Theca-interstitial Cells Research Articles

Localization of resistin and its possible roles in the ovary of a vespertilionid bat, Scotophilus heathi

The aim of the present study was to evaluate the expression and effect of resistin on ovarian activities of Scotophilus heathi. Immunohistochemical study showed marked variation in resistin immunostaining during different reproductive phases. Most intense immunostaining of resistin was noticed in thecal-interstitial cells in ovary during the period of delayed ovulation, the period of increased androgen synthesis and suppressed ovulation. The changes in ovarian resistin level also correlated positively with circulating leptin level and body white adipose tissue accumulation. The in vitro study showed that resistin alone preferentially stimulated progesterone synthesis, but with luteinizing hormone (LH) stimulated androgen secretion. Resistin alone dose-dependently increased expression of LH-receptor, steroidogenic acute regulatory protein and insulin receptor proteins in the ovary, whereas together with LH showed dose-dependent stimulatory effect on expression of androgen receptor and insulin receptor proteins in the ovary. In conclusion, during the period of fat accumulation increased ovarian resistin level may be responsible for increased androgen synthesis through insulin receptor mediated pathways in the ovary of S. heathi.

Obesity-Induced Infertility and Hyperandrogenism Are Corrected by Deletion of the Insulin Receptor in the Ovarian Theca Cell

Women with polycystic ovary syndrome (PCOS) exhibit elevated androgen levels, oligoanovulation, infertility, and insulin resistance in metabolic tissues. The aims of these studies were to determine the role of insulin signaling in the development and function of ovarian theca cells and the pathophysiologic effects of hyperinsulinism on ovarian function in obesity. We disrupted the insulin receptor (IR) gene specifically in the theca-interstitial (TI) cells of the ovaries (Cyp17IRKO). No changes in reproductive development or function were observed in lean Cyp17IRKO female mice, suggesting that insulin signaling in TI cell is not essential for reproduction. However, when females were fed a high-fat diet, diet-induced obesity (DIO) wild-type (DIO-WT) mice were infertile and experienced increased circulating testosterone levels, whereas DIO-Cyp17IRKO mice exhibited improved fertility and testosterone levels comparable to those found in lean mice. The levels of phosphorylated IRS1 and CYP17 protein were higher in the ovary of DIO-WT compared with DIO-Cyp17IRKO or lean mice. Ex vivo studies using a whole ovary culture model demonstrated that insulin acts independently or additively with human chorionic gonadotropin to enhance androstenedione secretion. These studies reveal the causal pathway linking hyperinsulinism with ovarian hyperandrogenism and the infertility of obesity.

Resveratrol potentiates effects of simvastatin on inhibition of rat ovarian theca-interstitial cells steroidogenesis

BackgroundPolycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, hyperplastic theca compartment and increased androgen production due to, at least in part, excessive expression of several key genes involved in steroidogenesis. Previously, our group has demonstrated that simvastatin, competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a rate-limiting step of the mevalonate pathway, reduces rat-theca interstitial cell steroidogenesis by inhibiting Cyp17a1 gene expression, the key enzyme of the androgen biosynthesis pathway. Recently, we demonstrated that resveratrol, a bioflavonoid abundant in red grapes, decreases rat theca-interstitial cell steroidogenesis and this suppressive effect is mediated through mechanisms independent of the mevalonate pathway. The present study evaluated the effect of combining simvastatin and resveratrol treatments on rat theca-interstitial cell steroidogenesis.MethodsRat theca-interstitial cells isolated from 30 day-old female rats were cultured for up to 48 h with or without simvastatin (1 μM) and/or resveratrol (3-10 μM). Steroidogenic enzymes gene expression was evaluated by quantitative real time PCR and steroid levels were measured by liquid chromatography-mass spectrometry. Comparisons between groups were performed using ANOVA and Tukey test.ResultsResveratrol potentiated inhibitory effects of simvastatin on androstenedione and androsterone production in theca-interstitial cells. This suppressive effect correlated with profound inhibition in Cyp17a1 mRNA expression in the presence of a combination of resveratrol and simvastatin.ConclusionsThe present findings indicate that resveratrol potentiates the simvastatin-induced inhibitory effect on theca-interstitial cell androgen production, raising the possibility of development of novel treatments of PCOS.

