Regulation of steroid production in cultured porcine thecal cells by transforming growth factor-beta.

Evidence that transforming growth factor-beta (TGF beta) is produced by porcine thecal cells and acts upon porcine granulosa cells suggests that this peptide may be a local regulator of follicular function in this species. The objective of the present study was to investigate the effects of TGF beta on steroidogenesis in thecal cells from 4-6 mm follicles of prepubertal gilts. In this culture system, cells undergo functional luteinization such that production of androstenedione, the major steroid product in 24 h incubations, declines, and in the presence of luteinizing hormone (LH) (250 ng/ml) and insulin (1 micrograms/ml), progesterone production increases over a 3-day culture period. TGF beta (0.1-10 ng/ml) had no effect on production of androstenedione from endogenous precursors in the presence or absence of LH, although there was a slight inhibition of androstenedione production in the presence of exogenous progesterone (up to 23%). As the cells luteinized in culture, the increase in progesterone production in response to LH increased (day 1, 4.4-fold; day 3, 13-fold). TGF beta at concentrations as low as 0.1 ng/ml caused marked (up to 90%) inhibition of LH-stimulated progesterone production in day 3 cultures. In the presence of TGF beta (10 ng/ml), the response to LH was completely abolished, and the response to dibutyryl cAMP was considerably attenuated (25% of controls). Since the primary site of action of TGF beta appeared to be distal to cAMP formation, the effect of TGF beta on conversion of exogenous 22-hydroxy-cholesterol and pregnenolone to progesterone was determined in day 3 cultures. 22-Hydroxycholesterol and pregnenolone restored progesterone production to at least 80% and 89% of controls, respectively. While the primary inhibitory action of TGF beta appears to be exerted distal to cAMP formation, neither cholesterol sidechain cleavage nor the 3 beta-hydroxysteroid dehydrogenase: delta 5-delta 4 isomerase reactions are primary targets of this factor. Together with evidence of thecal production of TGF beta, the results of this study indicate that this peptide may be an autocrine regulator of thecal steroidogenesis.     

Transforming growth factor-alpha and -beta differentially regulate growth and steroidogenesis of bovine thecal cells during antral follicle development

The actions and interactions of transforming growth factor-alpha (TGF alpha) and TGF beta on growth and differentiation of bovine thecal cells were investigated. Bovine thecal interna cells were isolated from small (less than 5 mm), medium (5-10 mm), and large (greater than 10 mm) antral follicles and cultured in the presence or absence of TGF alpha and/or TGF beta. Both [3H]thymidine incorporation and changes in cell number (i.e. DNA levels) were evaluated to determine effects on thecal cell growth. Short term treatment of cells with TGF alpha (18-24 h) stimulated thymidine incorporation, and longer term treatments (4 days) increased cell number. TGF beta suppressed thymidine incorporation below that observed in untreated cultures, but had no effect on cell number. When combined with TGF alpha, TGF beta suppressed the ability of TGF alpha to stimulate thymidine incorporation and increase cell number. The response to these growth factors was similar for cells isolated from the different stages of antral follicle development. The effects of TGF alpha and TGF beta on thecal cell differentiation were evaluated by quantitating changes in androstenedione and progesterone accumulation in cultures treated with TGFs in the absence (basal) or presence of hCG, estradiol (E2), or a combination of hCG and E2. E2 and hCG were included in this study because previous research has demonstrated that these hormones alter thecal cell steroidogenesis. Treatment with TGF alpha resulted in a suppression of basal and hormonally stimulated accumulation of androstenedione during days 0-3 of culture, whereas TGF beta did not significantly alter androstenedione accumulation. TGF alpha also suppressed progesterone accumulation during days 0-3 of culture in the absence or presence of hormones. In contrast, TGF beta stimulated accumulation of progesterone in cultures that did not contain E2, which suppressed progesterone during this period. Therefore, during days 0-3 of culture, TGF alpha appears to have suppressive effects on androstenedione and progesterone production, whereas TGF beta can stimulate progesterone production in the absence of E2. During days 3-6 of culture, thecal cell differentiation changes, and the capacity to produce androstenedione dramatically declines, while the capacity to produce progesterone increases. During this period, either TGF alpha or TGF beta slightly increased basal progesterone accumulation and partially suppressed the ability of hCG to stimulate progesterone. The effects of TGFs on thecal cell steroidogenesis were similar with cells isolated from the different stages of antral follicle development. Results from these studies provide evidence that THF alpha and TGF beta can modulate thecal cell growth and differentiation (i.e. steroidogenesis).(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidermal growth factor inhibits follicular response to human chorionic gonadotropin: possible role of cell to cell communication in the response to gonadotropin.

