Ovarian nerve extracts influence androgen production by cultured ovarian thecal cells

There is growing evidence that ovarian steroidogenesis is controlled not only by pituitary gonadotropins but also by ovarian nerves. Nerves reach the ovary via the plexus nerve and via the superior ovarian nerve (SON), which runs in the suspensory ligament, and innervate theca cells of all sizes of follicles. To investigate the role of ovarian nerves in steroidogenesis we have examined the effects of adding an extract of SON from adult rats on androgen production by cultured porcine theca cells. Addition of SON extract to cultured theca interna from 3 to 6 mm diameter follicles of prepubertal gilts significantly inhibited (p less than 0.05) LH-stimulated androstenedione production in a dose-dependent manner; significant inhibition (10.8%) occurred with the addition of the extract of 2 mg of SON/ml culture medium, and near maximal inhibition (83%) resulted when the SON extract was increased to 60 mg SON/ml. Extracts of sciatic nerves, used as non-ovarian nerve controls, failed to inhibit, and in fact significantly increased (p less than 0.05) androstenedione production over the same concentration range of neural tissue extract. The inhibitory effect of the SON extract was unaffected by chymotrypsin digestion or by the presence of the beta-adrenergic antagonist propranolol (10(-6) M), but was removed by charcoal treatment. These results suggest that the nervous system has the potential for modulation of follicular steroid biosynthesis via direct innervation of the ovaries, in addition to the well-established indirect mechanism of neural control exerted via the hypothalamic-pituitary system.     

Transforming growth factor beta inhibits steroidogenic acute regulatory (StAR) protein expression in human ovarian thecal cells

In this study, we investigated the effects of TGFβ1 on steroidogensis and expression of the steroidogenic acute regulatory (StAR) protein which regulates an important early step in the steroidogenic pathway. We utilized a human ovarian thecal like tumor (HOTT) cell model and investigated the effects of activin-A, inhibin-A, or TGFβ1 in the presence of forskolin and the effect of dibutyryl cyclic AMP (dbcAMP) on steroid accumulation in the culture medium. Cells were also treated with different concentration of TGFβ1 in the presence of forskolin, combined steroid production was measured at the end of 48 h and after 3 h incubation with 22R-hydroxycholesterol. In the presence of TGFβ1 there was a dose-dependent inhibition of androstenedione production. Inhibition in combined steroid production was apparent at the highest concentration of TGFβ1 tested. In the presence of 22R-hydroxycholesterol, combined steroid production was significantly inhibited at lower concentrations. TGFβ1 inhibited StAR protein expression in a concentration dependent manner. There was also a similar inhibition in StAR mRNA. These results suggest that the effect of TGFβ1 on steroid production and possibly follicular development may be in part due to its effects on StAR expression.     

Mediation of gonadotrophin-stimulated growth and differentiation of human granulosa cells by adenosine-3'',5''-monophosphate: one molecule, two messages