Comparison of Effects of Different Statins on Growth and Steroidogenesis of Rat Ovarian Theca-Interstitial Cells1

Statins are competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of the cellular production of cholesterol and other products of the mevalonate pathway. Statins exert hepatic and extrahepatic effects, modulating the function of various tissues and organs, including ovaries. Previously, we have demonstrated that simvastatin inhibited cellular proliferation and reduced androgen production by ovarian theca-interstitial cells. The above actions are of translational relevance to the most common endocrine disorder among women in reproductive age: polycystic ovary syndrome. However, different statins may have distinctly different profiles of effects on cholesterol and androgens. The present study was designed to compare the effects of several statins on growth and steroidogenesis of rat theca-interstitial cells. The cells were incubated in the absence (control) or in the presence of simvastatin, lovastatin, atorvastatin, or pravastatin. Assessment of effects of statins on cell growth was carried out by evaluation of DNA synthesis and by estimation of the number of viable cells. Effects on steroidogenesis were evaluated by quantification of steroid production and expression of mRNA for the key enzyme regulating androgen production: Cyp17a1. Among tested statins, simvastatin exerted the greatest inhibitory effects on all tested parameters. The rank order of the effects of the tested statins is as follows: simvastatin > lovastatin > atorvastatin ≥ pravastatin. While the lipophilicity is likely to play a major role in determining the ability of statins to act on nonhepatic cells, other factors unique to individual cell types are also likely to be relevant.

Growth differential factor-9 inhibits testosterone production in mouse theca interstitial cells

To explore the effect of growth differential factor-9 (GDF-9) alone on cell proliferation, cell viability, steroidogenesis, and hormone-stimulated gene expression in cultured mouse theca interstitial cells. Basic research. University hospital. Immature 3- to 4-week-old SPF KM mice obtained from the Laboratory Animal Center of Sun Yat-Sen University. Addition of GDF-9 at different dosages to primary culture of mouse theca interstitial cells. Cell number, cell viability, progesterone and testosterone levels, and hormone-stimulated gene mRNA abundance. Growth differential factor-9 mildly increased the number of mouse theca interstitial cells and cell viability in a dose-dependent manner and mildly inhibited the production of progesterone in mouse theca interstitial cells. Administration of GDF-9 at the dosages of 200 ng/mL and 400 ng/mL resulted in a significant decrease in the testosterone level compared with the control group by 60.42% and 68.76%, respectively. Growth differential factor-9 significantly suppressed Lhcgr mRNA by 47.36%, Cyp11a1 mRNA by 62.30%, and Cyp17a1 mRNA by 55.39%, but had only a mild effect on Star gene expression. Growth differential factor-9 can inhibit the production of testosterone in mouse theca interstitial cells and suppress the corresponding gene expression.

Bisphenol A Exposure during Adulthood Causes Augmentation of Follicular Atresia and Luteal Regression by Decreasing 17β-Estradiol Synthesis via Downregulation of Aromatase in Rat Ovary

Background: Bisphenol A (BPA) has been detected in human body fluids, such as serum and ovarian follicular fluids. Several reports indicated that BPA exposure is associated with the occurrence of several female reproductive diseases resulting from the disruption of steroid hormone biosynthesis in the adult ovary.Objective: We hypothesized that long-term exposure to low concentrations of BPA disrupts 17β-estradiol (E2) production in granulosa cells via an alteration of steroidogenic proteins in ovarian cells.Methods: Adult female rats received BPA for 90 days by daily gavage at doses of 0, 0.001, or 0.1 mg/kg body weight. We determined serum levels of E2, testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). We also analyzed the expressions of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase isomerase (3β-HSD), and aromatase cytochrome P450 (P450arom) in the ovary.Results: Exposure to BPA significantly decreased E2 serum concentration, which was accompanied by augmented follicular atresia and luteal regression via increase of caspase-3–associated apoptosis in ovarian cells. After BPA exposure, P450arom and StAR protein levels were significantly decreased in granulosa cells and theca-interstitial (T-I) cells, respectively. However, P450scc and 3β-HSD protein levels remained unchanged. The increase in LH levels appeared to be associated with the decreased synthesis of T in T-I cells after BPA exposure via homeostatic positive feedback regulation.Conclusions: BPA exposure during adulthood can disturb the maintenance of normal ovarian functions by reducing E2. The steroidogenic proteins StAR and P450arom appear to be targeted by BPA.

