Progesterone membrane receptor component 1 expression in the immature rat ovary and its role in mediating progesterone's antiapoptotic action.

Progesterone receptor membrane component-1 (PGRMC1) interacts with plasminogen activator inhibitor RNA binding protein-1 (PAIRBP1), a membrane-associated protein involved in the antiapoptotic action of progesterone (P4). In this paper, the first studies were designed to assess the ovarian expression pattern of PGRMC1 and PAIRBP1. Western blot analysis revealed that spontaneously immortalized granulosa cells (SIGCs) as well as granulosa and luteal cells express both proteins. Luteal cells were shown to express more PGRMC1 than granulosa cells. Immunohistochemical studies confirmed this and demonstrated that PGRMC1 was present in thecal/stromal cells, ovarian surface epithelial cells, and oocytes. PAIRBP1 was also expressed in thecal/stromal cells and ovarian surface epithelial cells but not oocytes. Furthermore, PAIRBP1 and PGRMC1 were detected among the biotinylated surface proteins that were isolated by avidin affinity purification, indicating that they localized to the extracellular surface of the plasma membrane. Confocal microscopy revealed that both of these proteins colocalize to the plasma membrane as well as the cytoplasm. The second studies were designed to assess PGRMC1's role in P4's antiapoptotic actions. These studies showed that overexpression of PGRMC1 increased 3H-P4 binding and P4 responsiveness. Conversely, treatment with a PGRMC1 antibody blocked P4's antiapoptotic action. Taken together, the present findings indicate that both PAIRBP1 and PGRMC1 show a similar expression pattern within the ovary and colocalize to the extracellular surface of the plasma membrane. At the plasma membrane, these two proteins interact to form a complex that is required for P4 to transduce its antiapoptotic action.

Placing progesterone in the apoptotic pathway.

Progesterone regulates the viability of cells of several different reproductive tissues, including the uterus, breast, corpus luteum, and ovarian follicle (that is, granulosa cells). Progesterone's antiapoptotic actions are thought to be mediated through the progesterone receptor because the progesterone antagonist RU 486 attenuates progesterone's action. Uterine, mammary, and luteal cells express the classic nuclear progesterone receptor, whereas granulosa cells of developing and mature follicles do not. This review presents data that suggest that progesterone maintains granulosa cell viability through a nongenomic mechanism.

Steroidogenic factor 1, an orphan nuclear receptor, regulates the expression of the rat aromatase gene in gonadal tissues.

In a concerted analysis of the genes encoding three mouse steroid hydroxylases, we identified and characterized a transcriptional regulatory protein, designated steroidogenic factor 1 (SF-1), that contributes to the coordinate expression in adrenocortical cells. SF-1, an orphan member of the nuclear receptor family, binds to PyCAAGGPyCPu motifs upstream of the steroid hydroxylases to regulate their expression. In the present study, we extend these findings by examining the role of SF-1 in regulation of the rat P450 aromatase gene in gonadal tissues. The 5'-flanking region of the rat aromatase gene was isolated by a polymerase chain reaction-based approach, using primers corresponding to the 5'- and 3'-ends of a published aromatase sequence. DNA sequence analysis revealed three differences between our sequence and the previously published sequence, including a 44-base pair (bp) insertion. Moreover, the transcription initiation site, as determined by primer extension analysis, differed from that previously proposed. The new transcription initiation site is located 23 bp 3' of a putative TATA box. When a revised rat sequence was compared to that of the human aromatase PII promoter by BEST-FIT analysis, a region of about 300 bp was identified that was 80% conserved between the two promoters. A potential SF-1 site, CCAAGGTCA, was identified at position -82 within this region. An oligonucleotide probe containing this putative SF-1 site was used in gel mobility shift assays. Consistent with previous studies, a specific complex was observed with nuclear extracts from gonadal steroidogenic tissues but was absent with nuclear extracts from nonsteroidogenic tissues. The role of SF-1 in this steroidogenic cell-specific complex was next addressed more directly. Bacterial extracts containing an SF-1-glutathione S-transferase fusion protein interacted specifically with the putative SF-1 site, and polyclonal antisera against SF-1-glutathione S-transferase specifically abolished the complex formed with nuclear extracts from rat ovaries or R2C rat Leydig tumor cells. Finally, the aromatase SF-1 element increased expression of an SV40 promoter/luciferase construct in transient transfection experiments in a steroidogenic cell-selective manner. Collectively, these studies implicate SF-1 in the regulation of steroid hydroxylase gene expression in nonadrenal tissues, significantly extending previous studies in adrenocortical cells.