OBJECTIVE: To determine how the second messenger adenosine-3',5'-monophosphate (cyclic AMP) is able to mediate divergent actions of FSH and LH on granulosa cell growth and differentiation in human ovaries. DESIGN: Human granulosa cells were cultured for 96 hours in serum-free medium 199 containing increasing doses of either FSH, LH or dibutyryl cyclic AMP. Extra and intra-cellular cyclic AMP levels were determined by radioimmunoassay. Tritiated thymidine uptake and cell number were measured as indices of cell growth, and spent medium was assayed for steroids (oestradiol and progesterone) to reflect differentiation. PATIENTS: 'Mature' granulosa cells were aspirated from preovulatory follicles in the ovaries of clomiphene-stimulated patients undergoing laparoscopic sterilization; 'luteinized' granulosa cells were aspirated from periovulatory follicles in the ovaries of gonadotrophin-stimulated in-vitro fertilization patients. RESULTS: LH consistently inhibited, whereas FSH maintained or stimulated, basal granulosa cell numbers. Steroidogenesis was dose-dependently increased by both gonadotrophins, with LH having the significantly greater effect over the entire dose-response range (1-100 micrograms/l). LH also induced significantly more cyclic AMP production than FSH, both intra and extra-cellularly, providing a basis for differential post-receptor signalling via a common second messenger. Addition of dibutyryl cyclic AMP, at low concentrations (10-250 mumol/l) to the cultured cells in the absence of gonadotrophins mimicked FSH effects with stimulation/maintenance of cell numbers and moderate steroidogenesis. High concentrations of dibutyryl cyclic AMP (500-1000 mumol/l) caused a significant inhibition of cell numbers together with maximal steroidogenesis, simulating LH action. CONCLUSIONS: These results suggest that granulosa cell maturation in the follicular phase of the menstrual cycle (controlled by FSH) is associated with a low cyclic AMP tone that favours cell growth and expression of aromatase activity in the developing preovulatory follicle. During the early luteal phase (dominated by LH), the intracellular cycle AMP tone increases to allow maximal progesterone production and inhibition of cell growth in the corpus luteum. Thereby one second messenger can mediate divergent gonadotrophic effects on granulosa cell growth and differentiation in the human ovary.     

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.     

Studies on the synergistic effect of androgen on the stimulation of progestin secretion by FSH in cultured rat granulosa cells: a search for the mechanism of action.

Cultures of granulosa cells from immature hypophysectomized DES-treated rats were unable to maintain progestin production of more than 48 h in medium without hormone supplementation or in the presence of FSH only. Production of progestin (20alpha-dihydroprogesterone, as measured by radioimmunoassay) remained unimpaired in the presence of androstenedione (Ad) and was markedly increased in the presence of both Ad and FSH. The combined treatment with FSH and Ad during the first 48 h of culture brought about persistent changes in the cultured cells, since progestin accumulation did not decline upon subsequent removal of these hormones during days 3 and 4 of culture. Dibutyryl cyclic AMP (DBC) was able to mimic the changes in steroidogenic capability induced by the combined action of FSH and Ad. The extent of [125I]-FSH binding, FSH-stimulable cAMP accumulation and cyclic 3',5'-nucleotide phosphodiesterase activity were not affected by addition of Ad to the culture medium. Ad synergized with DBC in the stimulation of progestin accumulation in granulosa cell cultures. It is suggested that androgen acts at a step in the regulation of progestin biosynthesis distal to cAMP production.     

Glucose Inhibition of Adenylate Cyclase in Intact Cells of Escherichia coli B

Previous studies in E. coli B have demonstrated an inverse correlation between the presence of glucose in the medium and the accumulation of cyclic AMP in the medium. This observation could not be explained by the action of glucose as a repressor of adenylate cyclase (EC 4.6.1.1) synthesis, as a stabilizer of cyclic AMP phosphodiesterase (EC 3.1.4.17) activity, or as a direct inhibitor of adenylate cyclase activity in cell-free preparations. The recent development of an in vivo assay for adenylate cyclase has provided a basis for further exploring the inhibitory action of glucose in intact cells. With this assay it has been possible to show that, while glucose does not affect adenylate cyclase in vitro, it rapidly inhibits the enzyme activity in intact cells. Extensive metabolism of glucose is not required, since alpha-methylglucoside also inhibits adenylate cyclase in vivo. When cells are grown on glucose as carbon source, some sugars (mannose, glucosamine) substitute for glucose as adenylate cyclase inhibitors while others (e.g., fructose) do not. Dose-response studies indicate that low concentrations of glucose lead to essentially complete inhibition of adenylate cyclase activity while only moderately decreasing intracellular cyclic AMP concentrations. The evidence presented suggests that the decreased cellular cyclic AMP levels resulting from glucose addition can be accounted for by inhibition of adenylate cyclase without any significant effect on cyclic AMP phosphodiesterase or the transport of cyclic AMP from the cells to the medium.     

