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.     

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)     

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)     

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)     

Effects of tumor necrosis factor-alpha in vitro on steroidogenesis of healthy and atretic follicles of the rat: theca as a target

Tumor necrosis factor-alpha (TNF), a pleiotropic cytokine localized within the ovary, alters follicular steroidogenesis. Preovulatory follicles dissected from ovaries of normal cyclic adult rats on the morning of proestrus exhibit steroidogenic and histological signs of atresia after 24 h of culture under the conditions of 5% CO2 and air. Follicles cultured for 24 h in 5% CO2 and 95% O2 appeared histologically and steroidogenically healthy. Under both culture conditions, human recombinant TNF (5 ng/ml) significantly increased the production of pregnenolone, progesterone, 20 alpha-dihydroprogesterone, and 17 alpha-hydroxyprogesterone by the follicles. Follicles cultured in 5% CO2 and air exhibited no change in androstenedione or estradiol production compared to control follicles incubated without TNF. In contrast, follicles cultured in 5% CO2 and 95% O2 responded to TNF with increased androstenedione and estradiol production. Separation of the thecal and granulosa compartments indicated that the increased progestin production observed in the whole follicle in response to TNF originated from the theca. TNF significantly inhibited basal and FSH-stimulated progesterone production from the granulosa of preovulatory follicles. Exogenous substrate added to whole follicles cultured in the presence or absence of TNF indicated that TNF enhanced the conversion of 25-hydroxycholesterol to pregnenolone. These studies reveal that TNF enhanced steroidogenesis in both healthy and atretic follicles and that this action of TNF is on the theca, where TNF increases the conversion of cholesterol to pregnenolone. The data imply that TNF has differential effects on thecal and granulosa steroidogenesis.     

Oxytocin inhibits LH-stimulated production of androstenedione by bovine theca cells

Oxytocin secretion by bovine granulosa cells increases dramatically after the LH/FSH surge. We have shown that oxytocin stimulates progesterone secretion and inhibits FSH-stimulated estradiol secretion in vitro by granulosa cells from bovine preovulatory follicles obtained before the LH/FSH surge. To determine if oxytocin regulates LH-stimulated steroid production by bovine theca interna cells, theca cells were isolated from preovulatory follicles obtained before the LH surge and were cultured for 4 days in the presence or absence of LH (2 or 4 ng/ml), without or with graded doses of oxytocin (125-1000 ng/ml). LH increased accumulation of androstenedione and progesterone. Oxytocin inhibited LH-stimulated androstenedione production, but had no effect on LH-stimulated progesterone production by cultured theca interna. The next objective was to determine if oxytocin regulates LH-stimulated steroidogenesis by modulating the levels of mRNA for steroidogenic enzymes and/or Steroidogenic Acute Regulatory protein (StAR). Low doses of LH alone increased the levels of mRNA for P450 17 alpha-hydroxylase (17 alpha-OH), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and cytochrome P450 side-chain cleavage, but not for StAR. In contrast, the effects of oxytocin on LH-stimulated androstenedione production were not associated with changes in the levels of mRNA for steroidogenic enzymes or StAR. These results suggest that oxytocin may play a paracrine role in regulating the follicular/luteal phase shift in steroidogenesis by decreasing androstenedione secretion by theca cells of the ovulatory follicle and that this effect is not mediated by changes in the levels of mRNA for steroidogenic enzymes and StAR.     

