Theca Cell Cultures Research Articles

Evaluation of Lipids and Lipid-Related Transcripts in Human and Ovine Theca Cells and an in Vitro Mouse Model Exposed to the Obesogen Chemical Tributyltin.

Exposure to obesogenic chemicals has been reported to result in enhanced adipogenesis, higher adipose tissue accumulation, and reduced ovarian hormonal synthesis and follicular function. We have reported that organotins [tributyltin (TBT) and triphenyltin (TPT)] dysregulate cholesterol trafficking in ovarian theca cells, but, whether organotins also exert lipogenic effects on ovarian cells remains unexplored. We investigated if environmentally relevant exposures to organotins [TBT, TPT, or dibutyltin (DBT)] induce lipid dysregulation in ovarian theca cells and the role of the liver X receptor (LXR) in this effect. We also tested the effect of TBT on oocyte maturation and neutral lipid accumulation, and lipid-related transcript expression in cumulus cells and preimplantation embryos. Primary theca cell cultures derived from human and ovine ovaries were exposed to TBT, TPT, or DBT (1, 10, or ). The effect of these chemical exposures on neutral lipid accumulation, lipid abundance and composition, lipid homeostasis-related gene expression, and cytokine secretion was evaluated using liquid chromatography-mass spectrometry (LC-MS), inhibitor-based methods, cytokine secretion, and lipid ontology analyses. We also exposed murine cumulus-oocyte complexes to TBT and evaluated oocyte maturation, embryo development, and lipid homeostasis-related mRNA expression in cumulus cells and blastocysts. Exposure to TBT resulted in higher intracellular neutral lipids in human and ovine primary theca cells. In ovine theca cells, this effect was dose-dependent, independent of cell stage, and partially mediated by LXR. DBT and TPT resulted in higher intracellular neutral lipids but to a lesser extent in comparison with TBT. More than 140 lipids and 9 cytokines were dysregulated in TBT-exposed human theca cells. Expression of genes involved in lipogenesis and fatty acid synthesis were higher in theca cells, as well as in cumulus cells and blastocysts exposed to TBT. However, TBT did not impact the rates of oocyte maturation or blastocyst development. TBT induced dyslipidemia in primary human and ovine theca cells, which may be responsible for some of the TBT-induced fertility dysregulations reported in rodent models of TBT exposure. https://doi.org/10.1289/EHP13955.

Single-Cell RNA-Seq Identifies Pathways and Genes Contributing to the Hyperandrogenemia Associated with Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by hyperandrogenemia of ovarian thecal cell origin, resulting in anovulation/oligo-ovulation and infertility. Our previous studies established that ovarian theca cells isolated and propagated from ovaries of normal ovulatory women and women with PCOS have distinctive molecular and cellular signatures that underlie the increased androgen biosynthesis in PCOS. To evaluate differences between gene expression in single-cells from passaged cultures of theca cells from ovaries of normal ovulatory women and women with PCOS, we performed single-cell RNA sequencing (scRNA-seq). Results from these studies revealed differentially expressed pathways and genes involved in the acquisition of cholesterol, the precursor of steroid hormones, and steroidogenesis. Bulk RNA-seq and microarray studies confirmed the theca cell differential gene expression profiles. The expression profiles appear to be directed largely by increased levels or activity of the transcription factors SREBF1, which regulates genes involved in cholesterol acquisition (LDLR, LIPA, NPC1, CYP11A1, FDX1, and FDXR), and GATA6, which regulates expression of genes encoding steroidogenic enzymes (CYP17A1) in concert with other differentially expressed transcription factors (SP1, NR5A2). This study provides insights into the molecular mechanisms underlying the hyperandrogenemia associated with PCOS and highlights potential targets for molecular diagnosis and therapeutic intervention.

Sphingosine-1-phosphate regulation of luteinising hormone-induced steroidogenesis and proliferation of bovine theca cells in vitro.