Epidermal growth factor (EGF) affects follicular steroidogenesis and expression of gonadotropin receptors. The effects of EGF on hCG-induced estradiol and progesterone secretion and ovulation were examined in the in vitro perfused rabbit ovary. We also examined the effects of EGF on hCG-induced progesterone secretion by isolated granulosa cells. In addition, distribution of hCG within the follicle was probed by immunohistochemical means 30 min after its administration to the in vitro perfused ovary. EGF significantly (P less than 0.05) reduced hCG-induced secretion of estradiol (control, 117 +/- 12 pg/min.follicle; 10 ng/ml EGF, 55 +/- 10) and progesterone (control, 18.2 +/- 1.2 ng/min.follicle; 10 ng/ml EGF, 11.9 +/- 0.8) by the perfused ovary. In contrast, EGF did not inhibit hCG-induced progesterone secretion by isolated granulosa cells. Ovulatory efficiency (number of ovulated ova per number of mature follicles x 100) when EGF was given 30 min before hCG was reduced dose-dependently from 58.2% with no EGF to 8.3% with 10 ng/ml EGF (P less than 0.001). Ovulation was not inhibited by EGF when it was given 30 min after hCG. Distribution of hCG in the preovulatory follicle was confined to the basement membrane, thecal cell layer, and a small fraction of the outer granulosa cell layer. These observations suggest that gonadotropin stimulates the follicle through the release of a secondary signal(s) from ligand-bound granulosa cells near the follicle wall to unexposed cells of the inner avascular area. EGF may inhibit the follicular response to hCG by attenuation of this cell to cell communication.     

Sites of interaction between epidermal growth factor and transforming growth factor-beta 1 in the control of steroidogenesis in cultured porcine Leydig cells.

The present study examines how the hormonal action of gonadotropin is modulated by transforming growth factor-beta 1 (TGF beta 1) and epidermal growth factor (EGF) in primary cultures of purified porcine Leydig cells. Although TGF beta 1 (1 ng/ml; 48 h) and EGF (10 ng/ml; 72 h) individually enhanced hCG-stimulated testosterone formation, the effects of EGF were more pronounced than those of TGF beta 1. When studied in combination, the effects of maximal concentrations of TGF beta 1 and EGF were additive on gonadotropin hormonal action. In the present study we demonstrate that their additive effects resulted from a complex interaction occurring at the levels of cholesterol substrate availability in the mitochondria and of 3 beta-hydroxysteroid dehydrogenase/isomerase activity (3 beta HSDI). First, TGF beta 1 (1 ng/ml; 48 h) and EGF (10 ng/ml; 72 h) were, respectively, shown to reduce and enhance dehydroepiandrosterone (DHEA) formation (evaluated in the presence of 10(-5) M WIN 24540, an inhibitor of 3 beta HSDI) in Leydig cells when acutely (3 h) stimulated with hCG (0.01-1 ng/ml), but not when incubated with 22R-hydroxycholesterol (3 micrograms/ml). Such findings indicate that TGF beta 1 and EGF did not affect cholesterol side-chain cleavage cytochrome P450 activity, but, respectively, decreased and increased cholesterol substrate availability for this enzyme in the mitochondria. Furthermore, when Leydig cells were treated with the combined factors, the formation of delta 5-steroid intermediates (such as DHEA) in untreated (control) and EGF-plus TGF beta 1-treated cells was not significantly different whether the cells were acutely stimulated with the gonadotropin or incubated with 22R-hydroxycholesterol. Such findings indicate that the effects of EGF and TGF beta 1 on cholesterol substrate availability in the mitochondria are antagonistic. Second, EGF, TGF beta 1, and EGF plus TGF beta 1 significantly (P less than 0.001) increased delta 5-steroid intermediate (i.e. pregnenolone and DHEA), but not delta 4-steroid intermediate (i.e. progesterone and androstenedione), conversion into testosterone, indicating that the growth factors increased, individually or in combination in an additive manner, 3 beta HSDI activity (respectively, 90.7 +/- 0.6%, 80.6 +/- 2.6%, and 164 +/- 4.5% increase in the presence of EGF, TGF beta 1, and EGF plus TGF beta 1). Together, the reciprocal suppression of the effects of TGF beta 1 and EGF on the mitochondrial cholesterol substrate availability coupled to their stimulatory additive actions on 3 beta HSDI activity provide an explanation of the additive actions of the two growth factors on gonadotropin-induced testicular androgen formation.     

