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.     

Effect of non-esterified fatty acids on bovine theca cell steroidogenesis and proliferation in vitro

Elevated serum non-esterified fatty acid (NEFA) levels associated with a negative energy balance (NEB) may affect ovarian function and hence reproductive performance in high-yielding dairy cows. We have investigated the individual and combined effects of the three major NEFAs on bovine theca cell proliferation and steroidogenesis in vitro. Theca cells from healthy large follicles (>8 mm) obtained from slaughterhouse ovaries were cultured in serum free medium in the presence of 0, 50, 150 and 200 microM of palmitic acid (PA; C16:0); 0, 50, 150 and 250 microM of stearic acid (SA; C18:0); and/or 0, 50, 150 and 250 microM of oleic acid (OA; C18:1). Progesterone and androstenedione concentrations were measured in spent medium after 48 h of culture and cell numbers were determined spectrophotometrically per culture well. Cell viability was assessed by annexin-V FITC/propidium iodide staining. Only the treatment with 200 microM of PA inhibited cell proliferation (P<0.001) when tested individually, both of the mixtures tested (M1=100 microM of PA, 130 microM of SA and 140 microM of OA; M2=200 microM PA, 260 microM of SA and 280 microM of OA) reduced cell numbers (P<0.001). Progesterone and androstenedione production, both per well and per 10(4) cells, were not affected by any of the treatments, with the exception of M2. This mixture reduced progesterone production per well and per 10(4) cells (P<0.05). The effects observed were most likely caused by the cytotoxic action of the NEFAs, as demonstrated by the increased percentage of early apoptotic (M1) and late apoptotic/necrotic cells (M1 and M2) in the combination treatments (P<0.05). When combined, elevated physiological concentrations of PA, SA and OA can modulate theca cell proliferation and steroidogenesis in vitro by reducing theca cell viability. These NEFAs may be one of the mediators through which NEB compromises ovarian functioning and thus fertility in high-yielding dairy cows.     

Production of tissue inhibitors of metalloproteinases (TIMPs) by pig ovarian cells in vivo and the effect of TIMP-1 on steroidogenesis in vitro

Precisely which ovarian cells produce tissue inhibitors of metalloproteinases (TIMPs) is unclear. Although granulosa cells are reported to produce TIMPs, thecal TIMP production has not been investigated nor has the influence of TIMPs on theca cells. Furthermore, although periovulatory follicles have been examined, little is known about smaller ovarian follicles. Follicles >/= 2 mm in diameter were collected from Large White hybrid gilts on the day before predicted oestrus (n = 3) or after hCG treatment (n = 3) and divided into 1 mm size classes. Small (2 to < 5 mm) follicles were kept intact, whereas follicles >/= 5 mm were separated into follicular fluid, granulosa and theca cell compartments. After homogenization, TIMP-1, -2 and -3 were detected by reverse zymography. Theca cells (50 x 10(3) per well) were cultured with TIMP-1 (10, 100 or 200 ng ml(-1) with or without long-R3 insulin-like growth factor I (IGF-I)) in a serum-free system to investigate the effect on steroidogenesis and the number of cells. Both large and small pig follicles produced TIMPs and TIMP-1, -2 and -3 were detected in follicular fluid, granulosa and theca cell samples. There was a phase x tissue type interaction for the presence of both TIMP-1 and -2 (P < 0.03, P < 0.05, respectively), and TIMPs were detected in more granulosa and theca cell samples after hCG than during the follicular phase. The concentrations were influenced by the type of tissue (TIMP-1, P < 0.005; TIMP-2, P < 0.005, TIMP-3, P > 0.05), and the highest concentrations occurred in the theca tissue. There were tissue type x follicle size interactions for the presence of both TIMP-1 and -2 (P < 0.001). In vitro, TIMP-1 increased thecal steroidogenesis after 144 h (oestradiol, P < 0.05, progesterone, P < 0.001) but reduced the number of viable cells (P < 0.001). In conclusion, TIMP-1, -2 and -3 were present in large and small pig follicles and were produced by both granulosa and theca cells, although concentrations differed with the type of tissue. Production was regulated by factors including follicle size and phase of the oestrous cycle. In addition to controlling tissue remodelling, TIMP-1 may also regulate steroidogenesis.     