Sensory denervation with capsaicin reduces ovarian follicular development and delays the onset of puberty in guinea pigs

Introduction: It has been documented that mammalian ovaries receive sympathetic, parasympathetic and sensory nerve fibers. The aim of this work was to investigate the effects of sensory denervation with capsaicin at the first vaginal opening (FVO) on follicular development and the expression of TRPV1 receptors in ovary cells as well as in the dorsal root ganglia (DRGs) and lumbar dorsal spinal cord neurons of guinea pigs. The DRGs and lumbar dorsal spinal cord neurons serve as a nerve connection from the ovaries to the CNS. Materials and Methods: Female guinea pigs received a subcutaneous injection of capsaicin (30 mM) at 10 days of age (P10), while control animals were injected with vehicle. Using light microscopy, we counted healthy preantral follicles (HPF), healthy antral follicles (HAF), atretic preantral follicles (APF), and atretic antral follicles (AAF) in the ovaries at the FVO, and the numbers of TRPV1-positive cells were counted in the ovarian follicles, DRGs, and lumbar dorsal spinal cord (L2-L4) neurons by immunohistochemistry. Results: Guinea pigs treated with capsaicin showed a significant delay of FVO in comparison with the control animals (36 vs. 44 days). In the ovaries, the number of preantral and antral follicles decreased significantly. Additionally, the number of TRPV1-positive thecainterstitial cells of the antral follicles was reduced significantly, and the number of TRPV1-positive neurons in the DRGs and lumbar dorsal spinal cord (L2-L4) decreased. Thus, we showed that TRPV1 receptors throughout the sensory fibers modulate ovarian follicular development and the onset of puberty in guinea pigs. Conclusion: Sensory denervation decreases ovarian follicular development and delays the onset of puberty of guinea pigs. Our data support the idea that through TRPV1 receptors, ovarian afferent fibers sense local stimuli that are sent to the CNS.

Stimulatory effect of insulin on theca-interstitial cell proliferation and cell cycle regulatory proteins through MTORC1 dependent pathway

The present study examined the effect of insulin-mediated activation of the mammalian target of rapamycin complex 1 (MTORC1) signaling network on the proliferation of primary culture of theca-interstitial (T-I) cells. Our results show that insulin treatment increased proliferation of the T-I cells through the MTORC1-dependent signaling pathway by increasing cell cycle regulatory proteins. Inhibition of ERK1/2 signaling caused partial reduction of insulin-induced phosphorylation of RPS6KB1 and RPS6 whereas inhibition of PI3-kinase signaling completely blocked the insulin response. Pharmacological inhibition of MTORC1 with rapamycin abrogated the insulin-induced phosphorylation of EIF4EBP1, RPS6KB1 and its downstream effector, RPS6. These results were further confirmed by demonstrating that knockdown of Mtor using siRNA reduced the insulin-stimulated MTORC1 signaling. Furthermore, insulin-stimulated T-I cell proliferation and the expression of cell cycle regulatory proteins CDK4, CCND3 and PCNA were also blocked by rapamycin. Taken together, the present studies show that insulin stimulates cell proliferation and cell cycle regulatory proteins in T-I cells via activation of the MTORC1 signaling pathway.