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.

Basic fibroblast growth factor maintains calcium homeostasis and granulosa cell viability by stimulating calcium efflux via a PKC delta-dependent pathway.

Previous studies have demonstrated that basic fibroblast growth factor prevents granulosa cell apoptosis. The following six observations provide insight into the mechanism by which basic fibroblast growth factor mediates its antiapoptotic action. First, loading granulosa cells with 1,2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an intracellular calcium chelator, prevented apoptosis when granulosa cells were deprived of basic fibroblast growth factor. Second, treatment with thapsigargin, an agent known to increase intracellular free calcium, induced granulosa cell apoptosis even in the presence of basic fibroblast growth factor. Third, an activator of PKC mimicked, whereas PKC inhibitors blocked, basic fibroblast growth factor's antiapoptotic action. Fourth, continuous basic fibroblast growth factor exposure maintained relatively constant levels of intracellular free calcium, and a PKC inhibitor induced a sustained 2- to 3-fold increase in intracellular free calcium. Fifth, granulosa cells, as well as spontaneously immortalized granulosa cells, were shown to express PKC delta, -lambda, and -zeta. Finally, the PKC delta-specific inhibitor, rottlerin, blocked basic fibroblast growth factor's antiapoptotic action in granulosa cells and spontaneously immortalized granulosa cells. These studies suggest that basic fibroblast growth factor regulates intracellular free calcium through a PKC delta-dependent mechanism and that a sustained increase in intracellular free calcium is sufficient to induce and is required for granulosa cell apoptosis. Additional studies demonstrated that in spontaneously immortalized granulosa cells, basic fibroblast growth factor increased PKC delta activity by 60% within 2.5 min compared with serum-free control levels. Rottlerin attenuated basic fibroblast growth factor's ability to stimulate PKC delta activity and to maintain intracellular free calcium. Further, intracellular free calcium levels in spontaneously immortalized granulosa cells transfected with a PKC delta antibody in the presence of basic fibroblast growth factor were 2-fold higher than those spontaneously immortalized granulosa cells transfected with IgG. Similarly, transfecting spontaneously immortalized granulosa cells with a specific PKC delta-substrate increased intracellular free calcium compared with spontaneously immortalized granulosa cells transfected with a specific substrate for PKC epsilon. Moreover, basic fibroblast growth factor increased and rottlerin attenuated (45)Ca efflux by 50% compared with that in basic fibroblast growth factor-treated cells. Finally, an inhibitor of the plasma membrane calciumadenosine triphosphatase pump suppressed (45)Ca efflux, elevated intracellular free calcium, and induced apoptosis. Collectively, these studies demonstrate that basic fibroblast growth factor activates PKC delta, which, in turn, stimulates calcium efflux, accounting in part for basic fibroblast growth factor's ability to maintain calcium homeostasis and, ultimately, granulosa cell viability.

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.

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.

Hepatocyte growth factor induces rat ovarian surface epithelial cell mitosis or apoptosis depending on the presence or absence of an extracellular matrix.

The present studies showed that sequential treatment with equine CG (eCG) and hCG not only induced an increase in ovarian weight, but also caused an estimated 4.6-fold increase in the number of ovarian surface epithelial cells. In addition, eCG-hCG treatment increased ovarian hepatocyte growth factor (HGF) messenger RNA levels. These studies also demonstrated that rat primary ovarian surface epithelial cells as well as a cell line derived from rat ovarian surface epithelium (i.e. ROSE-179 cells) do not express the LH (hCG) receptor. Both of these cells express c-Met, the receptor for HGF. To assess the effects of hCG and HGF on ovarian surface epithelial cell mitosis, ROSE-179 cells were cultured for 24 h in serum-supplemented medium on either glass or the synthetic fibronectin-like extracellular matrix protein, pronectin (RGD). The cells were then cultured for 24 h in serum-free medium in the presence or absence of hCG or HGF. The numbers of cells at 2, 24, and 48 h of culture were determined. The percentage of apoptotic cells was assessed by in situ DNA staining at 48 h of culture. In the serum-supplemented medium in the presence or absence of RGD, the number of ROSE-179 cells doubled. In serum-free medium, cell proliferation was reduced, and the percentage of apoptotic nuclei ranged between 10-15% regardless of the substrate. Neither mitosis nor apoptosis was influenced by hCG in the presence or absence of RGD. For ROSE-179 cells cultured in serum-free medium on RGD, HGF induced mitosis, resulting in a 2.8 +/- 0.2-fold increase in cell number compared with the 24 h control values. On a glass substrate in serum-free medium, HGF did not induce mitosis, but increased the percentage of apoptotic nuclei. Time-lapse photographic analysis revealed that on RGD, cells undergoing HGF-induced mitosis showed a transient reduction in cell contact. On glass, HGF caused many cells to completely lose contact and separate from each other. Collectively, these data suggest that in vivo gonadotropins stimulate HGF expression and ovarian surface epithelial cell proliferation. Based on in vitro studies, it is likely that the mitogenic action of hCG is mediated by HGF. However, HGF only induces mitosis in the presence of an extracellular matrix.