Ovarian action of leptin: effects on insulin-like growth factor-I-stimulated function of granulosa and thecal cells

To test the hypothesis that leptin signals metabolic information to the reproductive system in cattle by directly affecting IGF-I-induced ovarian cell function, granulosa and thecal cells from bovine ovarian follicles were cultured for 2 d in serum-free medium with added hormones. Recombinant human leptin at 30 and 300 ng/mL had no effect on basal thecal cell steroidogenesis or thecal cell numbers. However, 300 but not 30 ng/mL of leptin attenuated (p<0.05) luteinizing hormone-induced androstenedione production by 24% in the absence of IGF-I and by 16% in the presence of IGF-I. Leptin had no effect on IGF-I-induced estradiol production in the presence of follicle-stimulating hormone (FSH), but at 100 ng/mL, leptin inhibited (p<0.05) FSH plus IGF-I-induced progesterone production and granulosa cell proliferation by 29 and 31%, respectively. Leptin did not compete for ¹²⁵I-IGF-I binding to granulosa or thecal cells, whereas unlabeled IGF-I did. In conclusion, leptin has weak inhibitory effects on gonadotropin-and/or IGF-I-induced steroidogenesis of thecal and granulosa cells.     

On the mechanisms involved in the inhibitory and stimulating actions of transforming growth factor-beta on porcine testicular steroidogenesis: an in vitro study

By using immature porcine Leydig cells cultured in defined medium as a model, transforming growth factor-beta (TGF beta) was shown to exert a dramatic inhibitory effect on their basal and human chorionic gonadotropin (hCG) (or 8-bromo-cyclic AMP) stimulated dehydroepiandrosterone secretion, in the presence or absence of saturating concentrations of exogenous (low density lipoprotein) cholesterol substrate. In contrast, TGF beta exerted both a stimulating and inhibitory effect on testosterone secretion: while hCG-stimulated testosterone secretion was enhanced by low doses of TGF beta (0.06-0.4 ng/ml, 48 h), it was decreased with higher concentrations of TGF beta (2.5-10 ng/ml, 48 h). The data obtained show that the inhibitory action of TGF beta on testicular steroidogenesis was related to a decrease in pregnenolone formation by affecting a step(s) distal to cyclic AMP formation but before cholesterol association with cytochrome P-450 side-chain cleavage. As for the stimulatory effect of TGF beta on testosterone formation, this was mainly related to an increase (about 2-fold) in 3 beta-hydroxysteroid dehydrogenase/isomerase activity (ED50 0.05 ng/ml, 2 X 10(-13) M). The results indicate that the (short-term) steroidogenic stimulatory action of luteinizing hormone (LH)/hCG is antagonized by high concentrations of TGF beta by decreasing pregnenolone formation while it is enhanced by the stimulating action of low concentrations of TGF beta exerted on 3 beta-hydroxy steroid dehydrogenase/isomerase activity.     

Transforming growth factor-beta promotes differentiation of ovarian thecal-interstitial cells but inhibits androgen production

Evidence that transforming growth factor-beta (TGF beta) is produced by thecal-interstitial cells (TIC) has suggested the hypothesis that TGF beta may be an autocrine regulator of TIC function. The purpose of these studies is to begin to test this hypothesis. In the present experiments we tested the effects of TGF beta on steroid production by TIC isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation. When TIC (10(4) viable cells/well) were cultured in serum-free medium (0.2 ml in 96-well plates), low amounts of androsterone (less than 4 ng/ml) were produced at 2, 4, and 6 days. TGF beta (0.01-100 ng/ml) did not change basal androsterone production. Treatment with LH (50 ng/ml) stimulated a 100-fold increase in androsterone at 2 days and 60-fold increases at 4 and 6 days. Concomitant treatment with TGF beta (10 ng/ml) caused a 65% inhibition (ED50 = 2.3 +/- 0.7 ng/ml) of androsterone production at each time period. Analysis of key steroid metabolites demonstrated that androsterone and androstenedione were inhibited equally, while progesterone was significantly increased (ED50 = 1.2 +/- 0.2 ng/ml). Time-course studies revealed that TGF beta alone did not alter progesterone production at 2 days, but markedly increased progesterone (10-fold) above control levels at 4 and 6 days. Dose-response experiments showed that TGF beta did not alter the sensitivity of the TIC to LH stimulation, indicating that LH activation of the intracellular signaling pathway was not blocked by TGF beta. Treatment with insulin-like growth factor-I (IGF-I) together with LH caused a synergistic increase in androsterone production. The synergistic stimulation of LH action by IGF-I could be blocked by TGF beta. Interestingly, TIC were more sensitive to TGF beta in the presence of IGF-I (ED50 = 0.18 +/- 0.04 ng/ml). In contrast, TGF beta enhanced progesterone production only at the highest dose of TGF beta (10 ng/ml). To further elucidate the mechanism of TGF beta action, the effects of TGF beta on the TIC content of 17 alpha-hydroxylase/C17-20 lyase (P450(17)alpha) and cholesterol side-chain cleavage (P450scc) were analyzed by immunoblotting. TGF beta alone or in combination with LH stimulated an increase in P450scc content, but did not alter P450(17 alpha content. These results lead us to conclude that 1) the TIC are targets for TGF beta; 2) IGF-I increases the sensitivity of TIC to TGF beta action; and 3) TGF beta acts directly on TIC to stimulate progesterone while inhibiting androgen production.     

