Steroidogenesis in luteinized granulosa cell cultures varies with follicular priming regimen

During follicular development, a co-ordinated gonadotrophin and endocrine environment is believed to be essential for normal function of the resulting corpus luteum. Whether differences in the gonadotrophins used to promote follicular development can have lasting effects on granulosa cells after they have undergone luteinization and culture, remains to be studied. We measured steroid production under basal and human chorionic gonadotrophin (HCG) stimulation in short and long term cultures of luteinizing granulosa cells obtained from normal ovulatory women undergoing assisted folliculogenesis with either human menopausal gonadotrophin (HMG) or follicle stimulating hormone (FSH). Basal progesterone and oestradiol production by luteinized granulosa cells obtained from follicles stimulated to develop with FSH was significantly greater than that from HMG derived follicles (P < 0.001). In short term cultures, treatment with 10 IU HCG caused a 10-fold increase in progesterone release by cells from FSH stimulated follicles, whereas cells of HMG origin produced only 5-fold more progesterone (P < 0.0001). In cultures that were maintained for 2 weeks, progesterone secretion was reduced, but a similar trend in HCG responsiveness was observed. These experiments demonstrate that the composition of the gonadotrophins used to promote follicular development in vivo leads to differences in granulosa cell steroidogenesis which are evident after luteinization and culture. They additionally support the notion that the environment of follicular development will be reflected in the resulting corpus luteum.     

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We report the establishment and preliminary characterization of a stable steroidogenic granulosa cell line, JC-410. This cell line was obtained by spontaneous immortalization of a primary culture of porcine granulosa cells. Cultured JC-410 cells produced less progesterone than granulosa cells in primary culture. Progesterone synthesis by JC-410 cells was approximately 10% and 1% of the amount produced by granulosa cells from small and medium sized follicles, respectively. Although FSH and LH did not change progesterone levels in cultured JC-410 cells, forskolin and cholera toxin induced a 2.6- and 2.75-fold increase, respectively, versus control. The JC-410 cells responded to 0.1, 1 and 5 mM cAMP with an increase in progesterone synthesis of 2.5-, 28- and 49-fold versus control, respectively, after a 24 h incubation. No detectable levels of estradiol-17beta were found in JC-410 cells after 48 h in culture. However, addition of 0.01, 0.1 and 1 microM androstenedione elevated the levels of estradiol-17beta to 0.028, 0.3 and 1.21 pg/microg protein, respectively. The level of expression of 3betaHSD, aromatase and P450scc genes in JC-410 cells is of similar magnitude to the level of expression in granulosa cells in primary culture. The JC410 cells have been maintained in culture for more than one year during which their population doubled over 100 times. We conclude that JC-410 is a stable cell line that lost responsiveness to the gonadotropins during the process of immortalization, but retained its steroid biosynthetic capability and the expression of key steroidogenic genes. These characteristics may reflect features of cells arrested in an early stage of granulosa cell differentiation.     

Uncoupling between proliferation and differentiation of ovine granulosa cells in vitro

