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PubMed1989

Regulation of cholesterol side-chain cleavage and 17 alpha-hydroxylase/lyase activities in proliferating human theca interna cells in long term monolayer culture.

In this report we describe the development and characterization of a long term culture system to study regulation of the expression of 17 alpha-hydroxylase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase in human theca interna cells. Conditions have been established for the dispersal, growth, freezing, and storage of functional human theca interna cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Theca interna cells grown under these conditions have a doubling rate of 28-32 h and are morphologically distinct from human granulosa cells grown under the same conditions. Theca interna cells were grown, passed for successive passages, and transferred into serum-free medium containing forskolin, hCG, LH, or cAMP analogs. There was a time- and dose-dependent increase in 17 alpha-hydroxylase activity and progesterone synthesis from endogenous precursors. Added pregnenolone was converted to 17 alpha-hydroxypregnenolone, which was further converted primarily to dehydroepiandrosterone and, to a much lesser extent, androstenedione. Progesterone was converted to 17 alpha-hydroxyprogesterone and 16 alpha-hydroxyprogesterone. In studies using 17 alpha-hydroxyprogesterone as substrate, no metabolism to androstenedione or any other product was detectable. Similarly, 4-pregnen-20 alpha-ol-one (20 alpha-dihydroprogesterone) was not metabolized to any detectable products. Northern analysis performed on total RNA obtained from forskolin-stimulated theca interna cultures verified that the increase in 17 alpha-hydroxylase activity was associated with a corresponding increase in levels of mRNA specific for 17 alpha-hydroxylase cytochrome P-450. Message levels for cholesterol side-chain cleavage P-450 were similarly increased in cells treated with forskolin. No detectable mRNA encoding aromatase cytochrome P-450 was discerned. This procedure for the preparation and study of proliferating human theca internal cells provides an opportunity to study regulation of the expression of steroidogenic enzymes and other cellular processes unique to human ovarian cells.

Aldehyde-Lyases↗
PubMed2004

Progesterone receptor and the cell cycle modulate apoptosis in granulosa cells.

Our previous studies showed that exposure of bovine preovulatory follicles to the LH surge-induced resistance of granulosa cells, but not theca cells, to apoptosis. Here, the temporal development of resistance to apoptosis and potential roles of progesterone receptor (PR) and alterations in the cell cycle in mediating this effect were examined. Injection of cows with GnRH induced an LH surge within 2 h. Granulosa cells isolated 0, 6, and 10 h after GnRH were sensitive to Fas ligand-induced apoptosis, but cells isolated at 14 h were resistant. PR was first detectable in granulosa cells at 10 and 14 h and was not detectable in theca. Treatment of granulosa cells isolated 14 h after GnRH with the PR antagonist, RU486, induced susceptibility to apoptosis, an effect mediated by PR and not glucocorticoid receptor. After GnRH treatment, granulosa cells, but not theca cells, exited the cell cycle, expression of cyclin D2 was reduced, and p27(Kip1) was elevated. Treatment of granulosa cells isolated from small antral follicles with the G1 phase blocker, mimosine, reduced Fas ligand-induced killing, suggesting that nonproliferating cells are resistant to apoptosis. Treatment of granulosa cells isolated 14 h after GnRH with RU486 induced reentry of some cells into the cell cycle and reversed resistance to apoptosis, suggesting that cycling cells became susceptible to apoptosis. Treatment with mimosine prevented the ability of RU486 to promote susceptibility to apoptosis. In summary, the LH surge induces expression of PR by granulosa cells and withdrawal from the cell cycle, and these events promote resistance to apoptosis.

Animals↗
PubMed1994

Expression of inhibin alpha, beta A and beta B messenger ribonucleic acids in the normal human ovary and in polycystic ovarian syndrome.

We studied the cellular distribution of inhibin alpha, beta A and beta B mRNAs in the normal human ovary and in polycystic ovarian syndrome (PCOS) by in situ hybridization. Our results show that human granulosa cells express inhibin alpha, beta A and beta B subunit mRNAs, and theca cells express inhibin alpha and beta A subunit mRNAs. The co-localization of alpha and beta A mRNAs in theca cells supports the hypothesis that inhibin also has an autocrine function in these cells. We did not detect any inhibin subunit mRNA in the granulosa cells of atretic follicles, while theca cells also expressed alpha subunit mRNA in those follicles. The present findings suggest that the expression of inhibin subunits is regulated differently in human follicular granulosa and theca cells. It has been speculated that inhibin may be involved in the development of PCOS. Our results show that the cellular localization of inhibin subunit mRNAs is not disturbed in PCOS ovaries.