Sphingosine-1-phosphate (S1P) is synthesised by follicle granulosa cells under the influence of follicle-stimulating hormone and seems to be necessary for the biological effects of this gonadotrophin. To determine if luteinising hormone (LH) increases S1P production and if this sphingolipid, either induced by LH or added to culture media, regulates steroidogenesis and cell viability in bovine theca cells. We used bovine theca cell cultures treated with: S1P (0, 0.1, 1 and 10μM; Experiment 1), LH (0, 0.02, 0.2 and 2ngmL-1; Experiment 2) and LH (0.02ngmL-1) plus a sphingosine kinase inhibitor (SKI-178; 0, 5 and 10μM; Experiment 3). Treatment with S1P did not affect (P>0.05) theca cell viability or their ability to produce progesterone and testosterone. LH (0.02ngmL-1) increased (P<0.05) S1P production, and stimulated the expression of phosphorylated sphingosine kinase-1 (pSPHK1). However, the inhibition of SPHK1, by a specific SPHK1 inhibitor (SKI-178), reduced (P<0.05) cell viability and progesterone secretion. Additionally, the use of SKI-178 increased theca cell testosterone production (P<0.05). S1P added to culture media did not affect cell viability or steroid synthesis. However, LH stimulated the production of S1P, by increasing phosphorylation of SPHK1 in theca cells. This intracellular S1P was inhibitory on testosterone production but augmented progesterone and viable cell number. These results suggest a novel signalling pathway for LH in theca cells and underline the importance of S1P in the regulation of steroid synthesis.

Transcriptional regulation of CYP19A1 expression in chickens: ESR1, ESR2 and NR5A2 form a functional network

Aromatase, encoded by CYP19A1, is responsible for the conversion of androgen to estrogen, which plays a vital role in the development and function of the ovary and functions in many other physiological processes in both sexes. Instead of being expressed in ovarian granulosa cells, as in mammals, CYP19A1 is expressed in chickens in the theca cells of ovarian follicles, and the mechanism of CYP19A1 expression regulation remains unknown. Here, using immunofluorescence and western blotting assay, we first confirmed that CYP19A1 and FOXL2 (Forkheadbox L2) were coexpressed in pre-granulosa cells of female chicken embryonic gonads, while FOXL2 did not affect aromatase expression at embryonic stages. Second, our research showed that CYP19A1, ESR1 (estrogen receptor alpha), ESR2 (estrogen receptor beta) and NR5A2 (liver receptor homologue-1) were coexpressed in the theca cell layers of chicken small yellow follicles. There was cross-talk between CYP19A1 and candidate transcription factors (ESR1, ESR2 and NR5A2), which was identified by generating a reliable theca cell culture model. Using luciferase assays in theca cells and chicken embryonic fibroblast (DF-1) cells, the results suggested that ESR1 and NR5A2 had potential effects on CYP19A1 promoter activity in chickens. Overexpression of ESR1, ESR2 and NR5A2 in chicken embryonic fibroblast (DF-1) cells upregulated the protein expression of CYP19A1, mutually restricted each other and formed a potential regulatory network to coordinate the expression of CYP19A1. To conclude, our results indicated that FOXL2 cannot regulate the expression of CYP19A1 at chicken embryonic stages and after sexual maturity, ESR1, ESR2 and NR5A2 form a functional network to affect the expression of CYP19A1. These results laid a foundation for further research on the transcriptional regulation of chicken aromatase.

Lipopolysaccharide-induced mechanisms of ovarian dysfunction in cows with uterine inflammatory diseases.

Uterine inflammatory diseases commonly occur in postpartum dairy cows, resulting in reduced reproductive performance due to aberrant uterine and ovarian activity. Infection of the uterus with gram-negative bacteria results in the detection of lipopolysaccharide (LPS) in the plasma and follicular fluid of cows along with uterine inflammation. LPS acts on follicular components such as theca cells, granulosa cells, and follicle-enclosed oocytes, leading to impaired follicular activity. Follicles with a high LPS environment exhibit reduced follicular steroidogenesis due to the inhibition of steroidogenic enzyme transcription. Primary cell cultures of bovine granulosa and theca cells have shown that LPS acts on follicular cells to impair steroid production, which may disturb follicle growth and/or reduce their ability to ovulate. Even if ovulation occurs, cows with uterine inflammation are less likely to conceive because in addition to uterine damage, LPS also impairs the developmental competence of oocytes. LPS perturbs the nuclear and cytoplasmic maturation of bovine oocytes. Moreover, oocytes matured using LPS treatment are less likely to develop into the blastocyst stage. Such oocytes also have a reduced number of trophoblast cells in blastocysts. Therefore, the detrimental effects of LPS on ovarian activity may be partly responsible for infertility in cows with uterine inflammation. Novel treatment and prevention strategies for uterine inflammatory diseases can be developed by advancing our knowledge of the pathophysiology underlying ovarian dysfunction, and this can only be achieved by further research. The present review outlines the molecular pathogenesis of LPS-induced ovarian dysfunction.

Theca Cell INSL3 and Steroids Together Orchestrate the Growing Bovine Antral Follicle.