Letrozole increases ovarian growth and Cyp17a1 gene expression in the rat ovary

To evaluate the effects of letrozole on ovarian size and steroidogenesis invivo, as well as on proliferation and steroidogenesis of theca-interstitial cells alone and in coculture with granulosa cells using an invitro model. Invivo and invitro studies. Research laboratory. Immature Sprague-Dawley female rats. Invivo effects of letrozole were studied in intact rats receiving either letrozole (90-day continuous-release SC pellets, 400 μg/d) or placebo pellets (control group). In invitro experiments, theca cells were cultured alone or in coculture with granulosa cells in the absence or presence of letrozole. Deoxyribonucleic acid synthesis was determined by thymidine incorporation assay; steroidogenesis by mass spectrometry; and steroidogenic enzyme messenger RNA (mRNA) expression by polymerase chain reaction. Invivo, letrozole induced an increase in ovarian size compared with the control group and also induced a profound increase of androgen, LH levels, and Cyp17a1 mRNA expression. Conversely, a decrease in Star, Cyp11a1, and Hsd3b1 transcripts was observed in letrozole-exposed rats. Invitro, letrozole did not alter either theca cell proliferation or Cyp17a1 mRNA expression. Similarly, letrozole did not affect Cyp17a1 transcripts in granulosa-theca cocultures. These findings suggest that letrozole exerts potent, but indirect, effect on growth of rat ovary and dramatically increases androgen levels and Cyp17a1 mRNA expression, the key enzyme regulating the androgen biosynthesis pathway. The present findings reveal novel mechanisms of action of letrozole in the rat ovary.

Effects of resveratrol on growth and function of rat ovarian granulosa cells

To evaluate the effects of resveratrol on growth and function of granulosa cells. Previously, we demonstrated that resveratrol exerts profound proapoptotic effects on theca-interstitial cells. Invitro study. Research laboratory. Immature Sprague-Dawley female rats. Granulosa cells were cultured in the absence or presence of resveratrol. DNA synthesis was determined by thymidine incorporation assay, apoptosis by activity of caspases 3/7, cell morphology by immunocytochemistry, steroidogenesis by mass spectrometry, antimüllerian hormone (AMH), and vascular endothelial growth factor (VEGF) expression by polymerase chain reaction and Western blot. Resveratrol induced a biphasic effect on DNA synthesis, whereby a lower concentration stimulated thymidine incorporation and higher concentrations inhibited it. Additionally, resveratrol slightly increased the cell number and modestly decreased the activity of caspases 3/7 with no effect on cell morphology or progesterone production. However, resveratrol decreased aromatization and VEGF expression, whereas AMH expression remained unaltered. Resveratrol, by exerting cytostatic but not cytotoxic effects, together with antiangiogenic actions mediated by decreased VEGF in granulosa cells, may alter the ratio of theca-to-granulosa cells and decrease vascular permeability, and therefore may be of potential therapeutic use in conditions associated with highly vascularized theca-interstitial hyperplasia and abnormal angiogenesis, such as those seen in women with polycystic ovary syndrome.

Resveratrol reduces steroidogenesis in rat ovarian theca-interstitial cells: the role of inhibition of AKT/PKB signaling pathway

Previously, we reported that the bioflavonoid, resveratrol (RES,) inhibits proliferation of rat theca-interstitial cells. This study evaluated the mechanisms of actions of RES on steroidogenesis including possible roles in modulation of isoprenylation, activation of sirtuins and Akt/PKB signaling pathway.

Luteinizing Hormone/Human Chorionic Gonadotropin-Mediated Activation of mTORC1 Signaling Is Required for Androgen Synthesis by Theca-Interstitial Cells

LH triggers the biosynthesis of androgens in the theca-interstitial (T-I) cells of ovary through the activation of a cAMP-dependent pathway. We have previously shown that LH/human chorionic gonadotropin (hCG) activates mammalian target of rapamycin complex 1 (mTORC1) signaling network, leading to cell proliferation. In the present study, we provide evidence that the LH/hCG-mediated activation of the mTORC1 signaling cascade is involved in the regulation of steroidogenic enzymes in androgen biosynthesis. Treatment with LH/hCG increased the expression of downstream targets of mTORC1, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E as well as steroidogenic enzymes. LH/hCG-mediated stimulation of the steroidogenic enzyme mRNA was blocked by the mTORC1 inhibitor, rapamycin. This inhibitory effect was selective because rapamycin failed to block hCG-mediated increase in the expression of Star mRNA levels. Furthermore, pharmacological targeting of mTORC1 with rapamycin also blocked LH/hCG- or forskolin-induced expression of cAMP response element-binding protein (CREB) and steroidogenic enzymes (P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase type 1, and 17α-hydroxylase/17,20 lyase) but produced no effect on steroidogenic acute regulatory protein levels. These results were further confirmed by demonstrating that the knockdown of mTOR using small interfering RNA selectively abrogated the LH/hCG-induced increase in steroidogenic enzyme expression, without affecting steroidogenic acute regulatory protein expression. LH/hCG-stimulated androgen production was also blocked by rapamycin. Furthermore, the pharmacological inhibition of mTORC1 or ribosomal protein S6 kinase 1 signaling prevented the LH/hCG-induced phosphorylation of CREB. Chromatin immunoprecipitation assays revealed the association of CREB with the proximal promoter of the Cyp17a1 gene in response to hCG, and this association was reduced by rapamycin treatment. Taken together, our findings show for the first time that LH/hCG-mediated activation of androgen biosynthesis is regulated by the mTORC1 signaling pathway in T-I cells.