In vitro effects of follicle-stimulating hormone, luteinizing hormone, and prolactin on follicular deoxyribonucleic acid synthesis in the hamster

Follicles were dissected by hand or enzymatically from the ovary of the proestrous hamster at 0900 h and classified into 10 stages: stages 1-4, follicles with 1-4 layers of granulosa cells and no theca; stages 5-8, preantral follicles with 5 or more layers of granulosa cells and theca to small antral follicles; stage 9, intermediate-sized atretic antral follicles; and stage 10, healthy preovulatory antral follicles. Follicles were then incubated for 2 h with [3H]thymidine [( 3H]Tdr) in the absence or presence of gonadotropins and with incorporation of radionuclide into DNA as the end point. FSH (25 ng) significantly stimulated [3H]Tdr incorporation in all stages of follicular development with a latency of 2 h, and this effect was inhibited by 2 micrograms unlabeled Tdr. While FSH and PRL (25 and 100 ng) stimulated [3H]Tdr incorporation in all stages, LH (0.2-5 ng) action began from stage 5 onward, when definitive thecal cells and LH receptors started appearing. LH (5 ng) also suppressed 25 ng FSH-induced DNA synthesis in stages 5-10; however, stages 1-4 were unaffected. Significant increases in both intra- and extracellular cAMP levels occurred in follicles at stages 2-10 after FSH administration. In contrast, LH was active in stages 5-10, whereas PRL was ineffective. Follicular DNA synthesis increased markedly when stimulated by 8-bromo-cAMP (0.01-2 mM). These results show that gonadotropins act directly as a primary stimulus at the level of small primary and secondary follicles to regulate DNA synthesis and, thus, perhaps the growth and differentiation of granulosa and thecal cells; cAMP functions as one of the possible intracellular mediators of gonadotropin action in initiating DNA replication.     

Differential steroid production between theca interna and theca externa cells: a three-cell model for follicular steroidogenesis in avian species

In avian species, the granulosa cells produce progesterone (P), but not testosterone (T) or estradiol (E). The theca folliculi in avian species produces T and E and is anatomically comprised of the theca interna and the theca externa. It is not known, however, whether both T and E are produced by the same cell type. In the present study, preovulatory follicles of adult female turkeys were separated into their anatomical subdivisions, the granulosa, theca interna, and theca externa, and the relative steroidogenic contribution by each layer was investigated. Granulosa cells from the largest (F1) and fifth largest (F5) preovulatory follicles produced increasing levels of P in response to ovine LH (oLH; 0-10 micrograms/ml); however, T and E were not detectable. Theca interna and theca externa cells from F5 follicles were cultured alone or in combination. Theca interna cells produced P and T, but not E. Theca externa cells produced P, T, and E. E production was greatly enhanced by coincubation of cells from the two theca layers. oLH, (0-100 ng/ml) stimulated theca interna P and T production and E production by the coincubation of theca interna and theca externa cells. When theca interna and theca externa cells from the F1 and F5 follicles were tested, theca externa cells from F5, but not F1, converted androstenedione and T, but not P into E, indicating a loss of aromatase activity in the theca externa with follicular maturation. Neither F1 nor F5 theca interna cells converted P, A, or T into estradiol, providing additional evidence that aromatase activity is limited exclusively to the theca externa. Addition of oLH (0-100 ng/ml) did not increase conversion of T into E by the F5 theca externa cells, suggesting that production of E by the theca externa is substrate driven and not influenced by direct LH action. Avian ovarian steroidogenesis is described using a three-cell model, with the principle sources of P, T, and E being the granulosa, theca interna, and theca externa cells, respectively.     

Multiple steroidogenic cell populations in the thecal layer of preovulatory follicles of the chicken ovary

