Steroidogenesis In Theca Cells Research Articles

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.

Insulin Enhances Luteinizing Hormone-Stimulated Steroidogenesis by Porcine Theca Cells1

It has been shown recently that insulin enhances differentiation of rat, pig, and human granulosa cells. The present studies were done to determine if insulin also plays a role in the regulation of theca cell steroidogenesis. Theca cells were obtained from prepubertal gilts and cultured under serum-free conditions for 48 h. Theca cell androstenedione production under basal and luteinizing hormone (LH)-stimulated conditions was significantly increased by adding insulin (1 microgram/ml) to the culture medium. Treatment of basal and LH-stimulated cultures with increasing concentrations of insulin (0.001-10 micrograms/ml) caused dose- and time-dependent increments in androstenedione production, but the effect was independent of the dose of LH employed. The ability of insulin to enhance thecal cell androstenedione production was mimicked by somatomedin C, but not by relaxin. Studies to determine the mechanism(s) of action of insulin showed that insulin action is exerted, at least in part, at a site(s) proximal to cyclic adenosine 3'5'-monophosphate (cAMP) generation, since insulin enhanced both basal and LH-stimulated accumulation of extracellular cAMP in addition to increasing androstenedione production. This effect was further enhanced by 3-isobutyl-1-methyl xanthine, an inhibitor of phosphodiesterase activity. Insulin treatment also caused dose-dependent increments in forskolin- and prostaglandin E2-stimulated accumulation of extracellular cAMP and androstenedione. Insulin also increased both the basal and LH-stimulated production of progesterone and its precursor pregnenolone, in addition to the increases in androstenedione.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in content of cytochrome P450(17)alpha, cytochrome P450scc, and 3-hydroxy-3-methylglutaryl CoA reductase in developing rat ovarian follicles and corpora lutea: correlation with theca cell steroidogenesis.

The following study was undertaken to determine which hormones (luteinizing hormone, LH, and prolactin, PRL) and enzymes (cytochrome P450(17)alpha, nicotinamide adenine dinucleotide phosphate [NADPH]-cytochrome P450 reductase, 3-hydroxy-3-methylglutaryl [HMG] CoA reductase, cholesterol side-chain cleavage cytochrome P450 [P450scc], and adrenodoxin) were associated with the regulation of androgen biosynthesis by developing rat follicles and corpora lutea in vivo as well as by thecal explants maintained in culture. Immunoblots of soluble cell extracts of small antral (SA), preovulatory (PO), and luteinizing (PO + human chorionic gonadotropin [hCG], 7 h) follicles, newly formed corpora lutea (PO + hCG, 24 h), and corpora luteal isolated on Day 15 of pregnancy, demonstrated that cytochrome P450(17)alpha was low in SA follicles, selectively increased 4-fold in PO follicles, and decreased to less than 10% within 7 h after hCG. Filter hybridization assays using a 32P-labeled cytochrome P450(17)alpha cDNA probe demonstrated that changes in the content of P450(17)alpha mRNA exhibited a pattern similar to that of the enzyme. Conversely, immunoblots for other microsomal enzymes either exhibited no change (NADPH cytochrome P450 reductase) or a transient increase after the hCG surge (HMG CoA reductase), whereas the mitochondrial enzymes either increased markedly in association with luteinization (cytochrome P450scc) or were increased in a more transient manner (adrenodoxin). The LH-induced loss of cytochrome P450(17)alpha in vivo was not associated with loss of androgen biosynthesis when luteinizing theca were placed in culture in medium containing either LH or LH and PRL, suggesting that other hormones, or the presence of other cell types, are required to maintain the decrease in cytochrome P450(17)alpha in vivo. Conversely, the LH-induced increase in cytochrome P450scc in vivo was associated with the maintenance of elevated progesterone production by theca in culture, suggesting that cytochrome P450scc may be constitutively expressed in luteinized theca. Thus, thecal cell cytochrome P450(17)alpha and the regulation of its content and mRNA by LH are pivotal to the biosynthesis of androgens, the obligatory precursors for estradiol biosynthesis and the consequent development of preovulatory follicles. The molecular basis for the different effects of low versus elevated concentrations of LH on cytochrome P450(17)alpha, as well as cytochrome P450scc, remain to be determined.

Thecal cell 3-beta hydroxysteroid dehydrogenase activity: Modulation by human chorionic gonadotropin, progesterone, estradiol-17 beta and dihydrotestosterone

Thecal cell steroidogenesis plays a major role in folliculogenesis within the porcine ovary. Accorclingly, the effects of physiological concentrations of steroids on 3β-hydroxysteroid dehydrogenase activity (3β-HSD) were determined. Theca was excised from large porcine follicles and prepared in a monolayer culture in 1 ml of serum-free media. Cells were treated 24 h after culture as follows: (1) control, (2) hCG (5IU); (3) progesterone (P, 3μg); estradiol-17β (E, 4μg); 5β-dihydrotestosterone (DHT, 1 μg); (4) hCG + P or E or DHT. At 3, 6, 12, 24 and 48 h after treatment, media were assessed for P levels. For 3β-HSD activity, P formation by microsomal fractions incubated with 1 μM pregnenolone + 5 μM NAD+ for l h (37°C) was monitored. Thecal cell P secretion increased from 27 to 72 h. hCG significantly ( P < 0.05) increased P levels after 36 h compared to controls. E or E + hCG decreased P levels at 36, 48, and 72 h and DHT prevented the hCG-induced increase in P secretion. 3β-HSD activity in thecal microsomes increased significantly from 27 to 72 h. hCG had little effect on 3β-HSD activity compared with controls from 27 to 36 h, but significantly ( P < 0.05) decreased 3β-HSD activity at 48 and 72 h. However, P or P + hCG significantly ( P < 0.05) decreased 3β-HSD activity at all times. In addition, E or E + hCG significantly ( P < 0.05) decreased 3β-HSD activity at 48 and 72 h. DHT prevented the hCG-induced decrease in 3β-HSD activity. In conclusion, porcine thecal secretion of P and microsomal 3β-HSD activity increased during 72 h of culture. Paradoxically, the addition of hCG to cultures enhanced media P concentrations but inhibited 3β-HSD activity. Further, the addition of E to cultures decreased media concentrations of P while P or E decreased 3β-HSD activity. Therefore, paracrine/autocrine effects of locally produced steroids may play a role in modulating thecal cell steroidogenesis.