Granulosa cells of ovarian follicles both proliferate and undergo differentiation. In vivo, an inverse relationship between proliferation and steroidogenesis is observed. However, both processes can be enhanced by insulin-like growth factor-I (IGF-I) in vitro. Studies were undertaken in the ewe to understand the mechanisms controlling the balance between proliferation and differentiation in cultured granulosa cells from antral follicles better. For this purpose, granulosa cells from ovine small follicles (1-3 mm in diameter) and large follicles (5-7 mm in diameter) were compared for progesterone secretion, cytochrome P450 side-chain cleavage (P450scc) expression and their proportions of non-proliferating (G0) cells, in response to IGF-I and FSH stimulation in vitro. IGF-I mainly enhanced the proliferation of granulosa cells from small follicles but it strongly increased progesterone secretion and P450scc expression in granulosa cells from large follicles, in synergy with FSH. Blocking granulosa cell proliferation by the administration of colcemid or aphidicolin had no effect or a weak stimulating effect on progesterone secretion. At the beginning of the culture period, the proportion of non-proliferating cells, estimated by continuous [3H]thymidine labelling experiments, was clearly higher in large than in small follicles (91% vs 30%, P < 0.001). For both cell types, treatment with IGF-I in vitro reduced the proportion of non-proliferating cells at 72 h of culture (40% vs 70% respectively in IGF-I-stimulated and unstimulated cells from large follicles, P < 0.001, and 17% vs 30% respectively in IGF-I-stimulated and unstimulated cells from small follicles, P < 0.001). Treatment with FSH had no effect on the proportion of non-proliferating cells. As revealed by immunohistochemistry experiments, IGF-I, in synergy with FSH, clearly increased the percentage of cells expressing P450scc enzyme and the intensity of staining in granulosa cells from large follicles. Unexpectedly, heavily stained cells in mitosis were observed in IGF-I-stimulated cells from large follicles after 96 h of culture, suggesting that dividing cells might also produce progesterone. Overall, these results support the hypothesis that the growth-promoting and the cytodifferentiative effects of IGF-I are clearly distinct. Moreover, they suggest that uncoupling between proliferation and steroidogenesis may occur in cultured ovine granulosa cells. The loss of proliferative activity accompanying terminal follicular growth in vivo could be reversed in vitro. During terminal follicular growth in vivo, the existence of an active mechanism inhibiting granulosa cell proliferation, and unrelated to terminal differentiation, is therefore strongly suspected.     

Induction of Ad4BP/SF-1, steroidogenic acute regulatory protein, and cytochrome P450scc enzyme system expression in newly established human granulosa cell lines

We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 microM of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20alpha-hydroxy-4-pregnen-3-one (20alpha-OH-progesterone) comparable with amounts produced by highly differentiated primary human granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment the cAMP-stimulated progesterone production, whereas testosterone and PRL enhanced cAMP-induced progesterone synthesis only moderately. Estradiol, insulin-like growth factor I, and insulin showed no significant effect on cAMP-induced steroidogenesis. The phorbol ester TPA, and basic fibroblast growth factor, dramatically suppress cAMP-induced production of progesterone, whereas bovine corneal endothelial cell ECM (BCE/ECM) enhanced cAMP-induced progesterone and antagonized basic fibroblast growth factor suppression of cAMP-induced steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in control cells, and its expression was augmented by FK, whereas the steroidogenic acute regulatory protein showed low expression in the nonstimulated cells but was clearly elevated upon cAMP stimulation and was slightly decreased by TPA in cAMP-stimulated cells. Expression of the electron carrier adrenodoxin (ADX), which is a part of the cytochrome P450scc enzyme system, was very low in nonstimulated cells but was dramatically elevated in FK- and 8-Br-cAMP-stimulated cells, whereas no reduction of ADX was evident in cells costimulated with FK and TPA. Immunocytochemical studies revealed a weak staining of ADX in mitochondria of nonstimulated cells and intensive staining in highly clustered mitochondria of FK- or 8-Br-cAMP-stimulated cells. Only moderate reduction in ADX staining was evident in cells costimulated with FK and TPA. These unique cell lines can provide a useful model for the investigation of induced steroidogenesis in human granulosa cells.     

Differential responses of granulosa cells from small and large follicles to follicle stimulating hormone (FSH) during the menstrual cycle and acyclicity: effects of tumour necrosis factor-alpha