Alteration of follicular steroid secretion and thecal morphology after in-vitro exposure of individual pig follicles to follicle regulatory protein

Medium-sized (4-6 mm) pig follicles were incubated for 10 h and then examined via light microscopy. Treatment with pig FSH resulted in significantly increased concentrations of oestradiol, testosterone, androstenedione and progesterone in the medium. Follicle regulatory protein (FRP) alone (1 micrograms/ml) decreased follicular secretion of oestradiol (56%) and progesterone (53%) but stimulated the secretion of testosterone (226%) and androstenedione (139%). In the presence of 1 ng FSH/ml, the inhibitory effect of FRP on oestradiol secretion was enhanced (74%), progesterone values were unaffected and secretion of testosterone and androstenedione were reduced by 66% and 53%, respectively. All effects of FRP were fully overcome by 1 micrograms FSH/ml. The incidence of atresia, as defined by granulosa cell pycnosis, was similar in all treatment groups (1-3 of 10 follicles per group). The remaining follicles had intact granulosa cells. However, follicles treated with FRP (1 micrograms/ml) + FSH (1 ng/ml) had pycnotic nuclei in the theca interna cells, in the presence of an intact stratum granulosum. External exposure of follicles to FRP may not reflect physiological conditions since, in vivo, thecal pycnosis is never observed before granulosa cell pycnosis. However, the present results indicate that FRP is potentially capable of altering both follicular morphology and steroidogenesis. We suggest that FSH and FRP interact to affect follicular development.     

Effects of angiogenin on granulosa and theca cell function in cattle

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P<0.05) granulosa cell progesterone production and theca cell androstenedione production but did not affect (P>0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P<0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P<0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.     

Inhibition of thecal androstenedione production by exogenous progesterone in the cyclic hamster

Implants of progesterone on the day of dioestrus II in the hamster induced on the following day an increase in circulating levels of progesterone (6.0 +/- 0.7 ng/ml, N = 8; sesame oil controls, less than 0.5 ng/ml, N = 6) and a decline in serum levels of LH (5.3 +/- 0.4 ng/ml; controls 12 +/- 2 ng/ml) and oestradiol (10 +/- 2 pg/ml; controls 69 +/- 5 pg/ml). The production of androstenedione and oestradiol by antral follicles in vitro was reduced in progesterone-treated hamsters when compared with controls, but progesterone production was not affected. Aromatizing activities of antral follicles were the same in progesterone-treated and sesame oil-treated hamsters. Androstenedione production by theca was significantly less in progesterone-treated hamsters than in controls. On dioestrus II, LH replacement therapy (200 micrograms ovine LH by osmotic minipump inserted s.c.) prevented the decline in follicular androstenedione and oestradiol production induced by progesterone alone, and also prevented the decline in thecal androstenedione production in vitro. The results indicate that exogenous progesterone on dioestrus II lowers circulating levels of LH by the following day, inhibits thecal androstenedione production and thus reduces follicular oestradiol production without alteration in aromatizing ability.     

Bovine theca and granulosa cell interactions modulate their growth, morphology, and function

We have developed a culture system in which bovine granulosa and theca cells are allowed to attach to opposite sides of a collagen membrane. We studied the interaction between theca and granulosa cells by investigating the morphology, proliferation, and steroidogenesis of the cells. Granulosa cells cultured alone were flattened and polygonal and formed monolayer sheets. Granulosa cells cocultured with theca cells formed multilayer sheets. The apical surface of each cell appeared convex. Numerous filopodia spread over the cellular surface connecting cells. Theca cells cultured alone were thin, flat, and spindle-shaped. Theca cells cocultured with granulosa cells were also spindle-shaped; however, the apical surface appeared convex. Cocultured cells were more densely packed than theca cells cultured alone. The number of both granulosa and theca cells in the cocultured group increased approximately twofold compared to control cells cultured alone. Progesterone content per 1 x 10(5) granulosa cells in 24-h culture medium of the cocultured group was reduced to 40% of that of the control group. In contrast, androstenedione content per 1 x 10(5) theca cells of the cocultured group increased approximately threefold compared to androstenedione content of control group. These results indicate that communication between these two types of follicular cells results in reciprocal modulation of their proliferation, morphology, and function.     