Effects of adiponectin on ovarian folliculogenesis and steroidogenesis in the vespertilionid bat, Scotophilus heathi

The bat Scotophilus heathi undergo anovulation, known as delayed ovulation, coinciding with the period of seasonal adiposity due to fat accumulation. Because adiponectin is a well known fat cell product, it might be responsible for adiposity related anovulation in S. heathi. The aim of this study was thus to evaluate the effects of adiponectin treatment on ovarian activity in S. heathi. The bat during the period of delayed ovulation was treated with adiponectin (25μg/day) for 12days. Changes taken place in the ovarian activity of the treated bat was compared with the control bat and results obtained are presented here. Adiponectin treatment significantly decreased body mass by depleting adipose tissue. It acts directly upon the ovary and promotes antral follicular development by stimulating cell proliferation and modulating ovarian steroidogenesis. The bat treated with adiponectin showed significant decrease in circulating androstenedione (A4) and estradiol (E2) levels, but increase in circulating progesterone (P4) level. The inhibitory effect of adiponectin on androgen synthesis may be due to decreased thecal-interstitial cells differentiation in stroma by suppressing adiponectin receptor type 1 (AdipoR1), luteinizing hormone (LH) and insulin receptors in the ovary of S. heathi. The study provides evidences suggesting that the adiponectin treatment suppresses adiposity and restores normal ovarian activity.

Resveratrol Reduces Steroidogenesis in Rat Ovarian Theca-Interstitial Cells: The Role of Inhibition of Akt/PKB Signaling Pathway

Polycystic ovary syndrome is characterized by theca-interstitial hyperplasia and increased expression of steroidogenic genes, leading to excessive androgen production. Resveratrol, a natural polyphenol, promotes apoptosis and reduces rat theca-interstitial cell growth, in part by inhibiting the mevalonate pathway and decreasing the availability of substrates of isoprenylation [farnesyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP)]. This study evaluated the effect of resveratrol on rat theca-interstitial cell steroidogenesis. Because resveratrol may activate sirtuins, this study also investigated whether steroidogenesis was affected by sirtuin inhibitors (nicotinamide, sirtinol). Theca-interstitial cells were cultured with or without resveratrol (1-10 μm), GGPP (30 μm), FPP (30 μm), nicotinamide (1 mm), and/or sirtinol (10 μm). Resveratrol did not affect progesterone levels but reduced androgen production in a concentration-dependent fashion (androstenedione by up to 78% and androsterone by up to 76%). This inhibitory effect correlated with a decrease in mRNA expression of genes regulating androgen production, especially Cyp17a1 (by up to 73%). GGPP and FPP had no effect on androgen levels and Cyp17a1 mRNA levels and did not alter the effects induced by resveratrol. Similarly, sirtuin inhibitors did not reverse resveratrol-induced inhibition of steroidogenesis. However, resveratrol decreased activity of serine-threonine kinase/protein kinase B pathway, a cell-signaling pathway involved in ovarian steroidogenesis. The present findings indicate that resveratrol reduces androgen production primarily by inhibiting Cyp17a1 mRNA expression, and this inhibition may be mediated, in part, by blocking the activity of the serine-threonine kinase/protein kinase B pathway. These findings may be of clinical relevance to conditions associated with excessive production of androgens by theca cells, such as polycystic ovary syndrome.

