Regulation of steroidogenesis in cultured porcine theca cells by growth factors

Growth factors [insulin-like growth factors (IGF-I, IGF-II), transforming growth factor-beta (TGF beta), epidermal growth factors (EGF)], found in the ovary and known to alter granulosal function, were assessed for their ability to modulate porcine thecal steroidogenesis. Theca cells from large porcine follicles (8-10 mm) were plated (5 x 10(5) cells/ml.well) in serum-free M199, treated with increasing doses of growth factors: IGF-1 (0.1-50 ng/ml), IGF-II (0.5-200 ng/ml), EGF (0.021-100 ng/ml), TGF beta (0.001-40 ng/ml), or insulin (0.01-50 micrograms/ml), with or without human CG [(hCG); 20 ng/ml], and incubated for 72 h. Levels of steroids in media were determined by RIA. Insulin increased (P less than 0.05) basal and gonadotropin-induced secretion of androstenedione, progesterone, estradiol, and testosterone. IGF-I increased (P less than 0.05) the basal and hCG-induced secretion of progesterone and androstenedione at the highest doses, but did not affect basal secretion of estradiol or testosterone. IGF-II, at the highest doses, increased (P less than 0.05) thecal steroidogenesis, but only after administration of hCG. In contrast, TGF beta increased (P less than 0.05) basal and gonadotrophin-induced secretion of estradiol but inhibited thecal secretion of progesterone, androstenedione, and testosterone. EGF did not alter thecal secretion of progesterone, androstenedione, or testosterone but significantly (P less than 0.05) inhibited basal and hCG-stimulated secretion of estradiol. In conclusion, insulin IGF-I, IGF-II, EGF, and TGF beta can modulate steroidogenesis in porcine theca cells.     

The effect of transforming growth factor-beta on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells

Growth factors have been shown to modulate differentiation of cultured ovarian granulosa cells. Transforming growth factors (TGFs) constitute a family of polypeptide growth factors capable of reversibly inducing anchorage-independent growth in normal cells. Epidermal growth factor (EGF), which has significant structural homology with TGF alpha, has been shown to modulate differentiation of granulosa cells in vitro. Similarly, TGF beta (TGFB) has been found to have significant structural homology with ovarian follicular fluid inhibin. To examine whether TGFB might affect granulosa cell growth or differentiation, rat granulosa cells were cultured in serum-free medium containing insulin for up to 3 days with varying concentrations of TGFB in the presence or absence of FSH. TGFB caused a dose-dependent increase in FSH-stimulated LH/hCG receptor binding, but had no effect on binding in the absence of FSH; TGFB (10.0 ng/ml) further increased FSH-stimulated LH/hCG receptor binding by 48 +/- 8% (P less than 0.02). Similarly, FSH-stimulated progesterone production was increased by TGFB in a dose-dependent manner; TGFB (1.0-10.0 ng/ml) increased FSH-stimulated progesterone production 2- to 3-fold (P less than 0.02). In contrast, EGF (10.0 ng/ml) decreased FSH-stimulated LH/hCG receptor binding by 93 +/- 1% (P less than 0.02). Neither FSH-stimulated intracellular nor extracellular cAMP accumulations were affected by TGFB treatment. However, EGF (10.0 ng/ml) diminished extracellular and intracellular FSH-stimulated cAMP accumulation at 48 and 72 h of culture. Culture protein and DNA content were not significantly affected by TGFB. These results suggest that TGFB may enhance FSH-stimulated LH receptor induction and steroidogenesis by mechanisms that do not further increase net cellular cAMP accumulation; TGFB and EGF can have opposite effects on gonadotropin-dependent differentiation; and products of the TGFB/inhibin gene family may have a capacity for autocrine or paracrine modulation of granulosa cell differentiation.     