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.     

Human granulosa cells in vitro: characteristics of growth, morphology and influence of some cytokines on steroidogenesis.

Granulosa cells (GCs) were characterised morphologically by light and electron microscopy. The steroidogenic capability of GCs in vitro was estimated by radioimmunoassay (RIA): oestradiol (E2), progesterone (P) and androstenedione (A) secreted into the culture medium were measured. The influence of several culture media and anchorage of the cell either to plastic vessels (monolayer) or to collagen fibrils (in gel) were studied. As the various culture media were assayed with regard to their suitability for IVF, it was found that Ham's F10 is quite satisfactory (in agreement with other observations on embryo cultures). A chemically defined medium BM 86 was found to be inadequate. In addition to the two cell types which are known, a third cell type which can perform efficient aromatisation (E2 production) in vitro is characterised here. The influence of cytokines/growth factors (GF) like insulin-like GF (IGF-1), epidermal GF (EGF), platelet-derived GF (PDGF) and fibroblast GF (FGF) on steroidogenesis was tested either alone or with human chorionic gonadotrophin (hCG). Except for oestradiol (E2) from early GCs, hCG generally stimulated progesterone (P) and E2 secretion. EGF by itself enhanced the secretion of P but not of E2. EGF did not affect hCG stimulation of P, but reduced that of E2. In contrast, in pre-ovulatory GCs IGF-I reduced the stimulatory effect of hCG on both E2 and P. In early GCs IGF-I potentiated hCG stimulation of P. In early GCs, neither hCG nor IGF-I nor a combination of IGF-I with hCG had any effect on E2 production.     

Growth differentiation factor 9 (GDF9) stimulates proliferation and inhibits steroidogenesis by bovine theca cells: influence of follicle size on responses to GDF9

Ovarian follicular development is controlled by numerous paracrine and endocrine regulators, including oocyte-derived growth differentiation factor 9 (GDF9), and a localized increase in bioavailable insulin-like growth factor 1 (IGF1). The effects of GDF9 on function of theca cells collected from small (3-6 mm) and large (8-22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis, as measured by a (3)H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells, but had no effect on IGF1R, STAR, or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated similar to mothers against decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells, but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development.     

Leptin in the bovine corpus luteum: receptor expression and effects on progesterone production

In cattle, leptin has been implicated in the control of ovarian function and has been shown to modulate steroid production by theca and granulosa cells in a number of species. However, a direct effect of leptin on bovine luteal function has not been demonstrated. This study was conducted to determine if the leptin receptor (OB-R) is expressed in the bovine corpus luteum (CL), and to examine the effects of leptin on progesterone production by dispersed luteal cells in vitro. RT-PCR was used to detect the presence of OB-R and, more specifically, the long, biologically active isoform (OB-Rb), in CL, collected on days 2-18 of the oestrous cycle (n=18). The effects of leptin on progesterone production were investigated in dispersed luteal cells prepared from CL collected on days 5 and 8 (n=14) of the cycle. The dispersed luteal cells were cultured for 24 hr with recombinant human leptin and/or LR3-IGF-1 and/or LH. OB-Rs, in particular, OB-Rb, were expressed in the CL at all stages of development. Progesterone production by luteal cells was increased (P<0.001) by treatment with LH (10 ng/ml) but treatment with leptin alone had no effect. However, in the presence of IGF-1 (100 ng/ml), leptin (10 ng/ml) caused a significant (P<0.005) increase in progesterone production. In conclusion, we have shown that the leptin receptor is expressed in the bovine CL and have demonstrated a modulatory effect of leptin on luteal progesterone production in vitro.     