Insulin-like peptide 3 (INSL3) and its specific receptor RXFP2 are both expressed by theca interna cells of the growing antral follicle where they form an essential regulatory element in the production of the steroid precursor androstenedione. Using primary cultures of bovine theca cells from the mid follicular phase together with steroid agonists and antagonists we have examined how ovarian steroids modulate INSL3 expression. Transcript analysis shows that these cells express estrogen receptors α and β, androgen and progesterone receptors, besides the orphan nuclear receptors SF1 and nur77. Whereas, exogenous androgens have little or no effect, the androgen antagonist bicalutamide stimulates INSL3 production. In contrast, estrogen receptor agonists, as also progesterone, are stimulatory. Importantly, estrogen receptor signaling is convergent with the protein kinase A signaling pathway activated by LH, such that the estrogen receptor antagonist can inhibit the mild stimulatory effect of LH, and vice versa the PKA antagonist H89 blocks stimulation by estradiol. A significant finding is that the major steroid metabolite androstenedione appears to act predominantly as an estrogen and not an androgen in this system. Transfection of INSL3 gene promoter-reporter constructs together with various steroid receptor expression plasmids supports these findings and shows that steroid action uses non-classical pathways not requiring canonical steroid-responsive elements in the proximal promoter region. Together, the results indicate that increasing estrogens in the follicular phase stimulate a feedforward loop driving INSL3 signaling and thereby promoting steroidogenesis in the growing antral follicle until the LH surge which effectively switches off INSL3 expression.

Establishment of an in vitro culture model of theca cells from hierarchical follicles in ducks.

Theca cells, including theca interna cells and theca externa cells, are vital components of ovarian follicles. The aim of the present study is to identify a reliable method for the in vitro culture of theca cells from duck ovarian hierarchical (F4-F2) follicles. We improved the method for cell separation by using trypsin to further remove granular cells, and we increased the concentration of fetal bovine serum used in in vitro culture to improve cytoactivity. Cell antibody immunofluorescence (IF) showed that all inoculated cells could be stained by the CYP17A1/19A1 antibody but not by the FSHR antibody, which could stain granulosa cells. Furthermore, morphological differences were observed between the outlines of theca interna and externa cells and in their nuclei. Growth curve and CYP17A1/19A1 mRNA relative expression analyses suggested that the growth profile of theca interna cells may have been significantly different from that of theca externa cells in vitro. Theca interna cells experienced the logarithmic phase on d1–d2, the plateau phase on d2–d3, and the senescence phase after d3, while theca externa cells experienced the logarithmic phase on d1–d3, the plateau phase on d3–d5, and the senescence phase after d5. Taken together, these results suggested that we have successfully established a reliable theca cell culture model and further defined theca cell characteristics in vitro.

Adiponectin and Its Receptors in the Ovary: Further Evidence for a Link between Obesity and Hyperandrogenism in Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS), characterized by ovarian androgen excess, is the commonest endocrine disorder in women. Obesity increases androgen synthesis, a phenomenon attributed to the accompanying hyperinsulinemia. Our hypothesis was that adipokines, fat cell-derived hormones, play a direct role in modulating ovarian androgen secretion. Therefore, the aims of this study were to explore the effects of adipokines (in particular, adiponectin) on ovarian steroidogenesis and compare the expression of adiponectin receptors in ovaries from women with and without PCO. Sections of archived human ovaries (nine from women with normal ovaries and 16 with PCOS, classified histologically, with reference to menstrual history and ultrasound) were analysed by quantitative morphometry and the proportion of positive-labelling cells compared. In addition, studies of androgen production in relation to adipokine function in primary bovine theca cell culture were also performed. A significantly lower proportion of theca cells expressed adiponectin receptors 1 and 2 (AdipoR1, AdipoR2) in polycystic ovaries than in normal ovaries. In cultured theca cells, adiponectin suppressed androstenedione production and gene expression of LH receptor and key enzymes in the androgen synthesis pathway. Moreover, knockdown of genes for AdipoR1 and AdipoR2 was associated with increased androstenedione secretion by bovine theca cells. These results provide evidence for a direct link between fat cell metabolism and ovarian steroidogenesis, suggesting that disruption of adiponectin and/or its receptors plays a key role in pathogenesis of hyperandrogenism in PCOS.