Basic fibroblast growth factor inhibits apoptosis of spontaneously immortalized granulosa cells by regulating intracellular free calcium levels through a protein kinase Cdelta-dependent pathway.

Previous studies have shown that basic fibroblast growth factor (bFGF) inhibits primary granulosa cells from undergoing apoptosis. The present studies were designed to determine whether spontaneously immortalized granulosa cells (SIGCs) undergo apoptosis when deprived of growth factors and whether bFGF prevents apoptosis. In the absence of serum, the SIGCs lost cell contact and underwent apoptosis as indicated by the presence of annexin V binding, DNA ladders, and nuclear fragmentation. Basic FGF maintained cell contact and reduced the percentage of apoptotic cells. This antiapoptotic action was not observed ifbFGF was added 30 min after serum withdrawal. Further, intracellular free calcium ([Ca2+]i) levels gradually increased 3- to 4-fold within 10 min of serum withdrawal. This increase was inhibited by bFGF. The intracellular calcium chelator, BAPTA, completely prevented the SIGCs from undergoing apoptosis in the absence of serum. These observations suggest that bFGF's ability to regulate [Ca2+]i is an essential component of its antiapoptotic action. The phorbol ester TPA, an activator of protein kinase C (PKC), blocked apoptosis due to serum deprivation. Conversely, bisindolylmaleimide II, an inhibitor of PKC, completely attenuated, whereas bisindolylmaleimide V, an inactive bisindolylmaleimide analog, did not influence bFGF's antiapoptotic action. Also, treatment with the PKC inhibitor, chelerythrine chloride, interfered with bFGF's ability to maintain calcium homeostasis. Western blot analysis revealed that SIGCs expressed PKCdelta, tau, lambda, and zeta. Of these PKC isoforms, only PKCdelta has been shown to be activated by TPA. In apoptotic SIGCs, PKCdelta levels were depleted. When PKCdelta levels were reduced by pretreatment with 500 nM TPA, neither bFGF nor 10 nM TPA suppressed apoptosis. Collectively, these observations suggest that bFGF maintains [Ca2+]i and thereby SIGC viability through a PKCdelta-dependent pathway.

Follicle-stimulating hormone and insulin regulation of 17 beta-estradiol secretion and granulosa cell proliferation within immature rat ovaries maintained in perifusion culture.

The present study examined the effects of FSH and insulin (IN) on 17 beta-estradiol (E2) secretion and granulosa cell proliferation. For these studies, immature rat ovaries were maintained in perifusion culture and continuously exposed to FSH (0-800 ng RP-1 eq/ml) and /or IN (0.2 U/ml). At specific times, the ovaries were removed from perifusion, and the perifusate was assayed for E2 by RIA. The ovaries were then incubated with [3H]thymidine ([3H]T) in order to estimate granulosa cel mitotic activity. FSH increased E2 secretion in a dose-dependent manner (P less than 0.05), but did not enhance [3H]T incorporation (P greater than 0.05) after 48 h of perifusion. Conversely, IN increased [3H]T incorporation (P less than 0.05) without stimulating E2 secretion (P greater than 0.05) after 48 h of perifusion. FSH alone or in combination with IN stimulated [3H]T incorporation at 24 h of perifusion compared to both zero hour control (P less than 0.05) and IN treatments (P less than 0.05). The inability of FSH to stimulate [3H]T incorporation after 48 h did not appear to be due to increased E2, since IN stimulated [3H]T incorporation in the presence of 100 ng E2/ml. Further, continuous exposure to FSH was required to maintain E2 secretion, demonstrating that after 24 h, FSH loses its capacity to stimulate granulosa cell [3H]T incorporation, but not E2 secretion. Finally, when ovaries are pretreated with FSH for 48 h and then exposed to FSH and/or IN, [3H]T incorporation was stimulated, and E2 secretion inhibited. These data suggest that 1) initially, FSH acts to stimulate mitosis then promotes granulosa cell E2 secretion; and 2) once granulosa cells begin to secrete E2 they are still capable of mitosis, but their mitotic activity is no longer directed by FSH.

