Bovine theca and granulosa cells interact to promote androgen production

Pieces of theca interna or follicle wall (theca interna + attached granulosa cells), obtained from bovine preovulatory follicles prior to the surge of luteinizing hormone (LH) and cultured for 3 days, secreted androstenedione. Luteinizing hormone, but not follicle-stimulating hormone (FSH), increased production of androstenedione 3 to 4-fold. In both the presence and absence of LH, follicle wall preparations secreted about 4-fold more androstenedione than did equivalent amounts of theca interna tissue. Isolated granulosa cells produced only negligible quantities of androstenedione, which suggests that they may contribute to the greater production of androstenedione by follicle wall by supplying progestin precursor to the theca cells. The addition of pregnenolone or progesterone to isolated theca interna increased the secretion of androstenedione, but pregnenolone was by far the more effective precursor. This suggested that the delta 5 (delta 5) pathway is the preferred pathway for androstenedione synthesis by bovine theca cells and that granulosa cells might supply progestin precursor in the form of pregnenolone. Follicle wall and granulosa cell cultures secreted 2 and 7 times more pregnenolone, respectively, than did theca cultures. Luteinizing hormone, but not FSH, increased production of pregnenolone by the follicle wall, whereas the gonadotropins had no effect on secretion by either granulosa or theca cells. Since exogenous testosterone enhanced the production of pregnenolone by granulosa cells, thecal androgen (which is stimulated by LH) may increase the ability of granulosa cells to make pregnenolone and explain the stimulatory effect of LH on pregnenolone secretion by follicle wall.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

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.     

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.     

The source of follicular androgens in the hamster follicle

The comparative ability of granulosa cells and theca of the hamster preovulatory follicle to produce androgens in vitro from endogenous and exogenous substrates was assessed. The results indicate that theca are the major source of follicular androstenedione, but that the granulosa cells may be the major source of follicular testosterone. Theca and granulosa cells accumulate comparable amounts of dihydrotestosterone from exogenous androstenedione and testosterone and both may be a significant source of follicular DHT. LH stimulates the conversion of progesterone and 17 alpha-OH progesterone to androstenedione, testosterone and DHT in theca. LH does not stimulate the conversion of androstenedione to testosterone or DHT, and that of testosterone to DHT in either granulosa cells or theca. FSH, in granulosa cells but not in theca, stimulates the conversion of adrostenedione to testosterone but it has no effect in DHT accumulation from exogenous testosterone.     

Steroidogenesis by bovine theca interna in an in vitro perifusion system

The aims of these studies were: to examine the steroidogenic responses of perifused bovine theca interna to varying flow rates of media and varying amounts of luteinizing hormone (LH), and to compare the steroidogenic outputs of theca interna from follicles of differing size and health with those of other ovarian tissues. The results showed that the outputs of androstenedione by thecae interna from healthy but not atretic follicles, with or without stimulation by LH, were amplified by the flow rate of media. Steroidogenesis by perifused theca interna was also influenced by the mass and concentration of LH as well as by the duration of exposure to LH. When expressed on a per unit mass basis, the outputs of androstenedione from LH-primed thecae interna from small (2-5.5 mm diameter), medium (6-9.5 mm diameter) and large (greater than or equal to 10 mm diameter) healthy follicles were comparable. But when the above data were expressed per total mass of theca interna, the androstenedione output increased significantly with increasing follicular diameter (P less than 0.01). Under the experimental conditions employed, the fraction of androstenedione produced by thecal tissue as a percentage of the total output of progesterone, androstenedione, testosterone and estradiol was 82%, whereas the progesterone, testosterone and estradiol fractions were 1%, 15% and 2%, respectively. By contrast, the granulosa cell output of progesterone, androstenedione, testosterone and estradiol were 79%, 0%, 0% and 21%, respectively. When this cell type was supplied with saturating amounts of androstenedione, it contributed greater than or equal to 90% of the total quantity of estradiol by the two cell types in isolation.     