Effects of danazol on steroidogenesis and gonadotropic responsiveness in isolated human preovulatory follicular cells.

In order to investigate the influence of danazol on steroidogenesis and gonadotropic responsiveness of human follicular cells, granulosa and thecal cells of preovulatory follicles were isolated and separately incubated for short term periods. Human chorionic gonadotropin (hCG) (100 IU/ml), FSH (0.5 IU/ml) and danazol (10 micrograms/ml) alone or in combination were added to the incubation medium. Following incubation the cellular cyclic adenosine 3'5' monophosphate (cAMP) levels and the medium content of progesterone (P), androstenedione (A) and 17 beta-estradiol (E2) were determined. All follicles included in the study were classified as nonatretic and well developed, i.e. less than 3 days before ovulation. Human chorionic gonadotropin caused an increase in cAMP formation in both cell-types and this effect was significantly counteracted by danazol in vitro. In granulosa cells danazol tended to counteract a stimulatory effect of FSH on cAMP formation. No significant influence of danazol was found on the basal steroid formation of both cell types during short term incubation. On the other hand, danazol significantly counteracted the FSH stimulated P formation of the granulosa cells and the hCG stimulated A and E2 formation of the thecal cells. It is concluded that danazol inhibits gonadotropin-stimulated steroidogenesis locally in the human follicular cells and that this effect of danazol is mediated via the cyclic AMP system.

Interactions among endocrine control systems in the regulation of ovarian function

During sequential physiologic maturation of an individual follicle, the number of granulosa cells increases in excess of 1000-fold, while intra-ovarian concentrations of sex steroids escalate by 100-fold. The recent development of several in vitro ovarian systems has permitted a more extensive and direct assessment of specific mechanisms that control growth and steroidogenesis in granulosa and thecal cells. For example, estradiol and follicle stimulating-hormone seem to promote the production by ovarian cells of low-molecular-weight growth factors, that may participate in granulosa cell proliferation. Luteinizing hormone stimulates the de novo synthesis of prostacyclin by granulosa cells in vitro. Prostacyclin, in turn, may regulate the microvasculature of the maturing follicle and directly stimulate steroidogenesis. The effects of individual hormones are markedly modified by other intraovarian endocrine factors, and by the precise status of cytodifferentiation of the ovarian cells. For example, the actions of prolactin on granulosa-cell steroidogenesis are influenced strikingly by both agonistic and antagonistic interactions between prolactin and estradiol, as well as by the level of granulosa-cell cytodifferentiation attained in vivo. Similar bihormonal intrafollicular interactions are recognizable between estrogen and follicle-stimulating hormone in the early follicle, and between estradiol and luteinizing hormone in the maturing follicle. These interactions are susceptible to more precise examination under defined in vitro conditions. Overall, recent advances in biomedical research continue to elucidate basic molecular mechanisms of hormone action in ovarian cell physiology. Such advances are likely to continue to provide important insights into the pathophysiology of clinical disorders of human reproduction.

Obligatory role of luteinizing hormone (LH) in the initiation of preovulatory follicular growth in the pregnant rat: specific effects of human chorionic gonadotropin and follicle-stimulating of hormone on LH receptors and steroidogenesis in theca, granulosa, and luteal cells.

The changes in serum hormone concentrations accompanying preovulatory follicular development at the end of pregnancy in the rat suggest that follicular maturation is stimulated by subtle increases in serum LH and/or by a decrease in serum progesterone but not by increases in serum FSH. To test the ability of small antral nonovulatory follicles to respond to subtle increases in LH-like activity in the presence of elevated serum progesterone, hCG (0.25 to 3.0 IU), or the control, human FSH (hFSH) (0.88 to 4.4 IU) was administered twice daily to rats on days 14 and 15 of pregnancy. hCG stimulated follicular growth, increased granulosa cell and theca cell LH receptor content, and enhanced follicular estradiol accumulation in a time-related and dose-dependent manner. Follicles from animals treated twice daily for 2 days with 1.5 IU hCG (3.5 pmol) were by day 16 functionally identical to proestrous follicles isolated on day 23, and a bolus injection of 10 IU hCG was able to induce ovulation in these hCG-primed animals. Although 35.5 pmol hFSH caused granulosa cell LH receptor content and aromatase activity to increase, hCG was clearly much more effective on a “per mole” basis in evoking these responses. FSH failed to increase either theca cell LH receptor content or the inherent ability of follicles to produce estradiol when wholly dependent on endogenous androgen substrate. In addition, 2 days of treatment with hCG caused dose-dependent decreases in LH receptor content of luteal tissue but did not alter serum concentrations of progesterone, suggesting that luteal cell progesterone production may not be associated with the luteal LH receptor response system at this time. Results of this study suggest that, even in the presence of elevated serum progesterone, sustained increases in serum LH (but not FSH) activity will support the maturation of preovulatory follicles that are functionally indistinguishable from proestrous follicles. The dependence of follicular differentiation on LH appears to involve enhancement of theca cell function and synthesis of aromatizable androgens rather than stimulation of granulosa cell aromatase activity.