Thecal Androgen Research Articles

289 Postnatal Obesity Amplifies the Effect of Prenatal Testosterone Excess in Reducing the Progesterone Negative Feedback Sensitivity in First-Generation Ewes

Abstract Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women of reproductive age. Women with PCOS exhibit reduced sensitivity of the GnRH neuronal system to the inhibitory effects of both progesterone and estradiol. Additionally, obesity amplifies the severity of the PCOS phenotype. In sheep, testosterone (T) excess during gestational days (GD) 30-90 and 60-90 reduces neuroendocrine sensitivity to the progesterone negative feedback and recapitulates the PCOS phenotype. This study investigated the contribution of postnatal obesity in amplifying or mitigating the effects of mid-gestational T excess in disrupting the responsiveness to progesterone negative feedback in sheep. Suffolk ewes received T propionate (T; 100 mg i.m.) or corn oil (CO; vehicle) twice weekly from GD 60-90 (term = 147 d). At 5 mo of age, F1 T lambs were assigned randomly to either a maintenance (100% of NRC requirements) or overfed diet (130% of NRC requirements) and control lambs were fed the maintenance diet. At 22 mo of age, F1 ewes (n = 8/group) were synchronized with two injections of PGF2α 11 d apart. After the second PGF2α administration, blood samples were collected daily for 14 d and progesterone concentrations were determined via radioimmunoassay. Ewes that were confirmed to be cycling and in the mid-luteal phase based on progesterone concentrations (progesterone ≥ 1 ng/ml on day 10 and ≥ 0.5 ng/ml on days 8-9 and 11-12) were used for LH pulse characterization. On day 10 after PGF2α synchronization (mid-luteal phase), blood samples were collected every 15 min for 6 h to characterize LH pulsatile secretion via radioimmunoassay. Data were analyzed as one-way ANOVA with Tukey's HSD post-hoc analysis. Frequency of LH pulses was greater (P < 0.05) in T maintenance (3.75 ± 0.41 pulses/6h) and T overfed ewes (3.50 ± 0.34 pulses/6h) compared with controls (2.00 ± 0.37 pulses/6 h). Luteinizing hormone pulse amplitude was greater (P < 0.05) in T overfed (2.03 ± 0.15 ng/mL) compared with T maintenance (1.44 ± 0.17 ng/mL), but not to controls (1.74 ± 0.21 ng/mL). Also, average LH concentration was greater (P < 0.05) in T overfed (2.03 ± 0.24 ng/mL), but not T maintenance (1.31 ± 0.35 ng/mL), compared with controls (0.93 ± 0.30 ng/mLl). In conclusion, these findings demonstrate the activational role of postnatal obesity in amplifying prenatal T-induced alterations in tonic LH secretion during progesterone negative feedback. The activational effect of postnatal obesity in increasing LH secretion and consequently thecal androgen production is likely a contributor to the severity of hyperandrogenic status in women with PCOS. Research support: NIH-NICHD (R01HD099096).

Androgen production in response to LH is impaired in theca cells from nonovulatory dominant follicles in early-postpartum dairy cows

Most dairy cows develop a dominant follicle within two weeks postpartum, but 60% of these follicles fail to ovulate. In a previous study, we determined that cows destined to ovulate have higher LH pulse frequency and circulating estradiol. The latter characteristic provided a method for distinguishing ovulatory from nonovulatory follicles during development and we found that nonovulatory follicles have lower estradiol and androstenedione in their follicular fluid. We hypothesized that lower LH pulse frequency impairs androgen production by theca cells of nonovulatory cows, reducing their ability to make estradiol. In the present study, we applied our method for predicting follicle fate to collect dominant follicles from predicted ovulatory (n = 7) and nonovulatory (n = 3) follicles. Theca and granulosa cells were separated and cultured in the absence or presence of LH, FSH, and/or testosterone for three days, with daily collection of culture medium for steroid RIAs. Estradiol and progesterone production by granulosa cells were not different between ovulatory and nonovulatory follicles. By contrast, overall androstenedione production by theca cells from ovulatory follicles was significantly higher compared with nonovulatory follicles on all three days of culture and, as culture progressed, theca from nonovulatory follicles had increasingly poorer responses to LH. In the same cultures, the progesterone production by theca cells was similar in ovulatory and nonovulatory groups. In support of our hypothesis, the results show that estradiol production by granulosa cells from nonovulatory follicles is robust when androgen substrate is present, but that thecal androgen production in response to LH is impaired. This suggests that the initial defect in steroidogenesis in dominant follicles that fail to ovulate postpartum is lower production of androgen by theca cells.