This study determined effects of follicle stimulating hormone (FSH) alone and in combination with tumour necrosis factor (TNF), on granulosa cells from small (5-10 mm diameter) and large (>10-25 mm) follicles during follicular and luteal phases of the cycle and during periods of acyclicity. Granulosa cells were collected from ovaries of premenopausal women undergoing oophorectomy. The cells were cultured with human FSH (2 ng/ml) and testosterone (1 microM) in the presence or absence of human TNF-alpha (20 ng/ml). Media were removed at 48 and 96 h after culture and progesterone, oestradiol and cAMP in media were measured by radioimmunoassays. FSH stimulated the accumulation of oestradiol from granulosa cells of small follicles during the follicular and luteal phases but not during acyclicity; and TNF reduced oestradiol accumulation in the presence of FSH. Interestingly, in granulosa cells from small follicles, progesterone and cAMP secretion increased in response to FSH and neither was affected by TNF. Thus, TNF specifically inhibited the conversion of testosterone to oestradiol in granulosa cells from small follicles. FSH stimulated oestradiol production by granulosa cells of large follicles obtained only during the follicular phase of the cycle and TNF inhibited the FSH-induced oestradiol secretion. Granulosa cells obtained from large follicles during the luteal phase and during acyclicity did not accumulate oestradiol in response to FSH. However, FSH increased progesterone and cAMP secretion by granulosa cells obtained from large follicles during the follicular and luteal phases. During the luteal phase alone, TNF in combination with FSH increased progesterone accumulation above that of FSH alone. FSH did not increase progesterone, oestradiol or cAMP secretion by granulosa cells obtained from large follicles during acyclicity. Thus, FSH increases progesterone, oestradiol and cAMP secretion by granulosa cells of small follicles during the follicular and luteal phases and TNF appears to inhibit FSH-induced oestradiol secretion specifically in those cells. In large follicles, FSH-stimulated granulosa cell secretion of oestradiol is limited to the follicular phase and this effect can be inhibited by TNF. In addition, when granulosa cells of large follicles do not increase oestradiol secretion in response to FSH, TNF stimulates progesterone secretion.     

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Steroidogenic acute regulatory protein (StAR), a 30-kDa protein involved in the transport of cholesterol to inner mitochondrial membrane during stimulation of steroid hormone biosynthesis, has recently been cloned from human adrenals and MA-10 mouse Leydig tumor cells. We examined the regulation of StAR mRNA accumulation upon induction of steroidogenesis in immortalized rat granulosa cells. Granulosa cells were transfected with SV40 DNA alone (POGS5); with SV40 DNA and Ha-ras oncogene (POGRS1); with SV40 DNA, Ha-ras oncogene and LH/CG receptor (GLHR15) or with FSH receptor (GFSHR17) or with the beta 2-adrenergic receptor (G beta 2AR13) expression plasmids. Cells were cultured to confluency and then stimulated for 24 h with oFSH (4 nM), hCG (2.4 nM), isoproterenol (10 microM) or forskolin (50 microM). By quantitative RT-PCR, StAR mRNA was undetectable in non-steroidogenic cells (transfected with SV40 DNA alone, POGS5) either in the presence or in the absence of forskolin. In contrast, variable amount of the message was detected in all steroidogenic cell lines cotransfected with SV40 DNA and Ha-ras. Moreover, an increase in the StAR mRNA expression was evident in all steroidogenic cells upon stimulation with their respective agonists, concomitantly with enhanced progesterone production. The RT-PCR product was sequenced and the 379 base pairs of rat StAR were found to be 93% and 86% identical to mouse and human cDNA, respectively. The deduced 126 amino acid sequence was 95%, 88% and 88% identical to the mouse, human and bovine deduced protein sequences. We conclude that StAR message is expressed only in the steroidogenic rat granulosa cells and can be upregulated by FSH, hCG, isoproterenol and forskolin in the appropriate cell lines. In addition, we find that the rat StAR cDNA exhibit a high degree of homology with the mouse and human sequences.     

A bolus of recombinant human follicle stimulating hormone at midcycle induces periovulatory events following multiple follicular development in macaques

The efficacy of follicle stimulating hormone (FSH) as an alternative to luteinizing hormone (LH)/human chorionic gonadotrophin (HCG) for the initiation of periovulatory events in primate follicles is unknown. A single bolus of 2500 IU recombinant (r)-hFSH was compared to 1000 IU r-HCG for its ability to promote oocyte nuclear maturation and fertilization, granulosa cell luteinization and corpus luteum function following r-hFSH (60 IU/day) induction of multiple follicular development in rhesus monkeys. Following the r-hFSH bolus, bioactive luteinizing hormone concentrations were <3 ng/ml. Peak concentrations of serum FSH (1455+/-314 mIU/ml; mean+/-SEM) were attained 2-8 h after r-hFSH, and declined by 96 h. Bioactive HCG concentrations peaked between 2-8 h after r-HCG and remained > or = 100 ng/ml for >48 h, while immunoreactive FSH concentrations were at baseline. The proportion of oocytes resuming meiosis and undergoing in-vitro fertilization (IVF) were comparable for r-hFSH (89%; 47+/-19%) and r-HCG (88%; 50+/-17%). In-vitro progesterone production and expression of progesterone receptors in granulosa cells did not differ between groups. Peak concentrations of serum progesterone in the luteal phase were similar, but were lower 6-9 days post-FSH relative to HCG. Thus, a bolus of r-hFSH was equivalent to r-HCG for the reinitiation of oocyte meiosis, fertilization and granulosa cell luteinization, but a midcycle FSH surge did not sustain normal luteal function in primates.     