Female↗
PubMed1993

Involvement of protein kinase C and protein tyrosine kinase pathways in tumor necrosis factor-alpha-induced clustering of ovarian theca-interstitial cells.

Tumor necrosis factor-alpha (TNF) induces clustering of theca-interstitial cells (TIC) isolated from immature, hypophysectomized rats, while inhibiting luteinizing hormone (LH)-stimulated androstenedione in vitro. Stimulators of PKC, 1-oleoyl-2-acetyl-sn-glycerol (OAG, 50 and 100 microM) and phorbol-12-myristate-13-acetate (PMA, 50 nM), caused TIC clustering by 6 days in vitro. Clustering induced by these compounds resembled that induced by TNF. The protein kinase inhibitor, staurosporine at 1 and 10 nM, impaired TNF-induced TIC clustering for 6 days, as did the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride (H-7); conversely, the protein kinase inhibitor, chelerythrine chloride (0.1, 1.0 or 10 microM), did not attenuate TNF-directed clustering. The protein kinase inhibitors did not reverse the suppression of LH-stimulated androstenedione by TNF. Inhibitors of the EGF receptor PTK, A23 (10, 50, or 100 microM) and A46 (0.1, 1.0, 10, or 50 microM), impaired TNF-induced TIC clustering, while TNF suppression of LH-directed androstenedione was unaffected. EGF-induced TIC clustering was also impaired by A46, while A23 was less effective. Both A23 and A46 blocked EGF attenuation of LH-directed androstenedione after 4 days. When challenged with TNF (1 ng/ml) or PMA (50 nM), PKC activity increased in TIC. A23 (50 microM) and A46 (10 microM) each alone blocked the TNF-associated increase in PKC activity; however, PKC activity attributable to PMA was unaffected by A46. Together, these results suggest that TNF-induced TIC clustering involves activation of PTK which directs subsequent increases in PKC activity; however, mechanisms by which TNF inhibits LH-stimulated steroidogenesis remains elusive.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1983

Steroid metabolism by avian ovarian cells during follicular maturation.

The profiles of steroid hormones produced by ovarian cells from the domestic hen were examined. Theca cells from the immature, small white follicles (SWFT), the third largest (T3), and largest (T1) preovulatory follicles, and the ruptured, postovulatory follicle (POFT) were incubated for 3 h at 37 degrees with [3H] progesterone (Prog) or [3H] pregnenolone (Preg). Granulosa cells from the largest preovulatory follicle were incubated with [3H] Preg or were coincubated with theca cells and [3H] Preg. The production of specific steroid metabolites was determined on the basis of coelution of radioactivity with known standard compounds, using an isocratic high-pressure liquid chromatography (HPLC) technique. Granulosa cells converted 93% of [3H] Preg substrate to Prog. More Prog was utilized by T3 cells than by T1 and SWFT cells, either when [3H] Prog was the substrate or when coincubated with granulosa cells and [3H] Preg. The major metabolites of Prog were androstenedione, 17-hydroxyprogesterone, and an unidentified compound with an elution time of 53 min. The POFT cells metabolized [3H] Prog to the same extent as T3 cells did, but their profile of steroidogenesis favored production of the unidentified 53 min metabolite. SWFT cells utilized the least amount of [3H] Preg substrate. The results point to marked changes in enzyme activities in theca cells during maturation and following ovulation.

Animals↗
PubMed2000

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.

Analysis of Variance↗
PubMed1997

Localization of steroidogenesis and steroid receptors in human corpus luteum. Classification of human corpus luteum (CL) into estrogen-producing degenerating CL, and nonsteroid-producing degenerating CL.