Decreased androstenedione production with increased follicular maturation in theca cells from the domestic hen (Gallus domesticus)

Collagenase-dispersed theca cells from the 3rd and 4th largest ovarian follicles (T3) were responsive to LH stimulation of both oestrogen and androstenedione production, whereas theca cells from the largest follicle (T1) failed to respond to the gonadotrophin stimulation. Similarly, 8-bromo cAMP and forskolin were more effective in stimulating oestrogen and androstenedione production in T3 than in T1 cells, indicating that post-receptor events were involved in the decreased LH responsiveness of T1 cells. The C17-20-lyase activity, as measured by conversion of [3H]17-hydroxyprogesterone to androstenedione, was greatly reduced in T1 cells as compared to T3 cells. The results demonstrate that a decrease in C17-20-lyase activity, in addition to a decrease in aromatase activity, contributes to the loss of LH-stimulated steroidogenesis in mature theca cells.     

Development of a long-term serum-free culture system for immature granulosa cells from diethylstilboestrol-treated prepubertal rabbits: influence of androstenedione and fibronectin on FSH-induced cytodifferentiation

Granulosa cells from diethylstilboestrol-treated prepubertal rabbits were cultured for 6 days in M199 with FSH (1-100 ng ml(-1)) in uncoated or fibronectin-coated plates with or without androstenedione to define the time course profile of oestradiol and progesterone secretion, and the possible modulator role of androstenedione and fibronectin during FSH-induced rabbit granulosa cell differentiation. Every 48 h, cultures were photographed and samples of medium were collected and assayed by ELISA for oestradiol and progesterone. FSH increased oestradiol secretion in a dose-dependent manner. Androstenedione augmented FSH-stimulated oestradiol secretion, and led to a decrease in secretion of oestradiol with time in culture. FSH stimulated progesterone secretion in a dose-dependent manner. This was increased by androstenedione with 10 ng FSH ml(-1) (0-96 h) and 1 ng FSH ml(-1) (96-144 h). FSH-stimulated (100 ng ml(-1)) progesterone secretion decreased at 48-96 h. Fibronectin prevented this decrease, without affecting oestradiol or progesterone secretion at other time points. FSH caused cell reaggregation at 48 h. In conclusion, this serum-free culture system is appropriate for the study of mechanisms of rabbit granulosa cell differentiation. FSH induced cytodifferentiation and reaggregation of granulosa cells. Androstenedione appeared to act synergistically with FSH to promote steroidogenesis. Fibronectin sustained progesterone secretion during differentiation.     

Insulin enhances luteinizing hormone-stimulated steroidogenesis by porcine theca cells

It has been shown recently that insulin enhances differentiation of rat, pig, and human granulosa cells. The present studies were done to determine if insulin also plays a role in the regulation of theca cell steroidogenesis. Theca cells were obtained from prepubertal gilts and cultured under serum-free conditions for 48 h. Theca cell androstenedione production under basal and luteinizing hormone (LH)-stimulated conditions was significantly increased by adding insulin (1 microgram/ml) to the culture medium. Treatment of basal and LH-stimulated cultures with increasing concentrations of insulin (0.001-10 micrograms/ml) caused dose- and time-dependent increments in androstenedione production, but the effect was independent of the dose of LH employed. The ability of insulin to enhance thecal cell androstenedione production was mimicked by somatomedin C, but not by relaxin. Studies to determine the mechanism(s) of action of insulin showed that insulin action is exerted, at least in part, at a site(s) proximal to cyclic adenosine 3'5'-monophosphate (cAMP) generation, since insulin enhanced both basal and LH-stimulated accumulation of extracellular cAMP in addition to increasing androstenedione production. This effect was further enhanced by 3-isobutyl-1-methyl xanthine, an inhibitor of phosphodiesterase activity. Insulin treatment also caused dose-dependent increments in forskolin- and prostaglandin E2-stimulated accumulation of extracellular cAMP and androstenedione. Insulin also increased both the basal and LH-stimulated production of progesterone and its precursor pregnenolone, in addition to the increases in androstenedione.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kit ligand actions on ovarian stromal cells: effects on theca cell recruitment and steroid production