Modulation of steroidogenesis in choriocarcinoma cells by cholera toxin, phorbol ester, epidermal growth factor and insulin-like growth factor I

The effects of cholera toxin (CT), which stimulates adenylate cyclase, 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on progesterone (P) and estradiol (E2) secretion by human choriocarcinoma JEG-3 cells were studied. During a 48 h incubation, CT, TPA and EGF stimulated P production in a concentration-dependent manner, whereas IGF-I was without effect. CT (1.0 ng/ml), TPA (10 ng/ml) and EGF (10 ng/ml) stimulated P production maximally 4.3-, 3.3- and 2.3-fold over basal, respectively. When added together with CT, TPA and EGF stimulated P production 10.0- and 5.0-fold over basal production showing that the effects of CT plus TPA were more than additive but those of CT plus EGF less than additive. Time-course studies indicated that the effects were detectable at 12 h, and continued to increase up to 48 h. The conversion of added dehydroepiandrosterone sulfate (DHEAS) to E2 was stimulated by CT and TPA and inhibited by IGF-I in a concentration-dependent manner. By contrast, EGF had no effect. The maximal responses in E2 production were 3.2- and 2.0-fold over unstimulated cells by CT (1.0 ng/ml) and TPA (10 ng/ml), respectively. When both agents were added together, their effects on E2 production were additive with 5.5-fold increase over unstimulated cells. IGF-I (30 ng/ml) inhibited maximally basal and CT-stimulated E2 production by 33% and 42%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of the fetus and estrogen on maternal serum growth hormone, insulin-like growth factor-II, and epidermal growth factor concentrations during baboon pregnancy

In the present study we determined whether the fetus and estrogen affect maternal serum concentrations of GH, insulin-like growth factor-II (IGF-II), and epidermal growth factor (EGF) and placental IGF-II formation in pregnant baboons. The objective was to ascertain whether the previously reported increase in placental formation and serum concentrations of IGF-I induced by removal of the fetus and, thus, estrogen in pregnant baboons was mediated by GH and whether it was specific for IGF-I. On day 100 of gestation (term is 184 days), fetuses were removed, and placentas were left in situ, i.e. fetectomy. After fetectomy, baboons received pellets of aromatizable androstenedione (50-150 mg every 10 days, sc; n = 8), were injected with estradiol (E2) benzoate (0.50-2.5 mg/day, sc; n = 8), or were not further treated (n = 6) on days 101-159 of gestation. Placental cells obtained on day 160 were dispersed in 0.1% collagenase, isolated via 50% Percoll centrifugation, then incubated for 24 h at 37 C in medium 199. Maternal serum E2 concentrations increased with advancing gestation in intact baboons, were decreased by 79% after fetectomy and, thus, removal of adrenal C-19 steroid estrogen precursors, and restored by androstenedione or E2 treatment after fetectomy. Mean serum GH was 20.2 +/- 0.6 ng/ml on days 101-160 in untreated intact animals. Fetectomy decreased (P less than 0.001) GH levels to 12.1 +/- 0.5 ng/ml. Androstenedione or E2 treatment after fetectomy restored serum GH to 20.8 +/- 1.1 and 22.4 +/- 0.6 ng/ml, respectively. Serum IGF-II was 1406 +/- 54 ng/ml on days 101-160 in controls and decreased (P less than 0.001) rapidly after fetectomy to a value (305 +/- 16) that was 78% lower than that in untreated baboons. Androstenedione or E2 treatment after fetectomy had no effect on the fetectomy-induced decrease in IGF-II levels. In vitro secretion of IGF-II by placental trophoblasts of fetectomized baboons (10.3 +/- 0.6 ng/ml.24 h) was 88% lower (P less than 0.001) than that in controls (85.6 +/- 15.7). Despite androstenedione or E2 treatment after fetectomy, placental IGF-II production remained low (9.2 +/- 1.1 and 8.8 +/- 0.4 ng/ml.24 h, respectively). The overall mean maternal serum EGF concentration was 379 +/- 20 pg/ml in the second half of baboon pregnancy. Fetectomy or treatment with androstenedione or E2 had no effect on serum EGF levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of androstenedione production by adenosine 3',5'-monophosphate and phorbol myristate acetate in ovarian thecal cells of the domestic hen