In the analysis of the regulation of human corpus luteum, it is very important to localize the sites of specific steroid hormone production to obtain a better understanding of luteal function. We have examined expression of steroidogenic enzymes, steroid receptors, and adrenal 4 binding protein (Ad4BP), a transcription factor of steroidogenesis, in corpus luteum of normal cycling human ovary. Corpus luteum can be classified into four different stages from ovulation to complete regression or fibrosis based on these findings: (1) corpus luteum, (2) steroid-producing degenerating corpus luteum or SPDCL, (3) nonsteroid producing or NSPDCL, and (4) corpus albicans. Corpus luteum in the luteal phase is characterized as follows: (a) the expression of P450scc (cholesterol side chain cleavage), 3 beta HSD (hydroxysteroid dehydrogenase), and Ad4BP in almost all the luteinized granulosa and theca cells, consistent with active progesterone biosynthesis; (b) expression of estrogen-producing P450arom (aromatase) in luteinized granulosa cells, indicating active estrogen production and that of P450c17 (17 alpha hydroxylase) in luteinized theca cells, and (c) expression of progesterone receptor (PR) and androgen receptor (AR) in both luteinized granulosa and theca cells. SPDCL correspond to corpus luteum undergoing regression or degeneration in the following cycle and are characterized as follows: (a) absence of all the steroidogenic enzymes and Ad4BP in the luteinized granulosa cells, suggestive of hormonally inactive nature of these cells and (b) marked expression of P450scc, 3 beta HSD, P450c17 and Ad4BP in luteinized theca cells. NSPDCL is characterized as the absence of all the steroidogenic enzymes and sporadic expression of Ad4BP in luteinized theca cells. These findings indicate that luteal cells remain even after losing expression of steroidogenic enzymes, consistent with a prolonged process of degeneration or regression of human corpus luteum. In corpus albicans, all the cells were replaced by fibrosis and steroidogenic enzymes; steroid receptors and Ad4BP were not expressed at all. Localization of steroidogenesis in human corpus luteum has thus provided new insights into understanding of its biological features.

Corpus Luteum↗
PubMed2006

Insulin and oxidative stress modulate proliferation of rat ovarian theca-interstitial cells through diverse signal transduction pathways.

Insulin and moderate oxidative stress stimulate proliferation of ovarian theca-interstitial cells. The effects of these agents on selected signal transduction pathways were examined. PD98059 (inhibitor of MAP2K1, also known as MEK-1, upstream of extracellular signal-regulated protein kinases MAPK3/1, also known as ERK1/2), wortmannin (inhibitor of PIK3C2A, also known as PI3K), and rapamycin (inhibitor of FRAP1, also known as mTOR, upstream of RPS6KB1) each significantly decreased insulin and oxidative stress-induced proliferation of theca-interstitial cells. The greatest inhibition was observed in the presence of rapamycin; this effect occurred without a significant change in cell viability. Phosphorylation of AKT was stimulated by insulin only, while phosphorylation of MAPK3/1 and RPS6KB1 was increased by insulin and oxidative stress. Insulin-induced and oxidative stress-induced phosphorylation of RPS6KB1 was partly inhibited by wortmannin and partly by PD98059; the greatest inhibition was observed in the presence of a combination of wortmannin plus PD98059. Effects of insulin and oxidative stress on phosphorylation of RPS6KB1 were confirmed by kinase activity assays. These findings indicate that actions of insulin and oxidative stress converge on MAPK3/1 and RPS6KB1. Furthermore, we speculate that activation of RPS6KB1 may be in part induced via the MAPK3/1 pathway.

Androstadienes↗
PubMed2005

Quantitative analysis of estrogen receptor proteins in rat ovary.

The mRNAs of estrogen receptor beta (ERbeta), and its splice variant, ERbeta2, are abundant in granulosa cells in the ovary. With the use of antibodies, ERbeta protein has also been shown to be abundantly expressed, but to date no ERbeta2 protein has been demonstrated in the ovary. ERbeta2 has a peptide, 18 amino acids in length, inserted into its ligand-binding domain, resulting in a reported 35-fold reduction in its affinity for estrogen (E2). ERalpha, ERbeta1 and ERbeta2 were quantified by Western blotting and by RT-PCR and their cellular localization in the ovary was examined by immunohistochemistry. In 3- and 5-week-old virgin, pregnant, lactating and post-lactating rats, the level of ERalpha protein ranged between 1.6 and 3.8 fmol/microg total protein. That of ERbeta was 8.8-11.2 and of ERbeta2, in the same samples, 4.1-5.9 fmol/microg total protein. ERbeta2 and ERbeta1 proteins were, therefore, present in approximately equal amounts in the ovary throughout the various reproductive stages. The major ERbeta proteins in rat ovary, detected by their molecular weights on Western blots, were ERbeta1-530 and ERbeta2-548 (530+18 amino acids (aa)). Immunohistochemical staining revealed that ERbeta and ERbeta2 were expressed predominantly in granulosa cells of growing follicles, while ERalpha was found only in theca cells. In some theca cells, both ERalpha, ERbeta2 were expressed. The data suggests that in theca cells, where it is co expressed with ERalpha, ERbeta2 could function as a repressor of ERalpha. However, in granulosa cells where no ERalpha is detectable, and where E2 levels are high, ERbeta2, with its low affinity for E2, could be an important sensor through which E2 exerts regulatory control.