The thecal layer of the preovulatory follicle of the chicken ovary produces primarily androgens and estrogens. However, the precise cellular sites of androgen and estrogen synthesis in the thecal layer have not been identified. Therefore, our aims were 1) to identify steroidogenic cells in the thecal layer of the preovulatory follicles by localizing specific steroidogenic enzymes in these cells by immunocytochemistry, and 2) to confirm that these cells which contained the specific steroidogenic enzymes secreted the expected steroid in a short term cell incubation. Follicles were collected 2 h after oviposition and prepared for immunocytochemistry and short term cell incubation. Immunocytochemistry for cholesterol side-chain cleavage cytochrome P450 (P450SCC), 17 alpha-hydroxylase cytochrome P450 (P450C17), and aromatase cytochrome P450 (P450AROM) was performed to localize pregnenolone-, androgen-, and estrogen-producing cells, respectively, on frozen sections and paraffin sections of the five largest preovulatory follicles. Interstitial cells showed immunoreactivity for both P450SCC and P450C17, whereas a specific cell population of the theca externa, hereafter termed aromatase cells, showed immunoreactivity for P450AROM. Furthermore, fibroblasts in the theca externa indicated immunoreactivity for P450C17. The immunoreactivity of P450C17 and P450AROM enzymes in specific cells in the theca externa appeared to decrease with follicular maturation. The third largest, fourth largest, and fifth largest follicles were selected for short term cell incubations because the immunoreactivity for P450 enzymes in the thecal layer of these follicles was high. Isolated theca interna cells, theca externa cells, and a combination of interna and externa cells were incubated with/without ovine LH (oLH) for 3 h. The medium was assayed for progesterone, testosterone, and 17 beta-estradiol by RIAs. Incubation of theca interna cells with oLH increased progesterone and testosterone production in a dose-dependent manner. However, we did not observe any production of progesterone and testosterone by theca externa cells. Theca externa cells produced 17 beta-estradiol, and its production was increased significantly when theca interna and externa cells were coincubated in the present of oLH. Based on these data, we propose a multiple cell theory for steroidogenesis in the thecal layer of preovulatory follicles of the chicken ovary which states that interstitial cells in the theca interna produce progestins and androgens, fibroblasts in the theca externa may function as an additional site for the conversion of progestins to androgens, and aromatase cells in the theca externa require androgens as substrate to produce estrogens.     

Androgen inhibition of follicle-stimulating hormone-stimulated luteinizing hormone receptor formation in cultured rat granulosa cells

Since LH receptors are decreased in atretic follicles known to contain high androgen levels, we have studied the androgen modulation of LH receptor formation in vitro. Granulosa cells from hypophysectomized, diethylstilbestrol-treated rats were cultured for 3 days with FSH in the presence or absence of nonaromatizable androgens, dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol, or a synthetic androgen, R1881 (17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one). FSH increased LH receptor content in granulosa cells, while concomitant androgen treatment decreased LH receptor content in a dose- and time-dependent manner, without changing the equilibrium dissociation constant (Kd) for human CG. R1881 (10(-7) M), dihydrotestosterone (10(-6) M), and 5 alpha-androstane-3 alpha, 17 beta-diol (10(-6) M) inhibited LH receptor content by 68%, 65%, and 65%, respectively. Similar to earlier findings, these androgens enhanced FSH-stimulated progesterone biosynthesis and aromatase activity in the same cells. To study their LH responsiveness, androgen-treated cells were washed and reincubated for 2 more days with or without LH. Although basal progesterone production was elevated by R1881 pretreatment, the androgen-pretreated cells were less responsive to LH. Treatment with cyanoketone, an inhibitor of 3 beta-hydroxysteroid dehydrogenase, did not alter the inhibitory effects of R1881 on LH receptors, indicating that the androgen action is not mediated by endogenous progestins. Furthermore, R1881 inhibited the stimulation of LH receptor formation by forskolin, cholera toxin, and 8-bromo-cAMP, suggesting that androgens may inhibit LH receptor induction by affecting post-cAMP events. Estrogen treatment enhanced the FSH induction of LH receptor content, while concomitant addition of R1881 also suppressed the estrogen action. Thus, androgens inhibit FSH-induced functional LH receptors in cultured rat granulosa cells. The androgen effect is exerted, at least partially, at post-cAMP sites and is independent of changes in progestin biosynthesis.     