Inhibin removes the inhibitory effects of activin on steroid enzyme expression and androgen production by normal ovarian thecal cells

Activin and inhibin are important local modulators of theca cell steroidogenesis in the ovary. Using a serum-free primary theca cell culture system, this study investigated the effects of inhibin on theca cell androgen production and expression of steroidogenic enzymes. Androstenedione secretion from theca cells cultured in media containing activin, inhibin and follistatin was assessed by RIA over 144 h. Activin (1–100 ng/ml) suppressed androstenedione production. Inhibin (1–100 ng/ml) blocked the suppressive effects of added activin, but increased androstenedione production when added alone, suggesting it was blocking endogenous activin produced by theca cells. Addition of SB-431542 (activin receptor inhibitor) and follistatin (500 ng/ml) increased androstenedione production, supporting this concept. Infection of theca cells with adenoviruses expressing inhibitory Smad6 or 7 increased androstenedione secretion, confirming that the suppressive effects of activin required activation of the Smad2/3 pathway. Activin decreased the expression levels of steroidogenic acute regulatory protein (STAR), whereas STAR expression was increased by inhibin and SB-431542, alone and in combination. CYP11A was unaffected. The expression of CYP17 encoding 17α-hydroxylase was unaffected by activin but increased by inhibin and SB-431542, and when added in combination the effect was further enhanced. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly decreased by activin, while inhibin alone and in combination with SB-431542 both potently increased the expression of 3β-HSD. In conclusion, activin suppressed theca cell androstenedione production by decreasing the expression of STAR and 3β-HSD. Inhibin and other blockers of activin action reversed this effect, supporting the concept that endogenous thecal activin modulates androgen production in theca cells.

The configuration of the alpha and beta subunit domains in single-chain bovine LH analogs influences the secretion and steroidogenic response

The gonadotrophins LH, FSH and human (h) CG are non-covalent heterodimers composed of a common alpha and the hormone-unique beta subunit. LH regulates the production of androgens and progestins in the follicle, and the levels of these steroids are critical for the ovarian function. Structural features of the gonadotrophins involved in the steroidogenic response of the ovary are not completely understood. As an approach to address how the topology of the ligand affects steroidogenesis we exploited the single-chain (SC) gonadotrophin methodology because manipulating the relative position of the tethered subunit domains in SC hCG analogs enabled to change in the conformation, secretion, receptor binding and adenylyl cyclase activity. We genetically engineered a SC bovine LH analog with a linker derived from the CTP domain of the hCGbeta subunit, NH2-alpha-CTP-LHbeta-COOH (denoted as alphaCTPLHbeta; AB configuration) and evaluated the secretion form transfected CHO cells and steroidogenesis in follicular derived cells in comparison to the variant NH2-LHbeta-CTP-alpha-COOH (LHbetaCTPalpha; BA configuration). The secretion of the analogs from CHO cells was quantitative, and that of alphaCTPLHbeta was more efficient than that of LHbetaCTPalpha The experiments suggested that both variants were N- and O- glycosylated, though the posttranslational modifications are likely to be non-identical in the AB and BA analogs. The analogs stimulated progesterone secretion by immortalized rat granulosa cells that express the rat LH receptor but the EC50 of alphaCTPLHbeta (AB orientation) was higher by 20 fold, as compared to LHbetaCTPalpha (BA). In primary cultures of bovine theca cells, alphaCTPLHbeta stimulated progesterone release with a reduced sensitivity (by at least 50 folds) and smaller magnitude over the basal levels (about 3 folds) relative to LHbetaCTPalpha. In contrast, the accumulation of androstenedione in the media of the same primary cultures appeared to be nearly identical. As a result, the androstenedione/progesterone ratio for the alphaCTPLHbeta analog was significantly increased relative to LHbetaCTPalpha (2-3 folds). This unequal response suggests a distinct regulation of progesterone and androstenedione biosynthesis. Our data demonstrate major differences in steroid balance following stimulation of the receptor with structural LH analogs and provide further insight into gonadotrophin regulation of ovarian steroid production.

Comparison of effects of simvastatin and metformin in women with PCOS: a randomized trial