Effect of luteinizing hormone and cyclic adenosine 3',5'-monophosphate on steroidogenesis in the ovarian follicle of the rabbit.

Mature ovarian follicles have been isolated from estrus rabbits and the effects of gonadotropin and cyclic AMP on steroidogenesis in this tissue determined. Gonadotropins used include LH, FSH, and prolactin; follicular levels of progesterone, 17-hydroxyprogesterone, testosterone and 17beta-estradiol in all experiments were quantitated by radioimmunoassay. Incubation of follicles with LH in concentrations ranging from 0.05 to 50 mug/ml medium yielded increases in the total ng steroid/follicle. Five mug LH/ml gave a maximal response with no further increase in steroid concentration when the LH was raised to 50 mug/ml. Prolactin had no effect on follicular steroidogenesis while the stimulatory action of a FSH preparation could only be partially attributed to LH contamination. Cyclic AMP also proved to be a potent stimulatory agent in follicular steroidogenesis with maximal increases at 20 mumoles/ml and a decline in the ng steroid/follicle when cyclic AMP was raised to 30 and 40 mumoles/ml. The effects of LH and cyclic AMP proved to be nonadditive; incubation of follicles simultaneously with 5 mug LH/ml and 20 mumoles cyclic AMP/ml yielded steroid concentrations which were no different than levels following incubation with either agent alone. Taken together, these results demonstrate that both LH and cyclic AMP cause increases in radioimmunoassayable steroid in the rabbit follicle in vitro and that LH probably acts by way of a cyclic AMP intermediate.     

Steroidogenesis during postnatal development in the mouse ovary

In the mouse, follicular formation and development is largely postnatal. Changes in ovarian steroidogenesis during early postnatal life are likely, therefore, to reflect changes in follicular activity during early folliculogenesis. In this study, basal progesterone and androstenedione production and responsiveness to gonadotrophins, dibutyryl cyclic AMP (dbcAMP) and 22R-hydroxycholesterol have been measured following short-term in-vitro incubations of ovaries from mice aged 1, 3, 5, 7, 10 or 15 days. On the day of birth (day 1), basal and gonadotrophin-stimulated progesterone and androstenedione production were undetectable although a response to dbcAMP and a low level of cholesterol side-chain cleavage (CSCC) activity (measured using 22R-hydroxycholesterol) were present. On day 3 progesterone and androstenedione production were undetectable under all conditions. On day 5 only a low level of CSCC activity was detectable but on day 7 there was an increase in ovarian steroid production. Basal progesterone and androstenedione were detectable and LH, but not FSH, increased the production of both steroids. These changes were associated with a marked increase of more than 80-fold in CSCC activity. Basal steroid output increased from days 7 to 15 and LH continued to stimulate progesterone accumulation although no effect on androstenedione was seen. Addition of FSH had no effect on steroidogenesis on day 10 but significantly increased progesterone production on day 15. Ovaries from the mice used in these studies contained primordial follicles and stromal tissue on day 1. By day 5 primary and secondary follicles were present and the major increase in steroid production between days 5 and 7 was associated with an increase in secondary follicles and increased differentiation of the thecal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicle-stimulating hormone-induced prostaglandin E formation in isolated rat ovarian theca

