Participation of the cytoskeleton in avian granulosa cell steroidogenesis.

Cytochalasin B (CB) and the more specific cytochalasin D (CD), disruptors of microfilament polymerization, and colchicine, an inhibitor of microtubule polymerization, were studied for their effects on cAMP and steroid production in granulosa cells of domestic fowl. Each agent was incubated with freshly dispersed cells from the largest preovulatory follicle of laying hens taken 3-4 hr before expected ovulation. Total content (cells + medium) of cAMP and steroids was measured by established radioimmunoassays. CB dose dependently inhibited basal as well as LH- and forskolin-stimulated cAMP generation and diminished basal, LH- and 25-hydroxycholesterol (25-OHC)-supported progesterone production. Conversely, CD potentiated LH- and forskolin-promoted cAMP generation as well as LH- and 25-OHC-stimulated progesterone synthesis. Neither drug had any influence on metabolism of pregnenolone to progesterone. Colchicine had no effect on cyclic AMP generation, yet it suppressed progesterone synthesis by inhibiting the conversion of pregnenolone to progesterone. beta-Lumicolchicine, a colchicine analog that does not depolymerize microtubules, had no such effect. The results suggest that microfilaments are involved in steroidogenesis at two sites, namely, the adenylate cyclase-cAMP system, and cholesterol conversion to pregnenolone; whereas microtubules act on the conversion of pregnenolone to progesterone.     

Steroidogenesis by cultured granulosa cells aspirated from human follicles using pregnenolone and androgens as precursors

Human granulosa cells from Graafian follicles aspirated 3-4 h before the expected time of ovulation were incubated with various steroid substrates, including pregnenolone, androstenedione, testosterone and dehydroepiandrosterone (DHA). Steroid production after 3 and 10 h of incubation was determined by radioimmunoassay. Progesterone and 17alpha-hydroxyprogesterone were the major products of granulosa cells in control short-term cultures with endogenous substrates. The addition of pregnenolone increased the synthesis of progesterone and 17alpha-hydroxyprogesterone compared with the controls, although the response varied considerably between paired short-term cultures. Little or no oestradiol-17beta was produced from endogenous precursors or short-term cultures to which pregnenolone had been added; one follicle, however, produced similar amounts of oestradiol-17beta in the control cultures and after incubation with pregnenolone. When granulosa cells were cultured with various amounts of androstenedione, DHA or testosterone, large amounts of oestradiol-17beta were produced, especially in short-term cultures in which larger amounts of substrate were added. Progesterone production continued and progesterone was synthesized more rapidly or in greater amounts in some short-term test cultures than in the controls. The results indicate that human granulosa cells are one source of oestradiol-17beta during the preovulatory phase. The data support the two-cell theory for oestradiol synthesis, for granulosa cells do not appear to undertake steroid conversion via the 5-unsaturated pathway, but aromatize androgens known to be produced by thecal cells. It is also suggested that either androgens or oestradiol-17beta stimulate progesterone production by granulosa cells, at least in vitro.     

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.     

Steroidogenesis in ovarian cells of the Japanese quail (Coturnix coturnix japonica)

The influence of follicular maturation on steroidogenesis and steroid metabolism by isolated Japanese quail granulosa and theca cells was examined. When stimulated with LH, granulosa cells of the largest follicle (F1) responded with a sixfold increase over unstimulated progesterone levels, whereas progesterone production in cells of F3 less than doubled even when maximally stimulated. Forskolin stimulated progesterone synthesis in both F1 and F3 granulosa cells, but its effect was less pronounced than that of LH. Furthermore, F1 cells metabolized 25-hydroxycholesterol to a greater extent than did F3 cells. There was no appreciable metabolism of [3H]progesterone by granulosa cells. Theca cells from the smaller follicles (F3-F5) responded to LH stimulation with greater estrogen and androstenedione production than theca cells from F1. [3H]Progesterone was metabolized mainly to androstenedione in theca cells. Thus, the overall pattern of in vitro steroidogenesis in quail granulosa cells is similar to that described for the chicken and turkey even though the quantitative differences in the steroidogenic capacity between developing and mature follicles are more striking in the quail. Furthermore, although the LH-stimulated androstenedione and estrogen production appears similar in developing quail and chicken theca cells, the profile of [3H]progesterone metabolism is different in quail theca cells from that found previously in chicken theca cells.     