Statins are HMG-Co-A reductase inhibitors with pleiotropic effects including decreased cholesterol synthesis and reduction of oxidative stress. In ovarian theca cell cultures, statins inhibit proliferation, promote apoptosis and lower steroidogenesis reducing expression of CYP11 and 3ßHSD. Statins may also decrease oxidative stress inhibiting expression of NADPH subunits. Recently, we have evaluated the effects of simvastatin on women with polycystic ovary syndrome (PCOS), randomizing the subjects to the use of oral contraceptive pill (OCP) alone or in combination with simvastatin. In the presence of OCP, simvastatin reduced hyperandrogenism and improved lipid profile. This is the first trial comparing the use of simvastatin with metformin in women with PCOS not using any hormonal treatment. Prospective, randomized trial. Forty-one women with PCOS were enrolled and 34 completed the trial. Age of the women was 25.5 ± 4.3 (mean ± SD) and BMI was 23.6 ± 4.1. Patients were randomized to: simvastatin (S) alone (20 mg/day), metformin (M) alone (1,700 mg/day), and simvastatin+metformin (S+M). Evaluations were performed at baseline and after 3 months of treatment. All subjects were using barrier contraception. Data were analyzed using Student's t-test and Wilcoxon Sign-Rank test. S reduced total testosterone by 14% (P=0.005); M had no effect (decrease by 0.3%; P=0.9) while S + M decreased testosterone by 11% (P=0.02). Similarly, S decreased free testosterone by 12% (P=0.005) while M or M + S had no effect. In parallel, acne score improved following treatment with S by 46% (P=0.02); M or S + M had no effect. S also decreased total cholesterol by 22% while M had no effect (decrease by 2%). S + M reduced total cholesterol by 32%; this effect was greater than that of S alone (P<0.05). Similar effects of S were observed on LDL and triglycerides. Body mass index decreased minimally (by 2.5–3%) following each treatment (P<0.05). Insulin sensitivity index determined by a 2-hour glucose tolerance test increased following M (by 19%, P<0.05). This is the first study demonstrating that statin used alone significantly lowers hyperandrogenism and hyperandrogenemia in women with PCOS. Furthermore, as anticipated, simvastatin improves lipid profile. The effects of simvastatin are likely mediated by mechanisms distinctly different than those of metformin. We propose that statins use may represent a novel treatment of PCOS-related hyperandrogenism.

Effect of copper and thiomolybdates on bovine theca cell differentiation in vitro

Subfertility that will respond to appropriate copper supplementation is a widespread problem in the national dairy herd. The aims of this study were to determine the effect of copper and/or copper chelating thiomolybdates on LH-induced differentiation by looking at the effects on androgen production, steroidogenic enzymes (cytochrome P450 17alpha-hydroxylase and cytochrome P450 side-chain cleavage) and lysyl oxidase mRNA expression in cultured theca cells maintained under serum-free conditions. The effect of thiomolybdates and copper on LH differentiation was investigated by supplementing (ammonium) tetrathiomolybdate to optimum theca cell culture media at 0-100 microg/ml, copper (chloride) at equimolar concentrations (0-51.6 microg/ml) or equimolar combinations of both media. Lysyl oxidase mRNA expression was investigated with semi-quantitative RT-PCR, whilst expression of cytochrome P450 17alpha-hydroxylase and cytochrome P450 side-chain cleavage mRNA was examined using real time PCR. Both PCRs used bovine specific primers and cell lysates obtained from bovine theca cells cultured for 6 days and in the presence or absence of the 100 microg/ml dose of thiomolybdate and equimolar copper thiomolybdate. Thiomolybdate depressed androstenedione production in a dose-dependent manner at doses greater than 1 microg/ml at 96 h (P<0.05); doses above 20 microg/ml were all greatly reduced at all time points and at 192 h when related to numbers of cells (P<0.001). Copper alone had no effect at physiological doses, but the use of the equimolar copper thiomolybdate reversed the effect of tetrathiomolybdates on androstenedione production at the 20 microg/ml dose. Thiomolybdate supplementation, with and without copper, had no significant effect on the level of lysyl oxidase or cytochrome P450 side-chain cleavage expression. However, cytochrome P450 17alpha-hydroxylase expression was significantly increased (P<0.05) by tetrathiomolybdate, possibly due to a local regulatory system. In conclusion, these results demonstrate that thiomolybdates can prevent LH-induced differentiation of bovine theca cells in vitro and that these effects can be ameliorated by copper supplementation. Our results also indicate that it is unlikely that the effects of thiomolybdate are mediated at the transcriptional level and further work is required to determine if thiomolybdate exerts its effects through post-translation processing or some other unrelated mechanism. Overall, these data support the hypothesis that copper responsive subfertility results from perturbation of the normal pattern of ovarian steroidogenesis.