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.

Gonadotropin induction of c-fos and c-myc expression and deoxyribonucleic acid synthesis in rat granulosa cells.

Although gonadotropins stimulate ovarian granulosa cells to proliferate and differentiate into steroidogenic cells, little is known about the molecular mechanisms by which gonadotropins induce these fundamentally different responses. In this study the acute effects of PMSG on protooncogene expression, DNA synthesis, and steroid secretion were examined. The levels of c-fos, c-myc, and beta-actin mRNA were measured in total RNA samples from granulosa cells by quantitative polymerase chain reaction. PMSG increased the mRNA levels of c-fos, c-myc, and beta-actin within 15 min. Fos and myc proteins were localized within granulosa cells by immunocytochemistry. Less than 10% of granulosa cells stained for c-fos or c-myc proteins in the control samples. In contrast, approximately 40% of the cells stained for these protooncogene proteins 30 min after PMSG injection (P less than 0.05). These values declined to about 10% of the cells 60 min after PMSG injection. DNA synthesis, as estimated by [3H]thymidine incorporation, increased 30 and 60 min after PMSG (P less than 0.05). 17 beta-Estradiol and progesterone synthesis did not change within 60 min of PMSG injection. These data demonstrate that 1) c-fos and c-myc are expressed in ovarian granulosa cells; 2) the expression of the genes encoding c-fos, c-myc, and beta-actin is rapidly increased by gonadotropin; and 3) the increase in the corresponding products of the c-fos and the c-myc genes precedes an increase in DNA synthesis and steroid production. These data suggest that the expression of c-fos and c-myc may be a part of the molecular mechanism through which gonadotropins regulate granulosa cell function.

Involvement of an unnamed protein, RDA288, in the mechanism through which progesterone mediates its antiapoptotic action in spontaneously immortalized granulosa cells.

Progesterone (P4) inhibits apoptosis of rat granulosa cells and spontaneously immortalized granulosa cells (SIGCs), which were derived from rat granulosa cells. Defining the mechanism through which P4 mediates its action has been difficult because these cells do not express the classic nuclear P4 receptor. Previous studies have shown that a P4 receptor antibody, C-262, detects a 60-kDa protein that is involved in regulating P4's antiapoptotic action. Using a C-262 affinity column, this 60-kDa protein was isolated and sequenced by mass spectrometry. This analysis revealed that the C-262-detectable protein is an unnamed protein referred to as RDA288. This protein has several putative hyaluronic acid binding sites. Further hyaluronic acid antagonizes (3)H-P4 binding to SIGCs and mimics P4's action, whereas exogenous hyaluronic acid binding protein attenuates P4's actions. RT-PCR demonstrated that RDA288 mRNA was present in SIGCs, immature rat ovary, lung, and skeletal muscle but was not present in several other organs. Forced expression of RDA288 increased the capacity of SIGCs to bind and respond to P4. An antibody was also developed against RDA288. Using this antibody in a Western blot protocol, RDA288 expression was confirmed in both SIGCs and granulosa cells. An immunohistochemical study detected RDA288 in the cytoplasm and plasma membrane components of granulosa cells of antral follicles. Immunocytochemical studies on living nonpermeabilized SIGCs revealed that RDA288 was present on the extracellular surface of the plasma membrane. Finally, pretreatment with the RDA288 antibody blocked P4's antiapoptotic actions. Taken together, these data suggest that RDA288 plays a significant role in mediating P4's antiapoptotic action in granulosa cells.

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.

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.

Progesterone as an autocrine/paracrine regulator of human granulosa cell proliferation.