Theca cells and the regulation of ovarian androgen production

Theca cells are essential for female reproduction being the source of androgens that are precursors for follicular oestrogen synthesis and also signal through androgen receptors (AR) in the ovary and elsewhere. Theca cells arise from mesenchymal cells around the secondary follicle stage. Their recruitment, proliferation and cytodifferentiation are influenced, directly or indirectly, by paracrine signals from granulosa cells and oocyte although uncertainty remains over which are the critically important signals at particular stages. In a reciprocal manner, theca cells secrete factors that influence granulosa cell proliferation and differentiation at different follicle stages. Differentiated theca interna cells acquire responsiveness to luteinizing hormone (LH) and other endocrine signals and express components of the steroidogenic machinery required for androgen biosynthesis. They also express insulin-like peptide 3 (INSL3) and its receptor (RXFP2), levels of which increase during bovine antral follicle development. INSL3 signaling may play a role in promoting androgen biosynthesis since knockdown of either INSL3 or its receptor (RXFP2) in bovine theca cells inhibits androgen biosynthesis while exogenous INSL3 can raise androgen secretion. Bone morphogenetic proteins (BMPs) of thecal or granulosal origin suppress thecal production of both INSL3 and androgen. Inhibin, produced in greatest amounts by granulosa cells of preovulatory follicles, reverses these BMP actions. Thus, BMP-induced inhibition of thecal androgen production may be mediated by reduced INSL3-RXFP2 signaling. Activins also inhibit androgen production in an inhibin-reversible manner and recent evidence in sheep indicates that theca cells synthesize and secrete activin, implying an autocrine role in suppressing androgen biosynthesis in smaller follicles, akin to that envisaged for BMPs.

Diethylstilbestrol administration inhibits theca cell androgen and granulosa cell estrogen production in immature rat ovary

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

Follicular expression of pro-inflammatory cytokines tumour necrosis factor-α (TNFα), interleukin 6 (IL6) and their receptors in cattle: TNFα, IL6 and macrophages suppress thecal androgen production in vitro.

Pro-inflammatory cytokines secreted by macrophages and other cell types are implicated as intraovarian factors affecting different aspects of ovarian function including follicle and corpus luteum 'turnover', steroidogenesis and angiogenesis. Here, we compared granulosal (GC) and thecal (TC) expression of TNF, IL6 and their receptors (TNFRSF1A, TNFRSF1B and IL6R) during bovine antral follicle development; all five mRNA transcripts were detected in both GC and TC and statistically significant cell-type and follicle stage-related differences were evident. Since few studies have examined cytokine actions on TC steroidogenesis, we cultured TC under conditions that retain a non-luteinized 'follicular' phenotype and treated them with TNFα and IL6 under basal and LH-stimulated conditions. Both TNFα and IL6 suppressed androgen secretion concomitantly with CYP17A1 and LHCGR mRNA expression. In addition, TNFα reduced INSL3, HSD3B1 and NOS3 expression but increased NOS2 expression. IL6 also reduced LHCGR and STAR expression but did not affect HSD3B1, INSL3, NOS2 or NOS3 expression. As macrophages are a prominent source of these cytokines in vivo, we next co-cultured TC with macrophages and observed an abolition of LH-induced androgen production accompanied by a reduction in CYP17A1, INSL3, LHCGR, STAR, CYP11A1 and HSD3B1 expression. Exposure of TC to bacterial lipopolysaccharide also blocked LH-induced androgen secretion, an effect reduced by a toll-like receptor blocker (TAK242). Collectively, the results support an inhibitory action of macrophages on thecal androgen production, likely mediated by their secretion of pro-inflammatory cytokines that downregulate the expression of LHCGR, CYP17A1 and INSL3. Bovine theca interna cells can also detect and respond directly to lipopolysaccharide.

