7.2 ng/mL, respectively). In the presence of a maximally effective dose of FSH (30 ng/mL), the cells appeared to become more responsive to IGF-I (ED50 of IGF-I plus FSH, 1.09 +/- 0.29 ng/mL); however, this effect was not significant (P = 0.086). In the presence of a maximally effective dose of IGF-I (30 ng/mL), the stimulatory effect of FSH on E2 production was dramatically amplified, but the IGF-I did not significantly (P = 0.85) change the potency of FSH (ED50 of FSH plus IGF-I, 1.07 +/- 2.3 ng/mL). Treatment with IGF-II over the concentration range of 0.1-100 ng/mL had no effect on either control or FSH-stimulated E2 production. Treatment with insulin, either alone or together with FSH, increased the levels of E2, but the insulin effects were seen only at the highest doses tested (0.3-10 micrograms/mL). The results in these in vitro experiments with PCO granulosa cells indicate that 1) physiological concentrations of IGF-I are as effective as FSH in stimulating E2 production; 2) IGF-I and FSH act synergistically to control the level of E2 production; and 3) this synergy was not observed with insulin or IGF-II.

Follicle-stimulating hormone regulates low density lipoprotein metabolism by swine granulosa cells.

FSH amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine granulosa cells in a synergistic manner. The mechanisms subserving this synergism included the following. 1) FSH increased by 2.1-fold the number of specific high affinity low density lipoprotein (LDL) receptors on granulosa cells, with no change in apparent binding affinity. 2) FSH augmented by 1.8- and 3.6-fold the maximal rates of [125I]iodo-LDL internalization and degradation, respectively, without altering half-maximally effective concentrations of LDL supporting these processes. 3) FSH increased significantly the mass (micrograms) of free and esterified cholesterol (measured by fluorometry) contained in granulosa cells. 4) FSH stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester from exogenous [3H]cholesteryl linoleate-labeled LDL and amplified [3H]progesterone secretion by granulosa cells exposed to this source of lipoprotein-borne sterol. 5) The stimulatory effects of FSH on LDL degradation and cholesterol side-chain cleavage activity were mimicked by other activators of granulosa cell cAMP production, e.g. forskolin and cholera toxin. We conclude that FSH and LDL synergistically enhance progesterone biosynthesis by swine granulosa cells via cellular mechanisms that result in significantly augmented binding, internalization, and degradation of LDL in concert with increased effectual utilization of sterol substrate in the cholesterol side-chain cleavage reaction. These observations are consistent with a key trophic role for FSH in regulating the sterol-metabolizing ability and steroidogenic differentiation of granulosa cells and indicate that such actions of FSH can be accounted for by activation of the cAMP-effector pathway.

Hormonal control of epidermal growth factor receptors by gonadotropins during granulosa cell differentiation.

The hormonal induction of epidermal growth factor (EGF) receptor formation was analyzed during the maturation of granulosa cells obtained from diethylstilbestrol-implanted immature rats. In the absence of FSH, EGF receptors (as measured by the binding of [125I]iodo-EGF to the intact cells) rose by 50% at 6 h of culture, but then declined to about 25-40% of their initial levels at 24-96 h of culture. Scatchard analyses demonstrated the presence of high affinity EGF-binding sites in both freshly prepared cells and after FSH treatment. FSH stimulated a dose-dependent increase in the EGF receptor content of granulosa cells during a 96-h culture period. Concentrations of FSH as low as 2.5-5 ng/ml elevated EGF receptor levels 2- to 3-fold compared to those in untreated control cells, and 30 ng/ml FSH caused a maximal 15-fold rise. FSH increased EGF receptor levels approximately 2-fold in the first 6 h of culture and by up to 7-fold at 96 h compared to levels in freshly prepared cells. FSH treatment did not change the binding affinity (Kd = 5-6 X 10(-11) M) of the EGF receptor, but increased the total number of EGF-binding sites. The stimulatory effects of FSH on EGF receptor expression were mimicked by other cAMP-inducing ligands, including 8-bromo-cAMP, forskolin, and choleragen. Ligands known to inhibit granulosa cell function, including GnRH agonists and the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate, reduced the stimulation of EGF receptors by FSH. However, only 12-O-tetradecanoyl-phorbol 13-acetate suppressed the induction of EGF receptors by 8-bromo-cAMP. In granulosa cells cultured for 48 h with FSH, subsequent treatment with hCG for 24 h reduced EGF receptor content by 25%. Autoradiographic studies with [125I]iodo-EGF in ovarian thin sections demonstrated that EGF-binding sites were uniformly dispersed throughout the ovaries of diethylstilbestrol-implanted rats. Treatment with PMSG markedly increased EGF receptors in the outer walls of the growing follicles, while hCG treatment after PMSG caused a general decline in ovarian labeling. These results indicate that FSH maintains and increases the number of EGF receptors during granulosa cell differentiation, while LH/hCG reduces EGF-binding sites. Such changes in EGF receptors in the presence of endogenous growth factors may influence the number and selection of follicles destined for ovulation.

Hormonal regulation of granulosa cell inhibin biosynthesis.

The hormonal regulation of inhibin production by cultured granulosa cells from immature hypophysectomized, estrogen-treated rats was examined using a specific RIA which detects the N-terminal portion of the inhibin alpha-chain. The RIA measured bioactive inhibin of Mr about 32,000 in granulosa cell conditioned media fractionated by fast protein liquid chromatography. In the presence of 10(-7) M androstenedione, FSH stimulated inhibin production in a dose-dependent manner during a 2-day culture. Inclusion of a phosphodiesterase inhibitor decreased the EC50 for FSH from 2.6 to 0.8 ng/ml (n = 3). The stimulatory effect of FSH could be mimicked with forskolin (an adenyl cyclase activator) and with a cAMP analog, (Bu)2cAMP, consistent with FSH action mediated through a cAMP dependent pathway. Intracellular levels of inhibin were unmeasureable, suggesting that inhibin is not stored to any great extent by the granulosa cells. This finding was consistent with in vivo studies which showed that whereas FSH treatment for 2 days doubled serum inhibin levels when compared with basal levels, there was no increase in the concentration of extractable inhibin in ovarian tissue. Granulosa cells which had been exposed to 20 ng/ml FSH for 2 days to induce LH receptors produced inhibin in response to both LH and human CG during the subsequent 2-day culture, with the levels of inhibin equalling the amount inducible by FSH. In contrast, neither PRL nor terbutaline, a beta 2-adrenergic agonist, had any effect on inhibin production even though receptors for these hormones are also induced by FSH. GnRH was found to inhibit the FSH-stimulated production of inhibin (IC50, 10(-7) M), consistent with previous observations that GnRH can act at the ovarian level to inhibit granulosa cell differentiation. This inhibition by GnRH could be reversed by inclusion of a specific GnRH antagonist. On the other hand, another regulatory peptide, vasoactive intestinal peptide, slightly stimulated inhibin production. The effect of several growth factors was also tested. Insulin-like growth factor I raised not only FSH-stimulated inhibin levels, but basal levels as well. Insulin was also effective, but only at 100-fold higher concentration. Epidermal growth factor inhibited FSH-stimulated inhibin production (IC50 = 0.1 ng/ml), whereas fibroblast growth factor had no effect. Thus, granulosa cell inhibin secretion is regulated by FSH and LH but not by PRL, presumably via a cAMP-mediated pathway.(ABSTRACT TRUNCATED AT 400 WORDS)