Decay-accelerating factor in the periovulatory rat ovary

One of the most prominent inflammatory reactions is the activation of the complement system. The activated complement system does not distinguish between pathogens and the host cell. In order to prevent autologous complement-mediated attack, host cells express a variety of both membrane-bound and fluid-phase complement regulatory proteins which control activity of the complement cascade by acting on convertase enzymes or the membrane-attack complex. Although the process of ovulation is facilitated by the inflammatory reaction, this reaction has the potential to cause serious damage to growing follicles, ovulated follicles, and other important ovarian tissues. This study was undertaken to characterize the expression and regulation of decay-accelerating factor (DAF), a complement regulator, as a potential mediator of ovarian tissue protection from ovulatory inflammation. DNA microarray and Northern blot analyses showed that an ovulatory gonadotropin stimulus dramatically yet transiently induced DAF mRNA expression in the immature rat ovary. Northern blot and PCR analyses revealed that of the three known DAF isoforms, glycosylphosphatidylinositol (GPI)-, soluble-, and transmembrane-(TM) DAF, GPI-DAF was the predominant form. In situ hybridization localized GPI-DAF mRNA expression in the theca-interstitial cells of the periovulatory ovary. Neither the anti-progestin RU486 nor the cyclooxygenase inhibitor indomethacin significantly inhibited human chorionic gonadotropin (hCG)-induced GPI-DAF mRNA expression in vivo. In vitro theca cell culture studies indicated that hCG induces GPI-DAF mRNA expression through the protein kinase A pathway. This study suggests that gonadotropin-induced GPI-DAF may be involved in the protection of ovarian tissues from the potential attack by the complement system activated by the inflammatory response associated with ovulation.

Resistin Stimulation of 17α-Hydroxylase Activity in Ovarian Theca Cells in Vitro: Relevance to Polycystic Ovary Syndrome

A newly discovered hormone resistin has been shown to be increased in women with polycystic ovary syndrome (PCOS). The purpose of this study was to confirm increased resistin concentrations in women with PCOS and to test the direct effect of resistin on human theca cell androgen production. Resistin was measured in fasting serum samples by RIA. To test the direct effects of resistin on ovarian androgen biosynthesis, human theca cells were cultured with resistin for 3 d in the presence and absence of forskolin and insulin. Fasting serum samples were obtained from 45 women with PCOS and 74 regularly cycling premenopausal control women in the follicular phase of their menstrual cycles, and ovarian theca cell cultures were established from two control women. The mean serum resistin concentration was increased (40%) in women with PCOS. Serum resistin concentrations correlated positively with body mass index and testosterone in PCOS women but not in controls. There were no significant correlations between resistin and fasting insulin or indicators of insulin resistance when corrected for body mass index. In cultured human theca cells, basal 17alpha-hydroxylase activity was unchanged by resistin alone, but resistin enhanced 17alpha-hydroxylase activity in the presence of forskolin or a combination of forskolin plus insulin. Resistin (> or =1 ng/ml) augmented forskolin and forskolin plus insulin stimulation of CYP17 mRNA expression in a concentration-dependent manner. These data indicate that abnormal resistin secretion in PCOS may play a role in causing ovarian hyperandrogenism.

Requirement for proximal putative Sp1 and AP-2 cis-deoxyribonucleic acid elements in mediating basal and luteinizing hormone- and insulin-dependent in vitro transcriptional activation of the CYP17 gene in porcine theca cells.

The cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (CYP17) enzyme catalyzes the first committed step in androgen biosynthesis. In primary cultures of immature swine theca cells, LH and insulin induce CYP17 mRNA and incompletely processed heteronuclear RNA supraadditively over 2-6 h. To monitor in vitro transcriptional control by these two physiological signals, we cloned a -976 to +31-bp 5'-upstream region of the homologous CYP17 gene and fused it to a cytoplasmically targeted firefly luciferase minigene (CYP17/luc). LH and insulin individually stimulated transcriptional activity of transiently transfected CYP17/luc in theca cells by 2.7 +/- 0.31- and 2.5 +/- 0.24-fold over basal, respectively, at an optimal concentration (both 100 ng/ml) and time (6 h; both P < 0.01). Combined peptidyl agonists stimulated CYP17/luc by 6.6 +/- 1.2-fold (P < 0.001). To identify possible LH- and insulin-sensitive cis-acting DNA regulatory regions, we prepared four deletional constructs, -839, -473, -174, and -75/+35 bp 5' upstream of the transcriptional start site. Deletion from -976 to -839 bp decreased basal transcriptional activity by 89% and that stimulated by LH, insulin, and both effectors by 82%, 91%, and 78%, respectively (each P < 0.01). Further deletion to -473 bp conferred partial responsiveness to combined hormone stimulation, suggesting an intervening inhibitory sequence. Truncation to -174 bp and more proximally reduced basal CYP17/luc activity and hormonal action by more than 95% (P < 0.001). The marked loss of combined LH and insulin stimulation caused by deleting the region between -473 and -175 bp suggested the possible relevance of partially overlapping specificity protein-1 (Sp1) and activating protein-2 (AP-2)-like binding sites located between -193 and -180 bp. Point mutation of the proximal Sp1-like element in full-length -976/+31 CYP17/luc impaired basal transcription minimally (by 21%; P = NS) and stimulation by LH (76%), insulin (67%), and combined peptides (54%) significantly (each P < 0.05 vs. wild type). Mutation of the AP-2 site alone decreased basal CYP17/luc activity nonsignificantly (by 25%), but repressed stimulated transcriptional responses prominently, viz. to LH (57%), insulin (77%), and both effectors (82%; each P < 0.025 vs. wild type). Mutation of both sites inhibited basal and hormonally stimulated CYP17/luc activity by more than 95% (P < 0.001). At the level of second messenger signaling, insulin did not potentiate LH-enhanced cAMP accumulation, whereas a stable cAMP analog mimicked LH action and augmented insulin's stimulation of full-length and deletional fragments of CYP17/luc. In summary, LH and insulin stimulate transcriptional activity of a -976/+31 bp 5'-upstream cis-acting region of the (porcine) CYP17 gene individually and jointly in primary cultures of theca cells. Maximal transcriptional responsiveness to these peptide hormones requires proximal Sp1 and AP-2-like sequences -193 to -180 bp 5' upstream of the transcriptional start site. Exogenous cAMP mimics transcriptional up-regulation by LH and interacts analogously with insulin. These data are consistent with convergent drive of CYP17 gene expression by cAMP-protein kinase A and insulinsignaling pathways in untransformed theca cells.