The Role of Anti-Müllerian Hormone (AMH) During Follicle Development in a Monovulatory Species (Sheep)

Knockout studies in mice have suggested that anti-Müllerian hormone (AMH) modulates primordial follicle recruitment and the response of growing follicles to FSH. Little is known of the physiology of AMH in monovular species, despite intense clinical interest in this factor. Using sheep as a model, we sought to investigate the functional role of AMH in modulating follicle development in monovular species. In contrast to the rodent, the results indicate that AMH does not affect the rate of primordial follicle recruitment but appears to regulate the rate at which follicles progress through the gonadotropin-responsive phase, during which it is maximally expressed. Thus, knockdown of AMH bioactivity by active immunization lead to a decline in the population of gonadotropin-responsive preantral and small antral follicles (P < 0.01) and increases in both the number of gonadotropin-dependent antral follicles (P < 0.01) and ovulation rate (P < 0.05). These in vivo findings were consistent with the results of other studies examining the pattern of expression of AMH, which was negatively correlated with aromatase (P < 0.001), and in vitro supplementation experiments, which supported an inhibitory role for AMH in modulating the response of both theca and granulosa cells to LH and FSH, respectively. The elucidation of a functional relationship between AMH and LH-stimulated thecal androgen production may be significant in terms of the etiology of common forms of anovulatory infertility in women. Furthermore, the observed increase in both the number of recruitable antral follicles and ovulatory quota in response to AMH knockdown may have therapeutic value in women who respond poorly to ovarian stimulation.

Activin B is produced early in antral follicular development and suppresses thecal androgen production

Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (βA, βB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin βB mRNA levels decreased as follicle diameter increased from 1–3 to >6 mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin βA mRNA at 3–4 mm, and TCs more inhibin α mRNA at >4 mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1–3 mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin βA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s).

Enhanced Thecal Androgen Production Is Prenatally Programmed in an Ovine Model of Polycystic Ovary Syndrome

One of the hallmarks of polycystic ovary syndrome (PCOS) is increased ovarian androgen secretion that contributes to the ovarian, hormonal, and metabolic features of this condition. Thecal cells from women with PCOS have an enhanced capacity for androgen synthesis. To investigate whether this propensity is a potential cause, rather than a consequence, of PCOS, we used an ovine prenatal androgenization model of PCOS and assessed ewes at 11 months of age. Pregnant Scottish Greyface ewes were administered 100 mg testosterone propionate (TP) or vehicle control twice weekly from d 62 to 102 of gestation, and female offspring (TP = 9, control = 5) were studied. Prenatal TP exposure did not alter ovarian morphology or cyclicity, or plasma androgen, estrogen, and gonadotropin concentrations, at this stage. However, follicle function was reprogrammed in vivo with increased proportions of estrogenic follicles (P < 0.05) in the TP-exposed cohort. Furthermore, in vitro the thecal cells of follicles (>4 mm) secreted more LH-stimulated androstenedione after prenatal androgenization (P < 0.05), associated with increased basal expression of thecal StAR (P < 0.01), CYP11A (P < 0.05), HSD3B1 (P < 0.01), CYP17 (P < 0.05), and LHR (P < 0.05). This provides the first evidence of increased thecal androgenic capacity in the absence of a PCOS phenotype, suggesting a thecal defect induced during fetal life.

Oocyte-thecal Cell Regulatory Loop in the Control of Preantral Follicle Development

Oocyte-somatic cell interaction plays a crucial role in preantral folliculogenesis. Although topical research has focused on oocyte-granulosa cell interaction over the past decade, an oocyte-thecal cell regulatory loop may also play an important role during the preantral stage. Formation of the thecal cell layer is a key event that occurs during preantral folliculogenesis. Granulosal factors (e.g. IGF-I and KL) appear to stimulate the recruitment of thecal cells from stromal cells. Oocyte-derived GDF9 appears to indirectly modulate thecal cell differentiation, perhaps through regulating the granulosa/IGF-I and KL expression. Theca-produced androgens stimulate granulosa cell proliferation and preantral follicle growth. GDF9 enhances preantral follicle growth by up-regulating thecal androgen production, suggesting that a threshold level of androgens derived from thecal cells is necessary for preantral follicle growth, and that the androgen production may be controlled by the oocyte-derived GDF9. The challenge ahead is not to only understand the precise nature of these interactions, and to elucidate how dysregulation in these interactions may lead to ovarian pathologies such as polycystic ovary syndrome and gonadotropin poor-responsiveness.

Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production

We reported previously that bone morphogenetic proteins (BMPs) potently suppress CYP17 expression and androgen production by bovine theca interna cells (TC) in vitro. In this study, real-time PCR was used to analyse gene expression in TC and granulosa cell (GC) layers from developing bovine antral follicles (1-18 mm). Abundance of mRNA transcripts for four BMPs (BMP2, BMP4, BMP6, and BMP7) and associated type I (BMPR1A, BMPR1B, ACVR1 and ACVR1B) and type II (BMPR2, ACVR2A and ACVR2B) receptors showed relatively modest, though significant, changes during follicle development. BMP2 was selectively expressed in GC, while BMP6, BMP7 and betaglycan (TGFBR3) were more abundant in TC. Abundance of betaglycan mRNA (inhibin co-receptor) in TC increased progressively (fivefold; P<0.001) as follicles grew from 1-2 to 9-10 mm. This suggests a shift in thecal responsiveness to GC-derived inhibin, produced in increasing amounts as follicles achieve dominance. This prompted us to investigate whether inhibin can function as a physiological antagonist of BMP action on bovine TC in vitro, in a manner comparable to that for activin signalling. BMP4, BMP6 and BMP7 abolished LH-induced androstenedione secretion and suppressed CYP17 mRNA >200-fold (P<0.001), while co-treatment with inhibin-A reversed the suppressive action of BMP in each case (P<0.001). Results support a physiological role for granulosa-derived inhibin as an antagonist of BMP action on thecal androgen synthesis. A shift in intrafollicular balance between thecal BMP signalling (inhibitory for androgen synthesis) and betaglycan-dependent inhibin signalling (stimulatory for androgen synthesis) accords with the physiological requirement to deliver an adequate supply of aromatase substrate to GC of developing follicles.

Evidence for an inhibitory role of bone morphogenetic protein(s) in the follicular–luteal transition in cattle

Bone morphogenetic proteins (BMPs) and their receptors are expressed in ovarian theca cells (TC) and granulosa cells (GC) and BMPs have been implicated in the regulation of several aspects of follicle development including thecal androgen production and granulosal oestrogen production. Their potential involvement in luteal function has received less attention. In this study, we first compared relative abundance of mRNA transcripts for BMPs, activin-betaA and BMP/activin receptors in bovine corpus luteum (CL) and follicular theca and granulosa layers before undertaking functional in vitro experiments to test the effect of selected ligands (BMP6 and activin A) on luteinizing bovine TC and GC. Relative to beta-actin transcript abundance, CL tissue contained more BMP4 and -6 mRNA than granulosa, more BMP2 mRNA than theca but much less activin-betaA mRNA than both granulosa and theca. Transcripts for all seven BMP/activin receptors were readily detected in each tissue and two transcripts (BMPRII, ActRIIA) were more abundant in CL than either theca or granulosa, consistent with tissue responsiveness. In vitro luteinization of TC and GC from antral follicles (4-6 mm) was achieved by culturing with 5% serum for 6 days. Treatment with BMP6 (0, 2, 10, and 50 ng/ml) and activin A (0, 2, 10 and 50 ng/ml) under these conditions dose-dependently suppressed forskolin-induced progesterone (P4) secretion from both cell types without affecting cell number. BMP6 reduced forskolin-stimulated upregulation of STAR mRNA and raised 'basal' CYP17A1 mRNA level in theca-lutein cells without affecting expression of CYP11A1 or hydroxy-Delta-5-steroid dehydrogenase, 3 beta- and steroid Delta-isomerase 1 (HSD3B1). In granulosa-lutein cells, STAR transcript abundance was not affected by BMP6, whereas forskolin-induced expression of CYP11A1, HSD3B1, CYP19A1 and oxytocin transcripts was reduced. In both cell types, follistatin attenuated the suppressive effect of activin A and BMP6 on forskolin-induced P4 secretion but had no effect alone. Granulosa-lutein cells secreted low levels of endogenous activin A ( approximately 1 ng/ml); BMP6 reduced this, while raising follistatin secretion thus decreasing activin A:follistatin ratio. Collectively, these findings support inhibitory roles for BMP/activin signalling in luteinization and steroidogenesis in both TC and GC.