Although factors that regulate cAMP and steroid production in granulosa cells of hen preovulatory follicles have been well studied, much less is known of the mechanisms that control steroidogenesis in the adjacent thecal layer. These studies were conducted to examine the involvement and interaction of cAMP and protein kinase-C in modulating androstenedione output from isolated ovarian thecal cells collected from the second largest preovulatory follicle. Treatment of thecal cells with ovine LH (0.01-100 ng/tube) caused a dose-dependent increase in androstenedione secretion. Although coincubation of cells with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1 mM) potentiated the effects of LH on steroid production, cAMP levels increased only in response to the higher doses of LH (10-100 ng/tube). Small but significant increases in cAMP accumulation and androstenedione production were observed in response to vasoactive intestinal peptide (0.1 and 1.0 microM), but not to 100 ng/tube chicken FSH, in the presence of 0.1 mM 3-isobutyl-1-methylxanthine. Treatment of thecal cells with cholera toxin (0.001-100 ng/tube) or forskolin (0.001-10 microM) resulted in a dose-dependent increase in cellular cAMP levels and androstenedione secretion. Thecal cell androstenedione production was also stimulated by the cAMP analog 8-bromo-cAMP (0.1-1.0 mM). Incubation of thecal cells with phorbol 12-myristate 13-acetate (PMA; 0.32-162 nM) or 1-oleoyl-2-acetylglycerol (OAG; 2.5-126 microM) increased basal steroidogenesis (progesterone and androstenedione production) in the absence of a rise in cAMP levels. By contrast, the stimulatory effects of 1 ng/tube LH on androstenedione, but not progesterone, production were attenuated by the presence of PMA (3.2-162 nM) or OAG (25-126 microM). Only a high concentration of OAG (126 microM) suppressed cAMP accumulation stimulated by LH (50 ng/tube). Phorbol ester treatment (32-162 nM PMA) also inhibited androstenedione production in thecal cells stimulated by the presence of 8-bromo-cAMP (1 mM), indicating a post-cAMP effect of protein kinase-C activity on steroidogenesis. In contrast to the effects of PMA, phorbol 13-monoacetate (162 nM), a nontumor-promoting analog of PMA which does not activate protein kinase-C, did not alter basal steroidogenesis, nor did it affect androstenedione secretion stimulated by LH or 8-bromo-cAMP. Data from the present studies indicate that the adenylyl cyclase-cAMP pathway can mediate the induction of thecal cell steroidogenesis by extracellular signals (i.e. LH and vasoactive intestinal peptide), whereas activated protein kinase-C can both stimulate and inhibit androstenedione production, depending upon the hormonal environment.(ABSTRACT TRUNCATED AT 400 WORDS)     

The transfection-induced overexpression of IGF-binding protein-4 affects the secretory activity of porcine ovarian granulosa cells and their response to hormones and IGF-I

The aim of our studies was to examine whether IGF-binding protein (IGFBP)-4 is involved in the control of the secretion of various ovarian substances and also the mediation of the effects of several hormones and growth factors on this secretion. For this purpose, we carried out the transfection of porcine granulosa cells with a cDNA sense construct, increasing IGFBP-4 synthesis. We then compared the release of IGFBP-3, progesterone, oxytocin and IGF-I by control and transfected cells cultured with and without porcine LH (100 ng/ml), porcine GH (100 ng/ml), IGF-I (10 ng/ml), oxytocin (10 ng/ml) and estradiol-17beta (100 ng/ml). The concentration of IGFBP-4 produced was assessed using ligand blotting, and the release of progesterone, oxytocin, IGF-I and IGFBP-3 was evaluated using RIA/IRMA techniques. It was observed that GH, IGF-I, estradiol, LH and oxytocin alter the progesterone, oxytocin, IGF-I and IGFBP-3 release by porcine ovarian granulosa cells. Transfection of these cells with an IBFBP-4 cDNA expression construct significantly increased the IGFBP-4 accumulation in cell-conditioned medium. Furthermore, this transfection significantly reduced progesterone, oxytocin and IGFBP-3 release, and increased IGF-I output in cells cultured in the absence or presence of GH, IGF-I, estradiol and LH. The addition of oxytocin, but not of other tested substances, fully or partially prevented the effects of IGFBP-4 overexpression on IGFBP-3, IGF-I, but not on progesterone release. The present results suggested that IGFBP-4, as well as GH, IGF-I, estradiol, LH and oxytocin, is a potent regulator of porcine ovarian steroid (progesterone), nonapeptide hormone (oxytocin), growth factor (IGF-I) and growth factor-binding protein (IGFBP-3) release. IGFBP-4 is an inhibitor of basal progesterone, oxytocin and IGFBP-3 release and a stimulator of IGF-I output by porcine ovarian cells. The action of IGFBP-4 on the ovary can be mediated by (1) inhibition of oxytocin release, (2) suppression of receptor/postreceptor events induced by other hormones and IGF-I and (3) stimulation of IGF-I release.     