The aim of this study was to search for direct biochemical effects of highly purified FSH on isolated ovarian follicular theca in vitro. Granulosa cells (GC; approximately 1 x 10(5) cells per follicle) were flushed from isolated follicles of pro-oestrous rats. The remaining theca layer and the isolated GC were incubated with highly purified ovine FSH. Prostaglandin E (PGE) accumulation was measured by radioimmunoassay. Follicle-stimulating hormone induced a 15-fold increase in PGE accumulation over the basal level in the follicular theca, the stimulated rate exceeding threefold that observed in the GC fraction derived from the same follicle. Follicle-stimulating hormone caused no significant increase in cyclic AMP level or steroidogenesis in the theca layer, but was active on these parameters in the GC. In contrast, LH increased the accumulation of cyclic AMP, progesterone and testosterone, as well as of PGE, in follicular theca. Exogenous 8-bromo cyclic AMP or cyclic GMP also stimulated PGE production in follicular theca or GC, but FSH was without any effect on the level of endogenous cyclic GMP in GC or follicular theca. Antibodies to FSH prevented the effect of FSH (but not that of LH) on PGE formation by follicular theca and GC, while antibodies to the beta-subunit of LH blocked the effect of LH but not of FSH. We conclude that highly purified FSH has a stimulatory effect on PGE formation by the follicular theca.     

Bone morphogenetic protein inhibits ovarian androgen production

Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta superfamily, were recently shown to be expressed and to regulate steroidogenesis in rat ovarian tissue. The purpose of this study was to investigate the effect of BMP-4 on androgen production in a human ovarian theca-like tumor (HOTT) cell culture model. We have previously demonstrated the usefulness of these cells as a model for human thecal cells. HOTT cells respond to protein kinase A agonists by increased production of androstenedione and with an induction of steroid-metabolizing enzymes. In this investigation, HOTT cells were treated with forskolin or dibutyryl cyclic AMP (dbcAMP) in the presence or absence of various concentrations of BMP-4. The accumulation of androstenedione, progesterone, and 17alpha-hydroxyprogesterone (17OHP) in the incubation medium was measured by RIA. The expression of 17alpha-hydroxylase (CYP17), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cholesterol side-chain cleavage (CYP11A1), and steroidogenic acute regulatory (StAR) protein was determined by protein immunoblotting analysis using specific rabbit polyclonal antibodies. We also examined the expression of BMP receptor subtypes in our HOTT cells using RT-PCR. In cells treated with medium alone, steroid accumulation and steroid enzyme expression was unchanged. In cells treated with BMP alone there was a modest decrease in androstenedione secretion. In the presence of forskolin, HOTT cell production of androstenedione, 17OHP, and progesterone increased by approximately 4.5-, 35-, and 3-fold, respectively. In contrast, BMP-4 decreased forskolin-stimulated HOTT cell secretion of androstenedione and 17OHP by 50% but increased progesterone production 3-fold above forskolin treatment alone. Forskolin treatment led to an increase in CYP17, CYP11A1, 3betaHSD, and StAR protein expression. BMP-4 markedly inhibited forskolin stimulation of CYP17 expression but had little effect on 3betaHSD, CYP11A1, or StAR protein levels. Similar results were observed with the cAMP analog dbcAMP. In addition, BMP-4 inhibited basal and forskolin stimulation of CYP17 messenger RNA expression as determined by RNase protection assay. Other members of the transforming growth factor beta superfamily, including activin and inhibin, had minimal effect on androstenedione production in the absence of forskolin. In the presence of forskolin, activin inhibited androstenedione production by 80%. Activin also inhibited forskolin induction of CYP17 protein expression as determined by Western analysis. We identified the presence of messenger RNA for three BMP receptors (BMP-IA, BMP-IB, and BMP-II) in the HOTT cells model. In conclusion, BMP-4 inhibits HOTT cell expression of CYP17, leading to an alteration of steroidogenic pathway resulting in reduced androstenedione accumulation and increased progesterone production. These effects of BMP-4 seem similar to those caused by activin, another member of the transforming growth factor-beta superfamily of proteins.     