Metformin: direct inhibition of rat ovarian theca-interstitial cell proliferation

To determine whether metformin has direct effects on ovarian theca-interstitial (T-I) cell proliferation through activation of adenosine monophosphate-activated protein kinase (AMPK). In vitro experimental study. Academic medical center laboratory. Immature Sprague-Dawley female rats. Ovarian T-I cells were isolated, purified, and cultured in the absence (control) or presence of insulin (1 μg/mL) with or without metformin or other activators/inhibitors of AMPK (AICAR, compound C). Proliferation assessed by determination of expression levels of proteins involved in cell cycle progression, cyclin D3, and cyclin-dependent kinase 4 (CDK4) with Western blot analysis, and determination of DNA synthesis with bromodeoxyuridine (BrdU) incorporation assay; activation of AMPK, Erk1/2, and S6K1 determined by Western blot analysis with the use of antibodies specific for the phosphorylated (activated) forms. Metformin inhibited insulin-induced ovarian T-I cell proliferation and the up-regulation of the cell cycle regulatory proteins, cyclin D3 and CDK4. Metformin independently activated AMPK in a dose-dependent manner. Treatment with metformin inhibited insulin-induced activation of Erk1/2 and S6K1. This effect was reversed with the addition of compound C, a known AMPK inhibitor. Metformin directly inhibits proliferation of ovarian T-I cells via an AMPK-dependent mechanism. These findings further validate the potential benefits of metformin in the treatment of conditions associated with hyperinsulinemia and excessive growth of ovarian T-I cells (such as polycystic ovary syndrome).

Localization of adiponectin and its receptor and its possible roles in the ovary of a vespertilionid bat, Scotophilus heathi

The aim of this study was to evaluate the seasonal variation in serum adiponectin levels and ovarian expression of adiponectin and its receptor in the Scotophilus heathi bat and their relationship to the changes in the body fat mass, serum insulin and luteinizing hormone (LH) levels, and ovarian activity. A very high level of circulating adiponectin was found during recrudescence, whereas a very low level of circulating adiponectin was observed during pre-ovulatory period. The increased circulating adiponectin level coincided with fat deposition, insulin resistance and hyperinsulinemia (HI) in S. heathi. Immunocytochemical study in the ovary of bat showed localization of adiponectin mainly in thecal-interstitial cells (TICs), and adiponectin receptor 1 (AdipoR1) in the granulosa cells of growing follicles, but showed no staining in atretic follicle. Seasonal changes in ovarian adiponectin and AdipoR1 levels showed two peaks (during recrudescence and ovulatory phases) coinciding with two periods of follicular development. Adiponectin in the absence of LH, as occur during recrudescence, stimulates androstenedione (A4) synthesis in vitro by up-regulating the insulin receptor (IR). Adiponectin in presence of LH, as occur during pre-ovulatory phase, inhibits A4 synthesis in vitro by down-regulating androgen receptor (AR). Further, the in vitro study showed that adiponectin, in presence of LH, also promotes luteinizing hormone receptor (LH-R) and steroidogenic acute regulatory protein (StAR) level in the ovary, which might help in development of ovulatory follicles. In brief, adiponectin in the absence of LH induces increased A4 synthesis and consequently the condition of delayed ovulation, whereas in the presence LH suppresses both synthesis and action of A4 and thus induces preovulatory condition in the ovary of S. heathi.

Simvastatin Reduces Steroidogenesis by Inhibiting Cyp17a1 Gene Expression in Rat Ovarian Theca-Interstitial Cells1

Polycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, theca-interstitial hyperplasia, and increased androgen production by theca cells. Previously, our group has demonstrated that statins (competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, a rate-limiting step of the mevalonate pathway) reduce proliferation of theca-interstitial cells in vitro and decrease serum androgen levels in women with PCOS. The present study evaluated the effect of simvastatin on rat ovarian theca-interstitial cell steroidogenesis. Because actions of statins may be due to reduced cholesterol availability and/or isoprenylation of proteins, the present study also investigated whether steroidogenesis was affected by cell- and mitochondrion-permeable 22-hydroxycholesterol, isoprenylation substrates (farnesyl-pyrophosphate [FPP] and geranylgeranyl-pyrophosphate [GGPP]), as well as selective inhibitors of farnesyltransferase (FTI) and geranylgeranyltransferase (GGTI). Theca-interstitial cells were cultured for 12, 24, and 48 h with or without simvastatin, GGPP, FPP, FTI, GGTI, and/or 22-hydroxycholesterol. Simvastatin decreased androgen levels in a time- and concentration-dependent fashion. This inhibitory effect correlated with a decrease in mRNA levels of Cyp17a1, the gene encoding the key enzyme regulating androgen biosynthesis. After 48 h, GGPP alone and FPP alone had no effect on Cyp17a1 mRNA expression; however, the inhibitory action of simvastatin was partly abrogated by both GGPP and FPP. The present findings indicate that statin-induced reduction of androgen levels is likely due, at least in part, to the inhibition of isoprenylation, resulting in decreased expression of CYP17A1.