Factors that control recruitment of theca cells from ovarian stromal-interstitial cells are important for early follicle development in the ovary. During recruitment, theca cells organize into distinct layers around early developing follicles and establish essential cell-cell interactions with granulosa cells. Recruitment of theca cells from ovarian stromal stem cells is proposed to involve cellular proliferation, as well as induction of theca cell-specific functional markers. Previously, the speculation was made that a granulosa cell-derived "theca cell organizer" is involved in theca cell recruitment. Granulosa cells have been shown to produce kit-ligand/stem cell factor (KL). KL is known to promote stem cell proliferation and differentiation in a number of tissues. Therefore, the hypothesis was tested in the current study that granulosa cell-derived KL may help recruit theca cells from undifferentiated stromal stem cells during early follicle development. The actions of KL were examined using adult bovine ovarian fragment organ culture and isolated ovarian stromal-interstitial cells. In organ culture KL significantly increased the number of theca cell layers around primary follicles. Experiments using purified stromal-interstitial cell cultures showed that KL stimulated ovarian stromal cell proliferation in a dose-dependent manner. Stromal cell differentiation into theca cells was analyzed by the induction of theca cell functional markers (i.e., androstenedione and progesterone production). Bovine ovarian stromal cells produced low levels of androstenedione (5-40 ng/microg DNA) and progesterone (5-30 ng/microg DNA) in vitro that were approximately 20-fold lower than theca cells under similar conditions. Treatment with KL did not affect ovarian stromal cell androstenedione or progesterone production. Interestingly, hormones such as estrogen and hCG did stimulate stromal cell steroid production. The results in this study suggest that granulosa cell-derived KL appears to promote the formation of theca cell layers around small (i.e., primary) ovarian follicles. KL directly stimulated ovarian stromal cell proliferation but alone did not induce functional differentiation (i.e., high steroid production). Therefore, KL is proposed to promote early follicle development by inducing proliferation and organization of stromal stem cells around small follicles. Observations suggest that KL may act as a granulosa-derived "theca cell organizer" to promote stem cell recruitment of ovarian stromal cells in a manner similar to the way that KL promotes hematopoietic and lymphoid stem cells in bone marrow and the thymus.     

Growth differentiation factor-9 stimulates rat theca-interstitial cell androgen biosynthesis

Growth differentiation factor-9 (GDF-9) was shown recently to be essential for early follicular development, including the appearance of the theca layer. Theca cells provide the androgen substrate for aromatization and estrogen production by granulosa cells. Using biologically active recombinant GDF-9 (rGDF-9) and an androgen-producing immortalized theca-interstitial cell (TIC) line or primary TIC, we have examined the action of this paracrine hormone on theca cell steroidogenesis. The effect of GDF-9 on TIC progesterone synthesis was marginal and inconsistent in the primary cultures. In immortalized theca cells, GDF-9 attenuated the forskolin-stimulated progesterone accumulation. More significantly, this oocyte-derived growth factor enhanced both basal and stimulated androstenedione accumulation in the primary and transformed TIC cultures. The effects of GDF-9 on steroidogenesis by preovulatory follicles were relatively modest. Likewise, it did not affect the maturation of follicle-enclosed oocytes. The effect of GDF-9, an oocyte product, on TIC androgen production suggests a regulatory role of the oocyte on theca cell function and hence on follicle development and differentiation. This direct effect of GDF-9 on thecal steroidogenesis is consistent with its recently demonstrated actions on thecal cell recruitment and differentiation.     

Ovarian steroidogenesis during follicular maturation in the domestic fowl (Gallus domesticus)

