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J J Peluso

Publications and source records attributed to J J Peluso.

At least 19 recordsLinked to original sources
PubMed1999

Progesterone maintains large rat granulosa cell viability indirectly by stimulating small granulosa cells to synthesize basic fibroblast growth factor.

Ovarian follicles are composed of small and large granulosa cells (GCs). Since progesterone (P4) inhibits large GCs from undergoing apoptosis, studies were designed to determine whether both sizes of GCs bind P4. These studies revealed that fluorescein isothiocyanate-BSA-P4 bound only to the surface membranes of small GCs. This binding was steroid-specific and inhibited by an antibody directed against the ligand-binding domain of the nuclear P4 receptor (PR). In addition, a cell-impermeable derivative of P4, BSA-conjugated P4, was as effective as P4 in preventing apoptosis. Quantitative in situ hybridization studies showed that P4 increased the relative amount of basic fibroblast growth factor (bFGF) mRNA expressed per cell as well as the percentage of small GCs that expressed bFGF. To determine whether the anti-apoptotic action of P4 was mediated by bFGF, GCs were cultured in control medium supplemented with either P4, a neutralizing antibody to bFGF, or both P4 and the bFGF antibody. The results from this study demonstrated that P4 suppressed apoptosis and that this effect was attenuated in presence of the bFGF antibody. Basic FGF also prevented GC apoptosis, and its action was not influenced by either the PR antagonist (RU-486), an inhibitor of P4 synthesis (aminoglutethimide), or a PR antibody. Finally, FGF receptors were detected on both small and large GCs. Collectively, these data support the hypothesis that P4 acts through a putative membrane receptor on small GCs to stimulate the synthesis of bFGF. Basic FGF then activates its receptors within large GCs, and this initiates a signal transduction pathway that maintains large GC viability.

Aminoglutethimide↗
PubMed1998

Progesterone mediates its anti-mitogenic and anti-apoptotic actions in rat granulosa cells through a progesterone-binding protein with gamma aminobutyric acidA receptor-like features.

Progesterone (P4) inhibits small granulosa cell (GC) mitosis and large GC apoptosis. These actions are steroid specific and dose dependent and are inhibited by the progesterone receptor (PR) antagonist, RU-486. However, these cells do not express the nuclear PR but rather an ill-defined P4-binding protein (P4BP). This binding protein could function as a receptor and mediate P4's actions in GCs. Therefore, a series of studies was designed to characterize this P4BP. First, an antibody directed against the ligand-binding site of the nuclear PR was used in a Western blot analysis. This analysis revealed the presence of a 60-kDa P4BP within ovarian and GC lysates as well as within an ovarian membrane preparation. This protein was not observed in lysates of cells derived from the ovarian surface epithelium. In addition, this P4BP was immunoprecipitated by an antibody to the alpha1 chain of the gamma aminobutyric acidA (GABA(A)) receptor, suggesting that the P4BP could be the ovarian GABA(A) receptor. Since activation of the rat ovarian GABA(A) receptor increases intracellular cAMP levels, GCs were cultured with control medium supplemented with either 8-bromo-cAMP (8-br-cAMP), P4, or muscimol (a GABA agonist). Increases in cAMP were detected by monitoring the cAMP-dependent phosphorylation of cAMP response element-binding protein (CREB). Phosphorylated CREB was not observed in control or P4-treated cultures, but it was detected in the majority of both small and large GCs exposed to either 8-br-cAMP or muscimol. Since activation of the GABA(A) receptor with muscimol increases phosphorylated CREB but P4 does not, this study indicates that P4 does not activate the ovarian GABA(A) receptor. However, both bicuculline, a GABA(A) receptor antagonist, and the antibody to PR inhibited P4's ability to prevent both insulin-dependent mitosis and apoptosis. Collectively, these studies suggest that P4 mediates its anti-mitotic and anti-apoptotic effects through this 60-kDa P4BP, which has GABA(A) receptor-like properties and is localized within the surface membrane of GCs.

Animals↗
PubMed1998

Steroidogenic factor-1 regulates the rate of proliferation of normal and neoplastic rat ovarian surface epithelial cells in vitro.

