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PubMed2000

Expression and function of Fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia.

Fas antigen is a receptor that triggers apoptosis when bound by Fas ligand (FasL). A role for Fas antigen in follicular atresia was studied in follicles obtained during the first wave of follicular development during the bovine estrous cycle (estrus is Day 0). Granulosa and theca cells were isolated from healthy dominant follicles and the two largest atretic subordinate follicles on Day 5, atretic dominant follicles on Days 10-12, and preovulatory follicles on Day 1. Fas antigen mRNA levels were highest in granulosa cells from subordinate as compared to other follicles, and lowest in theca cells from healthy Day 5 dominant as compared to other follicles. FasL alone had no effect on viability of granulosa or theca cells but became cytotoxic in the presence of interferon-gamma (IFN). IFN has been shown to induce responsiveness to Fas antigen-mediated apoptosis in other cell types. In the presence of IFN, killing of granulosa cells by FasL was greater in subordinate compared to healthy dominant follicles on Day 5, did not differ between healthy and atretic dominant follicles, and was similar in theca among all follicles. Granulosa cells from preovulatory follicles, which had been exposed to the LH surge in vivo, were completely resistant to FasL-induced killing. In summary, Fas antigen expression, and responsiveness to Fas antigen-mediated apoptosis, vary during follicular development.

Animals↗
PubMed1999

Expression and regulation of relaxin-like factor gene transcripts in the bovine ovary: differentiation-dependent expression in theca cell cultures.

The relaxin-like factor (RLF) was recently discovered as a new member of the insulin-insulin-like growth factor-relaxin family of growth factors and hormones predominantly in the Leydig cells of the testis. In cattle, in contrast to other species, the RLF gene is also expressed to a high level in the ovary, where its expression pattern in the corpus luteum of the late cycle and pregnancy is similar to that of relaxin in the pig. The RLF gene was also transcribed to a high level in the theca cells of estrogen-rich, large antral follicles. Long-term primary cultures of bovine theca cells showed that expression was insulin dependent. After an initial decline in specific mRNA concentrations, there was a switch to a transcript with a longer poly(A) tail at about Day 6 of culture, which continued to increase to very high levels by Day 15 of culture. Addition of fetal calf serum to cultures caused a rapid and irreversible down-regulation of the RLF gene. Also, LH caused a decline in specific gene expression in long-term primary theca cell cultures. As in the Leydig cells of the testis, the pattern of RLF gene expression appears to reflect the differentiation state of the ovarian theca-luteal cell lineage, and should prove useful for mapping the fate of these cells under differing stimulation regimes.

Androstenedione↗
PubMed2002

Overexpression of theca-cell messenger RNA in polycystic ovary syndrome does not correlate with polymorphisms in the cholesterol side-chain cleavage and 17alpha-hydroxylase/C(17-20) lyase promoters.

OBJECTIVE: To determine whether overexpression of CYP17 or CYP11A messenger (m)RNA in theca cells from polycystic ovaries is related to polymorphic regions in the gene promoters that may increase transcription. DESIGN: Case-control study. SETTING: Research institute. PATIENT(S): Fifty-one women with PCOS and 280 regularly cycling controls underwent genotyping. Thecal cells were obtained from 23 women with PCOS and 51 controls. MAIN OUTCOME MEASURE(S): Ovarian tissue was obtained from women with PCOS undergoing wedge resection for treatment of their infertility and from controls undergoing ovariectomy for indications unrelated to the study. Expression of mRNA in theca cells was measured by using competitive reverse transcriptase polymerase chain reaction. Genotype analysis for polymorphisms in the CYP11A and CYP17 promoters was performed by using polymerase chain reaction. RESULT(S): Although expression of CYP11A and CYP17 mRNA was higher in women with PCOS, no significant dose effects of CYP11A or CYP17 alleles were observed with respect to serum testosterone; follicular fluid androstenedione, estradiol, and androstenedione-to-estradiol ratio; or CYP11A or CYP17 mRNA expression. CONCLUSION(S): Overexpression of CYP17 and CYP11A mRNA in theca cells from polycystic ovaries is explained by polymorphic differences in the gene promoters.