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)     

Hormonal regulation of thecal cell function during antral follicle development in bovine ovaries

The hormonal regulation of thecal cell function was investigated with cells isolated at various stages of antral follicle development. Bovine thecal cells were isolated from small antral, medium antral, and large Graffian follicles (small, medium, and large ovarian follicles). Serum-free cultures of thecal cells were established and viable for a minimum of 6-8 days of culture. The purity of the thecal cell population was characterized cytochemically and was found to contain less than 5% endothelial cell and/or granulosa cell contamination. The steroidogenic capacity of this purified population of thecal cells in serum-free culture was examined through an analysis of androgen and progesterone production. Androgen production was high during the first 3 days of culture, then declined to undetectable levels. Production of androstenedione was approximately 10-fold higher than production of testosterone. Progesterone production remained relatively constant throughout the 8-day culture period. hCG was found to stimulate androgen production during days 1-3 of culture, but had a negligible effect on progesterone production. In contrast, hCG stimulated progesterone production during days 3-6 of culture, but had a negligible effect on androgen production. Insulin stimulated progesterone production during days 3-6 of culture, but had no effect on androgen or progesterone production during days 1-3 of culture. The minimum effective concentrations of hCG and insulin required to stimulate steroidogenesis of the thecal cells ranged from approximately 1-10 ng/ml. Addition of serum to the cultures decreased androgen production and suppressed the hormone responsiveness of the cells. Thecal cells in culture appear to alter their steroidogenic capacity from an androgen-producing cell to a progesterone-producing cell. Analysis of the developmental regulation of thecal cell function revealed that androgen production and hormone responsiveness were relatively constant in small, medium, and large follicles. In contrast, progesterone production and hormone responsiveness were highest in small follicles, intermediate in medium follicles, and lowest in large follicles. A more general analysis of the developmental regulation of thecal cell function examined the secretion of radiolabeled proteins. A large number of radiolabeled proteins were secreted by thecal cells, ranging in molecular mass from 5-500 kDa. Interestingly, insulin and hCG had no major effect on secretion of proteins by cells isolated from any of the stages of development examined.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Effects of transforming growth factors and inhibin-related proteins on rat preovulatory graafian follicles in vitro

In view of recent reports on ovarian production and action of transforming growth factors (TGFs) and inhibin-related proteins (inhibin, activin, and follistatin), we have examined the effects of these hormones on the function of preovulatory follicles in vitro. Individual preovulatory follicles were obtained from PMSG-treated rats and incubated with these hormones in the absence or presence of LH. Oocyte maturation and progesterone production were monitored. Treatment with TGF alpha alone, but not with TGF beta or inhibin-related proteins, mimicked the action of LH on oocyte maturation by inducing the resumption of meiosis in follicle-enclosed oocytes (56.6% and 80.6% oocytes resumed meiosis in the presence of 0.5 and 1.0 microgram/ml TGF alpha, respectively). In follicle cultures treated with LH to induce oocyte maturation, cotreatment with inhibin and TGF beta (30-50 ng/ml), but not other related hormones, partially inhibited LH-induced meiosis in follicle-enclosed oocytes (from 82% mature ova in the presence of LH to 51% and 55% mature ova with TGF beta and inhibin, respectively). In contrast to follicle cultures, none of the hormones tested significantly affected the spontaneous maturation of rat oocytes explanted from their follicles and cultured within their cumulus mass for 4 h. Treatment with TGF alpha, but not with TGF beta, inhibin, activin, or follistatin, stimulated progesterone production. The present study demonstrated that TGF alpha, like LH, induces oocyte maturation and progesterone production in preovulatory rat follicles. Furthermore, inhibin and TGF beta suppressed LH-induced resumption of meiosis in follicle-enclosed oocytes. Because these growth factors and inhibin-related proteins are synthesized by follicle cells, they may play important roles in regulating follicular development and activity.     

Granulosa and thecal cells from human ovarian follicles under growth: steroid formation in vitro and responsiveness to human chorionic gonadotropin

In order to characterize the patterns of steroid production and gonadotropin responsiveness in growing human follicles, follicular thecal and granulosa cells were incubated for two hours in the presence or absence of human chorionic gonadotropin (hCG). After incubation, tissue cyclic AMP (cAMP) levels and medium content of progesterone (P), androstenedione (A) and estradiol-17 beta (E) were determined. A was the dominant steroid formed by the thecal cells, regardless if these were derived from small (diameter: 4-7.5 mm) or from large (diameter: 8-15 mm) follicles. Granulosa cells from small follicles formed minimal amounts of all steroids measured, while granulosa cells from large follicles produced considerable amounts of E in vitro. Thecal cells from both small and large follicles increased their production of cAMP in the presence of hCG. Steroid formation was significantly increased by hCG in thecal cells from large follicles only. Granulosa cells from large follicles responded to hCG in vitro with increased cAMP and steroid formation, while granulosa cells from small follicles appeared insensitive to hCG in vitro.     