Developmental changes in luteinizing hormone/human chorionic gonadotropin steroidogenic responsiveness in marmoset granulosa cells: effects of follicle-stimulating hormone and androgens

Factors regulating LH/hCG responsiveness in primate granulosa cells were examined in the marmoset monkey (Callithrix jacchus). Granulosa cells were isolated and pooled from small antral (0.5-1.0 mm) and large preovulatory (greater than or equal to 2 mm) follicles from mid- to late follicular phase ovaries of cyclic marmosets. The cells from small and large follicles were cultured in serum-free medium for 48 h in the absence or presence of increasing concentrations of hCG (0.1-100 ng/ml) with or without 0.1 microM androgen [testosterone or 5 alpha-dihydrotestosterone (DHT]). Granulosa cells from small follicles were also cultured in the absence or presence of a constant concentration of human FSH (30 ng/ml) with or without androgen for 48 h before exposure to hCG for an additional 48 h. Steroidogenic responsiveness was assessed by measuring progesterone accumulation in culture medium and aromatase activity in washed monolayers. Granulosa cells from large follicles showed dose-dependent increases in both progesterone accumulation and aromatase activity in response to treatment with hCG. In contrast, granulosa cells from small follicles were unresponsive to hCG. However, pretreatment of granulosa cells from small follicles for 48 h with FSH stimulated hCG responsiveness. The effects of both testosterone and DHT on hCG-stimulated aromatase activity and progesterone accumulation by granulosa cells from large preovulatory follicles were inhibitory. Testosterone and DHT also suppressed basal (no hCG) progesterone accumulation in these cells, but had no effect on basal aromatase activity. The effects of androgens on FSH-induced hCG responsiveness in immature granulosa cells were variable. The results show a development-related increase in marmoset granulosa cell responsiveness to LH/hCG and provide evidence that FSH and androgens interact to regulate the onset and expression of this critical event during preovulatory follicular development in the primate ovary.     

Influence of follicular atresia on LH-induced cAMP and steroid synthesis by bovine thecae interna

The aim of the present study was to examine the interrelationships between the luteinizing hormone (LH) receptor, the LH-induced changes in adenosine cyclic 3', 5'-monophosphate (cAMP) and steroid synthesis in theca interna tissue of large antral follicles (greater than or equal to 8 mm diameter) from oestrous cycling cows. Three distinct types of theca interna were identified (types I, II and III), all of which contained an LH receptor: type I was capable of secreting increased amounts of cAMP dehydroepiandrosterone, androstenedione and testosterone when exposed to LH; type II was capable of secreting increased amounts of cAMP and progesterone but not the androgens when exposed to LH; type III was incapable of cAMP or steroid synthesis when exposed to LH. Follicles with type I thecae contained: a full complement of granulosa cells; high intrafollicular concentrations of oestradiol; and granulosa cells with a high capacity to metabolise testosterone to oestradiol. These follicles were considered to be non-atretic structures. Follicles with types III thecae contained: fewer granulosa cells; low intrafollicular concentrations of oestradiol; and granulosa cells with a low capacity to metabolise testosterone to oestradiol. Moreover, follicles with type III thecae contained the highest concentrations of progesterone and the lowest concentrations of androstenedione and testosterone. These follicles were considered to be severely atretic structures. Follicles with type II thecae contained granulosa cell populations and progesterone, and androgen concentrations which were intermediate between those with thecae of types I and III. These follicles were considered to be at an intermediate stage of atresia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of testosterone production in isolated rabbit thecal tissue by LH/FSH, dibutyryl cyclic AMP, PGE2alpha, and PGE2.