Levels of messenger ribonucleic acid for cholesterol side-chain cleavage cytochrome P-450 and 3 beta-hydroxysteroid dehydrogenase in bovine preovulatory follicles decrease after the luteinizing hormone surge

During the follicular/luteal phase shift in steroidogenesis, follicular steroid production changes from predominantly estradiol and androgen secretion before the LH surge to decreased androgen and estrogen and increased progesterone after the LH surge. Our objective was to determine whether changes in progesterone production by the preovulatory follicle are effected via changes in mRNA levels for the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD). Bovine preovulatory follicles were obtained in the early follicular phase (n = 9 follicles), the midfollicular phase (n = 4), or the late follicular phase (after the LH surge, but before ovulation; n = 5). Total RNA extracted from granulosa cells and theca interna at the time of cell isolation or after 24 or 72 h of culture in control or LH-containing medium was subjected to Northern analysis, and autoradiographs were scanned densitometrically. P450scc mRNA levels in granulosa cells were high in the early follicular phase and decreased by 96% after the LH surge (P < 0.05). 3 beta HSD mRNA levels in granulosa cells were 4.2-fold higher in early vs. late follicular phase (P < 0.01). In theca interna, 3 beta HSD mRNA levels were 3.6- and 2.6-fold higher in the early vs. the mid- and late follicular phase (P < 0.05), but levels of P450scc mRNA did not differ significantly with stage of follicular development. After granulosa cells had been cultured for 24 h in control or LH-containing medium, P450scc and 3 beta HSD mRNA had declined dramatically compared to mRNA levels at the time of cell isolation during the early follicular phase (P < 0.01). However, after 72 h in control or LH-containing medium, an increase in P450scc and 3 beta HSD mRNA was observed relative to levels at 24 h (P < 0.01). After 72 h of culture, the signal for P450scc and 3 beta HSD mRNA in granulosa cells exposed to LH was higher than the signal detected in cultures without LH (P < 0.01). Similar changes in message for P450scc were observed in cultured thecal cells. Thus, the previously observed increases in production of progesterone by bovine theca interna and granulosa cells obtained after vs. before the LH surge cannot be explained by an increase in message for P450scc and 3 beta HSD.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of insulin on 3 beta-hydroxysteroid dehydrogenase expression in human luteinized granulosa cells

OBJECTIVE: We determined the relative effects of insulin and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human luteinized granulosa cells. METHODS: Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treated with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3 beta HSD were evaluated. RESULTS: Progesterone production was not affected by insulin alone but increased threefold in the presence of FSH (50 ng/microL) alone. The presence of FSH plus insulin (100 nmol/L) caused a significant increase in progesterone accumulation greater than that of FSH alone. The already high basal levels of 3 beta HSD activity were unaffected by insulin alone but increased 1.7-fold in the presence of FSH. The combination of FSH (50 ng/mL) and insulin (100 nmol/L) increased activity 1.3-fold over FSH alone (P < .02). Insulin (greater than 100 nmol/L) alone increased 3 beta HSD protein content as measured by Western analysis 1.8-2-fold over basal levels, whereas FSH alone increased protein content 2.8-fold, and was further augmented by the addition of insulin in a dose-related fashion up to 3.5-fold over basal levels. Insulin increased 3 beta HSD mRNA twofold over basal levels; FSH alone increased mRNA expression of 3 beta HSD 3.2-fold. In the presence of insulin plus FSH, 3 beta HSD mRNA expression increased 7.6-fold over basal levels. For comparison, insulin also stimulated cytochrome P450 aromatase activity, P450 aromatase protein, and mRNA but to a greater degree than that seen for 3 beta HSD. CONCLUSION: Insulin is a regulator of both 3 beta HSD and aromatase expression in human granulosa cells. Elevated insulin levels could therefore affect steroid production in human granulosa cells and presumably alter the menstrual cycle and fertility.     