Ovarian thecal cells produce transforming growth factor-beta which can regulate granulosa cell growth

Ovarian thecal cells in culture were found to synthesize and secrete transforming growth factor-beta (TGF beta). A component in thecal cell-conditioned medium was immunologically similar to TGF beta, as assessed with a RIA, and inhibited specific binding of TGF beta to its cell surface receptors. Thecal cell-secreted proteins also contained TGF beta biological activity, which was determined by stimulation of soft agar colony formation by AKR-2B indicator cells. Specific TGF beta antibodies precipitated a 25 K protein from radiolabeled thecal cell-secreted protein that comigrated with purified platelet-derived TGF beta. Both bovine thecal cell and rat thecal/interstitial cell preparations produced TGF beta, which required acid treatment to obtain fully active samples. The physiological significance of TGF beta production by thecal cells was addressed through an analysis of the effects of TGF beta on bovine granulosa cell growth. TGF beta inhibited epidermal growth factor stimulation of granulosa cell growth, but alone it had no apparent influence. Observations indicate that ovarian thecal cells produce TGF beta, which can regulate granulosa cell growth and differentiation. Discussion of thecal cell-granulosa cell interactions and the possible functions of TGF beta in the ovary is presented.     

Transforming growth factor-alpha and -beta are potent and effective inhibitors of GH4 pituitary tumor cell proliferation

The mechanisms that restrict cell proliferation play an important regulatory role in differentiation and tumorigenesis. The growth of PRL-secreting cells of the anterior pituitary is known to be highly estrogen dependent; however, estrogen may act indirectly via growth regulatory polypeptides. We have used the GH4C1 rat pituitary cell line to investigate the action of two classes of growth regulatory polypeptides, transforming growth factor-alpha (TGF alpha) and TGF beta. TGF alpha and TGF beta each inhibit GH4 cell proliferation, as measured by cell number and [3H]thymidine incorporation, and given together arrest GH4 cell proliferation. The growth inhibitory action of TGF alpha is concentration dependent (IC50 = 100 pM) and saturable. Activin-A, a TGF beta-related polypeptide, also inhibits proliferation, but is less effective than TGF beta. TGF alpha and TGF beta each alter GH4 cell cycle distribution by decreasing in the percentage of S phase cells (74% and 34%, respectively) and increasing proportionally G0-G1 phase cells. The growth inhibitory action of TGF alpha differs from that of TGF beta in that TGF alpha also causes a temporary accumulation of cells in G2-M phases. We next initiated experiments to evaluate the role of protein kinase-C in the growth inhibitory actions of TGF alpha and TGF beta. The alpha- and beta-isoforms of protein kinase-C were down-regulated by pretreatment with 12-O-tetradecanoylphorbol-13-acetate, yet TGF alpha and TGF beta still substantially inhibited GH4 cell proliferation. We next compared the actions of TGF alpha and TGF beta on two other well characterized prolonged GH4 responses. TGF alpha and TGF beta each increased GH4 cell adhesion, but differed in their effects on PRL production. This indicates that TGF alpha and TGF beta activate different signaling pathways in GH4 cells. Activin-A acted like TGF beta by enhancing cell-substratum adhesion and inhibiting PRL production, consistent with an interaction at a common receptor site. Taken together these results identify biological functions for TGF alpha, TGF beta, and activin-A on PRL cells and open the possibility that they may represent the direct in vivo mediators of estrogen action to regulate the growth of PRL cells in the anterior pituitary gland.     