The capacities of isolated rabbit theca and granulosa cells to secrete testosterone were studied in vitro. Large Graafian follicles (1-1.5 mm in diameter) were dissected intact from the ovaries of adult estrous rabbits. Granulosa cells from 4 follicles (50,000 cells) and theca tissue (16 pieces per dish, equivalent to 4 follicles) were cultured separately for 6 days either as controls (without exogenous hormones) or with one of the following agents: 1 lU/ml LH/FSH (Pergonal), 10-3M dibutyryl cyclic AMP (Bu2cAMP), 1 mug/ml prostaglandin F2alpha (PGF2alpha), or 1 mug/ml prostaglandin E2 (PGE2). The media were collected every 2 days, and the testosterone (T) was measured by radioimmunoassay. The control cultures of granulosa cells secreted small amounts of T (700 +/- 317 pg/culture: mean +/-SE) during the first 2 days in vitro, and the addition of LH/FSH, Bu2cAMP, PGF2alpha, or PGE2 did not significantly stimulate T production. After 2 days in vitro, very little T (greater than 200 pg/culture) was produced by control and prostaglandin-treated granulosa cells, whereas those incubated with LH/FSH and Bu2cAMP maintained their initial T production rates. Theca control cultures produced 3 +/- 0.4 ng of T (mean +/- SE) during the first 2 days in 13.6-fold by LH/FSH, 3.6-fold by Bu2cAMP, and 3-fold by PGF2alpha and PGE2- T was not detected in theca cultures after 2 days except in those treated with LH/FSH or Bu2cAMP, which produced 1.5 +/- 0.5 and 1.6 +/- 0.3 ng of T, respectively, at 4 days (mean +/- SE). These results suggest that under the present conditions, pieces of rabbit thecal tissue have a greater capacity to produce T de novo than do isolated granulosa cells, and indicate that T production is transiently stimulated by LH/FSH, Bu2cAMP, PGE2alpha, and PGE2.     

Preantral follicles stimulate luteinizing hormone independent differentiation of ovarian theca-interstitial cells by an intrafollicular paracrine mechanism

Ovarian thecal cells are thought to differentiate from fibroblast-like precursor cells in the stroma adjacent to developing follicles. Since the precursor cells do not contain LH receptors, a regulator other than LH must initiate thecal differentiation. These studies were designed to test the hypothesis that preantral follicles secrete substances that can stimulate thecal differentiation. Preantral follicles devoid of theca were obtained by limited enzymatic dispersal of 26-day old rat ovaries. Follicles were cultured (5 follicles/well) in 96-well plates containing serum-free medium to generate follicle-conditioned medium (FCM). Isolated theca-interstitial cells (TIC) were cultured (2 days) in 50% FCM, to bioassay for androgen-stimulating activity. Androsterone production was measured by RIA. FCM from follicles of increasing size with 1, 2, 3, 4 or 5 layers of granulosa cells (GC) stimulated increasing amounts of androsterone suggesting that secretion of androgen-stimulating activity is developmentally regulated in preantral follicles. The androgen-stimulating activity of 7.5-fold concentrated FCM was markedly increased above control levels or the levels stimulated by insulin-like growth factor-I (100 ng/ ml), transforming growth factor-α (100 ng/ml), transforming growth-factor-β1 (10 ng/ml), inhibin A (300 ng/ml), activin A (100 ng/ml), or Müllerian inhibiting substance (MIS; 300 ng/ml) suggesting that the bioactive substances were distinct from these intrafollicular growth factors. rFSH stimulated a > 10-fold increase in androgenstimulating activity demonstrating that the bioactivity is hormonally regulated. The bioactivity was sensitive to trypsin digestion but was not inhibited by indomethacin (10 μm) suggesting that it is peptide not prostaglandin in nature. Gel filtration chromatography indicated that the M of the bioactive peptides in FCM ranged from 19 500 to 23 600. Taken together our results demonstrate that preantral follicles secrete thecal differentiating factors (TDFs) that are developmentally and hormonally regulated by FSH. The properties of the TDFs are markedly different from known intrafollicular growth factors and may represent a new paracrine regulator in the ovary that can stimulate LH-independent thecal differentiation.     

Production of testosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and androsterone by dispersed testicular interstitial cells and whole testes in vitro.