Resveratrol inhibits the mevalonate pathway and potentiates the antiproliferative effects of simvastatin in rat theca-interstitial cells

To examine the mechanisms of action of resveratrol and its interaction with simvastatin on growth and the mevalonate pathway in rat theca-interstitial cells. Invitro study. Research laboratory. Immature Sprague-Dawley female rats. Theca-interstitial cells were cultured in the absence or presence of resveratrol, simvastatin, mevalonic acid, farnesyl pyrophosphate, and/or geranylgeranyl pyrophosphate. DNA synthesis was assessed by thymidine incorporation assay; 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) expression and activity were evaluated with the use of quantitative real-time polymerase chain reaction, Western blot analysis, and HMGCR activity assay. Cholesterol synthesis was determined by the conversion of [(14)C]-acetate to [(14)C]-cholesterol. Resveratrol potentiated the simvastatin-induced inhibition on cell proliferation in a concentration-dependent manner. Inhibitory effects of resveratrol were partly abrogated by the addition of mevalonic acid, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate. Resveratrol reduced HMGCR expression and activity, and decreased cholesterol synthesis. In contrast, simvastatin inhibited HMGCR activity with a compensatory increase in HMGCR expression. Resveratrol counteracted this effect of simvastatin on HMGCR expression but augmented the simvastatin-induced inhibition on HMGCR activity and cholesterol synthesis. Resveratrol inhibits the mevalonate pathway via distinctly different mechanisms than statins. These observations demonstrate a novel mechanism of action of resveratrol and underscore the potential translational/clinical relevance of resveratrol interactions with simvastatin.

Letrozole increases growth of rat theca-interstitial cells and CYP17A1 gene expression in the rat ovary

Letrozole (LET) is a non-steroidal aromatase inhibitor, blocking the conversion of androgens to estrogens in granulosa cells. However, little is known regarding the effect of LET on theca-interstitial cell function. This study evaluated the effect of LET in vivo and in cultures of theca-interstitial cells. In-vitro and in-vivo studies. Effects of LET were studied in intact rats receiving either LET (90-day-continuous-release sc pellets, 400 μg/day) or placebo pellets (control group). After 10 weeks, the animals were sacrificed, ovaries were isolated and blood samples collected. In vitro experiments evaluated theca-interstitial cells isolated from 30 day-old female rats and cultured for up to 48 hrs in the absence (control) or in the presence of LET (0.1-1μM). Cell proliferation was evaluated by quantification of DNA synthesis as determined by radiolabelled thymidine incorporation assay. CYP17A1 gene expression was evaluated by qrt-PCR using HPRT as a reference gene. Androstenedione levels in plasma were determined by liquid chromatography-mass spectrometry. Comparisons between groups were performed by ANOVA followed by post-hoc pairwise testing. LET, in vivo, induced an increase in ovarian size compared to the control group: 179.1 ± 9.46 mg vs. 115 ± 5.19 mg (P<0.05). LET also induced a 57-fold (P<0.001) increase in CYP17A1 gene expression and a 52-fold (P<0.001) increase in plasma androstenedione level. In vitro, LET stimulated theca-interstitial cell proliferation in a concentration-dependent fashion by up to 65% (P<0.01) at the highest concentration (1 μM). LET stimulates growth of the theca-interstitial cell compartment and increases expression of the key enzyme (CYP17A1) regulating the androgen biosynthesis pathway in the rat. The present findings reveal novel mechanisms of action of LET in the rat ovary.

Metformin inhibits cell proliferation and cell cycle regulatory proteins of ovarian theca-interstitial cells

Metformin inhibits cell proliferation and cell cycle regulatory proteins of ovarian theca-interstitial cells