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

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

Short term androgen production by rat ovarian follicles and long term steroidogenesis by thecal explants in culture

To characterize the aromatizable and 5 alpha-reduced androgens produced by developing ovarian follicles, small antral (SA) and preovulatory (PO) follicles, theca and granulosa cells were incubated for 4 h with or without 8-bromo-cAMP and androstenedione. In addition, thecal explants were cultured for 10 days with or without ovine LH (oLH) to determine if the hormone-induced changes in androgen synthesis by developing follicles could be mimicked in vitro. Short term incubations of SA and PO follicles, theca and granulosa cells in medium alone resulted in limited accumulation of androgen [testosterone, 5 alpha-androstan-17 beta-ol-3-one (DHT), 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha diol), and androsterone], as determined by RIA. In the presence of 8-bromo-cAMP, PO follicles produced large quantities of testosterone (3 ng), DHT (1 ng), 3 alpha diol (15 ng), and androsterone (14 ng), while SA follicles accumulated much less androgen (0.69, 0.05, 1.23, and 1.3 ng, respectively). In the presence of androstenedione and 8-bromo-cAMP, both SA and PO follicles and theca produced large amounts of aromatizable and 5 alpha-reduced androgens. SA and PO granulosa cells required the presence of the substrate androstenedione to produce androgens, primarily testosterone and 3 alpha diol. Therefore, progesterone, androstenedione, and 5 alpha-reduced androgens were used to monitor LH action on thecal cell function in culture. Small antral theca cultured in basic culture medium alone (containing 10% fetal calf serum) displayed an increased ability to accumulate androstenedione by day 6, approximately 3 times that observed on day 2. However, a 5-fold further increase in androstenedione accumulation was observed by day 6 for SA theca cultured in the presence of oLH. Maintenance of progesterone accumulation by SA theca throughout the culture period also was dependent on the presence of LH. In contrast, androstenedione accumulation by PO theca required the presence of LH in the culture medium, while progesterone accumulation in these cultures did not. Little or no 5 alpha-reduced androgen accumulated in the media of SA and PO theca cultured in basic culture medium alone. However, SA and PO theca cultured with oLH accumulated approximately 1 ng androsterone by day 10. We conclude that 1) SA and PO follicles, theca and granulosa cells possess the enzymes required to produce large amounts of 3 alpha diol and androsterone; 2) low concentrations of oLH are required to stimulate SA thecal steroidogenesis and to maintain PO thecal androstenedione accumulation in culture.(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.     

Rat prostate cancer cells contain functional receptors for transforming growth factor-beta

Clonally derived C-, D-, and T-family AXC/SSh rat prostate cancer cell lines contain transforming growth factor-beta (TGF beta) receptors. The content in C3, D1, T1, and T5 cells, respectively, was 8,560 +/- 1,450, 13,160 +/- 1,240, 2,425 +/- 490, and 10,540 +/- 1,025 sites/cell (mean +/- SEM). Respective Kd values were 160 +/- 48, 200 +/- 53, 24 +/- 3, and 115 +/- 15 pM (mean +/- SEM). T1 cell TGF beta receptor site content and Kd differed significantly from those of other prostate cancer cell lines (P less than 0.05). TGF beta is a bifunctional concentration-dependent modulator of T1 and T5 cell thymidine incorporation. At low concentrations, thymidine incorporation was inhibited, whereas as the medium TGF beta content was increased, T1 and T5 cell thymidine incorporation was stimulated. The concentrations of TGF beta causing half-maximum inhibition of T1 or T5 cell thymidine incorporation, respectively, were 0.11 and 0.24 pM, whereas the respective TGF beta concentrations causing half-maximum stimulation of thymidine incorporation were 14.4 and 134 pM. These findings establish that rat prostate cancer cell sensitivity to TGF beta inhibition of function is at least 2 orders of magnitude greater than that of most other mammalian cells. In contrast, the sensitivity of rat prostate cancer cells to TGF beta enhancement of function is comparable to that of other mammalian cells. TGF beta inhibited basic fibroblast growth factor (bFGF) stimulation of T1 and T5 cell thymidine incorporation. Because the concentration of bFGF required for half-maximum increase of T5 cell thymidine incorporation was independent of medium TGF beta content, the effect of TGF beta is distal to the T5 cell bFGF receptor. In contrast, the concentration of bFGF required for half-maximum increase in T1 cell thymidine incorporation increased 5-fold as the medium TGF beta content was increased; suggesting that the effect of TGF beta in T1 cells is proximal to the T1 cell bFGF receptor. Our studies establish that rat prostate cancer cells contain functional TGF beta receptors, imply the presence of functional bFGF receptors, and demonstrate that mitogen modulation of prostate cancer cell function is multifactorial. The finding that TGF beta is a bifunctional effector of prostate cancer cell DNA synthesis provides some insight into the potential complexity of mitogen modulation of prostate cancer cell proliferation. The mechanism by which these mitogens interact is unknown; however, our studies suggest that some interactive effects may be cell line specific.     