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To examine whether luteal phase defect is, in part, causally related to insufficient gonadotrophin stimulation, we compared the relation of the increment of serum progesterone concentrations in response to human chorionic gonadotrophin (HCG) with its basal level at mid-luteal phase. Thirty-eight naturally cycling infertile women aged between 27-41 years old were evaluated for hormonal responses to HCG injection at the mid-luteal phase. We measured luteinizing hormone (LH), follicle stimulating hormone (FSH), oestradiol and progesterone concentrations, before and 1, 2 and 3 h after the administration of HCG (5000 IU, i.m.) 7 days after ovulation verified by ultrasonography. Eleven out of 38 women exhibited progesterone concentrations below 10 ng/ml (low progesterone group), and those remaining showed progesterone concentrations of > or = 10 ng/ml (normal progesterone group). The basal LH, FSH and oestradiol concentrations were essentially the same in both groups. Progesterone concentrations rose significantly 1 h after the injection and levelled off thereafter. The increment of progesterone concentrations at 1 h in the normal progesterone group was 5.7 ng/ml on the average, whereas that in low progesterone group was 1.1 ng/ml. Furthermore, the percentage increase in progesterone concentrations at 1 h in the normal progesterone group was significantly greater than that in the low progesterone group. Both groups equally exhibited significant but marginal increases in oestradiol concentrations 1 h after the injection. LH and FSH concentrations at 3 h decreased significantly in both groups. In summary, HCG readily stimulates progesterone production in normally functioning corpus luteum whereas its stimulatory effect is minimal on malfunctioning corpus luteum. This suggests that luteal phase defect is not caused by inadequate gonadotrophin stimulation and, therefore, does not benefit from HCG administration.     

Heat shock-induced inhibition of acute steroidogenesis in MA-10 cells is associated with inhibition of the synthesis of the steroidogenic acute regulatory protein

The synthesis of heat shock proteins (HSPs) rapidly increases in cells under a broad range of stress conditions in addition to heat shock. Previous studies have shown that the induction of HSPs severely impairs the ability of steroidogenic cells to synthesize steroids in response to acute stimulation. De novo synthesis of the steroidogenic acute regulatory (StAR) protein has been shown to be indispensable for acute steroid hormone biosynthesis; however, the effect of HSP induction on the synthesis of the StAR protein has not yet been studied. In the present study we investigated whether HSP induction might influence the steroidogenic activity of MA-10 mouse Leydig tumor cells, and whether this effect may involve the synthesis of StAR protein. MA-10 cells exposed to 45 C for 10 min and allowed to recover for 2 h at 37 C displayed a 6-fold increase in HSP-70 at 3 h postrecovery and a 20-fold increase in this protein at 6 h postrecovery. This heat shock regimen also acutely inhibited both progesterone production and StAR protein synthesis in MA-10 cells in response to LH and cAMP analog stimulation. The activity and quantity of cytochrome P450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase were not affected by this heat shock treatment, indicating that the loss of steroidogenic capacity was not a result of inhibition of the enzymes involved in the conversion of cholesterol to progesterone. The results suggest that the previously observed antisteroidogenic effects of heat shock treatment may be due mainly to the acute inhibition of StAR protein synthesis.     