The effect of kaurenol on steroidogenesis and cyclic adenosine monophosphate production in rat granulosa cells

The effect of kaurenol (ent-kaur-16-en-15 beta-ol) on steroidogenesis and cyclic AMP production was examined in rat granulosa cells in short-term incubations (6 h). Kaurenol alone significantly augmented the production of progesterone in time- and concentration-dependent manner but attenuated the accumulation of the progesterone metabolite 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P). The steroidogenic effect of kaurenol is due to the acute stimulation of pregnenolone production from endogenous cholesterol and an inhibition in 20 alpha-hydroxysteroid dehydrogenase which catalyzes the metabolism of progesterone to 20 alpha-OH-P. Kaurenol had no appreciable effect on conversion of exogenous pregnenolone to progesterone. Although kaurenol was without effect on basal cyclic AMP generation, it inhibited the actions of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and forskolin on the production of the cyclic nucleotide. Kaurenol also significantly attenuated the LH-, FSH- and forskolin-stimulated progesterone and 20 alpha-OH-P production in a concentration-dependent manner. Because kaurenol induced steroidogenesis without increased cyclic AMP accumulation, it is concluded that its action on basal steroidogenesis is mediated by a mechanism independent of the cyclic nucleotide. Kaurenol may serve as a useful tool for elucidating cyclic AMP-independent action(s) of hormones in intact tissue/cells.     

Site of androgen inhibition of follicle-stimulating hormone-stimulated progesterone production in porcine granulosa cells

In granulosa cells derived from medium-sized porcine follicles, certain androgens have been shown to inhibit FSH-stimulated progesterone synthesis. To determine the site at which this inhibition takes place, the effects of androgens on FSH- and (Bu)2cAMP-stimulated pregnenolone and progesterone syntheses were examined. Granulosa cells were isolated from 4- to 6-mm follicles and cultured for 24 h in modified Eagle's Minimum Essential Medium, alone or with FSH (1 microgram/ml) or (Bu)2cAMP (0.5-4 mM) in the presence or absence of androstenedione or testosterone. (Bu)2cAMP stimulated progesterone production in a dose-dependent manner. Testosterone (5 microM) had a slight, but nonsignificant, inhibitory effect on basal progesterone production, but significantly inhibited the synthesis of progesterone in the presence of (Bu)2cAMP, suggesting that testosterone inhibits progesterone synthesis at a step distal to cAMP formation. In the absence of FSH, granulosa cells produced substantial quantities of pregnenolone. FSH caused a 3-fold stimulation of pregnenolone synthesis. The addition of androstenedione or testosterone (5 microM) markedly increased pregnenolone accumulation in FSH-treated cultures. To determine at what step androgens affected FSH-stimulated pregnenolone production, granulosa cells were cultured with (Bu)2cAMP and/or testosterone for 24 h. (Bu)2cAMP stimulated pregnenolone synthesis in a dose-dependent manner. Testosterone (5 microM) significantly increased pregnenolone synthesis in response to (Bu)2cAMP, suggesting that androgens acted at a step distal to cAMP formation. Since these concentrations of androgens markedly inhibited FSH-stimulated progesterone production by these preparations, these results suggest that androgens may affect the conversion of pregnenolone to progesterone.     

Age-related changes in endogenous steroids of human fetal testis during early and midpregnancy

The steroidogenic activity of human fetal testes during early and midgestation was monitored by analyzing 58 individual fetal testes (aged 6-20 weeks of pregnancy) for endogenous pregnenolone (P5), progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone (T) and estradiol. A clear increase in testicular steroid concentrations, especially in those of T and other 3-keto-4-ene steroids, occurred between 8-11 weeks of gestation and reached maximum between 11-14 weeks. The three steroids present in highest concentrations were P5, androstenedione, and T (maximum concentrations, 1.9-2.7 ng/mg wet tissue). The levels of all of the C-19 steroids measured decreased clearly between weeks 14-20 of gestation, whereas those of the C-21 steroids, P5, progesterone, and 17-hydroxyprogesterone, remained relatively high. Our results suggest that the metabolic reactions converting P5 to androgens are activated in the human fetal testis within a short time range between 8-11 weeks of gestation. The increased androgen production is possibly a consequence of increased 3 beta-hydroxysteroid dehydrogenase activity. Testicular androgen production decreases in the beginning of the second trimester of pregnancy, most likely due to a blockade in the C-21 steroid side-chain cleavage.     