The Src tyrosine kinase pathway regulates thecal CYP17 expression and androstenedione secretion

In order to evaluate the role of Src tyrosine kinase in thecal cell steroidogenesis, a pharmacological approach was utilized by treating enriched populations of mouse ovarian theca-interstitial cells in vitro with a direct Src kinase inhibitor, PP2. Inhibition of Src with PP2 increased both basal and forskolin-stimulated androstenedione secretion, and increased cytochrome P450 17-alpha hydroxylase-lyase (CYP17) promoter activity and steady state mRNA. PP2 did not change thecal levels of StAR mRNA. Inhibition of mitogen-activated protein kinase kinase, a downstream regulator of Src activity, using PD98059 also increased forskolin-stimulated secretion of androstenedione above forskolin alone, but had no effect on basal secretion of androstenedione. Src inhibition increased mitogen-activated protein kinase phosphatase-1 protein and decreased phosphorylation of SF-1, which correlated with increased CYP17 promoter activity and mRNA levels. These results implicate Src tyrosine kinase in the regulation of CYP17 and thecal androgen secretion.

Immune system involvement in the regulation of ovarian function and augmentation of cancer

Increasing evidence indicates a role for the immune system and mesenchymal-epithelial interactions in the regulation of ovarian function. Cytokines produced by mesenchymal cells can stimulate development and regression of ovarian structures. We report here that mesenchymal cells releasing surface molecules among epithelial cells--namely vascular pericytes and monocyte-derived cells (MDC)--and intraepithelial T lymphocytes are associated with oogenesis and formation of new primary follicles in both fetal and adult human ovaries. These activated mesenchymal cells interact with the ovarian surface epithelium, which appears to be a source of secondary germ cells and granulosa cells. Activated pericytes and MDC are also associated with stimulation of thecal development during selection of growing secondary follicles from the cohort of primary follicles. However, survival of the dominant follicle during mid-follicular phase selection is associated with a lack of activity of mesenchymal cells and retardation of thecal development, since immature granulosa cells lacking aromatase are unable to resist high levels of thecal androgens. Once the selected follicle matures (late follicular phase), it shows enhanced activity of thecal mesenchymal cells and advanced thecal development. Corpus luteum (CL) development is accompanied by a high activity of vascular pericytes and MDC. In mature CL and CL of pregnancy, luteal MDC and pericytes show a stable (inactive) state. Regression of the CL is associated with regression of pericytes, transformation of MDC into dendritic cells, infiltration by T lymphocytes, and binding of immunoglobulin G to the luteal cells. The immunoglobulin M (IgM) binds to young but not mature luteal cells. In the CL of pregnancy, IgM binds to luteal vessels, but not to luteal cells. Regressing CL shows IgM binding to both luteal cells and vessels. In ovarian cancers, highly activated MDC and sometimes activated pericytes (poorly differentiated carcinomas) are present. IgM binding is similar to that seen in the CL of pregnancy. These data indicate that vascular pericytes, MDC, T cells, and immunoglobulins may play an important role in the regulation of ovarian physiology and contribute to the augmentation of ovarian cancer growth.

The role of thecal androgen production in the regulation of estradiol biosynthesis by dominant bovine follicles during the first follicular wave1,2