Insulin drives transcriptional activity of the CYP17 gene in primary cultures of swine theca cells.

Insulin stimulates androgen biosynthesis and the accumulation of CYP17 mRNA and heterogeneous nuclear (hn) RNA in primary cultures of immature swine theca cells. To further assess insulinomimetic transcriptional control, we subcloned 1.007 kilobases (kb) of the 5'-upstream region of the CYP17 gene (-976 to +31 base pairs [bp] to the transcriptional start site) into a firefly-luciferase reporter construct. Insulin drove transcriptional activity of this probe in a time- and dose-dependent fashion, with maximal stimulation of 2.7- to 3.2-fold after insulin exposure (100 ng/ml) for 6 h. Progressive deletional constructs -839, -473, -174, and -75/+31 bp delineated expected reduction in responsiveness, except paradoxical gain of basal CYP17 promoter activity by the -473/+31-bp sequence. The latter suggests a possible intervening inhibitory sequence. Elimination of all sequences 5'-upstream to -174 bp markedly reduced basal transcriptional activity and abolished insulin action. Point mutation of a presumptive Sp1-like element located within -193/-180 bp inhibited basal and insulin-stimulated luciferase activity of the full-length promoter fragment by 40% and 67%, respectively. Disruption of a contiguous presumptive AP-2 site produced a comparable outcome. Combined mutation of the Sp1 and AP-2-like elements eliminated basal and insulin-potentiated CYP17 promoter activity. By Western analysis, insulin augmented cognate receptor phosphoprotein concentrations by 31-fold within 10 min. Chemical inhibitors of MEK-activated ERK1/2 attenuated insulin-enhanced CYP17 transcriptional activity by 76-80%. In summary, insulin drives transcriptional activity of a 5'-upstream regulatory sequence (-976 to +31 bp) of the swine CYP17 gene in primary cultures of theca cells, under a minimal requirement for combined activity of proximal (-193/180 bp) Sp1 and AP-2-like elements.

Valproate Potentiates Androgen Biosynthesis in Human Ovarian Theca Cells

In patients with epilepsy, treatment with valproate (VPA) has been reported to be associated with polycystic ovary syndrome-like symptoms including weight gain, hyperandrogenemia, and hyperinsulinemia. We examined the effect of VPA on androgen biosynthesis in ovarian theca cells isolated from follicles of normal cycling women to determine whether the hyperandrogenemia reported with VPA treatment could be a result of direct effects of VPA on the ovary. In long-term cultures of theca cells treated for 72 h with sodium valproate (30-3000 microm), we observed an increase in basal and forskolin-stimulated dehydroepiandrosterone (DHEA), androstenedione, and 17alpha-hydroxyprogesterone production compared with control values. In contrast, low doses of VPA treatment (i.e. 30-300 microm) had no effect on basal and forskolin-stimulated progesterone production, whereas higher doses of VPA (1000-3000 microm) inhibited progesterone production. The most pronounced effect of VPA on androgen biosynthesis was observed in the dose range of 300-3000 microm, which represent therapeutic levels in the treatment of epilepsy and bipolar disorder. Western analyses demonstrated that VPA treatment increased both basal and forskolin-stimulated P450c17 and P450scc protein levels, whereas the amount of steroidogenic acute regulatory protein was unaffected. In transient transfection studies, VPA was found to increase P450 17alpha-hydroxylase and P450 cholesterol side-chain cleavage promoter activity, whereas steroidogenic acute regulatory protein promoter activity was unaffected. Consistent with the ability of VPA to act as a histone deacetylase (HDAC) inhibitor in other cell systems, VPA (500 microm) treatment was observed to increase histone H3 acetylation and P450 17alpha-hydroxylase mRNA accumulation. The HDAC inhibitor butyric acid (500 microm) similarly increased histone H3 acetylation and DHEA biosynthesis, whereas the VPA derivative valpromide (500 microm), which lacks HDAC inhibitory activity, had no effect on histone acetylation or DHEA biosynthesis. These data suggest that VPA-induced ovarian androgen biosynthesis results from changes in chromatin modifications (histone acetylation) that augment transcription of steroidogenic genes. These studies provide the first biochemical evidence to support a role for VPA in the genesis of polycystic ovary syndrome-like symptoms, and establish a direct link between VPA treatment and increased ovarian androgen biosynthesis.