Regulation of steroidogenesis in jc-410, a stable cell line of porcine granulosa origin

We investigated the regulation of steroidogenesis in a cell line of porcine granulosa origin, JC-410. Cells responded to the protein kinase-A activators, cholera toxin and forskolin, with increased accumulation of intracellular cAMP. Histochemically, cells were shown to contain 3beta-HSD, the enzyme which converts pregnenolone to progesterone. The JC-410 cells produced progesterone and responded to the protein kinase-A activators with an increase in progesterone synthesis. Progesterone levels were also increased by 25-hydroxycholesterol, pregnenolone, estradiol and androstenedione. Follicle-stimulating hormone and luteinizing hormone had no effect on cAMP or progesterone accumulation. Androstenedione was required for the synthesis of estradiol by JC-410 cells. Steady-state levels of mRNA for the steroidogenic enzymes 3beta-HSD and P450scc were increased by treatment with cholera toxin, whereas P450arom was not changed. These cells express the steroidogenic enzymes genes in a similar fashion to primary cultures of porcine granulosa cells. The JC-410 cells may represent a valuable model to study second messenger regulation and the molecular mechanisms involved in steroidogenesis in granulosa cells.     

Trifluoperazine inhibits progesterone and cyclic AMP production in granulosa cells of the hen (Gallus domesticus)

Unstimulated (basal) as well as luteinizing hormone (LH)-promoted progesterone production in collagenase-dispersed hen granulosa cells was inhibited in a dose-related manner by two phenothiazines, trifluoperazine (TFP) and chlorpromazine (CP), both of which are known calmodulin antagonists. Using TFP, the more potent antagonist of the two, it was found that LH-stimulated cyclic AMP production was also suppressed. Moreover, TFP attenuated the steroidogenic effects of both 8-bromo-cyclic AMP and isobutylmethylxanthine but had no effect on the conversion of pregnenolone to progesterone. The inhibitory effects of TFP on steroidogenesis were reversible. It is concluded that phenothiazines inhibit steroidogenesis in ovarian granulosa cells by acting at multiple sites both proximal and distal to cyclic AMP generation without influencing the enzyme complex responsible for the conversion of pregnenolone to progesterone. The results are discussed in relation to calmodulin- and non-calmodulin-mediated actions of phenothiazines.     

Mevinolin (lovastatin) inhibits androstenedione production by porcine ovarian theca cells at the level of the 17 alpha-hydroxylase:C-17,20-lyase complex

Mevinolin, putatively a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme-A reductase, was used to assess the contribution of de novo synthesized cholesterol to androgen production by ovarian thecal cells in vitro. Enzymatically dispersed thecal cells from 3- to 6-mm follicles of prepubertal gilts were incubated at 150,000 cells/ml with a maximally effective dose of LH (250 ng/ml) for 24 h. Mevinolin (3-50 microM) caused dose-dependent inhibition of androstenedione production. Addition of 25-hydroxycholesterol (0.025-25 microM) failed to restore androstenedione production to levels seen in the absence of mevinolin, suggesting an additional site of action of mevinolin beyond 3-hydroxy-3-methylglutaryl coenzyme reductase. The site of this inhibitory effect was determined by measuring steroid products formed in the presence of relevant steroid precursors. Mevinolin (12 microM) inhibited the production of 17 alpha-hydroxyprogesterone from progesterone and that of androstenedione from 17 alpha-hydroxyprogesterone, while 25-hydroxycholesterol to progesterone and pregnenolone to progesterone conversions were unimpaired. That mevinolin did not affect 3 beta-hydroxysteroid dehydrogenase:delta 5-delta 4-isomerase reactions was confirmed by demonstrating that conversions of pregnenolone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone to progesterone, 17 alpha-hydroxyprogesterone, and androstenedione, respectively, were not affected by 12 microM mevinolin. These results indicate that mevinolin has an additional inhibitory action at the level of the 17 alpha-hydroxylase:C-17,20-lyase complex. The degree of inhibition of androstenedione production was not decreased with increased concentrations of progesterone or 17 alpha-hydroxyprogesterone substrate, suggesting that the inhibition was not competitive in nature. As the dose of mevinolin was increased up to 50 microM, progesterone accumulation was unaffected, but pregnenolone concentrations in medium greatly increased. While the mechanism of this effect is unclear, this finding suggests that preformed intracellular cholesterol, rather than that synthesized de novo, is supplying steroidogenic substrate in these cells.     