Transforming growth factor beta and follicle-stimulating hormone promote rat granulosa cell proliferation

Rat granulosa cells isolated from the ovaries of diethylstilbestrol-primed immature rats were treated with estrogen, FSH, and growth factors to determine those factors that were required to promote DNA synthesis. Estrogen and FSH, previously shown to stimulate the incorporation of [3H]thymidine into rat granulosa cell DNA in vivo, were ineffective in vitro. Epidermal growth factor, insulin-like growth factor 1 (IGF1), and fibroblast growth factor did not influence DNA synthesis whereas transforming growth factor beta (TGF beta) alone had a significant effect. Neither estradiol-17 beta (5 X 10(-8)-5 X 10(-6) M) nor IGF1 augmented the actions of TGF beta and FSH. FSH did not influence the actions of epidermal growth factor or IGF1 but dramatically augmented the effect of TGF beta on DNA synthesis. FSH and TGF beta also stimulated [3H]thymidine incorporation into the DNA of granulosa cells isolated from immature rats not treated with diethylstilbestrol. The increase in [3H]thymidine incorporation into DNA stimulated by TGF beta and FSH resulted subsequently in an increase in cell number. The response of the cells to TGF beta in the presence of a constant level of FSH (10 ng/ml) was dose dependent, 2.5 ng/ml being the minimal effective concentration. In the presence of antibody specific for TGF beta the bioactivity of the TGF beta was neutralized indicating that the growth promoting activity was due to TGF beta and not due to contaminants. In this paper, we have shown that the combined actions of FSH and TGF beta influence DNA synthesis and the proliferation of rat granulosa cells. Interactions between FSH and TGF beta may be important in regulating aspects of rat granulosa cell growth in addition to exerting pronounced effects on cytodifferentiation.     

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.     

Epidermal growth factor influences growth and differentiation of rat granulosa cells

The interactions of epidermal growth factor (EGF) with transforming growth factor beta (TGF beta), insulin-like growth factor-I (IGF-I), and FSH in the modulation of DNA synthesis and differentiated functions were examined in cultures of granulosa cells isolated from the ovaries of immature rats primed with diethylstilbestrol. EGF alone or in the presence of FSH had no effect on [3H]thymidine incorporation into the DNA of granulosa cells; however, EGF inhibited FSH plus TGF beta-induced DNA synthesis. In contrast, when FSH was omitted from the culture medium, EGF acted in concert with TGF beta, and TGF beta plus IGF-I, to promote DNA synthesis. EGF therefore has opposing actions on DNA synthesis; it inhibits or stimulates depending upon the presence or absence of FSH and consequently upon the endocrine environment in the follicle. As shown previously EGF alone had no effect on basal aromatase activity. EGF however inhibited FSH-induced and FSH plus IGF-I-induced aromatase activity. In this paper we show that EGF also inhibited the FSH-induced aromatase activity in the presence of TGF beta, which augmented FSH action on this system. The action of EGF on 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was different from the effect of EGF on aromatase. In the absence of FSH, EGF induced 3 beta-HSD activity in the presence or absence of TGF beta. EGF augmented the action of FSH on 3 beta-HSD, and this interaction was further enhanced by TGF beta. These observations emphasize the multifunctional nature of EGF in influencing the growth and differentiation of immature rat granulosa cells. EGF can inhibit or stimulate growth and differentiated functions (aromatase and 3 beta-HSD), the response depending on the context of the signals that the cell receives from its endocrine and microenvironment.     