Adult↗
PubMed2005

Granulosa theca cell tumor with luteoma in the ovary of a bonnet monkey (Macaca radiata).

A mass was identified on the left caudal region of the abdomen in a 13-year-old bonnet monkey (Macaca radiata). The mass was excised and diagnosed as granulosa theca cell tumor accompanied with luteoma based on the microscopic findings. Morphologically it appeared pink, round, firm multilobulated measured approximately 5 x 3 x 2.5 cm in dimension. Histologically the luteoma composed of polyhedral cells with pale strained vacuolated cytoplasm, centrally located nuclei with distinct cytoplasmic borders. Granulosa theca cell tumor appeared as densely packed spindle shaped fusiform cells arranged in interlacing bundles and whorled pattern with neoplastic cells appearing irregular shaped solid sheets. The concomitant development of granulosa theca cell tumor with luteoma in a single ovary is very rare and is the first reported case in a bonnet macaque to our knowledge.

Animals↗
PubMed1991

The effect of leuprolide acetate on steroidogenesis by granulosa and theca cells in vitro.

Enhancement of follicular response to controlled ovarian hyperstimulation for human in vitro fertilization (IVF) has been suggested following pretreatment with leuprolide acetate (LA). However, additional human menopausal gonadotropin (hMG) is required to achieve follicle maturity in the presence of LA. We studied the effect of LA on steroidogenesis of granulosa and theca cells in vitro. Human granulosa cells obtained from IVF follicular fluid aspirations were cultured for 14 days in the presence and absence of human chorionic gonadotropin (hCG). hCG significantly enhanced progesterone (P) and estradiol (E2) production by the cells, however, the addition of LA in concentrations of 10, 100, and 1000 ng/ml had no effect. Porcine granulosa cells were cultured for 48 hr in the presence and absence of follicle stimulating hormone (FSH) with the addition of LA at the same doses. LA did not affect the FSH-induced increase in P production. Porcine theca cells were cultured for 48 hr in the presence and absence of hCG. The addition of LA did not affect androstenedione (A) production by these cells. We conclude that in this dynamic model in vitro, LA does not inhibit or stimulate P or E2 production by granulosa or A production by theca cells.

Androstenedione↗
PubMed1997

The effect of microtubule and microfilament-disrupting drugs on prolactin-stimulated progesterone synthesis and secretion by cultured porcine theca cells.

The effect of microtubule- and microfilament-disrupting drugs (colchicine, cytochalasin B and D) on basal and prolactin (Prl)-stimulated progesterone synthesis and secretion was studied. Microtubules were visualized immunocytochemically using a monoclonal antibody against the alpha-subunits of tubulin, while microfilaments were detected using a polyclonal antibody against actin. The second antibody was conjugated with FITC. Progesterone, androgen and estradiol concentration were detected in tissue and culture medium by appropriate radioimmunoassays. In Prl-treated cultures microtubules formed a network with more radial organisation than in the controls. After colchicine-treatment the cells were round, regular in shape and microfilaments were disintegrated and replaced by punctate aggregates. Actin filaments formed typical stress fibers in theca cells (Tc). In Prl-treated cells some microfilaments were broken resulting in a diffuse immunofluorescent pattern. After treatment with cytochalasin B and D many of the stress fibers disappeared, the cells became rounded and diffuse microfilaments were seen. Prl added to the culture medium increased synthesis of all investigated steroids and additionally stimulated progesterone secretion. Exposure of theca cells to colchicine caused an increase of basal progesterone secretion into the incubation medium which simultaneously decreased the steroid content in the cells. Colchicine suppressed Prl-stimulated synthesis of all three steroids studied and did not have any effect on their secretion. Exposure of theca tissue to cytochalasin B increased basal progesterone and androgen synthesis but drastically decreased basal estradiol synthesis by these cells. The opposite effect was observed after the addition of cytochalasin D, when estradiol synthesis increased and progesterone and androgen synthesis by theca cells was not affected. Microtubule and microfilaments-disrupting drugs had no effect on the secretion of the investigated steroids in Prl-treated cells. Our results suggest that microtubules and microfilaments are involved in Prl-stimulated steroid synthesis but not in steroid secretion.