The first wave of follicular development following ovulation in cattle is characterized by selection and growth of a large, estrogenic dominant follicle. After the follicle becomes morphologically dominant, concentrations of estradiol in its follicular fluid decrease abruptly. The purpose of this study was to determine whether this decrease in estrogen production is caused by an insufficient supply of androgen from theca interna or decreased aromatization of androgen precursor by granulosa cells. Dominant follicles were collected from Holstein heifers on d 4, 6, or 8 of the first follicular wave (n = 5/d). Amounts of 17alpha-hydroxylase mRNA in theca interna were sevenfold higher (P < 0.01) on d 4 than on d 8. After 3 h in culture, secretion of androstenedione by theca interna collected on d 4 (236 +/- 44 pg/microg of protein) tended to be lower (P = 0.055) compared with d 6 (517 +/- 162 pg/microg protein) and was lower (P < 0.05) compared with d 8 (387 +/- 51 pg/microg of protein). In granulosa cells, amounts of aromatase mRNA decreased (P < 0.05) on d 8 compared with d 6 but not d 4. In vitro secretion of estradiol was higher in granulosa cells collected on d 4 (3.5 +/- 0.8 ng/[10(5) cells x 3 h]) compared with d 6 (1.8 +/- 0.6 ng/[10(5) cells x 3 h]; P < 0.05) and tended to be higher on d 4 than on d 8 (2.2 +/- 0.2 ng/[10(5) cells x 3 h]; P = 0.058). We conclude that the decrease in estradiol production observed during atresia of the dominant follicle is not due to lack of androgen substrate for aromatization or downregulated expression of the aromatase gene, but may be the direct result of decreased activity of the aromatase enzyme within granulosa cells.

Bone Morphogenetic Proteins (BMP) -4, -6, and -7 Potently Suppress Basal and Luteinizing Hormone-Induced Androgen Production by Bovine Theca Interna Cells in Primary Culture: Could Ovarian Hyperandrogenic Dysfunction Be Caused by a Defect in Thecal BMP Signaling?

We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and -7), whereas bovine granulosa cells and oocytes express BMP-6. Therefore, BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, -6, and -7 potently suppress both basal (P < 0.0001; respective IC(50) values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced (P < 0.0001; respective IC(50) values, 5.00, 0.55, and 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semiquantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17. Levels of mRNA encoding steroidogenic acute regulatory protein, P450scc, and 3beta-hydroxy- steroid dehydrogenase were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein after BMP treatment (P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by coincubating cells with BMPs and several potential BMP antagonists, chordin, gremlin, and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and -7, respectively, and the observation that both antagonists enhanced (P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP-4 and -7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be a result of a defective autoregulatory pathway involving thecal BMP signaling.

Recombinant follicle-stimulating hormone stimulates ovarian androgen synthesis in down-regulated ovulatory women

In order to study androgen secretion during controlled ovarian hyperstimulation for in-vitro fertilization-embryo transfer ,an open randomized study comparing the response to recombinant or urinary follicle-stimulating hormone (FSH) in down-regulated cycles was performed. During FSH administration significant increases in testosterone ,androstenedione and dehydroepiandrosterone sulfate (DHEAS) levels were observed. During the same period a slight decrease in luteinizing hormone (LH) levels was seen. At all times during the stimulation period a significant correlation between estradiol and testosterone or androstenedione levels was observed. We conclude that FSH ,through granulosa derived paracrine factors ,initiates thecal androgen synthesis and secretion.

Potential local regulatory functions of inhibins, activins and follistatin in the ovary.

The changing pattern of granulosa cell expression of inhibin/activin subunits and follistatin during follicle development and their differential regulation by extrinsic and intraovarian factors supports evidence from functional studies, mostly in vitro, that these proteins have important roles in folliculogenesis, oocyte maturation and corpus luteum function. Gonadal inhibins function as negative feedback hormones to regulate the synthesis and secretion of pituitary FSH, a key determinant of follicle development, but there is little supportive evidence for a peripheral endocrine role for ovary-derived activins or follistatin in this regard. However, activins and follistatin are expressed in numerous other tissues, including anterior pituitary, and they are firmly implicated as local intrapituitary regulators of FSH secretion. Intraovarian actions of granulosa cell-derived activins include the promotion of granulosa cell proliferation and upregulation of FSH receptors, P450arom, oestrogen synthesis, granulosa cell LH receptors and enhancement of oocyte maturation. Through its activin-binding role, follistatin can reverse each of these activin-induced responses. In addition to their endocrine feedback role, granulosa-derived inhibins can sensitize theca cells to LH, thereby enhancing the production of androgens, an essential requirement for follicular oestrogen synthesis. Activins can oppose this effect and suppress thecal androgen production. Granulosa cells overproduce inhibin a subunit precursor relative to betaA/betaB subunit precursors and evidence indicates that different parts of the inhibin a subunit precursor have intrinsic biological activities distinct from inhibin alphabetaA/B dimer, and serve as additional local modulators of follicle and corpus luteum function.