The production of 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol), androsterone and testosterone by whole rat testes and testicular interstitial cells dispersed with collagenase was studied in vitro. Luteinizing hormone stimulated the production of each of the androgens by cells prepared from 31- to 34-day-old rats. Half maximum stimulation of the production of each androgen occurred with approximately 3.5 ng NIH-LH-B9/ml medium. Androstanediol was the predominant product then androsterone and then testosterone. Luteinizing hormone stimulated the production of testerone, but not androstanediol or androsterone by dispersed interstitial cells from 200-day-old rats. The time-course of production and the effect of the concentration of cells on the production of these androgens suggested that in dispersed testicular interstitial cells from immature animals androstanediol and androsterone are formed, at least partially, by the metabolism of testosterone. In these experiments LH-stimulated testosterone production increased during incubation for 15--60 min and then remained constant up to 180 min. The concentrations of androstanediol and androsterone increased in a linear manner during incubation for 60--180 min. Varying the number of cells incubated yielded a positive correlation between cell concentration and the ratio 5 alpha-reduced androgen : testosterone produced. Luteinizing hormone stimulated production of each androgen by whole tests obtained from rats at 30--175 days of age. The serum concentration of testosterone in these rats increased abruptly at 50 days of age. Significant changes in androgen production in vitro also observed at this age included: (1) increased production of the three steroids when incubated in either the presence or absence of LH and (2) testosterone production, either in the presence or absence of LH, which represented a greater percentage of the total production of the three androgens.     

Evidence that granulosa cell aromatase induction/activation by follicle-stimulating hormone is an androgen receptor-regulated process in-vitro

The role of androgen in aromatase induction/activation by follicle-stimulating hormone (FSH) was studied in cultured granulosa cells from estrogen-pretreated, immature rat ovaries. Aromatase activity was measured in washed cell monolayers after a 48-h culture in medium containing hFSH and/or various sex steroids or their analogues. Culture with hFSH (100 ng/ml) plus 10(-7) M testosterone (T) stimulated aromatase activity to a level similar to that of granulosa cells from preovulatory follicles in the cyclic adult on the morning of proestrus. But if T was omitted, or replaced by estrogen (DES) or progesterone (P), the response to hFSH was at least 90% lower. The abilities of T, androstenedione, five nonaromatizable 5 alpha-reduced androgens, an aromatase reaction intermediate (19-hydroxyandrostenedione), and a pharmacological competitive aromatase inhibitor (delta 1-testoloalactone) to stimulate the aromatase response to hFSH were proportionate to their stimulatory effects on P production during the culture. By both criteria T was the most potent androgen while 19-hydroxyandrostenedione and delta 1-testololactone were completely inactive. The stimulatory effect of 10(-7) M T on the aromatase response to FSH was inhibited by the nonsteroidal antiandrogen SCH 16423 (ID50 = 3.6 x 10(-6) M). These results indicate that granulosa cell aromatase induction/activation by hFSH is an androgen receptor-regulated process in vitro.     

Inhibitory action of follicular fluid on progesterone and prostaglandin synthesis in bovine follicles.

The present studies examined the inhibitory effect of mid-cycle and preovulatory bovine follicular fluid (bFF) on the secretion of progesterone and prostaglandin F 2 alpha (PGF 2 alpha) by bovine theca and granulosa cells cultured in tissue culture Medium 199. The inhibitory effect of bFF on the secretion of PGF 2 alpha by incubated and cultured hemipituitary glands of male rats was also studied. Addition of 5, 10 or 20% charcoal-extracted, mid-cycle bFF to the culture medium caused a twofold decrease in the accumulation of both progesterone and PGF 2 alpha by the cultured granulosa cells (P less than 0.01). In contrast, when preovulatory bFF was used, there was no inhibitory effect on secretion of progesterone and PGF 2 alpha. The addition of 10% mid-cycle bFF to the culture medium caused a two- to fourfold decrease in the accumulation of both PGF 2 alpha and progesterone by the cultured theca (p less than 0.008). However, in the presence of 1 microgram LH/ml, the inhibitory effect of mid-cycle bFF on progesterone and PGF 2 alpha secretion was abolished. The secretion of PGF 2 alpha was significantly (p less than 0.03) decreased in male rat hemipituitary glands after 5 h of incubation or 18 h of culture. These findings suggest that bFF from mid-cycle follicles inhibits prostaglandin synthetase as well as luteinization. The inhibition disappeared with bFF from preovulatory follicles.     