Alternative Splicing↗
PubMed1990

Insulin-like growth factor-I enhances luteinizing hormone binding to rat ovarian theca-interstitial cells.

We tested the hypothesis that insulin-like growth factor-I (IGF-I) stimulates ovarian androgen production by increasing theca-interstitial cell luteinizing hormone (LH) binding affinity and/or binding capacity. We then investigated the role of transcriptional and translational events in mediating these actions of IGF-I. LH bound to saturable, high affinity binding sites on rat ovarian theca-interstitial cells. Preincubation with LH produced a decrease in LH binding capacity with no effect on LH binding affinity. Treatment with IGF-I, both in the absence and presence of LH, increased LH binding capacity 1.5- to 2-fold with no change in LH binding affinity. Androgen production was increased progressively by LH, suggesting that LH-stimulated steroidogenesis is not tightly coupled to LH receptor downregulation. IGF-I increased androgen synthesis in proportion to its upregulation of LH binding capacity. Transcriptional inhibition with dichlorobenzimidazole riboside inhibited the IGF-I-mediated increase in LH binding capacity but had no effect on androgen production. Translational inhibition with cycloheximide inhibited both the IGF-I-mediated increase in LH binding and stimulation of androgen synthesis. We conclude that IGF-I increases theca-interstitial cell LH binding capacity and reverses the LH-induced downregulation of LH binding sites. The enhancement of LH binding by IGF-I is compatible with transcriptional mediation whereas the effect of IGF-I on androgen synthesis appears to be mediated by a direct effect of the peptide on the translational process(es) involved in steroidogenesis.

Androsterone↗
PubMed1998

Developmental and hormonal regulation of hepatocyte growth factor expression and action in the bovine ovarian follicle.

Ovarian hormones (i.e., estrogen and LH) may promote folliculogenesis by regulating the local production of mesenchymal "inducer proteins" that mediate theca cell-granulosa cell interactions. Theca cells produce hepatocyte growth factor (HGF) that can stimulate granulosa cell growth. In order to investigate the physiological role of HGF in the ovarian follicle, the developmental and hormonal regulation of HGF was examined during follicular development in the bovine ovary. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to examine HGF expression in theca cells and the HGF receptor (HGFR or c-met) in granulosa cells. Both HGF and HGFR were detected throughout follicular development in small (< 5 mm)-, medium (5-10 mm)-, and large (> 10 mm)-sized follicles. Steady-state levels of HGF and HGFR mRNAs were determined using sensitive quantitative RT-PCR assays. Developmental regulation of HGF in theca cells and HGFR in granulosa cells was analyzed in freshly isolated small-, medium-, and large-sized follicles. Observations demonstrate that expression of HGF (in theca cells) and HGFR (in granulosa cells) was highest in large-sized follicles. Hormonal regulation of HGF was analyzed in hormone-treated theca cell cultures. Steady-state levels of HGF mRNA in theca cells were increased by treatment with hCG (an LH agonist), but estradiol had no effect. These results suggest that LH may promote ovarian follicular growth (i.e., granulosa cell proliferation) in part by stimulating the local production of HGF by theca cells. Effects of HGF on granulosa cell differentiated functions were examined. Treatment with HGF reduced basal and FSH-stimulated levels of aromatase activity in bovine and rat granulosa cells. In addition, HGF inhibited the ability of hCG to stimulate progesterone production by granulosa cells. The inhibition of granulosa cell steroid production by HGF is proposed to be the indirect effect of promoting cellular proliferation. Therefore, HGF directly stimulates granulosa cell proliferation and indirectly inhibits granulosa cell differentiated functions. The developmental and hormonal regulation of HGF and HGFR during folliculogenesis provides evidence that HGF may be important for hormone-induced granulosa cell proliferation. As a result, HGF may be essential for establishing the granulosa cell population and microenvironment required for oocyte maturation in the female.