Estrogen regulation of thecal cell steroidogenesis and differentiation: thecal cell-granulosa cell interactions

Estrogen regulation of thecal cell steroidogenesis and differentiation was investigated with cells from ovarian antral follicles. Bovine theca interna cells were isolated and cultured in serum-free conditions to evaluate the effects of estradiol on thecal cell production of androstenedione, testosterone, and progesterone. Estradiol increased thecal cell androgen production throughout a 6-day culture period; however, the basal and stimulated levels of androgen production diminished after day 3 of culture. Androstenedione accumulation was approximately 10-fold greater than that of testosterone. In contrast to the stimulatory effects that estradiol had on androgen production, estradiol suppressed progesterone production throughout the 6-day culture period. Comparison of the effects of estradiol and hCG on thecal cells from small (less than 5 mm), medium (5-10 mm), and large (greater than 10 mm) antral follicles demonstrated that estradiol stimulated androgen production to a greater extent than hCG with cells from all of these stages of follicle development. Estradiol stimulation of androstenedione was greater in theca from small follicles than in theca from medium or large follicles. In contrast, suppressive effects of estradiol on progesterone were most apparent on thecal cells from medium and large follicles and less apparent on theca from small follicles. Estradiol stimulated androstenedione production in a dose-dependent fashion, with a minimum effective concentration of 10(-9) M and a maximum effective concentration of 10(-7)-10(-6) M. Concentrations greater than 10(-6) M estradiol resulted in a decline in the stimulatory response and may be important in the preovulatory follicle to suppress thecal cell androgen production and initiate the process of luteinization. Progesterone production was slightly stimulated by 10(-9) M estradiol, whereas higher concentrations (10(-7)-5 x 10(-6) M) resulted in a dose-dependent suppression of progesterone production. Interestingly, combined treatment of thecal cells with estradiol and hCG resulted in a greater than additive stimulation of androstenedione production, and estradiol decreased the ability of hCG to stimulate progesterone production. Observations demonstrate that estradiol can dramatically alter thecal cell production of steroids and support a hypothesis that steroid-mediated interactions between granulosa and thecal cells play an important role in regulating cellular function within follicles. The data provide evidence that a local feedback loop may exist in ovarian follicles, where androgens produced by thecal cells are used as a substrate for granulosa cell aromatization into estrogens, which, in turn, may feed back to stimulate thecal cell production of androgens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differentiation of rat ovarian thecal cells: evidence for functional luteinization

To determine if thecal cells of rat preovulatory (PO) follicles become functionally luteinized, theca from small antral (SA) and PO follicles were isolated before and 8 h after iv injection of an ovulatory dose (10 IU) of hCG. Thecal explants were cultured for 30 days in Dulbecco's Modified Eagle's Medium-Ham's F-12 medium containing 1% fetal calf serum (FCS) with or without 5 ng/ml ovine LH or 10 microM forskolin. Whereas theca from SA, hCG-treated SA, and PO follicles were dependent on LH or forskolin to maintain progesterone (greater than 10 ng/ml) and androstenedione (greater than 10 ng/ml) accumulation, luteinizing theca (hCG-treated PO) accumulated more than 10 ng/ml progesterone and more than 2 ng/ml androstenedione with or without LH or forskolin for 30 days. Granulosa cells were isolated from these same follicles and cultured under similar conditions, including 10 ng/ml testosterone and 25 ng/ml ovine FSH. Only granulosa cells isolated from luteinizing follicles (hCG-treated PO) maintained progesterone (greater than 20 ng/ml) and estradiol (10 ng/ml) accumulation with or without FSH or forskolin for 30 days. Basal concentrations of cAMP were 5 to 10-fold higher in thecal and granulosa cells from luteinizing follicles than in these tissues isolated from SA or PO follicles. We conclude that thecal cells as well as granulosa cells of rat PO follicles respond to the LH/hCG surge by becoming functionally luteinized, less dependent on LH, and capable of maintaining an increased accumulation of basal cAMP. Furthermore, the data suggest that one luteinizing thecal explant produces a similar amount of progesterone as one follicle equivalent of luteinizing granulosa cells. Thus, luteinized theca have the potential of contributing significantly to progesterone secretion by the mature rat corpus luteum.     

Epidermal growth factor directly stimulates steroidogenesis in primary cultures of porcine Leydig cells: actions and sites of action