Actin Cytoskeleton↗
PubMed1998

Androstenedione and progesterone production in vitro by the inner or the outer theca cells in preovulatory follicles of gonadotropin stimulated calves.

This study reports some of the steroidogenic characteristics of the interna and externa theca cells taken from young and eCG primed calves. These cells were isolated from large healthy follicles. The were separately cultured for 3 days in absence or in presence of steroid substrates. Androstenedione (A4) and progesterone (P4) were measured by radioimmunoassay (RIA). In control conditions, A4 levels, higher in interna than in externa cells (P < 0.001), decreased during cultures (P < 0.01). In both cell types, A4 increased in presence of 17 alpha-hydroxypregnenolone (17OHP5), pregnenolone (P5) and 22R-hydroxycholesterol (22R-chol)(*P < 0.05) but not with P4 or 17 alpha-hydroxyprogesterone (17OHP4)(P > 0.05). The most efficient substrate was dehydroepiandrosterone (DHEA) (P < 0.005). In control conditions, P4 levels increased in both cell types. They were higher in externa than in interna cells on day 1, the reverse was observed on day 3. P4 levels increased after addition of 22 R-chol and P5 (P < 0.005) but not with 17OHP5, 17OHP4 and DHEA (P > 0.05) from day 1 in externa cells and only on day 3 in internal cells. P4 levels measured on day 1 were lower than the quantity of P4 added as a substrate. These results, obtained with theca cells from young calf follicles, indicate: 1/A4 is synthesized by the delta5 pathway and 17 alpha-hydroxylase activity decreases in vitro, 2/externa and interna cells differ by the quantities of A4 and P4 produced, 3/both lack precursors to produce A4 and P4 but their 3 beta-hydroxysteroid dehydrogenase activity subsists, 4/P4 could be metabolized during the first 2 days in both cell cultures.

17-alpha-Hydroxypregnenolone↗
PubMed2002

Growth differentiation factor-9 inhibits 3'5'-adenosine monophosphate-stimulated steroidogenesis in human granulosa and theca cells.

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor superfamily, modulates the development and function of granulosa and theca cells. Targeted deletion of GDF-9 in the mouse revealed that GDF-9 was essential for the establishment of the thecal cell layer during early folliculogenesis. During later stages of follicular development, the roles of GDF-9 are less well understood, but it has been postulated that oocyte-derived GDF-9 may prevent premature luteinization of follicular cells, based on its ability to modulate steroidogenesis by rodent ovarian cells. In the rodent, GDF-9 is expressed solely by the oocyte from the early primary follicular stage through ovulation. Recent studies in the rhesus monkey demonstrated that granulosa cells express GDF-9, suggesting a broader role for this protein in ovarian function in primates. We examined the effect of recombinant GDF-9 on proliferating human granulosa and thecal cell steroidogenesis and the expression of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage, and P450 aromatase. We also examined granulosa cell GDF-9 expression by quantitative RT-PCR and by Western analysis. GDF-9 inhibited 8-Br-cAMP-stimulated granulosa progesterone synthesis by approximately 40%, but did not affect basal progesterone production. Concordant with reduced steroid production, 8-Br-cAMP-stimulated StAR protein expression was reduced approximately 40% in granulosa cells, as were expression of StAR mRNA and StAR promoter activity. Additionally, GDF-9 inhibited 8-Br-cAMP-stimulated expression of P450 side-chain cleavage and P450 aromatase. Human granulosa cells expressed GDF-9, as determined by RT-PCR and Western analysis. Treatment of human thecal cells with GDF-9 blocked forskolin-stimulated progesterone, 17alpha-hydroxyprogesterone, and dehydroepiandrosterone synthesis. Thecal cells exhibited greater sensitivity to GDF-9, suggesting that this cell may be a primary target of GDF-9. Moreover, GDF-9 increased thecal cell numbers during culture, but had no effect on granulosa cell growth. Our findings implicate GDF-9 in the modulation of follicular steroidogenesis, especially theca cell function. Because GDF-9 mRNA and protein are detectable in granulosa-lutein cells after the LH surge, the concept of GDF-9 as a solely oocyte-derived luteinization inhibitor needs to be reevaluated.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Oestradiol feedback stimulation of androgen biosynthesis by human theca cells.