In vitro differentiation of bovine theca and granulosa cells into small and large luteal-like cells: morphological and functional characteristics

This study was undertaken to investigate whether bovine granulosa and theca interna cells could be luteinized in vitro into luteal-like cells. Granulosa and theca cells were cultured for 9 days in the presence of forskolin (10 microM), insulin (2 micrograms/ml), insulin-like growth factor I (100 ng/ml), or a combination of these agents. During the first day of culture, granulosa and theca cells secreted estradiol and androstenedione, respectively; progesterone rose only after 3-5 days in culture and reached a maximum on the ninth day of culture. Cells incubated in the presence of forskolin plus insulin exhibited morphological and functional characteristics of luteal cells isolated from the corpus luteum. It was found that cell diameter, basal and stimulated progesterone secretion, and pattern of cell replication for both cell types were comparable to those of luteal cells. Numerous lipid droplets and intensified mitochondrial adrenodoxin staining also indicated active steroidogenesis in luteinized cells. After 9 days in culture, stimulants were withdrawn, and the culture proceeded in basal medium for an additional 5 days; elevated progesterone levels were maintained by luteinized granulosa cells (LGC), whereas in contrast a dramatic drop in progesterone production was observed in luteinized theca cells (LTC). On Day 9, cells were challenged for 3 h with LH (10 ng/ml), forskolin (10 microM), or cholera toxin (100 ng/ml), resulting in a 4-fold increase in progesterone secretion by LTC; the same treatments failed to stimulate progesterone in LGC.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro steroidogenesis by dissociated rat follicles, primary to antral, before and after injection of equine chorionic gonadotropin.

Prepubertal female rats were injected s.c. with 5.0 IU eCG, and ovaries were collected 24 and 48 h post-eCG, on Day 25, as well as from an untreated group also on Day 25. Large antral follicles were manually dissected, and the ovarian remnants were incubated with collagenase overnight to liberate preantral follicles from adhering stromal cells. The viability of the follicles was established by normal histology and lack of pyknotic granulosa cells (GCs) and by their ability to secrete steroids. After a 1-h baseline incubation, either 10 ng LH or 100 ng FSH was added for an additional hour, and the media-before and after gonadotropin administration-were used to measure progesterone, androstenedione, and estradiol by RIA. A distinct hierarchy existed in steroid synthesis, with the maximal production by the largest (700 microm) antral follicles. The major steroid that had accumulated after addition of LH at 48 h post-eCG was androstenedione (1099 pg/follicle per hour), followed by equal amounts of progesterone (155 pg/follicle per hour) and estradiol (191 pg/follicle per hour). There was a precipitous drop in steroid production by 550-microm and 400-microm antral follicles, especially in estradiol for the latter-sized follicles (0.08 pg/follicle per hour). Preantral follicles also produced progesterone and androstenedione after addition of LH. For example, follicles 222 microm in diameter with 4-5 layers of GCs and well-developed theca responded to LH at 48 h post-eCG by accumulating androstenedione (37 pg/follicle per hour) and progesterone (6 pg/follicle per hour) but negligible estradiol. The smallest follicles secreting steroids, 110-148 microm in diameter, had 2-4 layers of GCs. However, primary follicles (1 layer of GCs and no theca) did not synthesize appreciable amounts of any steroid. Although small preantral follicles were consistently stimulated by LH, FSH was ineffective. This result differs from findings in the hamster showing that intact preantral follicles with 1-4 layers of GCs and no theca respond to FSH by secreting progesterone in vitro (Roy and Greenwald, Biol Reprod 1987; 31:39-46). The technique developed to collect intact rat follicles should be useful for numerous investigations.     

The acceleration of reproductive aging in Nrg1flox/flox;Cyp19‐Cre female mice

Irregular menstrual cycles, reduced responses to exogenous hormonal treatments, and altered endocrine profiles (high FSH/high LH/low AMH) are observed in women with increasing age before menopause. In this study, because the granulosa cell‐specific Nrg1 knockout mice (gcNrg1KO) presented ovarian and endocrine phenotypes similar to older women, we sought to understand the mechanisms of ovarian aging and to develop a new strategy for improving fertility in older women prior to menopause. In the ovary of 6‐month‐old gcNrg1KO mice, follicular development was blocked in bilayer secondary follicles and heterogeneous cells accumulated in ovarian stroma. The heterogeneous cells in ovarian stroma were distinguished as two different types: (i) the LH receptor‐positive endocrine cells and (ii) actin‐rich fibrotic cells expressing collagen. Both the endocrine and fibrotic cells disappeared following long‐term treatment with a GnRH antagonist, indicating that the high levels of serum LH induced the survival of both cell types and the abnormal endocrine profile to reduce fertility. Moreover, follicular development to the antral stages was observed with reduced LH and the disappearance of the abnormal stromal cells. Mice treated with the GnRH antagonist regained normal, recurrent estrous cycles and continuously delivered pups for at least for 3 months. We conclude that endocrine and matrix alternations occur within the ovarian stroma with increasing age and that abolishing these alternations resets the cyclical release of LH. Thus, GnRH antagonist treatments might provide a new, noninvasive strategy for improving fertility in a subset of aging women before menopause.     