Steroidogenic factor-1 (SF-1) is a transcription factor that is expressed by many cell types within the ovary and has been shown to inhibit granulosa cell proliferation. The present studies were designed to determine whether: 1) SF-1 is expressed by primary and transformed rat ovarian surface epithelial cells (i.e. ROSE cells); and 2) SF-1 expression effects the proliferation of both normal and neoplastic ROSE cells. These studies used immature, gonadotropin-primed and mature rat ovaries, as well as ROSE-179 cells from early passages (EP) and late passages (LP), T-sv-40 transformed ROSE cells, and T-ras transformed ROSE cells. In situ hybridization studies demonstrated that SF-1 was detected in the surface epithelium of rat ovaries, independent of age or gonadotropin treatment. Further, Northern blot and quantitative in situ hybridization studies revealed that significant amounts of SF-1 messenger RNA (mRNA) were present in EP-ROSE-179 cells but not in the other cell lines. Interestingly, EP-ROSE-179 cells proliferated at a significantly slower rate than the other cell lines. Further, SF-1 mRNA levels were higher in EP-ROSE-179 cells in the G0/G1 stage than in the S-, G2/M stage of the cell cycle. These observations suggest that a cause and effect relationship exists between the level of SF-1 expression and cell proliferation. To test this hypothesis, LP, T-sv-40, and T-ras ROSE cells were transfected with either control vector or SF-1 expression vector. Forty-eight hours after transfection, SF-1 expression was assessed by in situ hybridization, and the fold increase in cell number/24 h was determined. For each cell line, about 30% of the cells were successfully transfected. The fold increase in the number of cells observed after transfection with the SF-1 expression vector was significantly less than the increase in cell number after transfection with the control vector (P < 0.05). To confirm that the forced expression of SF-1 prevented proliferation, LP cells were cotransfected with a green fluorescent protein (GFP) expression vector and either control vector or SF-1 expression vector. This study demonstrated that virtually none of the GFP/SF-1-transfected cells proliferated over a 24-h period, whereas GFP/Control vector-transfected cells proliferated. Further, approximately 40% of the GFP/SF-1-transfected cells underwent apoptosis after 24 h of culture in serum-supplemented medium. These data demonstrate that: 1) normal ovarian surface epithelial cells express SF-1; 2) SF-1 is also expressed by EP-ROSE-179 cells, but its expression seems to be suppressed when the cells enter the cell cycle; 3) LP-, T-sv, and T-ras ROSE cells do not express SF-1 mRNA; and 4) the inability to express SF-1 is associated with an increase in cell proliferation. Finally, forced SF-1 expression interferes with serum-induced proliferation and leads to apoptosis.

Animals↗
PubMed1997

Putative mechanism through which N-cadherin-mediated cell contact maintains calcium homeostasis and thereby prevents ovarian cells from undergoing apoptosis.

To date most of the studies involving the maintenance of ovarian cell viability have focused on the endocrine, paracrine, and autocrine factors that inhibit these cells from undergoing programmed cell death or apoptosis. Recently, studies have demonstrated that cell contact also prevents ovarian cells from dying via an apoptotic mechanism. In this commentary, the role that homophilic binding of the cell adhesion molecule, N-cadherin, plays in maintaining ovarian cell viability is presented. These studies showed that N-cadherin homophilic binding (1) is part of the mechanism through which cell contact maintains cell viability, (2) results in the activation (i.e. tyrosine phosphorylation) of the fibroblast growth factor (FGF) receptor, and (3) prevents a sustained elevation in intracellular free calcium ([Ca2+]i) which triggers apoptosis. These studies also revealed that hepatocyte growth factor (HGF), also known as scatter factor (SF), disrupts cell contact, which leads to a sustained increase in [Ca2+]i levels and ultimately to cell death. Based on these studies, this commentary presents a putative mechanism that relates the cellular and molecular mechanism through which basic FGF, N-cadherin, and HGF/SF interact to regulate [Ca2+]i levels and ultimately ovarian cell survival.

Animals↗
PubMed1997

Basic fibroblast growth factor and N-cadherin maintain rat granulosa cell and ovarian surface epithelial cell viability by stimulating the tyrosine phosphorylation of the fibroblast growth factor receptors.

Both granulosa cells (GCs) and ovarian surface epithelial cells undergo apoptosis in vivo. Although basic fibroblast growth factor (bFGF) and N-cadherin-mediated cell contact inhibit GC apoptosis, little is known about the factors that influence rat ovarian surface epithelial (ROSE) cell apoptosis. The present studies were designed to determine whether bFGF and N-cadherin maintain the viability of both GC and ROSE cells by stimulating separate signaling pathways. For the GC studies, large GCs were collected from immature rat ovaries after Percoll gradient centrifugation and placed in serum-free culture for 24 h. These studies confirmed that about 10% of the aggregated GCs and more than 50% of single GCs were apoptotic after culture. bFGF reduced the percentage of apoptotic single GCs, but did not influence aggregated GCs. A neutralizing antibody to bFGF blocked bFGF's antiapoptotic action, but did not alter the percentage of apoptotic aggregated GCs. The antibody to N-cadherin not only increased the percentage of aggregated apoptotic GCs, but also blocked bFGF's ability to maintain the viability of single GCs. The effect of the FGF receptor antibody was similar to that of the N-cadherin antibody. Like GCs, ROSE cells also undergo apoptosis in serum-free medium. Exposure to either the N-cadherin or FGF receptor antibody, even in the presence of serum, increased the percentage of apoptotic aggregated ROSE cells. As tyrosine kinase activity is involved in maintaining cell viability, the pattern of tyrosine-phosphorylated proteins was examined after culture in control (ascites) or N-cadherin antibody-supplemented medium. Exposure to the N-cadherin antibody altered the pattern of tyrosine-phosphorylated proteins, decreasing the tyrosine phosphorylation of proteins in the 130- to 180-kDa range and increasing the tyrosine phosphorylation of one or more proteins of about 50 kDa. The identity of the 50-kDa protein is unknown. However, immunoprecipitation studies demonstrated that the N-cadherin antibody reduced the amount of tyrosine-phosphorylated FGF receptor in both GCs and ROSE cells by 50%. This decrease corresponds to an increase in apoptosis among aggregated cells. Taken together, these data suggest that homophilic N-cadherin binding and bFGF-FGF receptor binding activate signal transduction pathways that converge at the level of the FGF receptor and subsequently promote the viability of both GC and ROSE cells.

Animals↗
PubMed1997

Opposing actions of hepatocyte growth factor and basic fibroblast growth factor on cell contact, intracellular free calcium levels, and rat ovarian surface epithelial cell viability.

Previous studies demonstrated that cell-to-cell contact stimulates a tyrosine phosphorylation signal transduction pathway that prevents rat ovarian surface epithelial (ROSE) cells from undergoing apoptosis. Hepatocyte growth factor (HGF), also know as scatter factor (SF), is expressed by ovarian stromal and thecal cells and has been shown to reduce cell contact in nonovarian tissues. The present studies were designed to determine whether HGF/SF promotes ROSE cells to dissociate and subsequently become apoptotic. Because an increase in intracellular free calcium ([Ca2+]i) is often an early event in the apoptotic cascade, the effects of HGF/SF on [Ca2+]i levels were also assessed. ROSE cells were cultured in serum-free medium with HGF/SF, basic fibroblast growth factor (bFGF), thapsigargin, Bay K, actinomycin D, cycloheximide, and/or BAPTA depending on the experimental design. Cell contact was assayed by time-lapse photography; [Ca2+]i levels were measured with Fluo-3, and apoptosis was assessed by in situ DNA staining. HGF/SF decreased cell contact within 1 h, increased [Ca2+]i levels by 3 h, and induced apoptosis by 6 h of culture. bFGF inhibited these HGF/SF-induced responses. The increase in [Ca2+]i appears to represent a point in the apoptotic cascade that commits ROSE cells to die. This concept is based on the observations that: 1) in the presence of the calcium chelator BAPTA, HGF/SF decreased cell contact but did not increase [Ca2+]i or apoptosis; 2) bFGF blocked HGF/SF-induced increase in [Ca2+]i; 3) bFGF did not attenuate HGF/SF's apoptotic action if exposed to cells after the increase in [Ca2+]i; and 4) RNA and protein synthesis were required for HGF/SF to increase [Ca2+]i, whereas the thapsigargin- and Bay K-induced increase in [Ca2+]i and apoptosis were independent of RNA/protein synthesis. These observations indicate that the components of the apoptotic cascade distal to the increase in [Ca2+]i are present within ROSE cells and are activated by a sustained elevation of [Ca2+]i. The present studies also show that when ROSE cells establish contact with 3T3 cells that express N-cadherin, [Ca2+]i levels are maintained at low basal levels. In contrast, cell contact with 3T3 cells that do not express N-cadherin results in elevated [Ca2+]i levels. Similarly, a synthetic N-cadherin peptide, which inhibits homophilic N-cadherin binding, increases [Ca2+]i levels. Taken together, these data indicate that homophilic N-cadherin binding between adhering cells plays an important role in maintaining calcium homeostasis. Further, these data support the concept that HGF/SF's ability to promote the dissociation of ROSE cells accounts in part for its ability to increase [Ca2+]i levels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Apoptosis as a function of ovarian reserve in women undergoing in vitro fertilization.

OBJECTIVE: To test the hypothesis that preovulatory follicles from women with diminished ovarian reserve contain fewer numbers of luteinized granulosa cells and have a greater percentage of cells undergoing apoptosis as compared with women with uncompromised ovarian reserve undergoing superovulation for IVF-ET. DESIGN: Comparative study. SETTING: Academic clinical practice. PATIENTS: Luteinized granulosa cell counts were determined from follicular aspirates harvested from 19 women with day 3 serum FSH < or = 6 mIU/mL (conversion factor to SI unit, 1.00) and from 15 women with FSH levels > or = 10 mIU/mL. The second part examined the percent of proliferating and apoptotic cells harvested from nine women with day 3 serum FSH < or = 6 mIU/ mL and from eight women with FSH levels > or = 10 mIU/mL. MAIN OUTCOME MEASURES: Cell counts by hemacytometer and percent of proliferating and of apoptotic granulosa cells by flow cytometry. RESULTS: The mean number of viable cells isolated per follicle were 2.7 x 10(5) (95% confidence interval [CI] 2.3 to 3.1 x 10(5) from the low FSH group and 1.8 x 10(5) (95% CI 1.4 to 2.3 x 10(5) from the high FSH group. Although the percentage of proliferating cells was the same, the mean percentage of apoptotic cells was 0.5% (95% CI 0.2% to 1.4%) from the low group and 2.0% (95% CI 1.1% to 3.7%) from the high group. CONCLUSIONS: Women undergoing IVF with day 3 serum FSH > or = 10 mIU/mL have preovulatory follicles with fewer luteinized granulosa cells and an increase in the percentage of cells undergoing apoptosis as compared with women with day 3 serum FSH < or = 6 mIU/mL.

Adult↗
PubMed1996

Steroidogenic factor-1 expression is transiently repressed and c-myc expression and deoxyribonucleic acid synthesis are induced in rat granulosa cells during the periovulatory period.

The expression of steroidogenic factor 1 (SF-1), cytochrome P450 aromatase (P450arom), and cytochrome P450 cholesterol side-chain cleavage (P450scc) was examined during the periovulatory period. Immature rats were injected with eCG to induce development of ovarian follicles to the preovulatory stage. At 48 h after the eCG injection, the LH surge was simulated by an injection of an ovulatory dose of hCG, and RNA was isolated at 0, 2, 4, 6, 8, and 24 h after hCG injection. The mRNA levels for SF-1, P450arom, and P450scc were relatively high in total ovarian RNA samples from eCG-treated rats. Levels of SF-1 and P450arom mRNA decreased within 2 h after injection of hCG. The SF-1 mRNA levels gradually increased from 4 to 24 h. Aromatase levels remained undetectable until 24 h after hCG. P450scc mRNA levels increased throughout the 24-h period after hCG. Levels of SF-1 and P450arom, but not P450scc, mRNA were also reduced in RNA samples from isolated granulosa cells at 4 h after hCG treatment relative to those from eCG-treated rats. In situ hybridization analysis also revealed that hCG uniformly suppressed SF-1 mRNA levels an all granulosa cells compared to those of eCG-treated controls. The relationship of SF-1 expression to immediate/early gene expression and cell cycle traverse was also examined. C-myc mRNA levels were induced by up to 10-fold at 4 h after hCG injection. Similarly, DNA synthesis, as measured by the percentage of granulosa cells that incorporated 5'-bromodeoxyuridine, was increased from 16 +/- 4% in eCG-treated rats to 61 +/- 7% at 4 h after hCG treatment (p < 0.05). This study provides the novel finding that SF-1 expression is transiently repressed to very low levels in response to the LH surge. Further, these studies suggest that granulosa cells traverse the cell cycle before becoming terminally differentiated luteal cells.

Animals↗
PubMed1996

Steroidogenic factor-1 as a positive regulator of rat granulosa cell differentiation and a negative regulator of mitosis.

Ovarian follicles contain small nonaromatase-expressing and large aromatase-expressing granulosa cells (GCs). The present studies were designed to determine whether small GCs can differentiate into large GCs and/or express aromatase. Additional studies were conducted to assess the role of steroidogenic factor-1 (SF-1), an orphan nuclear receptor, in regulating GC differentiation and proliferation. For these studies, small GCs were isolated from immature rats by Percoll gradient centrifugation and cultured for up to 48 h with FSH and/or 8-bromo-cAMP (8-br-cAMP). FSH/8-br-cAMP induced a 2-fold increase in SF-1 messenger RNA levels within 4 h. This increase was maintained throughout the culture period. By 24 h culture, FSH/8-br-cAMP increased the percentage of large GCs. It was not until 48 h of culture with FSH and 8-br-cAMP that aromatase expression increased. This increase was detected by both Western blot and quantitative immunocytochemistry. 8-br-cAMP alone did not promote GC differentiation. Small GCs were then cultured with FSH/8-br-cAMP in the presence or absence of an antisense oligonucleotide complementary to the putative SF-1 ligand-binding site (SF-1 AS). As a control, small GCs were cultured with FSH/8-br-cAMP and an 18-mer nonsense oligonucleotide (SF-1 NS). The SF-1 AS, but not the SF-1 NS, prevented FSH/8-br-cAMP from increasing 1) SF-1 messenger RNA levels, 2) transcription of a SF-1(x2) promoter/luciferase construct, 3) GC size, and 4) aromatase expression. In a third series of experiments, small GCs were cultured for 24 h in 1) control media supplemented with 2) a mitogen, phorbol ester [12-O-tetraphorbol acetate (TPA)], 3) FSH/8-br-cAMP, or 4) both. TPA increased the number of GCs by 51 +/- 9%. FSH/8-br-cAMP completely blocked TPA-induced mitosis. When small GCs were cultured with FSH/cAMP, TPA, and SF-1 AS, the number of GCs increased by 50 +/- 7%. This increase was not observed with SF-1 NS. Taken together, these data demonstrate that SF-1 is expressed in both small and large GCs, and enhanced SF-1 expression is part of the molecular mechanism associated with GC differentiation. Interestingly, SF-1 not only regulates differentiation, but also inhibits TPA-induced GC mitosis.

Animals↗
PubMed1996

N-cadherin-mediated cell contact inhibits granulosa cell apoptosis in a progesterone-independent manner.

Previous studies have shown that both progesterone and cell contact inhibit granulosa cell (GC) apoptosis in vitro. Since the progesterone concentration associated with aggregated GCs may be higher than that of single GCs, experiments were conducted to differentiate progesterone's action from that of cell contact. For these studies, GCs were isolated from immature rats. Large GCs were collected after Percoll gradient centrifugation and placed in serum-free culture for 24 h. These studies confirmed that the rate of apoptosis was 2-3 times higher for single GCs than for aggregated GCs. This relationship was observed in the presence of aminoglutethimide, where progesterone concentrations were 3 ng/ml or less. A dose-response studied revealed that a minimum of 100 ng/ml progesterone were required to suppress apoptosis of single GCs. In addition, a single cell contact was shown to be sufficient to suppress apoptosis, with a small nonsteroidogenic GC being as effective as a large steroidogenic GC. Taken together, these data support the concept that cell contact blocks apoptosis in a progesterone-independent manner. GC contact is due to the presence of gap and adhesion-type junctions. To assess which, if either, of these junctions is involved in mediating the antiapoptotic action of cell contact, cocultures were set-up between GCs and R2C cells. Contact with R2C cells inhibits GC apoptosis, but does not result in the formation of functional gap junctions. This demonstrates that gap junctions are not essential to maintain GC viability. Adhesion-type junctions result from a homophilic binding of N-cadherin, which is expressed by both GCs and R2C cells. When this binding is inhibited by treatment with either an antibody to N-cadherin or a synthetic N-cadherin peptide, cell aggregation is attenuated. For those cells that form cell contacts in the presence of these N-cadherin-binding inhibitors, the percentage of apoptotic cells is increased compared to that in controls. These observations suggest that homophilic binding of N-cadherin molecules on the surface membranes of adjacent GCs initiates a signal transduction cascade that ultimately inhibits apoptosis.

Animals↗
PubMed1995

Cellular and molecular mechanisms that mediate insulin-dependent rat granulosa cell mitosis.

Rat ovarian follicles are composed of small and large granulosa cells (GC). The present studies demonstrate that small GCs undergo insulin- or phorbol ester-dependent mitosis in vitro. In order to examine the cellular and molecular events that account for insulin's mitogenic action, small GCs were cultured with either insulin, phorbol ester (TPA), or both insulin and TPA. Insulin and TPA increased GC numbers by 21 +/- 3% and 20 +/- 2% over control values, respectively (p < 0.05). Simultaneous addition of insulin and TPA increased GC numbers by 20 +/- 3% (p < 0.05). In a second experiment, small GCs were exposed to control medium, insulin, staurosporine (a protein kinase C [PKC] inhibitor), or both insulin and staurosporine. These studies revealed that insulin induced a 21 +/- 5% increase in GC numbers and that staurosporine blocked insulin's mitogenic action. These observations suggest that insulin mediates its mitogenic action through a PKC-dependent mechanism. Since the proto-oncogenes, c-fos and c-jun, are expressed during GC mitosis, studies were undertaken to determine whether or not the expression of these two proto-oncogenes products was enhanced by insulin. The expression of c-fos and c-jun proteins was assessed by immunocytochemistry. These studies showed that after 5 h, insulin increased the percentage of cells that stained for c-fos and c-jun by 15 +/- 2% and 19 +/- 4, respectively (p < 0.05). The expression of these proto-oncogenes was blocked by staurosporine. Both progesterone and 8-br-cAMP, which block insulin-dependent GC mitosis, also inhibited the expression of c-fos and c-jun.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Effect of in vivo gonadotropin treatment on the ability of progesterone, estrogen, and cyclic adenosine 5'-monophosphate to inhibit insulin-dependent granulosa cell mitosis in vitro.

The ability of progesterone (P4), estradiol-17 beta (E2), and 8-bromo (br)-cAMP to inhibit small granulosa cells (GCs) from undergoing insulin-dependent mitosis was examined. Small GCs were isolated from immature and eCG-primed rats and separated by Percoll fractionation. Small GCs were cultured for 24 h with various combinations of insulin, steroids, steroid receptor antagonists, and 8-br-cAMP. Before and after culture, the number of GCs was counted. Small GC proliferation was expressed as a percentage increase over the initial value P4 inhibited insulin-dependent mitosis of small GCs isolated from both immature and eCG-primed rats. The effects of P4 were dose-dependent, steroid-specific, and reversed by the progesterone antagonist RU486. E2 inhibited insulin-dependent mitosis of small GCs isolated from immature but not eCG-primed rats. The action of E2 was dose-dependent and inhibited by the estrogen antagonist tamoxifen. Additional studies were conducted in which small GCs from immature rats were cultured with insulin in the presence of both P4 and E2 and their respective antagonist. Both antagonists were required for insulin to induce GC mitosis in the presence of P4 and E2. Further, the ability of P4 to suppress insulin-dependent mitosis was reduced if it was not present during the first 6 h of culture. In contrast, E2 could be added up to 12 h after insulin exposure and still completely prevent GC mitosis. 8-br-cAMP also prevented insulin-dependent GC proliferation. The actions of 8-br-cAMP could not be reversed by aminoglutethimide or RU486.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Proliferative and steroidogenic capabilities of rat granulosa cells of different sizes.

Equine chorionic gonadotrophin stimulates both rat granulosa cell mitoses and oestradiol secretion. However, the mitotic potential of oestradiol-secreting granulosa cells is not known. In the first study, granulosa cells of different sizes were isolated and their ability to secrete oestradiol and proliferate in vitro was determined. Granulosa cells were harvested from equine chorionic gonadotrophin-primed immature rats, separated on a 15-45% Percoll gradient, and collected in 12 fractions. An enriched population of small granulosa cells (44 +/- 1 micron2) was collected in fractions 3 and 4 and an enriched population of large granulosa cells (97 +/- 2 microns2) in fractions 6-8. When granulosa cells from each fraction were cultured for 24 h in the presence of testosterone, the large cells secreted 50% more oestradiol than did the small cells (P < 0.05). Aromatase was shown, by immunocytochemistry, to be expressed mainly by granulosa cells larger than 73 microns2, with the relative amount of aromatase expressed per cell increasing with increasing cell size. However, not all large granulosa cells expressed aromatase. To test proliferative capacity, cells from each fraction were cultured with testosterone and the mitogen, insulin. This study showed that only small cells were able to undergo insulin-induced mitosis. In a second study, follicles of different sizes were isolated from immature and equine chorionic gonadotrophin-primed immature rats and the granulosa cell size distribution determined for each follicle size. This study confirmed that equine chorionic gonadotrophin altered the size distribution from principally small mitotically competent cells to large oestradiol-secreting cells.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

The steroidogenic and morphological effects of paclitaxel on cultured ovarian cancer cells.

We have studied the biochemical effects of paclitaxel (trade name Taxol) in three ovarian cancer (OV Ca) cell lines and JEG-3 choriocarcinoma cells. Paclitaxel (1 microgram/ml) was cytotoxic to approximately 80% of OV Ca cells, and the ED50 ranged from 6 to 9 ng/ml. Paclitaxel was also cytotoxic to JEG-3 cells (ED50 40 ng/ml), but even at 1 microgram/ml, 40-50% of the cells survived paclitaxel treatment. Paclitaxel increased 17 beta-estradiol secretion 3-4 fold in all three OV Ca cell lines with an ED50 range of 3-13 ng/ml. Similarly, paclitaxel increased estradiol secretion from JEG cells with an ED50 of 50 ng/ml. Colchicine also increased estradiol secretion significantly from OV Ca cells at 2 microM while reducing cell number approximately 40% (beta-lumicolchicine, an inactive isomer, was ineffective at this concentration). Paclitaxel (1 microgram/ml) treatment of BR OV Ca cells produced alterations in morphology leading to "rounding" of cells within 6 h of treatment. Simultaneously, paclitaxel increased immunohistochemical staining for aromatase, and this increase was coincident with morphological alterations. The present results demonstrate that low concentrations of paclitaxel can have significant steroidogenic, as well as cytotoxic, effects on OV Ca cells, suggesting that paclitaxel may activate signal transduction pathways in addition to disrupting microtubule function. These findings further suggest that paclitaxel could be efficacious at submicromolar concentrations.

Adenocarcinoma↗
PubMed1994

Epidermal growth factor inhibits large granulosa cell apoptosis by stimulating progesterone synthesis and regulating the distribution of intracellular free calcium.

The initial study was designed to determine whether all granulosa cells (GCs) undergo apoptosis in vitro. GCs were isolated from immature rat ovaries and separated on a 15-45% Percoll gradient. Twelve fractions were collected, and GCs were pooled according to size: small GCs (approximately 50 mu 2; fractions 2-5) and large GCs (> or = 75 mu 2; fractions 6-8). GCs were cultured in serum-free medium for 24 h. After 24 h of culture, fragmented DNA, detected by in situ end labeling of the 3'OH ends of DNA fragments, was observed within 70-80% of large GCs. Similarly, in situ DNA staining demonstrated that at least 50% of large GCs possessed apoptotic nuclei. These degenerative changes in DNA were observed within < or = 5% of small GCs. These studies demonstrate that in serum-free medium, most large GCs die via an apoptotic mechanism within 24 h. Subsequent studies focused on the mechanism by which epidermal growth factor (EGF) inhibits large GC apoptosis. EGF reduced the percentage of large GCs with apoptotic nuclei from 47 +/- 1% for controls to 18 +/- 2% (p < 0.05). EGF also increased progesterone (P4) secretion from large GCs (6.3 +/- 0.7 for controls vs. 18.7 +/- 1.0 ng/ml for EGF treatment; p < 0.05). The effect of EGF on apoptosis was mimicked by P4 and attenuated by the P4 antagonist, RU 486, and aminoglutethimide (AG), an inhibitor of P4 synthesis. The effect of AG was overridden by P4. Therefore, EGF reduces large GC apoptosis by stimulating P4 synthesis, with P4 mediating its action through its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoglutethimide↗
PubMed1993

Control of rat granulosa cell mitosis by phorbol ester-, cyclic AMP-, and estradiol-17 beta-dependent pathways.

The present studies examined the effect of 8-bromo-cAMP (8-Br-cAMP), phorbol ester (TPA), and estradiol-17 beta (E2) on the capacity of rat granulosa cells (GC) to undergo mitosis. In the first series of experiments, GC were either maintained within immature rat ovaries in perifusion culture or isolated and placed in tissue culture. GC were cultured for 24 h with 1) control medium, 2) 8-Br-cAMP, 3) TPA, or 4) 8-Br-cAMP plus TPA in the presence of 3H-thymidine (3H-T). In perifusion culture, 8-Br-cAMP stimulated both 3H-T incorporation (p < 0.05) and E2 secretion (p < 0.05) while TPA increased 3H-T (p < 0.05) without altering E2 secretion (p > 0.05). Simultaneous exposure to 8-Br-cAMP and TPA enhanced 3H-T incorporation and suppressed E2 as compared to 8-Br-cAMP treatment alone (p < 0.05). In tissue culture, 8-Br-cAMP did not increase 3H-T incorporation or cell number. TPA increased both 3H-T incorporation (p < 0.05) and cell number (p < 0.05), while 8-Br-cAMP suppressed both of these TPA-induced responses. In the presence of testosterone, 1) TPA's mitogenic action was also observed, 2) basal E2 secretion ranged between 30 and 35 pg/ml, 3) neither 8-Br-cAMP nor TPA stimulated E2 secretion over basal levels, and 4) Rp-cAMP, a cAMP antagonist, blocked TPA-induced cell proliferation. E2 at 250 pg/ml also blocked TPA's mitogenic action. In a second series of experiments, GC were collected from eCG-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

The role of progesterone in regulating human granulosa cell proliferation and differentiation in vitro.

To further elucidate the role of progesterone in regulating granulosa cell function, human granulosa and luteal cells were obtained from follicular aspirates of women undergoing in vitro fertilization and placed in culture. Cells plated at 5 x 10(3) cells/mL doubled after 3 days. In contrast, cells plated at 50 x 10(3) cells/mL did not proliferate, but differentiated, secreting high levels of progesterone. Cells plated at 5 x 10(3) cells/mL and cultured with spent medium from cells plated at 50 x 10(3) cells/mL did not increase in number over 3 days of culture. The growth-inhibiting action of the spent medium was removed by either RU 486 (a progesterone receptor antagonist) or charcoal extraction, but not by heat inactivation. The addition of progesterone to fresh medium also prevented cell proliferation. Progesterone's ability to inhibit cell division was attenuated by either RU 486 or aminoglutethamide, which blocked progesterone synthesis. Further, epidermal growth factor (EGF) stimulated cell proliferation, and continuous exposure to progesterone blocked EGF-induced mitosis. When progesterone was added 2 h after EGF, it did not block EGF-stimulated cell proliferation. Progesterone also increased the percentage of granulosa cells and decreased the percentage of large luteal cells present after 3 days of culture, indicating that progesterone inhibited differentiation. Progesterone's effect on differentiation was dose dependent, reversible, and could be overridden by hCG or 8-bromo-cAMP. These observations suggest that progesterone acts directly on granulosa cells through its receptor to inhibit mitosis and that progesterone mediates its antiproliferative effects within 2 h of mitotic stimulation. Progesterone also blocks differentiation, but this effect of can be overcome by hCG or cAMP analogs. These data indicate that progesterone plays a major role in controlling the number of luteal cells that ultimately develop within a corpus luteum by regulating both granulosa cell proliferation and differentiation.

Aminoglutethimide↗