The actions and the mechanisms of action of epidermal growth factor (EGF) in testicular steroidogenesis were investigated using a model of primary culture of purified porcine Leydig cells from immature intact animals. EGF decreased (1.7-fold) human CG (hCG)-induced dehydroepiandrosterone (DHEA) accumulation in the medium whereas it enhanced (2.5-fold) that of testosterone. The maximal and half-maximal effects on both DHEA and testosterone secretions were observed at similar concentrations which were, respectively, 3 (5 x 10(-10) M) and 0.7 (11 x 10(-11) M) ng/ml EGF, after 72-h treatment. EGF effect on DHEA and testosterone secretion was similarly observed whether the cells were acutely (3 h) stimulated with hCG (1 ng/ml) or with 8-bromo-cAMP (10(-3) M). To further localize the steroidogenic biochemical steps affected by EGF, the growth factor action on steroidogenic enzyme activities was investigated. EGF increased delta 5 steroid intermediate (i.e. pregnenolone and DHEA) formation [evaluated in the presence of 10(-5) M of WIN 24540, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/iosomerase (3 beta-HSDI) activity]. However, this stimulation was observed in cells when acutely (3 h) stimulated with hCG (0.01-1 ng/ml) but not when incubated with 22R-hydroxycholesterol (0.01-10 micrograms/ml). Such findings indicate that EGF did not affect cholesterol side chain cleavage cytochrome P450 activity but probably increased cholesterol substrate availability for this enzyme in the inner mitochondria. Moreover, EGF significantly (P less than 0.001) increased delta 5 steroid intermediate (i.e. pregnenolone and DHEA) but not delta 4 steroid intermediate (i.e. progesterone and androstenedione) conversion into testosterone, indicating that EGF enhances 3 beta-HSDI activity. Such effects of EGF are directly exerted on Leydig cells since EGF receptors (Kd = 16 x 10(-11) M) are present in primary cultures of purified porcine Leydig cells. Together, the present findings show that in Leydig cells from intact animals, EGF enhances the gonadotropin action on testosterone formation through an increase in the availability of cholesterol substrate in the mitochondria as well as an increase in the activity of 3 beta-HSDI.     

Steroidogenesis by equine preovulatory follicles: relative roles of theca interna and granulosa cells

Estrous cycles in mares have several unique characteristics, including the presence of a long period of estrus and the absence of a typical LH surge. Like follicles of other species, equine preovulatory follicles are characterized by their ability to secrete large amounts of 17 beta-estradiol, but it is not clear which follicular cell type is responsible for estradiol synthesis in mares. To better understand the relative roles of theca interna and granulosa cells in follicular steroidogenesis, presumptive ovulatory follicles were obtained from mares during early estrus (first or second day of estrus; n = 4) and during late estrus (fourth or fifth day of estrus; n = 4). Preparations of theca interna and granulosa cells were cultured for 3 days in medium with or without equine LH, FSH, LH plus FSH, or CG (100 ng/ml) in the presence or absence of 0.5 microM testosterone, and culture media were assayed for progesterone, androstenedione, and 17 beta-estradiol. Progesterone was the predominant steroid secreted by granulosa cells in the absence of exogenous testosterone. Its accumulation was significantly higher in cultures of granulosa cells from late vs. early estrus (P less than 0.05), and all gonadotropins stimulated progesterone secretion at both stages of follicular development (P less than 0.05). In contrast, granulosa cells secreted very low amounts of androstenedione in vitro, and only very small amounts of 17 beta-estradiol were produced when cells were cultured in medium without testosterone. However, the addition of testosterone caused a 170-fold increase over control values in estradiol accumulation over 3 days of culture (P less than 0.0001), clearly indicating the presence of a very active aromatase enzyme system in equine granulosa cells. Steroid secretion by theca interna differed in several respects from secretion by granulosa cells. Theca interna from early and late estrous follicles secreted negligible amounts of progesterone in vitro, and equine gonadotropins had no effect on its secretion. Also, theca interna secreted only small amounts of estradiol in vitro, and its accumulation was not increased by the addition of exogenous testosterone. Also, in contrast to granulosa cell cultures, androstenedione was the predominant steroid secreted by theca interna from early and late estrous follicles. In conclusion, this study does not support the current model of equine follicular steroidogenesis, which holds that 17 beta-estradiol biosynthesis derives primarily from the theca interna layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Luteinization of porcine thecal cells in vitro

The objective of this study was to determine the requirements for the functional luteinization of porcine thecal cells in vitro. In serum-free incubations with luteinizing hormone (LH) (250 ng/ml) androstenedione concentrations increased up to 14 h, after which time no further accumulation occurred; progesterone accumulation was low, and did not increase after 4 h. In the presence of 1% fetal bovine serum and LH, androstenedione production declined, but progesterone production per day increased over a 4-day period, while cellular protein remained constant. LH was required for both the induction and maintenance of elevated progesterone production. Insulin (maximal response at 500 ng/ml) in the presence of 1% serum enhanced the response to LH, causing a dramatic increase in progesterone production, an effect which became greater with time in culture. Dose-response curves for insulin and insulin-like growth factor I (IGF-I) were parallel, but IGF-I was approximately 23-fold more potent than insulin, suggesting that insulin was acting through IGF-I receptors. Our results show that porcine thecal cells, in the presence of LH, insulin or IGF-I, and 1% serum, undergo functional luteinization in vitro, such that androstenedione production declines, and the rate of progesterone production increases with time in culture.     