Significance of the delta 5 and delta 4 steroidogenic pathways in the hamster preovulatory follicle

Isolated hamster granulosa cells and theca from preovulatory follicles were incubated in vitro for 2 and 6 h in the absence/or presence of LH and steroid substrates. The purpose of the experiments was to determine, in theca, the relative activities of the delta 5 and delta 4 pathways under controlled conditions, and to compare the ability of granulosa cells and theca to form progesterone from exogenous pregnenolone. The results of the experiments show that the delta 5 pathway in theca predominates before and up to 2 h after LH stimulation. The delayed effect of LH after 2 h is a switch from delta 5 to delta 4 as the major metabolic pathway. Progesterone formation from exogenous pregnenolone is 7 to 10 times greater in unstimulated granulosa cells than in theca. Acute effects of LH lead to increased conversion of exogenous pregnenolone to progesterone in granulosa cells but not theca. LH does, however, acutely stimulate the thecal conversion of DHEA to androstenedione. The longer term effect of LH in both cell types is to increase pregnenolone conversion to progesterone.     

The regulation of steroidogenesis by opioid peptides in porcine theca cells

The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488). Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells. The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH. All delta receptor agonists depressed basal progesterone (P4) output. However, the influence of these agents on LH-treated cells was negligible. Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release. The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory. Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells. All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells. Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output. Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids. Basal secretion of E2 was also suppressed by kappa agonists. This inhibitory effect was not observed when the cells were additionally treated with LH. In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis.     

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.     

In-vitro effects of angiotensin II on steroid production by hamster ovarian follicles and on ultrastructure of the theca interna

Summary Angiotensin II (AII) is present in the mammalian ovary and has been correlated with atresia in follicles. Since the theca interna may be one site at which atresia is intiated, we wished to determine whether AII exerts an effect on theca interna from explanted ovarian follicles of hamsters. Hamsters were sacrified on the morning of proestrus, and ovaries were removed. Preovulatory follicles were excised from the ovaries, and cultured with one of the following components: medium alone (control); medium plus AII (1x10^-6 M); the AII-receptor antagonist [Sar¹, Ile⁸] AII (1x10^-4 M); or AII plus antagonist. After 72 h, the follicles were processed for transmission electron microscopy (to determine quantities of theca interna organelles involved in the steroid synthetic pathway) or for protein determination (to normalize steroid production rates). The incubation medium was drawn off and analyzed by radioimmunoassay for progesterone, androstenedione, or estradiol-17. There was a significant positive correlation (r=0.92, P<0.01) between follicular androstenedione secretion and area comprising theca interna smooth endoplasmic reticulum. In the theca interna, AII induced a two-fold and 1.6-fold increase in lipid droplet number and area comprising smooth endoplasmic reticulum, respectively (P<0.05). Excess antagonist negated the increase in cell or-ganelles and also reduced androstenedione secretion compared with AII alone (P<0.05). Most importantly, AII significantly augmented the ratio of androstenedione: estradiol-17 secretion by 44% over that of control. The ultrastructural changes observed in this study and the increase in the andostenedione: estradiol-17 production ratio are consistent with atresia-like changes in ovarian follicles. We believe, therefore, that AII is involved, possibly at its membrane receptor, in an aspect of the overall process of follicular atresia, operating in part at the level of the theca interna.     