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.     

Implications of progesterone metabolism in MA-10 cells for accurate measurement of the rate of steroidogenesis.

In virtually all studies with MA-10 cells, progesterone RIAs have been used to measure steroid synthesis. To test whether progesterone is a stable end product, we investigated the metabolism of added tritiated progesterone and pregnenolone in MA-10 cells over a period of 3 h. Steroids were then extracted, separated by HPLC, and identified by GC/MS. We found that more than 70% of radiolabeled steroids were converted to at least five different metabolites. A major metabolite (40%) was 5 alpha-pregnan-3 alpha or 3 beta-ol-20one. Similar studies, using radiolabeled T, demonstrated conversion to dihydrotestosterone and two forms of 5 alpha-androstane-diols. These data indicate the presence of active 5 alpha-reductase and 3 alpha- and/or 3 beta-hydroxysteroid dehydrogenase activities in MA-10 cells. Because these results suggest that progesterone is an unstable end product, to gauge the level of active metabolism, we incubated cells in the presence of inhibitors of pregnenolone metabolism and assessed pregnenolone levels by RIA. We discovered that basal levels of steroidogenesis in MA-10 cells were considerably higher than previously estimated. Moreover, dibutyryl cAMP-stimulated steroid production was linear over more than 13 h, in contrast to previous findings that measured progesterone levels. Other consequences of inaccurate assessment of steroidogenic activity in MA-10 cells because of the application of the progesterone assay are discussed.     

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 luteinizing hormone withdrawal on Leydig cell smooth endoplasmic reticulum and steroidogenic reactions which convert pregnenolone to testosterone

Depletion of endogenous LH with sc implants of testosterone-17 beta-estradiol (T-E) caused a reduction in the Leydig cell smooth endoplasmic reticulum (SER) over a 10-day treatment period. Decreases also occurred in some, but not all, of the testicular steroidogenic reactions responsible for the conversion of pregnenolone (PREG) to testosterone. The conversions of progesterone (PROG) to 17 alpha-hydroxyprogesterone, 17 alpha-hydroxyprogesterone to androstenedione, and androstenedione to testosterone were significantly correlated (P less than 0.05) with the loss of Leydig cell SER. In contrast, the testicular conversion of PREG to PROG in rats deprived of endogenous LH for up to 10 days was identical to that in intact controls. Similar results were obtained when rats were hypophysectomized for 10 days. These results indicate that the Leydig cell enzyme activities responsible for converting PREG to PROG are distributed in the Leydig cell SER fraction which remains in Leydig cell cytoplasm 10 days after LH withdrawal, and thus, the bulk of these enzyme activities are sequested in a SER compartment that is resistant to LH withdrawal.     

Testicular steroidogenesis in the testicular feminized (Tfm) mouse: loss of 17 alpha-hydroxylase activity.

Testicular feminized (Tfm) mice are totally insensitive to androgen and may be used to study the role of the androgen receptor in normal development and function. We have examined testicular and Leydig cell steroidogenesis in Tfm mice. Serum bioactive LH was high in Tfm mice but serum testosterone was low and this was associated with a severe reduction in testicular testosterone production in vitro. Examination of [3H]pregnenolone metabolism by testes of Tfm mice indicated that progesterone, rather than testosterone, was the major steroid produced. Leydig cells were isolated from normal and Tfm mice and from normal mice in which testicular descent was surgically prevented before puberty. As in whole testes, androgen production in response to human chorionic gonadotrophin was severely reduced in Leydig cells from testes of Tfm mice compared with normal or cryptorchid groups. In contrast, progesterone production by Leydig cells from testes of Tfm mice was markedly increased in comparison with other groups. Total steroid production (progesterone plus androstenedione plus testosterone), however, was only 24% of normal in Leydig cells from Tfm mice. The pattern of steroid production by Leydig cells from cryptorchid testes was similar to control, although total steroid production was reduced to about 50% (this was significantly higher than the Tfm group, P less than 0.05). The high progesterone/androgen ratio in testes from Tfm mice suggested that 17 alpha-hydroxylase was depleted in these animals. To confirm this, activity of the four major steroidogenic enzymes associated with the smooth endoplasmic reticulum was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms subserving calcium's modulation of luteinizing hormone action in isolated swine granulosa cells