Animals↗
PubMed1988

Adrenergic regulation of ovarian androgen biosynthesis is mediated via beta 2-adrenergic theca-interstitial cell recognition sites.

Acting alone or in concert with pituitary gonadotropins, catecholamines have recently been shown to enhance androgen production by ovarian theca-interstitial cells. It is the objective of the in vitro studies reported herein to further characterize this catecholaminergic activity as well as to type and subtype the putative adrenergic recognition sites mediating this phenomenon. Treatment of collagenase-processed whole ovarian dispersates or highly enriched (greater than 90%) theca-interstitial cells from immature rats with norepinephrine (10(-6) M) resulted in a 2.0-fold increment in the accumulation of androsterone (3 alpha-hydroxy-5 alpha-androstane-17-one), the main androgenic steroid identified in culture medium by HPLC. Qualitatively similar stimulation was obtained using beta (isoproterenol)- but not alpha (methoxamine)-selective adrenergic agonists. Moreover, combined treatment with both norepinephrine (10(-6) M) and hCG (1 ng/ml) unmasked a synergistic interaction subject to stereospecific blockade by beta (propranolol)- but not alpha (phentolamine)-selective adrenergic antagonists. Further probing with subtype-selective adrenergic ligands revealed terbutaline (a beta 2-selective agonist) to enhance androgen biosynthesis, with dobutamine (a beta 1-selective agonist) having little or no effect. Moreover, a beta 2 (ICI-118406)- but not a beta 1 (ICI-89406)-selective adrenergic antagonist yielded dose-dependent inhibition of the isoproterenol effect. Unaccounted for by either enhanced cellular growth or an alteration of the overall steroidogenic pattern, catecholaminergically stimulated androgen biosynthesis proved time and dose dependent but independent of the hCG dose (0.1-10 ng/ml) employed. Binding of [125I]iodocyanopindolol to highly enriched theca-interstitial cells proved stereoselective and saturable, displaying a single class (Hill coefficient = 0.96 +/- 0.01) of high affinity (Kd = 5.6 X 10(-11) M), low capacity (1219 +/- 317 sites/cell) binding sites. The rank order of competitive potencies of selective adrenergic ligands (beta 2 greater than beta 1 greater than alpha), was consistent with a beta 2-adrenergic receptor subtype. Taken together, these findings suggest that catecholaminergic stimulation of ovarian androgen biosynthesis is mediated via beta 2-adrenergic recognition sites, the role of which may now be studied.

Androgens↗
PubMed1992

Localization of preovulatory expression of plasminogen activator inhibitor type-1 and tissue inhibitor of metalloproteinase type-1 mRNAs in the rat ovary.

Proteinases and their inhibitors control follicular connective tissue remodeling associated with follicular rupture. We examined the regulation and cellular localization of plasminogen activator inhibitor type-1 (PAI-1) and tissue inhibitor of metalloproteinase type-1 (TIMP-1) mRNAs by in situ hybridization. [35S]UTP-labeled RNA probes were hybridized to ovarian sections of eCG-primed immature rats treated with hCG. Before hCG stimulation of ovulation, very low expression of PAI-1 mRNA was observed in theca cells. After hCG administration, expression of PAI-1 mRNA was increased in theca cells of most antral follicles, whereas expression in granulosa cells was limited to preovulatory follicles and only to areas where the basal membrane was dissociated. Before hCG treatment, low expression of TIMP-1 mRNA was observed in theca cells, but not in granulosa cells. After hCG treatment, TIMP-1 mRNA was greatly stimulated in theca cells irrespective of follicle size, while the expression in granulosa cells was limited to large antral follicles. The present study demonstrates cell-specific expression of PAI-1 and TIMP-1 mRNAs in the LH/hCG-stimulated ovary, thus confirming the localized control of preovulatory proteolysis by coexpression of both enzymes and their respective inhibitors.

Animals↗
PubMed2001

Relationship of Fas ligand expression and atresia during bovine follicle development.