Cyclic adenosine monophosphate (cAMP) production in avian theca cells during follicular maturation

LH was used to stimulate cAMP production in theca cells from the 5 largest preovulatory follicles of hens and this was related to LH-stimulated androstenedione production in the same cells. cAMP production was stimulated by LH to the same extent in theca cells from each follicle. However, LH was not effective in stimulating androstenedione production in theca cells from the largest follicle (T1), although androstenedione production was greatly increased by LH in the smaller follicles (T2-T5). Effects similar to those of LH on cAMP production were observed in response to forskolin, indicating that the intrinsic adenylate cyclase activity was similar in theca cells from each follicle. In addition, forskolin was unable to stimulate androstenedione production by T1 cells. Our results provide evidence that the levels of receptor-mediated and non-receptor-mediated cAMP production are similar in theca cells from the 5 largest follicles. We conclude that the step that restricts the ability of T1 cells to produce androgen is distal to cAMP generation.     

Effect of progesterone on the GnRH-induced secretion of oestradiol and androstenedione from the autotransplanted ovary of the anoestrous ewe.

Two experiments were conducted during the anoestrous period in Border Leicester x Merino ewes with ovarian autotransplants to study the effects of a single injection of 20 mg progesterone on follicular steroid secretion. The aim of these experiments was to determine whether pretreatment with a 20 mg intramuscular injection of progesterone could reduce GnRH-induced ovarian steroid secretion in anoestrous ewes. In both experiments, an injection of 150 ng GnRH induced an LH pulse in all ewes with a maximum concentration 10 min (the first post-injection sample) after injection. Oestradiol and androstenedione secretion increased progressively after the GnRH-induced LH pulse and reached maximum rates of secretion between 60 and 90 min before decreasing slowly to pre-injection rates at 150 min. There were no differences in the pattern of secretion of oestradiol (measured in both experiments) or androstenedione (measured only in Expt 2). In Expt 1, the injection of progesterone 72 h before the challenge with GnRH had no effect on the maximum rate of oestradiol secretion from the autotransplanted ovary. However, in Expt 2, when progesterone was given either 36 or 60 h before GnRH, there was a significant suppression in the maximum rate of secretion of both oestradiol and androstenedione between 60 and 90 min after GnRH injection. These data show that pretreatment of anoestrous sheep with progesterone can suppress LH-stimulated steroid secretion from the ovary and indicate that progesterone may have a direct effect on oestrogenic follicles in sheep.     

Moderate grade hyperammonemia activates lactate dehydrogenase-4 and 6-phosphofructo-2-kinase to support increased lactate turnover in the brain slices

Interleukin 8 (IL-8) is a chemoattractant associated with ovulation in the mammalian ovary. This chemokine is also involved in the recruitment and activation of neutrophils. Using bovine tissue, we examined the possible role of IL-8 in steroid production by theca cells of the large ovarian follicles. IL-8 promoted progesterone production and stimulated StAR expression in cultured theca cells. The inhibitor of p38 did not disturb the P4 production and StAR expression in IL-8-treated theca cells. On the other hand, the inhibitor of MEK disturbed the P4 production and expression of StAR in theca cells treated with IL-8. These results suggest that IL-8 is associated with progesterone production in bovine theca cells via the MEK pathway.     

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.     

Immunolocalization of MHC-II+ cells in the ovary of immature, young laying and old laying hens Gallus domesticus

The aim of this study was to localize major histocompatibility complex class II positive (MHC-II+) cells in the hen ovary, and to determine the effects of ageing and sex steroids on their frequency. Cryostat sections of ovarian tissues of immature, young laying and old laying hens and those of immature hens treated with or without diethylstilboestrol or progesterone were prepared. Sections were immunostained for MHC class II antigens using mouse anti-chicken MHC class II monoclonal antibody and observed under a light microscope. Positive cells were counted using a computer-assisted image analyser. MHC-II+ cells were localized in the ovarian stroma and theca layer of primary follicles in all birds examined. The frequency of MHC-II+ cells in the stroma and theca of primary follicles (approximately 400-600 microns in diameter) was significantly greater in young laying hens than it was in immature and old laying hens (P < 0.01). In the stroma and the theca of primary follicles of diethylstilboestrol-treated birds, the frequency of MHC-II+ cells was significantly greater than it was in the stroma and theca of control and progesterone-treated birds (P < 0.01). Progesterone had no significant effect when compared with controls. These results indicate that both the ovarian stroma and theca of follicles in the hen ovary contain MHC-II+ cells, the frequency of MHC-II+ cells increases in association with sexual maturation and decreases thereafter during ageing, and oestrogen may be one of the factors enhancing the induction of MHC-II+ cells in the ovary.     

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.