Regulation of porcine granulosa cell steroidogenic acute regulatory protein (StAR) by insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist

The transfer of cholesterol from the outer to the inner mitochondrial membrane, where side-chain cleavage occurs to form pregnenolone, is a crucial event in the regulation of steroidogenesis and recently has been demonstrated to be mediated by steroidogenic acute regulatory protein (StAR). We generated a partial porcine StAR complementary DNA (280 bp) by RT-PCR and used the corresponding antisense riboprobe to quantify the control of StAR gene expression by FSH and insulin-like growth factor I (IGF-I) in hormonally responsive swine granulosa cells, which typically manifest synergistic steroidogenic stimulation by these two dominant intrafollicular regulators. RNase protection assays were implemented to investigate the time course of the actions of FSH (100 ng/ml), IGF-I (100 ng/ml), and FSH plus IGF-I on StAR messenger RNA accumulation in serum-free cultures granulosa cells. Treatment with FSH (1.6-fold) or IGF-I (2.7-fold) alone had a small but consistent stimulatory effect on StAR message accumulation (corrected for 18S ribosomal RNA in each lane) at 48 h, whereas only IGF-I stimulated StAR protein expression (at least 6-fold as assessed by Western blot). Notably, the combined effect of FSH plus IGF-I was strongly synergistic and already significant by 24 h and maximal at 48 h (P < 0.001). Protein kinase A agonist, 8-bromoadenosine 3',5'-cAMP (8-bromo-cAMP) (1 mM) alone elicited a 3.5-fold increase in StAR message and more than 3.7-fold increase in StAR protein expression by 48 h. The combination of IGF-I and FSH or 8-bromo-cAMP evoked a 26- to 40-fold (P < 0.001) synergistic rise in StAR message accumulation. StAR protein also showed a similar synergistic pattern of expression driven by IGF-I and FSH or 8-bromo-cAMP, namely a greater than 56- to 60-fold increase. In summary, two distinct first messenger regulatory molecules, FSH and IGF-I, interact synergistically to induce amplification of StAR messenger RNA and protein expression in serum-free monolayer cultures of immature (swine) granulosa cells.     

Interleukin-6 production by rat granulosa cells in vitro: effects of cytokines, follicle-stimulating hormone, and cyclic 3',5'-adenosine monophosphate

In the present study we examined the influence of FSH as well as a number of well-established cytokines on interleukin (IL)-6 by rat granulosa cells in culture. Increasing concentrations of FSH, IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF alpha), and lipopolysaccharide (LPS) were incubated for 48 h with undifferentiated granulosa cells obtained from diethylstilbestrol-primed immature rats. The results demonstrate that FSH, IL-1 alpha, IL-1 beta, and LPS, but not TNF alpha, caused significant concentration-dependent increases in IL-6 release. We also examined the effects of dibutyryl-cAMP, forskolin, and 3-isobutyl-1-methyl-xanthine (IBMX) on IL-6 release by granulosa cells. Each of these agents caused a significant concentration-dependent increase in IL-6 production by granulosa cells in either the absence or presence of FSH. Taken together, these results show that the granulosa cell is not only a likely source of IL-6 but that the release of IL-6 can be regulated. Moreover, evidence suggests that cAMP may serve as a second messenger for the stimulated secretion of IL-6 by undifferentiated granulosa cells.     

Differential modulation of porcine theca, granulosa, and luteal cell steroidogenesis in vitro by tumor necrosis factor

The role of tumor necrosis factor alpha (TNF alpha) in ovarian function was investigated using in vitro culture of theca and granulosa cells isolated from gilt follicles (4-6 mm) and small (SLC) and large (LLC) luteal cells from mid-cycle corpora lutea. TNF alpha did not affect basal accumulation of progesterone (P) by theca cells after 72 h of culture. However, TNF alpha (0.1-100 ng/ml) caused a marked dose-dependent noncytotoxic inhibition (p < 0.05) of LH or LH+insulin (I)-stimulated P accumulation by theca cells after 72 h. Maximal inhibitions averaged 87 +/- 6% at 5 ng/ml TNF alpha for LH-stimulated P and 69 +/- 4% at 50 ng/ml TNF alpha for LH+I-stimulated P. The inhibitory effect of TNF alpha, evident by 24 h after culture, progressively increased on Days 2 and 3 of culture. The effect of TNF alpha on theca cells was mediated by cAMP generation as evidenced by TNF alpha inhibition of LH-induced cAMP accumulation and P accumulation in response to LH and forskolin but not dibutyryl cAMP. Consistent was this, TNF alpha had no effect on increased P accumulation by theca cells in the presence of 22-hydroxycholesterol or pregnenolone alone, but inhibited further increases in P accumulation stimulated by LH plus sterol substrates. Unlike that in theca cells, FSH-induced P accumulation in granulosa cell cultures was slightly enhanced (p < 0.05) by low doses of TNF alpha (0.1, 0.5, and 1.0 ng/ml) after 72 h, while higher doses (5-50 ng/ml) did not alter P accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of progesterone and estradiol production by mouse cumulus and mural granulosa cells by A factor(s) secreted by the oocyte