The Fas antigen (Fas) is a cell surface receptor that may be involved in the initiation and progression of follicle cell apoptosis during atresia. Fas initiates apoptosis in sensitive cells after binding Fas ligand (FasL). Other experiments have shown that expression of Fas mRNA and responsiveness to Fas-mediated apoptosis vary in bovine granulosa and theca cells during follicle development. In the present study, FasL mRNA content was measured and Fas and FasL protein expression was examined in bovine granulosa and theca cells of healthy dominant follicles and the two largest atretic subordinate follicles on day 5 of the oestrous cycle (day 0 = oestrus), and of dominant follicles from the first wave of follicle development after they had become atretic and showed no growth for 4 days. FasL mRNA content was higher in granulosa cells from atretic compared with healthy follicles. FasL mRNA content was also higher in theca cells from atretic subordinate compared with healthy dominant follicles on day 5, but did not differ between theca cells from healthy and atretic dominant follicles. Immunohistochemical staining for FasL was more intense in theca compared with granulosa cells and in atretic compared with healthy follicles. Immunohistochemical staining for Fas was more intense in granulosa compared with theca cells and in atretic subordinate compared with healthy dominant follicles on day 5. Immune cells, known to express Fas and FasL, were localized in the theca, but not the granulosa, cell layer of all follicles. Higher concentrations of Fas and FasL in cells from atretic follicles, together with the previous demonstration of increased responsiveness of granulosa cells from subordinate follicles to FasL-induced apoptosis, support a potential role for FasL-mediated apoptosis during ovarian follicle atresia.

Animals↗
PubMed2000

Estrogen receptor-beta expression in relation to the expression of luteinizing hormone receptor and cytochrome P450 enzymes in rat ovarian follicles.

Changes in mRNA expression for estrogen receptor (ER beta) in relation to mRNAs for LH receptor (LHr) and cytochrome P450 enzymes were examined in granulosa and theca cells from proestrous rat ovarian follicles. Of the 30 ovaries harvested from 15 adult rats, 24 were processed for in situ hybridization, and the remaining were used for reverse transcription-polymerase chain reaction. Messenger RNAs for ER beta, LHr, cytochrome P450 side-chain cleavage enzyme (P450(scc)), 17 alpha-hydroxylase (P450(c17)), aromatase (P450(arom)), and steroidogenic acute regulatory protein (StAR) were localized in cross sections of ovaries by in situ hybridization and quantified in granulosa and theca cell layers by a computer-image analyzing system. Ovarian follicles were classified as healthy or atretic. Healthy follicles were divided into four size groups: very small (40-100 microm), small (101-275 microm), medium (276-450 microm), and large (451-850 microm). Atretic follicles were divided into medium (276-450 microm) or large follicles (451-850 microm). A low level of ER beta mRNA expression was first detected in granulosa cells of very small healthy follicles, and the expression increased progressively up to medium-sized follicles. The expression of ER beta mRNA was highest (P < 0.01) in medium-sized follicles that was followed by a decrease (P < 0.01) in large follicles. Messenger RNAs for LHr, P450(scc), and P450(arom) were first detected in granulosa cells of medium-sized healthy follicles, while mRNAs for LHr, P450(scc), P450(c17), and StAR were first detected in theca cells associated with very small follicles. The highest expression of LHr, P450(scc), P450(c17), P450(arom), and StAR was seen in granulosa and/or theca cells of large healthy follicles. In atretic follicles, level of gene expression was relatively low in both granulosa and theca cells. In conclusion, stage-specific expression of ER beta mRNA was observed in granulosa cells during follicular development. The increased expression of ER beta and a concomitant initiation of LHr, P450(scc), and P450(arom) expression in granulosa cells of medium follicles may signify a role for estrogen in follicular development. Also, a strong correlation between ER beta mRNA expression in granulosa cells, and the expression of mRNAs for LHr, P450(scc), P450(c17), and StAR in theca cells associated with growing follicles suggests a possible role for estrogen in steroidogenesis.

Animals↗
PubMed1986

Ultrastructural evidence for estradiol synthesis in the ovary of persistent-estrous rats exposed to continuous illumination.