This study analysed the effect of oestradiol on basal and LH-stimulated production of androstenedione and progesterone by human theca cells in monolayer culture. Incubations were carried out for either 2 days (seven experiments) or 4 days (four experiments), in the presence or absence of luteinizing hormone (LH), oestradiol (10(-9)-10(-6) M) or inhibin. Medium collected at 48 and 96 h was stored until radioimmunoassay for steroid content. Theca pooled from small follicles (<10 mm) was used in all but two experiments; in these, ovaries were obtained from ovulatory women in the mid-follicular phase of their cycle and theca from small and large follicles was pooled. Oestradiol inhibited progesterone production in a dose-dependent manner in all experiments, irrespective of follicle size, ovulatory status and ovarian morphology, with maximum effect at 10(-6) M. At this dose, oestradiol had no effect on androstenedione production by theca from four anovulatory women with polycystic ovaries but produced a significant augmentation of both basal and LH-stimulated androstenedione production in theca from five of the seven ovulatory women, with maximal response in theca from the two pre-ovulatory subjects. During the 48-96 h period of incubation, oestradiol augmented androstenedione production in all four experiments and had a greater stimulatory effect than the physiological dose of inhibin (10 ng/ml). This is the first report of oestradiol regulating human theca cell steroidogenesis in a dose-dependent manner.

Adult↗
PubMed1999

Comparison of cyclic AMP production in response to LH by granulosa and theca cells from Meishan and Large-White hybrid gilts.

Previous experiments have demonstrated differences in various follicular characteristics between the prolific Chinese Meishan (MS) pig and European Large-White (LW) hybrids and the present experiment was designed to compare the cAMP response to LH by granulosa and theca cells in vitro between the two breeds. Ovaries were recovered from MS (n = 7) and LW hybrid (n = 8) gilts on the day before predicted oestrus and the 12 largest follicles dissected. Quadruplicate aliquots of granulosa cells or minced theca tissue were incubated for 1 h in the presence of 0, 0.1, 1.0, 10, 100 or 1000 ng/ml LH and cAMP production measured. Follicles from MS gilts were smaller (5.9 vs. 7.7 mm; P < 0.001), contained less fluid (81.5 vs. 177.4 microl; P < 0.001), had fewer granulosa cells (3.8 vs. 5.3 x 10(6); P < 0.01) and less theca tissue (30.3 vs. 50.5 mg; P < 0.05) than those from LW hybrid animals. Mean follicular fluid oestradiol concentration was > or = 149 ng/ml in all animals and tended to be higher in the MS follicles (P = 0.07). LH stimulated cAMP production by granulosa and theca cells from both breeds (P < 0.001). Although there was no overall breed effect for the granulosa cells, there was a significant (P < 0.001) interaction between LH dose and breed in the granulosa cells, whether cAMP production was expressed per 10(6) cells or per follicle. In the theca incubations, cAMP production by MS tissue was higher (P < 0.01) when results were expressed per mg tissue and again there was an interaction (P < 0.001) between LH dose and breed whether cAMP production was expressed per mg tissue or per follicle. For both tissue types, MS follicles produced more cAMP at the higher LH doses. In conclusion, this study has shown that MS granulosa and theca tissue respond differently to increasing doses of LH in terms of cAMP production in vitro compared to LW hybrid tissue and this supports previous suggestions of enhanced maturity of MS follicles in the late follicular phase.