The present study examined the role of progesterone in regulating human granulosa cell proliferation. Human granulosa and luteal cells were obtained from follicular aspirates of women undergoing in vitro fertilization. Cells were plated at 5, 10, or 50 x 10(3) cells/mL and cultured for up to 6 days. At specific times, cells were harvested and assessed for cell number and morphology. The medium was assayed for progesterone. Cells plated at 50 x 10(3) cells/mL did not increase in number after 3 or 6 days of culture, but rapidly differentiated, secreting high amounts of progesterone (> or = 320 nmol/L). Conversely, cells plated at 5 x 10(3) or 10 x 10(3) cells/mL doubled in number over the first 3 days of culture and subsequently differentiated. The addition of 100 ng/mL progesterone or more to the medium inhibited proliferation. Aminoglutethamide blocked progesterone secretion and increased the number of cells present after 3 days of culture. The antiproliferative effects of progesterone were not mimicked by estradiol, testosterone, dihydrotestosterone, or dexamethasone and could not be overridden by epidermal growth factor, a potent mitogen. These data suggest that progesterone plays an autocrine/paracrine role in regulating granulosa cell proliferation.

Effects of ovariectomy and steroid replacement on hypothalamic LHRH content in aging female rats.

To examine the role of age on the hypothalamic LHRH response to ovariectomy (ovx) and steroid replacement, young cycling (3-4 months) and old constant estrous (18-20 months) rats were ovariectomized. Two weeks later, rats were treated for 5 days with estradiol benzoate (E, 5 micrograms/kg), progesterone (P, 10 mg/kg), E + P (5 micrograms E + 10 mg/kg) or corn oil, after which time they were killed for determination of hypothalamic LHRH content. The young and old ovx rats had similar levels of LHRH in the medial basal (MBH) and anterior (AH) hypothalamus, but E treatment was only effective in increasing MBH-LHRH content in the young animals. There was no significant effect of P alone or in combination with E. In the second experiment, similar results were seen using a single dose of E (1 microgram/rat) in young and old ovx rats. In addition, the radioimmunoassay of LHRH using two different antibodies binding to different portions of LHRH gave similar results in young and old rats, suggesting that the LHRH peptide was being processed similarly in the two age groups. In conclusion, it appears that hypothalamic LHRH content of young and old ovx rats does not differ with age despite a marked attenuation of serum LH levels with age. The hypothalamus from old rats, however, is less responsive to steroid stimulation of LHRH content.

Sex hormones in the aging female.

Aging of the human ovary results in a gradual diminution in ovarian steroid production, followed by an abrupt and almost complete cessation of both estrogen and progesterone production at the menopause. Although carefully controlled quantitative studies have not been published, it appears that the human reproductive axis still retains its ability to respond to steroid action. However, it still remains to be determined if alterations in cycle length and regularity preceding the menopause are due to changes in ovarian function or to subtle changes in the hypothalamic pituitary axis. More experimental evidence is available to describe the etiology of age-related reproductive dysfunction in laboratory rodents. From these studies, it appears that hypothalamic dysfunction is the principal cause for cessation of reproductive cycles. Recent evidence suggests that aging of the hypothalamus is due to cumulative effects of estrogen exposure over the course of the reproductive life span.

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.

Progesterone as a regulator of granulosa cell viability.

Progesterone (P4) prevents numerous cells, including uterine, mammary and ovarian cells, from undergoing apoptosis. Interestingly, P4 prevents apoptosis of ovarian granulosa cells (GCs), which do not express the classic nuclear P4 receptor. This review presents data that support a non-genomic action of P4 in granulosa cells. These studies were conducted using both primary rat granulosa cells and rat spontaneously immortalized granulosa cells (SIGCs). Specifically, these studies reveal that (1) 3H-P4 specifically binds to SIGCs; (2) an antibody directed against the ligand binding domain of the nuclear P4 receptor (C-262) detects a 60kDa protein, which localizes to the plasma membrane and binds P4; and (3) treatment with C-262 blocks P4's ability to maintain granulosa cell viability. Additional studies demonstrate that a protein kinase G (PKG) activator, 8-br-cGMP, mimics and PKG antagonists, Rp-8-pcCPT-GMP and KT5823, attenuate P4's action. These studies support the concept that the 60kDa P4 binding protein functions as membrane receptor for P4 which activates a PKG-dependent mechanism to regulate granulosa cell survival.