The relation between sex hormone levels in blood and ultrastructural changes of ovarian follicles was examined in persistent-estrous rats exposed to continuous illumination (LL) for 100 days. Plasma LH showed a tonic level secretory pattern, and circulating estradiol and estrone concentrations were relatively high, while both levels of FSH and progesterone were low. Various stages of growing and degenerating follicles were observed in the ovary of the LL-exposed rats. The early stage of antral follicle did not seem to possess the ability of steroidogenesis. Theca cells around mature antral follicles contained prominent Golgi apparatuses, plenty of smooth endoplasmic reticulum (ER), abundant free ribosomes and many round-mitochondria. A few newly formed lipid droplets were seen in some of theca cells. Granulosa cells contained much distended rough ER, well-developed mitochondria, several lipid droplets and microfilaments. The theca cells of abnormal follicles with hyperplastic and infolded layers of granulosa cells contained many lipid droplets. However, the development of the smooth ER became hindered with increasing lipid droplets in the theca cell. On the other hand, granulosa cells of abnormal follicles contained greater numbers of lipid droplets than those of antral mature follicles, and were equipped with well-developed cytoplasmic organelles as were those of mature antral follicles. Theca interna cells of abnormal follicles may be more involved in the secretion of androgen, which has already been accumulated in the lipid droplets, than the cells involved in the active synthesis of the hormone, while the granulosa cells may convert its androgen to estrogen. The present findings suggest that both follicles of mature and abnormal types in the LL-exposed rat retain enough capacity of estradiol production and participate in the continued elevation of circulating estradiol, probably resulting in the stimulation of the theca cells by the tonic level of LH and of the granulosa cells by the levels of FSH, which are lower than the basal values during the normal 4-day estrous cycle.

Animals↗
PubMed1996

Growth hormone stimulates androsterone synthesis by rat theca-interstitial cells.

The aim of this study was to assess the possible role of growth hormone (GH) on androsterone synthesis. This effect was analyzed in theca-interstitial cells obtained from immature female rats. The addition of GH to the cultures significantly stimulated androsterone (A) synthesis in a dose- and time-dependent way and this effect was not due to a cellular number increase. When added to the hCG cultures, GH significantly enhanced androgen production even though it did not synergyze with the chorionic gonadotropin. The addition of antibodies anti-IGF-I to the GH cultures did not modify the growth hormone effect suggesting that GH probably does not require IGF-I to achieve its effect on A production. Finally, no effect of GH on cAMP levels were observed in the cultures at the end of the treatment. Our results demonstrate that GH is able to significantly induce A synthesis by rat theca-interstitial cells. Since the presence of GH and its receptors in the ovary is now well established the present data strongly suggest a potential relevance of GH in reproductive biology.

Androsterone↗
PubMed2004

Mevastatin inhibits ovarian theca-interstitial cell proliferation and steroidogenesis.

OBJECTIVE: Statins reduce cardiovascular risks by improving hypercholesterolemia, reducing vascular smooth muscle proliferation, and ameliorating inflammation. Polycystic ovary syndrome (PCOS) is associated with increased cardiovascular risks and is characterized by ovarian theca-interstitial hyperplasia and hyperandrogenism. This study tested the hypothesis that mevastatin limits theca-interstitial proliferation and decreases steroidogenesis. DESIGN: In vitro study. SETTING: Academic laboratory. PATIENT(S): None. INTERVENTION(S): Effects of mevastatin on cultured theca-interstitial cells. MAIN OUTCOME MEASURE(S): Proliferation was evaluated by determination of DNA synthesis using thymidine incorporation assay and by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay. Production of P and T was determined by specific radioimmunoassays. RESULT(S): Mevastatin induced a profound concentration-dependent inhibition of DNA synthesis. At the highest concentration (30 microM), mevastatin inhibited DNA synthesis by 92%. Similarly, in the MTT proliferation assay, mevastatin induced a concentration-dependent decrease in cell number. Mevastatin decreased production of P (by up to 49%) and T (by up to 52%); these effects remained significant when the effect on cell culture protein content was accounted for. CONCLUSION(S): Mevastatin inhibits proliferation of theca-interstitial cells; it also inhibits P and T production independently of the effects on cell growth. These findings provide a foundation for studies evaluating statins as potential therapeutic agents in the treatment of ovarian mesenchymal hyperplasia and hyperandrogenism characteristic of PCOS.

Animals↗