Animals↗
PubMed1985

Analysis of steroid metabolites produced by theca cells from the adult domestic hen.

In a previous study on steroid metabolism by hen ovarian cells we reported on the production of 17-hydroxyprogesterone (17OH), androstenedione (A), testosterone (T), and oestrogens from [3H]progesterone (P) by theca cells. The present study examines further the metabolism of P by theca cells from the preovulatory follicles of the hen. The results show that the major metabolite of P is 20 beta-hydroxy-4-pregnen-3-one (20 beta-DHP), representing up to 40% of the recovered radioactivity. In addition, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha-DHP) and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta) were identified as metabolites of P, comprising 1 and 3% of the recovered radioactivity, respectively. This is the first evidence that the allylic steroid, 3 alpha-DHP, can be produced by avian ovaries.

Animals↗
PubMed2004

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.

Animals↗
PubMed1996

The effect of microtubule-disrupting drugs on morphology, progesterone and prorenin secretion of bovine cultured ovarian theca cells.

The effect of three microtubule-disrupting drugs (vinblastine, colchicine and nocodazole) on basal and LH-stimulated secretion of progesterone and prorenin by bovine theca cells was studied. Microtubules were visualized immunocytochemically using a monoclonal antibody against the alpha-subunit of tubulin and a secondary antibody conjugated with rhodamine. Progesterone and prorenin secretion were detected by adequate radioimmunoassays. Theca cells treated with LH alone or with a combination of LH, colchicine and vinblastine, showed round shape and disorganization of microtubules which were more obvious than after treatment with the two disrupting drugs alone. Nocodazole or LH treatment alone resulted in the formation of numerous cell processes, conspicuously different in shape from those in the controls. All three drugs increased basal progesterone secretion independently on the dose, but were without effect on basal prorenin secretion. LH-stimulated progesterone secretion was significantly suppressed by each of the three drugs while LH-stimulated prorenin secretion was decreased only by high doses of vinblastine and nocodazole. Colchicine in all three doses used did not exert any significant effect. The results obtained suggest that microtubules are involved in progesterone but not in prorenin secretion.

Animals↗
PubMed1990

Effect of several growth factors on plasminogen activator activity in granulosa and theca cells of the domestic hen.

This study was conducted to evaluate the effects of several growth factors on plasminogen activator (PA) activity in granulosa and theca cells collected from the largest preovulatory follicle in the hen ovary and to determine the involvement of cyclic adenosine monophosphate (cAMP) or protein kinase C, or both, in mediating the actions of epidermal growth factor (EGF) on granulosa PA activity. The granulosa cells were treated with increasing concentrations of: EGF (.33 to 16.4 nM); insulin-like growth factor I (IGF-I, 2.61 to 131 nM); fibroblast growth factor (FGF, .15 to 7.5 nM); or platelet-derived growth factor (PDGF, .02 to 1 nM). The treatments resulted in a dose-dependent increase in both cell-associated and secreted enzyme activity. The stimulatory effects of IGF-I (131 nM), however, were not mimicked with an equimolar concentration of the related peptide, insulin-like growth factor II. By contrast, theca cell PA activity was not significantly altered by EGF (16.4 nM), IGF-I (131 nM), FGF (7.5 nM), or PDGF (1 nM). Accumulation of cAMP was measured following exposure of granulosa cells to luteinizing hormone (LH, 10 ng, used as a positive control) or to EGF (16.4 and 164 nM) in the presence of .1 mM isobutylmethylxanthine. A 5-fold increase in cAMP levels was observed in response to LH; however, granulosa cell cAMP production was not altered by the presence of EGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Mitogenic effects of fibroblast growth factors on chicken granulosa and theca cells in vitro.