The effect of calcium on the potentiation of LH-stimulated steroidogenesis and inhibition of LH-stimulated cyclic AMP production by LHRH agonist (ICI 118630) in rat Leydig cells

A luteinizing hormone-releasing hormone (LHRH) agonist (ICI 118630) potentiated the effects of luteinizing hormone (LH) and dibutyryl cyclic AMP on steroidogenesis during 4 h incubations with rat Leydig cells. LH-stimulated cyclic AMP levels were decreased by the addition of the LHRH agonist. The potentiation of the LH-increased steroidogenesis was dependent on Ca2+; maximum effects required at least 2.5 mM Ca2+ in the incubation medium. The calcium ionophore A23187 negated the potentiation in a dose-dependent manner (ED50 = 0.2-0.3 microM), but had no effect on LH-induced steroidogenesis, despite a 90% decrease in cyclic AMP production. The latter decrease was found to be dependent on the Ca2+ concentration. In the presence of the phosphodiesterase inhibitor methylisobutylxanthine (MIX), the ionophore A23187 induced a dose-dependent decrease in both LH and LH plus LHRH agonist-stimulated steroidogenesis and cyclic AMP production. The results obtained indicate that calcium, rather than cyclic AMP, is the mediator of the potentiating effects of LHRH agonist on LH-increased steroidogenesis in rat Leydig cells. The marked inhibition of the synergism in the presence of calcium ionophore A23187 suggests that Leydig cell calcium homeostasis must be intact for LHRH agonist action to occur. LHRH agonist causes a Ca2+-dependent decrease in LH-stimulated cyclic AMP production.     

Cyclic AMP and cyclic GMP: studies utilizing immunohistochemical techniques for the localization of the nucleotides in tissue.

Antibodies to the cyclic nucleotides initially were utilized in radioimmunoassays for cyclic AMP and cyclic GMP which might be present in mammalian tissues. allowed measurement of the nucleotides on small amounts of tissue in physiologic studies. To gain further insight into the relative roles of cyclic AMP and cyclic GMP in cell function, these antibodies have been applied to immunohistochemical studies for the localization of the cyclic nucleotides in tissues and cells. This methodology is useful for determining in which cell type in a heterogeneous tissue increases in cyclic nucleotide concentrations occur. In addition, within individual cells, staining patterns for cyclic AMP and cyclic GMP are usually quite distinct. Cyclic GMP in canine thyroid is located to the follicular cell membrane while cyclic AMP is ubiquitously distributed in follicular cell cytoplasm. In both rat adrenal cortex and testis, there is prominent nuclear localization of cyclic GMP, suggesting a role for the nucleotide in growth regulation. These studies provide histologic evidence suggesting diverse roles for cyclic AMP and cyclic GMP in mammalian physiology. It is anticipated that this technique will also be useful in the ultrastructural localization of the cyclic nucleotides and for the identification of other cyclic nucleotides which might be present in mammalian tissues.     

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.     

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.     

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.     

LH-induced inhibition of follicular androgen formation requires intact steroidogenesis

LH exerts a biphasic effect on rat pre-ovulatory follicular steroidogenesis: an initial (1–4h) overall stimulation followed by a later (4–6 h) occurring inhibition of androgen synthesis. Because exogenous steroids may inhibit androgen formation, we investigated whether the steroids produced initially in response to LH are involved in the late inhibition of androgen synthesis. Isolated pre-ovulatory rat follicles were incubated for 6 h with and without ovine LH and 1 of 3 inhibitors of steroidogenesis (aminoglutethimide, cyanoketone, Su 10603). Accumulation of androstenedione and testosterone in a subsequent 2-h incubation in the presence of exogenous 17-hydroxyprogesterone was measured. LH treatment alone caused inhibition of apparent 17,20-lyase activity. The inhibitors had no effect on basal 17,20-lyase activity but were able to prevent the LH-induced inhibition of this enzymic activity. The results suggest that the physiological decline in pre-ovulatory androgen formation may in part be mediated by local action of follicular steroids.     

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.     