The steroidogenic potential of various physiological compartments within the ovary of the hen were examined using in vitro systems. Three-hour incubations of individual whole small follicles (less than 1 mm-1 cm) or 100,000 collagenase-dispersed theca cells of the five largest ovarian follicles (F1-F5) were conducted in 1 ml of Medium 199 at 37 degrees C in the presence and absence of luteinizing hormone (LH) (0.39, 0.78, 1.56, 3.13 and 6.25 ng), progesterone (5 ng), and dehydroepiandrosterone (DHEA, 5 ng). Steroid output was measured by radioimmunoassay of incubation media. Progesterone was not produced by small follicles although they are a major source of DHEA and estradiol and a significant source of androstenedione. Output of DHEA, androstenedione and estradiol was highly stimulated by LH. The substrate for androstenedione and estradiol in small follicles is probably DHEA. Output of DHEA and androstenedione in theca cells of F2-F5 was stimulated by LH in a dose-related manner. A dose-response relationship between estradiol output and the concentration of LH in media was not apparent in theca cells from F2-F5. Steroidogenesis in theca tissue of large follicles occurs predominantly via the delta 4 pathway. The ability of these theca cells to metabolize progesterone to androstenedione is lost between 36 and 12 h before ovulation. Their ability to metabolize DHEA to androstenedione is still present 12 h before ovulation. Aromatase activity is significantly reduced between 36 and 12 h before ovulation. These data indicate that both large and small follicles can be stimulated by LH. The small follicles are the major source of estrogen. As the large yolky follicles mature, steroidogenesis shifts from the delta 5 to the delta 4 pathway. By 12 h before ovulation, the F1 follicle has lost the ability to convert progesterone to androstenedione. The inability of the largest ovarian follicle to convert progesterone to androstenedione contributes at least in part to the preovulatory increase in the plasma concentration of progesterone that generates the preovulatory LH surge by positive feedback.     

Steroid production by isolated theca and granulosa cells after initiation of atresia in the hamster

Hypophysectomized PMSG-primed hamsters were injected with PMSG antiserum and the theca and granulosa cells of the resulting atretic follicles were incubated in vitro. In the absence of added hormone, 17 alpha-hydroxyprogesterone and oestradiol production was not detectable in granulosa cells collected and incubated at 0, 12 and 24 h after antiserum. Progesterone production was not detected in control incubations at 0 h but was measurable with cells collected at 12 h after PMSG antiserum. When incubated with androstenedione or pregnenolone (10 ng/ml for each) 17 alpha-hydroxyprogesterone and progesterone production by granulosa cells were significantly increased at 0, 12 and 24 h after antiserum. Granulosa cells were capable of aromatizing androstenedione to oestradiol at all times examined. At 0 and 12 h after antiserum to PMSG, isolated thecal shells produced androstenedione. LH stimulation caused increased androstenedione production in all thecae at 0 h, in 50% of the thecae at 12 h and in none at 24 h after antiserum. Thecal shells produced 17 alpha-hydroxyprogesterone in response to LH at 0, 12 and 24 h after antiserum, and produced progesterone at all times examined. Thecae also responded to LH with increased progesterone production up to 72 h after antiserum. These experiments demonstrate that one important steroidogenic event in atresia may be the loss of activity of C 17,20 lyase in the theca leading to loss of substrate (androstenedione) for granulosa cell aromatization, although aromatase activity is present until at least 24 h after the induction of atresia.     

Inhibition of androgen and oestrogen production by clomiphene citrate in avian theca cells

Isolated theca cells (2 X 10(5)/ml) were pre-incubated for 1 h in the presence or absence of clomiphene citrate (10(-12)-10(-4) M). Ovine LH (50 ng/ml) was added and cells were incubated for an additional 3 h. A 50% inhibition of LH-stimulated androstenedione and oestrogen production was obtained with doses of 10(-8) M and 2 X 10(-7) M clomiphene, respectively. Furthermore, the effect of clomiphene on LH-stimulated androstenedione production was reversed by washing clomiphene from the cells before stimulation with LH. In subsequent experiments, the effects of clomiphene on C17-20-lyase and aromatase activities were examined. Conversion of [3H]17-hydroxyprogesterone to androstenedione was inhibited by 50% when theca cells were pretreated with 10(-5) M-clomiphene. In addition, conversion of testosterone to oestrogen by theca cells was inhibited in a dose-dependent manner by clomiphene, with 50% inhibition occurring at a dose of 5 X 10(-6) M. The results show that clomiphene treatment in vitro inhibits androgen and oestrogen production in theca cells by inhibitory effects on the activities of C17-20-lyase and aromatase. In addition to the widely-accepted effects of clomiphene on the hypothalamic-pituitary axis, the present findings add further support to the suggestion that clomiphene exerts direct effects on ovarian steroidogenesis.