Ovulation induction with human menopausal gonadotropin compared to human urinary follicle-stimulating hormone results in a significant shift in follicular fluid androgen levels without discernible differences in granulosa-luteal cell function

Follicular fluid estradiol, progesterone, testosterone, and androstenedione levels were compared in 2 groups of spontaneously ovulatory women undergoing ovulation induction with human menopausal gonadotropin (hMG; which contains equal amounts of LH and FSH) or human urinary FSH (huFSH). The results were correlated with the ratios of embryo cleavage and pregnancy. Although significantly more FSH [1268 +/- 38 (+/- SEM) vs. 953 +/- 38 IU; P less than 0.05] was required for equivalent hyperstimulation in hMG compared to huFSH cycles, the number of oocytes retrieved and fertilized and the number of embryos transferred were similar for the 2 ovulation induction protocols. Forty-two follicles from 21 women stimulated with hMG and 38 follicles from 15 women stimulated with huFSH were examined and found to be representative of the total cohort of aspirated follicles. Follicular fluid estradiol and progesterone levels were similar, but hMG-stimulated follicles contained significantly more testosterone [7.83 +/- 0.52 (+/- SEM) vs. 6.30 +/- 0.42 ng/ml; P less than 0.03] and less androstenedione (24.4 +/- 3.6 vs. 37.8 +/- 5.0 ng/ml; P less than 0.03) than did huFSH-stimulated follicles. Embryonic cleavage rates were similar for all fertilized oocytes from both hMG- and huFSH-stimulated cycles, although pregnancy rates were significantly higher in huFSH cycles (40% vs. 9.5%; P less than 0.05). In addition, aromatase activity, progesterone production, and [125I]hCG-binding activity were compared in granulosa-luteal cells isolated from some of these women. Cells from 21 follicles from 9 women stimulated with hMG and 24 follicles from 9 women stimulated with huFSH were studied. There were no significant differences in aromatase activity, progesterone production, or [125I]hCG binding. Thus, the presence or absence of exogenous LH during ovulation induction with FSH has little direct effect on granulosaluteal cell function. However, the presence of LH during ovulation induction with FSH does appear to alter thecal androgen metabolism, resulting in higher testosterone and lower androstenedione levels in follicular fluid. Such a shift in androgen milieu may impair oocyte development and successful implantation.     

Effects of transforming growth factor-beta on the production of immunoreactive insulin-like growth factor I and progesterone and on [3H]thymidine incorporation in porcine granulosa cell cultures

Transforming growth factor-beta (TGF beta) has been reported to enhance many FSH-stimulated functions in rat granulosa cell cultures. We therefore, investigated the actions of TGF beta on cultured porcine granulosa cells. We evaluated the production of immunoreactive insulin-like growth factor I (iIGF-I) and progesterone in short term (3-day) and in longer term (7-day) cultures using porcine TGF beta 1 (pTGF beta 1). TGF beta had a biphasic effect on epidermal growth factor (EGF)-stimulated iIGF-I production in short term cultures. A modest stimulatory effect was apparent at 10 pg/ml; however, this end point was completely inhibited by 1-10 ng/ml. TGF beta also had a slight stimulatory effect on basal iIGF-I production at 1 pg/ml, but not at higher levels. In longer term cultures TGF beta did not have a significant effect on either basal or FSH-stimulated iIGF-I production. In both short and longer term cultures TGF beta markedly inhibited basal and FSH-stimulated progesterone production. We also evaluated the effects of TGF beta on the incorporation of [3H]thymidine into DNA and found that basal and growth factor-stimulated [3H]thymidine incorporation were inhibited. No stimulatory effects of TGF beta on progesterone production or [3H]thymidine incorporation could be detected over the dose range tested (1 pg/ml to 10 ng/ml). The effects of human TGF beta 1 and pTGF beta 2 were compared with those of pTGF beta 1 on basal and EGF-stimulated [3H]thymidine incorporation. Effects of the peptides were qualitatively similar, but pTGF beta 2 was somewhat less inhibitory to EGF-stimulated [3H]thymidine incorporation than pTGF beta 1. The present studies show that in contrast to the well documented stimulatory actions of TGF beta in cultured rat granulosa cells, this growth factor is a predominantly negative regulator of porcine granulosa cells. With the exception of a modest stimulation of iIGF-I production at very low doses, the effects of TGF beta were to potently inhibit both growth and differentiated function. The inhibitory nature of TGF beta should not be overlooked when considering the possible role of this peptide in ovarian development and differentiation.     

Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells

In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), bone morphogenetic protein-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1-5 mm) and large (8-22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150-600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P < 0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P < 0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P < 0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P > 0.10). Treatment with 600 ng/ml GDF-9 increased (P < 0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P < 0.05) progesterone production (by 28%), increased (P < 0.05) cell numbers (by 60%), and had no effect (P > 0.10) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13-48%) and estradiol (by 20-51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P < 0.05) steroidogenesis by 27-42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation.