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PubMed2001

Secretion of matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases into follicular fluid during follicle development in equine ovaries.

Extensive tissue remodelling is required in equine ovaries for follicle growth and development and also migration of the follicle to the ovulatory fossa, where ovulation occurs. The mechanisms for these processes are largely unexplored. Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) are important for control of breakdown of extracellular matrix during tissue remodelling. The aims of this study were to determine the pattern and sites of secretion of the gelatinases MMP-2 and -9 and TIMPs into follicular fluid during follicle development in mare ovaries. The predominant gelatinase detected in follicular fluid was MMP-2, which was present in similar amounts throughout follicular development, as demonstrated by zymography. MMP-9 was also present in follicular fluid and secretion increased significantly (P < 0.05) with development of follicles from < 10 mm to 11-20 mm in diameter. Follicular fluid also contained TIMP-1, TIMP-2, unglycosylated and glycosylated TIMP-3, and TIMP-4, as shown by reverse zymography. The abundance of TIMPs remained largely unchanged during follicle development. MMP-2 and -9 were localized by immunohistochemistry to stromal cells and granulosa and theca cells. TIMP-1, -2, -3 and -4 were present in granulosa and theca cells of the follicle and in stromal cells and also associated with extracellular matrix of the ovarian stromal tissue. The MMPs and TIMPs are likely to be involved in the regulation of the breakdown of extracellular matrix during tissue remodelling for follicle development and migration to the ovulation fossa in mares.

Animals↗
PubMed1988

Evidence for protein kinase C regulation of ovarian theca-interstitial cell androgen biosynthesis.

The hypothesis that protein kinase C may be an important regulator of ovarian theca-interstitial cell steroidogenesis was tested by using phorbol-12-myristate-13-acetate (PMA) and phorbol-12, 13-dibutyrate (PDB) to directly stimulate protein kinase C activity. Collagenase-dispersed cells (4 x 10(5) viable cells/dish) form ovaries of hypophysectomized immature rats were cultured in serum-free medium in the presence and absence of 0-100 ng/ml of luteinizing hormone (LH), PMA (0-100 nM), and/or PDB (0-100 nM). Treatment with 100 ng/ml LH stimulated androsterone production 100-fold at Day 4 of culture. The presence of 100 nM PMA or PDB had no effect on basal androsterone production; however, treatment with increasing concentrations of PMA or PDB (0-100 nM) caused a dose-related inhibition (maximum 70%) of LH-stimulated androsterone synthesis (ID50 = 1.8 nM and 2.4 nM, respectively). PMA and PDB did not significantly alter DNA, protein, or cell viability, indicating that their inhibitory effects were not due to changes in cell number or viability. Cells treated with LH and 100 nM 4 alpha-phorbol didecanoate (4 alpha-PDD; a phorbol ester that does not activate protein kinase C) failed to show significant decreases in androsterone production. Time-course studies revealed that when PMA treatment was delayed until Day 2 or 4 of culture, dramatic inhibitory effects on LH-stimulated androsterone production were still observed. These results suggest that the biological activity of protein kinase C is retained after the cells have expressed their differentiated state.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed1994

Effect of interactions between granulosa and thecal cells on meiotic arrest in bovine oocytes.

An in vitro culture system in which two types of cell from ovarian follicular walls were attached to opposite surfaces of a collagen membrane was developed. The effect of granulosa-theca cell communication on oocyte maturation was investigated using this culture system. Bovine cumulus-oocyte complexes were incubated with granulosa cells, with or without theca cells. The time course of germinal vesicle breakdown was assessed. When cultured with granulosa cells alone, oocytes showed 100% germinal vesicle breakdown after incubation for 6 h. However, when cultured with granulosa and theca cells, germinal vesicle breakdown was significantly delayed for up to 12 h. Breakdown was further delayed by 12 h when cumulus-oocyte complexes were incubated with FSH-treated granulosa and thecal cultures. FSH did not exert this inhibitory effect when in culture with granulosa cells alone. When cumulus-oocyte complexes were incubated with thecal cells alone, all oocytes underwent germinal vesicle breakdown within 6 h; the addition of FSH to the culture medium did not affect the time course of breakdown. It is concluded that a signal(s) from thecal cells augments the meiosis-arresting activity of granulosa cells and that FSH increases the meiosis-arresting activity of granulosa cells under the influence of thecal cells.

Animals↗
PubMed1994

Immunohistochemical distribution of progesterone, androgen and oestrogen receptors in the human ovary during the menstrual cycle: relationship to expression of steroidogenic enzymes.

In order to characterize immunohistochemically the possible in-situ effects of gonadal steroid hormones in the human ovary during the menstrual cycle, we immunolocalized progesterone (PR), androgen (AR) and oestrogen (ER) receptors in 50 normal cycling human ovaries, and examined the relationship between these findings and the cellular localization of steroidogenic enzymes including cytochrome P-450 cholesterol side-chain cleavage (P-450scc) enzyme, 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), cytochrome P-450 17 alpha-hydroxylase (P-450c17) and cytochrome P-450 aromatase (P-450arom). A large number of stromal cells were positive for AR, regardless of the distance from a follicle. No steroidogenic enzymes were observed in the stromal cells. In the pre-antral follicle, AR was observed in the theca cells. P-450scc, 3 beta HSD and P-450c17 were sporadically expressed in the theca cells in relatively large-sized pre-antral follicles. ER was positive in the granulosa cells only in the P-450arom-positive antral or pre-ovulatory follicle, which is likely to be a selected follicle. In the corpus luteum, in the period from ovulation to the mid-secretory phase, PR immunoreactivity was observed in a large number of both the luteinized granulosa and the theca cells. All steroidogenic enzymes were observed in all corpora lutea, but ER was negative in any corpus luteum. In the atretic follicle, AR was present in the theca interna cells. P-450scc, 3 beta HSD and P-450c17 were observed in the theca interna cells in some atretic follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Tumor necrosis factor-alpha induces clustering in ovarian theca-interstitial cells in vitro.

Tumor necrosis factor-alpha (TNF) has been implicated in the regulation of steroidogenesis in theca-interstitial cells (TIC). The purpose of this study was to evaluate any change in TIC morphology during the time course of TNF-induced inhibition of LH-stimulated androstenedione production. Ovaries from immature hypophysectomized rats were enzymatically digested and highly purified TIC were obtained by density gradient centrifugation. TIC treated with TNF (0.1-10 ng/ml) demonstrated distinct clustering in the presence and absence of LH (50 ng/ml). The number of clusters and the mean area per cluster were greatest after 4 days as a result of treatment with 1 or 10 ng TNF/ml. In addition, a dose-dependent inhibition of LH-supported androstenedione production was induced by TNF. TNF also inhibited LH-induced androstenedione in TIC after 2, 4, or 6 days of continuous LH treatment, and TIC clustering still occurred. TIC clustering was impeded by the protein kinase inhibitor H7 at 10 microM; however, the protein kinase inhibitor, HA 1004 (5 microM), did not inhibit TNF-induced clustering in TIC. Since H7 blocked TNF induced clustering, but did not block TNF inhibition of LH stimulated androstenedione synthesis, it is suggested that alternate signal transduction pathways for TNF induced inhibition of LH-stimulated androstenedione and stimulation of clustering of TIC may exist. The results also indicate that the TNF-induced TIC clustering may be independent of the TNF-induced inhibition of LH-stimulated androstenedione production and states of LH-induced differentiation of TIC.

Androstenedione↗
PubMed1988

Purification of ovarian theca-interstitial cells by density gradient centrifugation.

The study of the biochemical mechanisms regulating the differentiation of the ovarian androgen-producing cells has been difficult because of the lack of a method for isolating the theca-interstitial cells (TIC) from the granulosa and other cell types. We report here a simple, rapid and reproducible method for obtaining large numbers of highly enriched TIC using density gradient centrifugation. When dispersed cells from ovaries of hypophysectomized immature rats were centrifuged in a Percoll gradient (20-70%), the cells were separated into five incompletely resolved bands. Of these, band V contained the androgen-producing cells. The band V cells produced 3.2- and 3.9-fold more cAMP and androgen, respectively, than the whole ovarian cells and contained 3.8- and 3.5-fold more 125I-hCG binding and 3 beta-HSDH labeled cells, respectively. These data indicated that the TIC were 65% pure. In order to improve the degree of purification, a discontinuous density procedure was used. When the ovarian cells were centrifuged in d = 1.055 g/ml Percoll, the purified TIC produced 4.7- and 5.9-fold more cAMP and androgen than the whole ovarian cells and they contained 5.3-fold more 125I-hCG binding. The TIC were cleanly separated from the granulosa cells and histochemical staining for 3 beta-HSDH activity revealed that the TIC were 93.0 +/- 1.3% pure. This method provides, for the first time, the opportunity to study TIC differentiation under defined conditions without granulosa cell contamination.

Animals↗
PubMed1989

Differential steroid production between theca interna and theca externa cells: a three-cell model for follicular steroidogenesis in avian species.

In avian species, the granulosa cells produce progesterone (P), but not testosterone (T) or estradiol (E). The theca folliculi in avian species produces T and E and is anatomically comprised of the theca interna and the theca externa. It is not known, however, whether both T and E are produced by the same cell type. In the present study, preovulatory follicles of adult female turkeys were separated into their anatomical subdivisions, the granulosa, theca interna, and theca externa, and the relative steroidogenic contribution by each layer was investigated. Granulosa cells from the largest (F1) and fifth largest (F5) preovulatory follicles produced increasing levels of P in response to ovine LH (oLH; 0-10 micrograms/ml); however, T and E were not detectable. Theca interna and theca externa cells from F5 follicles were cultured alone or in combination. Theca interna cells produced P and T, but not E. Theca externa cells produced P, T, and E. E production was greatly enhanced by coincubation of cells from the two theca layers. oLH, (0-100 ng/ml) stimulated theca interna P and T production and E production by the coincubation of theca interna and theca externa cells. When theca interna and theca externa cells from the F1 and F5 follicles were tested, theca externa cells from F5, but not F1, converted androstenedione and T, but not P into E, indicating a loss of aromatase activity in the theca externa with follicular maturation. Neither F1 nor F5 theca interna cells converted P, A, or T into estradiol, providing additional evidence that aromatase activity is limited exclusively to the theca externa. Addition of oLH (0-100 ng/ml) did not increase conversion of T into E by the F5 theca externa cells, suggesting that production of E by the theca externa is substrate driven and not influenced by direct LH action. Avian ovarian steroidogenesis is described using a three-cell model, with the principle sources of P, T, and E being the granulosa, theca interna, and theca externa cells, respectively.

Animals↗
PubMed2001

Type 5 17beta-hydroxysteroid dehydrogenase: its role in the formation of androgens in women.

Type 5 17beta-HSD, one of the seven types of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) so far characterized in humans, catalyzes the transformation of 4-androstenedione (4-dione) into testosterone (T). This reaction is also catalyzed by type 3 17beta-HSD which is responsible for pseudohermaphroditism in deficient man but is asymptomatic in deficient women. Since type 3 17beta-HSD is not found in the ovary, whereas type 5 is, it is suggested that the latter is involved in the conversion of 4-androstenedione to testosterone in the ovary. The comparison of type 5 17beta-HSD of different species shows that the human enzyme shares 95 and 78% identity with the monkey and mouse enzymes respectively. In addition, the human and monkey enzymes are labile and are destroyed upon homogenization of the transfected cells, whereas the mouse enzyme is not. Human type 5 17beta-HSD also possesses a high 20alpha-HSD activity that inactivates progesterone, whereas the monkey and mouse enzymes do not have such high 20alpha-HSD activity. Since the endocrine ovary is composed of two types of cells, one producing androgens (theca cells) and the other producing progesterone and estrogens (granulosa cells), it is tempting to suggest that the role of the high 20alpha-HSD activity of type 5 17beta-HSD is to protect the theca cells against the progesterone produced by the granulosa cells. The double activity of type 5 17beta-HSD in the female reproductive tissues is probably necessary to the control of the optimal level of progesterone and sex steroids.

17-Hydroxysteroid Dehydrogenases↗
PubMed2000

Expression and activity of the Fas antigen in bovine ovarian follicle cells.

The Fas antigen is a cell surface receptor that triggers apoptosis when bound to Fas ligand (FasL). Studies were undertaken to determine whether the cow provides a suitable model to study the role of the Fas pathway in inducing apoptosis of ovarian cells during follicular atresia. Expression of Fas antigen mRNA and responsiveness to FasL-induced killing in vitro were measured. Effects of the cytokines tumor necrosis factor (TNF)-alpha and interferon-gamma (IFN) were studied because of previous demonstrations of their role in Fas-mediated apoptosis in other cell types. Fas antigen mRNA was detectable in cultured granulosa and theca cells, and expression was increased by treatment with IFN but not TNF. Granulosa and theca cells were resistant to FasL-induced killing unless pretreated with IFN. TNF had no effect on FasL-induced killing. Granulosa and theca cell cultures in which killing occurred in response to FasL stained positively for annexin V, an early marker for cells undergoing apoptosis. These results provide a basis for further studies using the bovine ovary to examine the role of the Fas antigen in follicular atresia.

Animals↗
PubMed1997

Hepatocyte growth factor regulates ovarian theca-interstitial cell differentiation and androgen production.

During ovarian follicle growth, precise regulation of the onset of androgen production by ovarian theca-interstitial cells (TIC) is necessary for maintaining follicle viability. Thus, temporary suppression of TIC androgen production in preantral follicles is the key to promoting follicle development. Evidence indicates that this process is coordinated via intraovarian growth factors. Hepatocyte growth factor (HGF) can induce granulosa cell (GC) proliferation and suppress follicular atresia, indicating a role for HGF in promoting follicle growth and viability. To determine whether HGF could reversibly suppress androgen production, this study investigated the effect of HGF on TIC differentiation and steroid production. Twenty-six-day-old rats were used in all studies. HGF messenger RNA (mRNA) expression in TIC and GC was determined by reverse transcription-PCR. Agarose gel electrophoresis of the PCR products yielded a single band corresponding to the 290-bp HGF product for both TIC and GC. HGF expression in cultured TIC and GC was not blocked by gonadotropins or HGF. To investigate the effects of HGF on TIC steroidogenesis, TIC were isolated from the ovaries of hypophysectomized rats. TIC (3.0 x 10(4) cells/well) were cultured with LH (0-3 ng/ml) and/or HGF (0-100 ng/ml) for 48 h, and androsterone levels were measured by RIA. HGF did not alter androsterone levels in the absence of LH; however, HGF reversibly impaired LH-dependent androsterone production by as much as 57% (IC50 = 1.5 +/- 0.01 ng/ml). LH (0.3 ng/ml) stimulated progesterone (P4) synthesis by TIC (1201 +/- 190 pg/ml) compared to that by control cells (210 +/- 30 pg/ml). HGF stimulated basal P4 production, and LH-dependent P4 synthesis was augmented 2.6-fold by HGF (ED50 = 0.3 +/- 0.01 ng/ml). The DNA content and cell viability in TIC cultures were not affected by HGF. The effect of HGF on steroidogenic enzyme gene expression in TIC was also investigated via PCR. HGF did not alter the level of basal or LH-induced P450 side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase mRNAs; however, LH-dependent P45017 alpha hydroxylase/C17,20 lyase mRNA content was reduced 4.5 fold in the presence of HGF. Thus, HGF is expressed in both TIC and GC obtained from the immature rat ovary, suggesting its presence in growing follicles. In TIC, HGF stimulated P4 synthesis, but impaired androgen production, concurrent with a down-regulatory effect on P45017 alpha hydroxylase/C17,20 lyase gene expression. Collectively, these results indicate that HGF reversibly impairs LH-stimulated androgen production in TIC. Such effects may help promote folliculogenesis.

Androgens↗
PubMed1990

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

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

Androstenedione↗
PubMed1994

Computed tomography, diagnosis, staging and follow-up of pure granulosa cell tumour of the ovary.

Granulosa theca cell tumours represent the largest group of hormone-secreting ovarian tumours and comprise approximately 1.5% of all ovarian malignancy. However, the prognosis and behaviour of pure granulosa cell tumours have been particularly difficult to evaluate, because in previous reports they have been grouped with granulosa theca cell tumours. Consequently, their radiological features and the possible contribution of computed tomography (CT) to clinical management has not been evaluated. We report the CT appearances of 15 patients with pure granulosa cell tumours and their clinical outcome. The results suggest the possibility of a positive relationship between histological grade and FIGO stage at presentation. Large tumour volume, extensive lymph node involvement and ascites on CT are adverse prognostic features. However, patients with abdominal masses less than 9 cm in diameter, absent or small volume lymph node disease and peritoneal and liver metastases achieved complete remission, suggesting that CT can assist in identifying those patients likely to respond best to current therapeutic regimens.

Adult↗
PubMed2005

Effects of luteinizing hormone and human chorionic gonadotropin on corpus luteum cells in a spheroid cell culture system.