We have investigated the role that fibroblast growth factors (FGFs) may play in the rapid growth of preovulatory ovarian follicles in chickens. Granulosa and theca cells, dissected from the follicles of laying hens, were cultured in vitro and treated with FGF-1, FGF-2, FGF-5, and FGF-7. The synthesis of DNA by cultured cells was measured by incorporation of [(3)H]thymidine, which was added to the cultures. FGF-1 and -2 increased the synthesis of DNA in a dose-dependent manner in both cell types; however, FGF-5 and -7 had no effect in this respect. When genistein, a tyrosine kinase inhibitor, was added to these cultures, the synthesis of DNA due to FGF-2 was abolished. Treatment of cells with the glycosaminoglycans heparan sulphate and chondroitin sulphate had no effect on FGF-2-induced mitogenesis, while heparin inhibited it. Addition of a glycosaminoglycan antagonist, hexadimethrine bromide, to FGF-2-treated cultures inhibited DNA synthesis due to FGF-2, although not completely. Our data show that FGF-1 and FGF-2 are mitogenic for chicken granulosa and theca cells, and indicate that the actions of FGF-2 may be mediated via both tyrosine-kinase-type and glycosaminoglycan-type receptors on the surface of these cells.

Animals↗
PubMed1991

Cytochrome P450 side-chain cleavage (P450scc) in the hen ovary. I. Regulation of P450scc messenger RNA levels and steroidogenesis in theca cells of developing follicles.

We have recently shown that granulosa cells from hen ovarian follicles, collected at a stage of development 2-3 wk prior to ovulation (e.g. 6-8 mm in diameter) are steroidogenically inactive. Therefore, the hypothesis tested in the present studies was that theca cells from follicles at this stage of development must contain sufficient levels of functional cytochrome P450 side-chain cleavage (P450scc) enzyme to produce the progestin precursor required for the synthesis of androgens and estrogens. Northern blot analysis of total theca RNA collected from 6-8-mm follicles indicated the presence of a single P450scc mRNA transcript of approximately 2 kb whose expression was increased following an 8-h preincubation with 200 ng/ml ovine LH (oLH) or 10 microM forskolin. Western blot analysis of crude mitochondrial protein revealed a band of immunoreactive P450scc protein of approximately 53 kDa that was determined to be capable of converting 25-hydroxycholesterol to pregnenolone in a cell-free system. In the second set of studies, conducted to examine the cellular regulation of steroidogenesis in isolated theca cells of 6-8-mm follicles, theca cells were found to produce measurable basal levels of cAMP, progesterone, androstenedione, and estradiol following a 3-h incubation of 5 x 10(5) cells. Furthermore, significant dose-dependent increases in steroidogenesis were observed in response to oLH (0.2-200 ng/ml), chicken FSH (cFSH; 20-200 ng/ml), cholera toxin (0.002-20 ng/ml), and 8-bromo-cAMP (0.1-3.33 mM). Phorbol 12-myristate 13-acetate (PMA; 10-167 nM) also stimulated dose-dependent increases in basal progesterone, androstenedione, and estradiol production. In addition, while PMA had no effect on oLH (200 ng/ml)-promoted cAMP accumulation, or on oLH (20 ng/ml)- or 8-bromo-cAMP (1 mM)-stimulated progesterone production, it attenuated oLH-induced and 8-bromo-cAMP-induced androstenedione and estradiol accumulation. We conclude that theca cells from 6-8-mm follicles possess mRNA and immunoreactive protein coding for functional P450scc. Furthermore, basal steroidogenesis is increased by both the protein kinase A and protein kinase C pathways, whereas evidence suggests that protein kinase C inhibits LH-induced androstenedione production at a site distal to cAMP and progesterone production, most likely by decreasing C17,20-lyase activity.

Androstenedione↗
PubMed2003

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.

17-alpha-Hydroxyprogesterone↗