Effects of cyclic AMP elevation on the levels of insulin receptors in glial C6 cells

The regulation of the insulin receptor by cAMP has been examined in glial C6 cells. Incubation for 48 h with dibutyryl cyclic AMP produced a dose-dependent inhibition of 125I-insulin binding to the cells. Other agents that elevate intracellular cAMP levels such as forskolin and cholera toxin mimicked the effect of this cyclic AMP analog. With all compounds the maximal decrease of binding was found between 24 and 48 h and normally varied between 40 and 60%. Forskolin, cholera toxin and dibutyryl cyclic AMP also affected C6 cell proliferation, and the dose-response for decreasing the receptor was very similar to that observed for the inhibition of cell growth, suggesting a relationship between both phenomena. Scatchard analysis showed that cAMP did not produce major changes in the affinity of the receptor for insulin, but rather decreased receptor number. An accumulation of receptors at the cell surface was observed in the absence of de novo protein synthesis, since cycloheximide caused a significant increase in insulin binding to the cells. This inhibitor almost totally blocked the cAMP effect both when added simultaneously to the cells with the agents which increase cAMP and when added to cells pre-treated for 48 h with the same compounds.     

Differences in LH receptor down-regulation between rat and mouse Leydig cells: effects of 3',5'-cyclic AMP and phorbol esters.

The role of cyclic AMP and phorbol esters in luteinizing hormone (LH) receptor down-regulation in Leydig cells has been studied. Dibutyryl cyclic AMP (db-cAMP) (0.01, 0.1 and 1 mM), forskolin (80 microM) and cholera toxin (1.19 nM) caused a 30-50% loss of [125I]hCG binding sites and an inhibition of receptor-[125I]hCG complex internalization in mouse tumour Leydig (MA10, MLTC-1) cells during 2 h. In contrast, db-cAMP had no effect on the level of binding sites or internalization of the hormone receptor complex in rat testis Leydig cells or a rat tumour (R2C) Leydig cell. Phorbol 12-myristate 13-acetate (PMA) at concentrations from 10(-9) to 10(-5) M had no effect on hormone binding or hormone-receptor complex internalization in any of the Leydig cells. In contrast a 2 h preincubation of MLTC-1 cells with 10(-7) M PMA caused a loss of subsequent LH-stimulated cyclic AMP and pregnenolone production. These results indicate that LH receptor down-regulation is mediated by cyclic AMP dependent kinase, but not protein kinase C, in mouse Leydig cells. No down-regulation of rat Leydig cell LH receptor occurs with either kinase.     

Antigonadotropic and antisteroidogenic actions of peroxide in rat granulosa cells.

Reactive oxygen species are produced in the ovary. In luteal cells, peroxide abruptly inhibits LH-sensitive cAMP and progesterone production, and may serve a role as a mediator of luteolysis by such mechanisms. The objective of the present studies was to evaluate the acute actions of peroxide in rat granulosa cells. Peroxide at concentrations in the low micromolar range produced a marked and dose-dependent inhibition of FSH-sensitive cAMP accumulation and progesterone production, and depleted cell levels of ATP within 1 min. Longer treatment with peroxide (60 min) caused complete abrogation of the actions of FSH. Peroxide-induced depletion of ATP was prevented by 3-aminobenzamide, an inhibitor of DNA repair, but maintenance of cell levels of ATP did not prevent the anti-FSH effects of peroxide. Peroxide also abrogated cAMP accumulation and progesterone production in response to LH in granulosa cells. Unlike that seen with LH, inhibition of FSH-sensitive cyclic AMP accumulation by peroxide was partially reversed with isobutylmethyl xanthine, an inhibitor of cyclic AMP phosphodiesterase. Although peroxide inhibited cAMP accumulation in response to cholera toxin, it did not inhibit this same response to forskolin, which indicates that peroxide may interfere with G-protein-dependent activation of adenylate cyclase. Peroxide inhibited steroidogenesis in response to cholera toxin, forskolin, and 8-bromo-cAMP. The marked inhibitory actions of peroxide on gonadotropic hormone action and steroidogenesis in granulosa cells raise the possibility that peroxide may mediate events associated with loss of follicular function.