The human corpus luteum (CL) is a highly vascularized, temporarily active endocrine gland and consists mainly of granulosa cells (GCs), theca cells (TCs), and endothelial cells (ECs). Its cyclic growth and development takes place under the influence of gonadotropic hormones. If pregnancy does occur, human chorionic gonadotropin (hCG) takes over the function of luteinizing hormone (LH) and, in contrast to LH, extends the functional life span of the CL. In this study, we investigated the effects of hCG and LH in a spheroidal cell culture model of CL development. Our data indicate that GCs secrete factors under the control of hCG that increase sprout formation of EC-spheroids. We demonstrate that the most prominent of these factors is VEGF-A. Furthermore, we found that both LH and hCG decrease sprout formation of GC-spheroids. After forming EC-GC coculture spheroids and consequently bringing GCs and ECs in close contact, sprouting increased under the influence of hCG, however not under LH. These experiments provide evidence for an hCG dependent functional switch in the GCs after coming in contact with ECs. Moreover, it demonstrates the considerably different effects of hCG and LH on GCs although their signaling is transmitted via the same receptor.

Cells, Cultured↗
PubMed1992

Effects of methoxychlor on the reproductive system of the adult female mouse: 2. Ultrastructural observations.

The purpose of this study is to examine the effects of the pesticide methoxychlor (MXC) on the ultrastructural appearance of the different cellular components of the mouse ovary. Sexually mature (7- to 8-week) virgin female CD-1 mice were exposed to 5.0 mg MXC (50% technical grade) via oral gavage for 5 consecutive days each week for 4 weeks. Control groups received either 0.025 mg estradiol-17 beta (E-17 beta) or the sesame oil vehicle for the same time period. Twenty-four hours following the final exposure, animals were sacrificed. Ultrastructural observations revealed increased lipid accumulation in interstitial cells and theca cells of both estradiol-treated and 5.0 mg MXC-treated mice. This would suggest that these cells are unable to synthesize and secrete steroids. Thus, this commonly employed pesticide appears to closely mimic those effects on the female ovary induced by estrogen.

Animals↗
PubMed2004

Spontaneous tumors of small mammals.

The most common tumor of guinea pigs is bronchogenic papillary adenoma; of hedgehogs is mammary gland adenocarcinoma; of hamsters is adrenal cortical adenoma; of gerbils is ovarian granulosa cell and theca cell tumors; of mice is pulmonary carcinoma; and of rats is mammary fibroadenoma. A relatively low incidence of tumors is described for chinchillas and hamsters, whereas the incidence of tumors is high for gerbils, hedgehogs, mice, and rats. Limited literature regarding neoplasia exists for prairie dogs, sugar gliders, and chinchillas.

Animals↗
PubMed2002

Anti-macrophage inhibitory factor antibody inhibits PMSG-hCG-induced follicular growth and ovulation in mice.

PURPOSE: To investigate the effect of an anti-MIF antibody on PMSG-hCG-induced murine follicular growth and ovulation and to determine whether MIF plays an essential role in this process. METHODS: Mice were primed with an intraperitoneal injection of pregnant mare serum gonadotropin (PMSG) and were treated with an anti-rat MIF antibody and human chorionic gonadotropin (hCG) to induce ovulation. After that, the ovulated ova were counted. The ovaries were studied using standard histological procedures. RESULTS: Ovaries treated with the anti-MIF antibody showed reduced numbers of growing follicles surrounded by granulosa cells and theca cells with a little proliferation compared with the control. The average numbers of ova collected from mice treated with the anti-MIF antibody were reduced compared with those collected from control mice. CONCLUSIONS: Anti-MIF antibody inhibits the follicular growth and ovulation in mice, and MIF may play an important role in the inflammatory reactions during follicle growth and ovulation.

Animals↗
PubMedDate not supplied

Mixed gonadal dysgenesis: endogenous hormonal effects in the endometrium and histogenesis of germinoma.

The clinicopathologic features of two patients with mixed gonadal dysgenesis are presented, with specific reference to the relationship between endogenous sex hormones and the endometrium and the development of neoplastic disease. One patient, whose immature gonad contained granulosa cells and theca cells, had elevated serum estrogen levels and an endometrium with frequent ciliated metaplasia and squamous metaplasia. Another patient had elevated serum testosterone levels and atrophic endometrium. Both had gonadal tumors, more specifically, germinomas, which contained many calcified nodules within the tumor. These findings suggest that these germinomas arose from a gonadoblastoma.

Adult↗
PubMed2003

Microvascular endothelial cells of the corpus luteum.

The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of eosinophils and macrophages. The review highlights areas for future investigation of ovarian microvascular endothelial cells. The potential clinical applications of research directed on corpus luteum endothelial cells are intriguing considering reproductive processes in which vascular dysfunctions may play a role such as ovarian failure, polycystic ovary syndrome (PCOS), and ovarian hyperstimulation syndrome (OHSS).

Angiogenesis Inducing Agents↗