Recombinant follicle-stimulating hormone stimulates ovarian androgen synthesis in down-regulated ovulatory women

In order to study androgen secretion during controlled ovarian hyperstimulation for in-vitro fertilization-embryo transfer ,an open randomized study comparing the response to recombinant orurinary follicle-stimulating hormone (FSH) in down-regulated cycles was performed. During FSH administration significant increases in testosterone ,androstenedione and dehydroepiandrosterone sulfate (DHEAS)levels were observed. During the same period a slight decrease in luteinizing hormone (LH) levels was seen. At all times during the stimulation period a significant correlation between estradiol and testosteroneor androstenedione levels was observed. We conclude that FSH ,through granulosa derived paracrine factors ,initiates thecal androgen synthesis and secretion.

Roles of KIT and KIT LIGAND in ovarian function.

Evidence from mouse mutants indicates that the Kit gene encoding KIT, a receptor present on the oocyte and theca cells, and the Mgf gene encoding KIT LIGAND, the ligand of KIT, are important regulators of oogenesis and folliculogenesis. Recently, in vitro cultures of fetal gonads, of follicles and of oocytes have identified specific targets for the KIT-KIT LIGAND interaction. In fetal gonads, an anti-apoptotic effect of KIT-KIT LIGAND interactions on primordial germ cells, oogonia and oocytes has been demonstrated. In postnatal ovaries, the initiation of follicular growth from the primordial pool and progression beyond the primary follicle stage appear to involve KIT-KIT LIGAND interactions. During early folliculogenesis, KIT together with KIT LIGAND controls oocyte growth and theca cell differentiation, and protects preantral follicles from apoptosis. Formation of an antral cavity requires a functional KIT-KIT LIGAND system. In large antral follicles, the KIT-KIT LIGAND interaction modulates the ability of the oocyte to undergo cytoplasmic maturation and helps to maximize thecal androgen output. Hence, many steps of oogenesis and folliculogenesis appear to be, at least in part, controlled by paracrine interactions between these two proteins.

Differential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serum-free culture

The regulation of pig theca cell steroidogenesis was studied by the development of a physiological serum-free culture system, which was subsequently extended to investigate potential theca-granulosa cell interactions. Theca cells were isolated from antral follicles 6-9 mm in diameter and the effects of plating density (50-150x10(3) viable cells per well), LH (0.01-1.0 ng ml(-1)), Long R3 insulin-like growth factor I (IGF-I) (10, 100 ng ml(-1)) and insulin (1, 10 ng ml(-1)) on the number of cells and steroidogenesis were examined. The purity of the theca cell preparation was verified biochemically and histologically. Co-cultures contained 50x10(3) viable cells per well in granulosa to theca cell ratio of 4:1. Wells containing granulosa cells only were supplemented with 'physiological' doses of androstenedione or 100 ng ml(-1). Oestradiol production by co-cultures was compared with the sum of the oestradiol synthesized by granulosa and theca cells cultured separately. Oestradiol and androstenedione production continued throughout culture. High plating density decreased steroid production (P < 0.01). LH increased androstenedione (P < 0.001) and oestradiol (P < 0.05) synthesis and the sensitivity of the cells increased with time in culture. Oestradiol production was increased by 10 ng IGF-I ml(-1) (P < 0.001) but androstenedione required 100 ng ml(-1) (P < 0.001). Co-cultures produced more oestradiol than the sum of oestradiol synthesized by theca and granulosa cells cultured separately (P < 0. 001), irrespective of the androstenedione dose. This serum-free culture system for pig theca cells maintained in vivo steroidogenesis and gonadotrophin responsiveness. Thecal androstenedione and oestradiol production were differentially regulated and were primarily stimulated by LH and IGF-I, respectively. Theca-granulosa cell interactions stimulated oestradiol synthesis and this interaction was mediated by factors additional to the provision of thecal androgen substrate to granulosa cells.