Possible role of transforming growth factor-beta as a mediator of luteotropic action of prolactin in rat luteal cell cultures

PRL is known to be a hormone carrying luteotropic action in rats and enhances progesterone secretion by suppressing 20 alpha-hydroxysteroid dehydrogenase (20 alpha HSD) activity in the corpus luteum. In this study, we investigated the effects of PRL and transforming growth factor-beta (TGF beta) on the 20 alpha HSD activity of rat luteal cells in vitro and examined whether TGF beta is involved in the luteotropic action of PRL. 20 alpha HSD activity of luteal cells (from midpseudopregnant rats), which had been suppressed by PRL in vivo, increased when the cells were cultured for 48 h without PRL addition. TGF beta (0.01, 0.1, 1.0, and 10.0 ng/ml) as well as PRL (2, 20, 200, and 2000 ng/ml) suppressed this increase in a dose-dependent manner. Furthermore, the suppressive effect of PRL on 20 alpha HSD activity was significantly attenuated by anti-TGF beta antibody. Activin, having homology with TGF beta in its chemical structure, also suppressed the increase in enzyme activity, although the effect was much less marked than that of TGF beta. TGF beta or PRL did not affect total progestin (progesterone plus 20 alpha-dihydroprogesterone) secretion, but induced reduction in 20 alpha-dihydroprogesterone secretion during a 48-h culture period, without any alteration of DNA or protein content per culture dish. These results indicate that TGF beta, like PRL, can suppress luteal 20 alpha HSD activity without producing nonspecific cell damage, and that the luteotropic action of PRL is at least in part mediated by TGF beta or an immunoreactive TGF beta-like substance(s).     

The production of transforming growth factor-beta in the porcine ovary and its secretion in vitro

Porcine granulosa cells isolated from small (1-3 mm in diameter) follicles proliferate rapidly in culture in response to 10% fetal bovine serum (FBS) and epidermal growth factor (EGF) (10 ng/ml). Transforming growth factor-beta (TGF beta) inhibits FBS/EGF-stimulated proliferation in a dose-dependent manner. We have used this proliferation inhibitory property of TGF beta to assay qualitatively, the presence of this growth factor in conditioned medium from cultured follicle cells as well as in partially purified preparations from porcine ovarian compartments. In addition, the concentration of TGF beta in the theca cell conditioned medium was quantitatively estimated by generating a TGF beta-dose-response curve (inhibition of FBS/EGF-stimulated proliferation of granulosa cells in monolayer culture) using authentic human TGF beta-1. Ovarian thecal cells isolated from small and large size follicles in the pig ovary secrete TGF beta-like activity in vitro. Medium conditioned by thecal cells in primary monolayer culture contains a latent form of TGF beta which can be activated by heat or acid treatment. In contrast, and unlike rat granulosa cells, porcine granulosa cells in primary monolayer culture do not secrete detectable levels of TGF beta-like activity in the medium. Incubation of heat-activated thecal cell conditioned medium with a TGF beta-neutralizing antibody (which recognizes TGF beta-1 and 2) but not nonimmune serum attenuated the TGF beta-like activity in thecal cell conditioned medium suggesting that this activity is due to authentic TGF beta. Since many cell types secrete latent TGF beta in the medium when cultured in vitro, we next investigated whether thecal cell secretion of latent TGF beta was a function of cell culture or whether the ovarian thecal compartment actually contained detectable levels of TGF beta-like activity. To this end, we used an acid-ethanol extraction procedure to isolate thecal proteins from fresh-frozen tissue. The acid-ethanol extracted protein fraction was mixed with trace amounts of 125I-TGF beta for detection and chromatographed on Bio-Gel P-60 column under acidic conditions. Elution of TGF beta bioactivity from the Bio-Gel P-60 column as measured by inhibition of granulosa cell proliferation correlated with the elution of radioiodinated authentic TGF beta. Preincubation of TGF beta-like activity-containing fractions with TGF beta-neutralizing antibody attenuated the proliferation-inhibitory activity in these fractions. TGF beta activity was also observed in fractions extracted from porcine corpora lutea.(ABSTRACT TRUNCATED AT 400 WORDS)