Microanalysis of hormone responsive ovarian interstitial gland cells in miniature culture

Small cell aggregates of functional interstitial tissue were isolated from ovaries of 21- to 24-day-old rats, and their biochemical properties were studied in miniature cultures of 500-4000 cells. The isolated interstitial cells expressed high amounts of the mitochondrial cholesterol side-chain cleavage cytochrome P-450, visualized by immunofluorescent staining. Freshly isolated cells also expressed high activities of 17 alpha-hydroxylase and 17:20-lyase, which were assayed by TLC analysis of [3H]progesterone metabolites. The TLC technique revealed the immediate conversion of progesterone to 5 alpha-reduced progestins. Consequently, no aromatizable androgens were produced but, accumulation of androsterone resulted instead from the direct action of 17 alpha-hydroxylase on 3 alpha-hydroxy-5 alpha-pregnane-20-one (pregnanolone). Both the immunoreactive levels of cholesterol side-chain cleavage cytochrome P-450 and the rate of the 17:20-lyase activity declined rapidly during culture. However, addition of LH to the medium restored both enzymes, indicating the presence of functional LH receptors. The latter were also demonstrable by their ability to evoke cAMP formation in response to LH, but not to FSH. The activity of 5 alpha-reductase in the interstitial cells was much higher (3-fold) than its activity in the granulosa cells. Unlike 17:20 lyase, the activity of 5 alpha-reductase did not decay in culture, nor was it affected by LH. We thus established a novel and sensitive experimental method to isolate and study a minute population of ovarian cells which, unlike the follicular granulosa and theca cells, are enigmatically differentiated at the early stages of ovarian development.     

Effects of oxytocin on steroidogenesis by bovine theca and granulosa cells

Oxytocin (OT) is secreted during the final stages of bovine follicular development. To test OT's potential role as a regulator of follicular steroidogenesis, theca and granulosa cells were isolated from bovine preovulatory follicles 48 h after initiation of luteolysis with prostaglandin F2 alpha, and cultured with graded doses of OT (0, 0.5, 5, 50, and 500 mIU/ml). Granulosa cells were cultured with testosterone (0.5 microM) in either defined medium or medium containing 10% fetal bovine serum in the presence or absence of FSH (300 ng/ml); medium was collected and replaced daily for 5 days. In defined medium, oxytocin alone significantly increased progesterone production by granulosa cells (P less than 0.001) in a dose-dependent manner; over 5 days, doses of 0.5, 5, 50, and 500 mIU/ml OT caused 1.7-, 2.0-, 2.2-, and 2.6-fold increases. FSH enhanced progesterone 5-fold, but no dose of OT increased progesterone in the presence of FSH. OT also elevated progesterone in serum-containing medium (P less than 0.005), but the magnitude of its effects was lower (1.07-, 1.1-, 1.2-, and 1.4-fold increases with 0.5, 5, 50, and 500 mIU/ml OT). OT had little effect on estradiol secretion by granulosa cells cultured with or without FSH. To test the specificity of OT's effects on progesterone production by granulosa cells, granulosa cells were treated with graded doses of an OT antagonist (0, 1, 10, 100, and 1000 ng/ml) in the presence or absence of OT (5 and 50 mIU/ml). Progesterone production by granulosa cells in the presence of the antagonist alone was similar to production in control cultures. The stimulatory effects of 5 and 50 mIU OT were completely abolished in the presence of 100 or 1000 ng antagonist, respectively (P less than 0.01). Preparations of theca interna were cultured in defined medium with graded doses of OT (0, 0.5, 5, 50, and 500 mIU/ml) in the presence or absence of LH (300 ng/ml), with collection and replacement of medium at 3, 6, 12, 24, 48, and 72 h. LH alone increased both progesterone (12-fold) and androstenedione (4-fold) production over controls. However, no dose of OT significantly affected either progesterone or androstenedione production. These results show that OT stimulates progesterone production by granulosa cells, and thus, suggest that OT regulates steroidogenesis in bovine granulosa cells in vivo.