Effect of PCB 126 and PCB 153 on incidence of apoptosis in cultured theca and granulosa cells collected from small, medium and large preovulatory follicles

The aim of the presented study was to evaluate the effects of PCB 126 and PCB 153 on granulosa and theca cell apoptosis. Granulosa and theca cells were collected from small, medium, and large preovulatory porcine follicles and cultured as monolayers. Cells were initially cultured for 24 h to allow attachment to the plates. Media were changed and 100 pg/ml PCB 126 or 100 ng/ml PCB 153 were added. After 48 h, granulosa and theca cells were fixed for assessment of the number of apoptotic cells utilizing a Hoechst staining technique or frozen for measurement of caspase-3 activity. Media were collected for testosterone concentration analysis from theca cell cultures or estradiol from granulosa cell cultures. Neither PCB 153 nor PCB 126 had an effect on testosterone secretion by theca cells collected from small and medium size follicles, while both PCBs decreased testosterone secretion by large follicles. The decrease in testosterone secretion by large follicles under the influence of both PCBs was paralleled by a suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. Neither of the PCBs had an effect on estradiol secretion by granulosa cells collected from small and medium size follicles, while both PCBs increased estradiol in granulosa cells collected from large follicles. PCB-associated increased estradiol secretion by granulosa cells collected from large follicles was accompanied by suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. In conclusion, we have presented evidence that in preovulatory follicles PCBs inhibit both theca and granulosa cells apoptosis. Therefore, an exposure to PCBs may cause alterations in the pattern of terminal differentiation of follicles and attenuate spontaneous elimination of atretic follicles.

Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development.

The ovary contains a pool of primordial follicles containing oocytes arrested in meiosis that are the source of developing follicles for the female. Growth and differentiation factor-9 (GDF-9) is a member of the transforming growth factor beta superfamily of growth factors, and follicles of GDF-9 knockout mice arrest in the primary stage of development. The effect of GDF-9 treatment on the primordial to primary follicle transition and on subsequent follicle progression was examined using a rat ovary organ culture system. Ovaries from 4-day-old rats were cultured under serum-free conditions in the absence or presence of growth factors. GDF-9 treatment caused a decrease in the proportion of stage 1 early primary follicles and a concomitant increase in the proportion of stage 2 mature primary follicles. GDF-9 did not effect primordial follicles or stage 0 to stage 1 follicle transition. GDF-9 also did not influence stage 3 or 4 secondary follicle numbers. Isolated antral follicle granulosa and theca cell cultures were used to analyze the actions of GDF-9. GDF-9 treatment did not directly influence either granulosa or theca cell proliferation. The ability of GDF-9 to influence the expression of another growth factor was examined. GDF-9 treatment increased kit ligand (KL) mRNA expression in bovine granulosa cells after 2 days of culture. Ovaries from 4-day-old rats were also cultured with or without GDF-9 treatment, and total ovary expression of KL mRNA was increased by GDF-9. In summary, GDF-9 was found to promote the progression of early primary follicle development but did not influence primordial follicle development. The actions of GDF-9 on specific stages of follicle development may in part be mediated through altering the expression of KL.

Strategies to elucidate the mechanism of excessive theca cell androgen production in PCOS

Polycystic ovary syndrome (PCOS) is characterized by increased ovarian androgen production. We have developed a strategy to identify differentially transcribed genes in PCOS theca cells. Using long-term cultures of normal and PCOS theca cells, we are characterizing the complete repertoire of differentially transcribed genes, the common transcription factor mediators, and the signal transduction cascade(s) that are abnormal in PCOS theca cells. The latter will be interrogated for mutations/genetic variants linked to PCOS.