We studied the mechanism(s) by which calcium ions modulate progesterone biosynthesis by isolated swine granulosa cells incubated in chemically defined medium in vitro. In selectively calcium-deficient incubations, the capacity of 8-bromo-cAMP to stimulate pregnenolone synthesis from endogenous sterol substrate was significantly impeded. This effect of calcium ions was specific, because calcium ions did not influence basal pregnenolone production or alter progesterone production in response to exogenously supplied cholesterol substrate. Moreover, calcium ions did not modify other biosynthetic processes in granulosa cells, such as de novo synthesis of cholesterol from [14C]acetate or the aromatization of testosterone to 17 beta-estradiol. The possible role of calmodulin in mediating calcium's actions in pig granulosa cells was tested by measuring the calmodulin content of these cells and assessing the functional responses to classical calmodulin antagonists. By immunoassay, swine granulosa cells contained high concentrations of calmodulin, viz. 4.21-4.88 micrograms calmodulin/mg protein. Moreover, calmodulin antagonists inhibited LH-stimulated progesterone production with the following rank order of potencies [estimated by half-maximally inhibitory concentrations (ID50)]: penfluridol (1 microM), trifluoroperazine (9 microM), chlorpromazine (95 microM), and trifluoperazine sulfoxide (greater than 300 microM). In addition, the nonphenothiazine calmodulin antagonist W7 inhibited stimulated progesterone production with an ID50 of 16.7 microM. W5 was less active. None of these antagonists significantly suppressed LH-stimulated cAMP generation at the low concentrations capable of inhibiting progesterone production. The effects of calcium ions seemed to depend upon the availability of intracellular pools of calcium, because TMB-8, an inhibitor of intracellular calcium mobilization, effectively suppressed LH-stimulated progesterone production (ID50, 18 microM). However, even 100 microM TMB-8 failed to alter basal progesterone production or suppress LH-stimulated cAMP generation in these cells. In summary, the present studies indicate that calcium ions significantly modulate LH's stimulation of pregnenolone biosynthesis from endogenous cholesterol substrate in swine ovarian cells. Calcium does not influence basal pregnenolone production, estrogen synthesis from androgen substrate, de novo biosynthesis of cholesterol from [14C]acetate, or progesterone production from exogenously supplied sterol substrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of thyroid hormone on steroidogenic enzyme induction in porcine granulosa cells cultured in vitro

In order to elucidate the mechanism of thyroid hormone action on the ovary, direct effects of L-thyroxine (T4) or L-triiodothyronine (T3) on steroidogenic enzyme induction were investigated in vitro using a monolayer culture system of porcine granulosa cells obtained from small follicles. The cells were cultured in the absence or presence of porcine FSH (20ng/ml) for 6 days, with or without T4 or T3, under sparsely (4%) serum supplemented condition. The mechanism of thyroid hormone action on the granulosa cells was studied by testing the capability of thyroid hormone to enhance the steroidogenesis in response to exogenously provided substrates. Concomitant treatment with FSH (20ng/ml) and T4 (10(-7) M) caused a further increased production of progesterone in response to the addition of pregnenolone compared to that in the absence of pregnenolone. The same treatment with FSH and T4 also caused a further increased production of estrone in response to the addition of androstenedione. Concomitant treatment with 10(-9) MT3 demonstrated similar stimulatory effects on the steroidogenesis in cultured granulosa cells. T4 or T3 alone without FSH was incapable of exhibiting these stimulatory effects. Furthermore, aromatase activity in cultured granulosa cells assessed by the release of tritiated water from [1 beta-3H, 4-14C] androstenedione was significantly higher in the cells treated concomitantly with FSH (20ng/ml) and T4 (10(-7) M) than that in the cells treated with FSH alone. These results suggest that thyroid hormone synergizes with FSH and increases FSH-mediated induction of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and aromatase activity in immature granulosa cells. Since the effective dose of T4 and T3 observed in our studies is in the physiological range of circulating total levels of T4 and T3, it can be concluded that the synergism between FSH and thyroid hormone is of physiological importance to the full expression of FSH actions in the functional differentiation of immature granulosa cells.     

Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor

We established a steroidogenic human ovarian granulosa-like tumor cell line, designated KGN, from a patient with invasive ovarian granulosa cell carcinoma. KGN had a relatively long population doubling time of about 46.4 h and had an abnormal karyotype of 45,XX, 7q-, -22. A steroid analysis of the cultured medium by RIA performed 5 yr after the initiation of culture showed that KGN was able to secrete pregnenolone and progesterone, and both dramatically increased after stimulation with (Bu)(2)cAMP. However, little or no secretion of 17alpha-hydroxylated steroids, dehydroepiandrosterone, androstenedione, or estradiol was observed. The aromatase activity of KGN was relatively high and was further stimulated by (Bu)(2)cAMP or FSH. These findings showed a pattern similar to that of steroidogenesis in human granulosa cells, thus allowing analysis of naturally occurring steroidogenesis in human granulosa cells. Fas-mediated apoptosis of KGN was also observed, which mimicked the physiological regulation of apoptosis in normal human granulosa cells. Based on these findings, this cell line is considered to be a very useful model for understanding the regulation of steroidogenesis, cell growth, and apoptosis in human granulosa cells.     

Metabolism of 25-hydroxycholesterol by rat luteal mitochondria and dispersed cells

The metabolism of 25-hydroxycholesterol (25-OH-cholesterol) to progestins by mitochondria and dispersed cells prepared from ovaries of PMSG-hCG-primed rats was studied. Mitochondria converted [3H]25-OH-cholesterol into [3H]pregnenolone and [3H]progesterone. Unlabeled 25-OH-cholesterol also stimulated mitochondrial steroidogenesis in a dose-dependent, saturable fashion. A direct relationship between rates of steroid synthesis in the presence of 25-OH-cholesterol and mitochondrial cytochrome P-450 levels was found. Although steroid production and cytochrome P-450 content per milligram protein were higher in mitochrondia prepared from ovaries removed on day 8 post hCG than on either day 1 or day 14, steroid production per nanomole cytochrome P-450 was similar. Treatment of rats with hCG 1 h before killing significantly increased mitrochondrial steroid synthesis from endogenous substrate but had no effect on metabolism of 25-OH-cholesterol. Dispersed cells increased progestin production by 6-fold when incubated with 25-OH-cholesterol. The effects of 25-OH-cholesterol were dose dependent and saturable. While both LH and (Bu)2cAMP stimulated progestin synthesis from endogenous substrate, secretion of progestins with these agents reached levels only 60% of those observed in the presence of 25-OH-cholesterol. Neither LH nor (Bu)2cAMP altered the metabolism of the dydroxysterol by the cells nor did cycloheximide, which substantially inhibited progestin secretion in the absence of the hydroxysterol. However, animoglutethimide did block the stimulation of steroidogenesis by 25-OH-cholesterol. We conclude that 25-OH-cholesterol is an effective steroidogenic substrate for rat luteal tissue. With its use, information regarding the maximal capacity of luteal tissue to produce progestins in vitro can be obtained.     

Steroid biosynthesis by ovarian follicles of Xenopus laevis in vitro during oogenesis

In vitro steroid metabolism was investigated in Xenopus laevis vitellogenic follicles (0.8 mm ? diameter ? 1.0 mm) and full-grown follicles (diameter ? 1.2 mm); in both classes of follicles pregnenolone was transformed to progesterone and progesterone itself was further metabolized to 4-en-3-ketosteroids. The 3β-hydroxysteroid dehydrogenase activity was restricted to follicular cells. Xenopus laevis follicles catalyze the conversion of androstenedione and testosterone to estradiol-17β and estrone, respectively. Generally 10 to 50 times more estrogens were found to be associated with follicular envelopes. Ovarian follicles also metabolized estrone to estradiol-17β; there was only minimal conversion of estradiol to estrone. In vitro purified Acipenser gonadotropic hormone induced a 60% inhibition of the conversion of androstenedione to estrogens.