Mouse oocytes secrete a factor(s) that inhibits progesterone and enhances estradiol production by cumulus granulosa cells. The purpose of this study was to investigate the mechanisms by which the production of these steroids is modulated. Mouse oocyte-cumulus cell complexes (intact) and complexes from which the oocytes were removed microsurgically (oocytectomized; OOX) were cultured for up to 48 h in the presence of FSH (150 ng/ml) and testosterone (5 x 10(-7) M). For these experiments, all cells were obtained from antral follicles of 24- to 26-day-old mice primed with eCG. Intact complexes produced primarily estradiol, with significant accumulation occurring between 24 and 48 h. In contrast, OOX complexes produced little estradiol but, starting at 18 h of culture, released significantly more progesterone than did intact complexes. Progesterone accumulation in cocultures of denuded oocytes with either OOX complexes or monolayers of mural granulosa cells was significantly reduced compared to that with OOX complexes or mural granulosa cells cultured alone. If dibutyryl cAMP replaced FSH in the cocultures, similar results were obtained, suggesting that the oocyte-secreted steroid-regulating factor acts downstream of the generation of cAMP to inhibit progesterone production. Since estradiol can inhibit progesterone production by granulosa cells, we investigated the possibility that the increased progesterone released by OOX complexes was secondary to the lower estradiol production. Intact complexes cultured in the presence of the nonaromatizable androgen, 5 alpha-dihydrotestosterone, or steroidal (4-hydroxyandrostenedione) or non-steroidal (CGS 16949A) aromatase inhibitors produced little estradiol; however, progesterone production by these complexes was no different from that of estradiol-producing intact complexes. These results suggest that the steroid-regulating factor(s) secreted by occytes acts to regulate granulosa cell production of estradiol and progesterone by independent mechanisms.     

An aminopeptidase inhibitor, bestatin, enhances progesterone and oestradiol secretion by porcine granulosa cells stimulated with follicle stimulating hormone in vitro

We examined the presence of cell surface aminopeptidase on cultured porcine granulosa cells by employing the aminopeptidase assay using alanine-p-nitroanilide and histochemical staining using L-leucyl-beta-naphthylamide. Porcine granulosa cells obtained from follicles 4-5 mm in diameter were cultured for 7 days. The aminopeptidase assay showed that the porcine granulosa cell culture had aminopeptidase activity and that this activity was inhibited in a dose-dependent manner by bestatin which binds to cell surfaces and inhibits cell surface aminopeptidases. Histochemical staining also indicated that cultured granulosa cells had aminopeptidase activity. Porcine granulosa cells were cultured in the presence or absence of porcine follicle stimulating hormone (FSH, 3.125 nmol/l) and/or bestatin (0.4, 4.0 and 40.0 micrograms/ml) for 7 days, and the production of progesterone and oestradiol was measured. In the presence of porcine FSH, the production of progesterone and oestradiol by granulosa cells was increased significantly by approximately 5- and 2-fold respectively. These increases were enhanced further by bestatin (40.0 micrograms/ml). In the absence of porcine FSH, progesterone production was enhanced by bestatin (40.0 micrograms/ml), whereas no significant effect of bestatin on oestradiol secretion was observed. These findings indicate that the inhibition of membrane-bound aminopeptidase(s) on the cell surfaces affects the steroidogenesis of granulosa cells, and that these aminopeptidase(s) are important regulators of granulosa cell differentiation.