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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 7 3441-3451
Copyright © 2002 by The Endocrine Society

Other Original Articles

Stimulation of Apoptosis in Human Granulosa Cells from in Vitro Fertilization Patients and Its Prevention by Dexamethasone: Involvement of Cell Contact and Bcl-2 Expression

Ravid Sasson and Abraham Amsterdam

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel

Address all correspondence and requests for reprints to: Dr. Abraham Amsterdam, Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: . abraham.amsterdam{at}weizmann.ac.il

Abstract

Human granulosa cells obtained from in vitro fertilization patients are highly luteinized, but can still be stimulated by LH/cAMP for production of progesterone. This stimulation involved enhancement of apoptosis. Incubation of the cells with dexamethasone (Dex) reduced the apoptotic incidence compared with nontreated cells and completely abolished the increase in apoptosis stimulated by LH or forskolin, concomitantly with a pronounced increase in progesterone production. Organization of the actin cytoskeleton was dramatically reduced after LH/forskolin stimulation. In contrast, Dex prevented disorganization of the actin filament networks. LH and forskolin also decreased the organization of gap junctions, which could be prevented by Dex. However, the intracellular level of connexin 43 was elevated in the presence of LH, forskolin, and Dex. Endogenous levels of the survival gene protein Bcl-2 were significantly elevated in all cultures treated with Dex compared with either nonstimulated cultures or cultures stimulated with LH and forskolin. Our data suggest that LH/cAMP can stimulate steroidogenesis even during the initial stage of apoptosis of human granulosa cells, whereas Dex, which blocks apoptosis, could further elevate progesterone production. Moreover, the integrity of gap junctions and the actin cytoskeleton as well as elevated levels of Bcl-2 may play an important role in the suppression of apoptosis of human granulosa cells.

HUMAN GRANULOSA cells obtained from in vitro fertilization (IVF) patients heavily stimulated with FSH and hCG have been intensively and extensively studied in the last 18 yr (1, 2). These cells become refractory to gonadotropin/cAMP stimulation when analyzed within 1 or 2 d in primary cultures (3, 4). However, they become responsive to gonadotropin/cAMP if maintained for several days in culture deprived of gonadotropins. Unfortunately, a high apoptotic rate in such cultures, even without stimulation, makes it difficult to study factors that control their death or survival (5).

No apoptosis was detected in dominant human follicles (6). However, degeneration of the corpus luteum is regulated by apoptotic mechanisms (6), involving caspase-1 and caspase-3 activation, DNA fragmentation, and apoptotic protease factor-1 activation (7, 8). In addition, up-regulation of p53 and Bax and down-regulation of clusterin were observed during apoptosis of granulosa-lutein cells (9, 10, 11). Attempts were made to use ultrastructural characteristics (12) and incidence of apoptosis in human granulosa cells obtained from IVF patients as possible indicators of IVF outcome. It was concluded that fewer granulosa-lutein cells are apoptotic in women who have an ongoing pregnancy after IVF treatment than in women who do not conceive (13).

There is only partial information on the molecular determinants that control apoptosis and particularly on molecular mechanisms that control apoptosis in human granulosa-lutein cells. Gonadotropin/cAMP signals are considered to act as survival factors in mammalian granulosa cells during folliculogenesis (14, 15). However, LH/hCG and substances elevating intracellular cAMP, such as forskolin (FK) and 8-bromo-cAMP (8Br-cAMP) were found to be proapoptotic in primary cultures of rat and human granulosa-lutein cells (5, 9, 10, 14, 16). GnRH agonist was demonstrated to directly increase the incidence of apoptosis in porcine and human granulosa cells (17). Therefore, it was suggested that the clinical dosage of GnRH agonist and hCG should always be considered with regard to the pro- or antiapoptotic effect during the treatment of IVF patients (17).

Progesterone and glucocorticoids, such as dexamethasone (Dex) and hydrocortisone, inhibit apoptosis in primary cultures and immortalized human granulosa cells, respectively (18, 19, 20). Moreover, we demonstrated that the effect of glucocorticoids involves up-regulation of the survival gene bcl-2 that may be responsible for their antiapoptotic effects (20). There is conflicting evidence on the role of Bcl-2 in protection against human granulosa cell apoptosis. Matsubara et al. (21), using an immunocytochemical method, reported the absence of Bcl-2 in human granulosa-lutein cells. In contrast, Rodger et al. (22) clearly demonstrated the existence of Bcl-2 in the human corpus luteum by the same method. Therefore, we decided to investigate the presence of Bcl-2 and its possible modulation in pro- and antiapoptotic stimuli for apoptosis in human granulosa-lutein cells in primary cultures. Because cortisone was found to be converted to active cortisol during ovulation and follicular rupture through activation of 11ß-hydroxysteroid dehydrogenase (11ßHSD), which may play an important role in the antiinflammatory process during ovulation (23, 24), we also investigated the effect of glucocorticoids in controlling apoptosis in human granulosa-lutein cells.

Cell-cell contacts and intercellular communications play important roles in granulosa cell development, differentiation, and luteinization (25, 26, 27, 28, 29, 30). However, their involvement and their fate during apoptosis are not completely understood. Growing human and rat granulosa cells on extracellular matrix in the form of the native basement membrane enhanced the development of gap junctions (31) and prevented ovarian granulosa cells from undergoing apoptosis (32). N-cadherin and E-cadherin, the major building blocks of adherence junctions, were also found to be involved in preventing apoptosis of rat granulosa cells (33, 34, 35). In the present paper we examined the distribution and integrity of gap junctions using connexin 43 (CX-43), a major building block of human granulosa cell gap junctions, as a marker to study the response to anti- or proapoptotic signals in primary human granulosa cells. Here we demonstrate for the first time that modulation of CX-43, Bcl-2, and the actin cytoskeleton by LH/cAMP and Dex play an important role in regulation of apoptosis in human granulosa cells.

Materials and Methods

Antibodies

Polyclonal rabbit antihuman Bcl-2 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Goat antirabbit IgG coupled to horseradish peroxidase was obtained from BioMakor (Rehovot, Israel). Dr. A. Mayerhofer provided polyclonal antibody against CX-43 (Anatomical Institute, Technical University of Munich, Munich, Germany) (36). Antihuman steroidogenic acute regulatory protein (StAR) antibodies were provided by Dr. J. F. Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA).

Reagents

FK (a potent activator of adenylate cyclase), Dex, 4',6-diamido-2-phenylindole hydrochloride (DAPI; for DNA staining), and fluorescein isothiocyanate (FITC)-labeled-phalloidin were purchased from Sigma (Rehovot, Israel). Human LH (hLH) was provided by the NIH and Dr. A. Parlow of the National Hormone and Pituitary Program.

Cell culture

Primary granulosa cells were obtained from women undergoing IVF at Sheba Medical Center (Tel-Hashomer, Israel). Patients received a GnRH analog in combination with FSH or human menopausal gonadotropin, followed by administration of hCG (12, 31). Granulosa cells were isolated from aspirated follicular fluid after ovum retrieval. For each set of experiments luteinizing granulosa cells from three women were pooled. Each set of experiments was repeated twice in triplicate plates (total number of patients, 6). The follicular fluid was centrifuged at 300 x g for 5 min to separate granulosa cells from red blood cells. The resulting pellet was resuspended in 10 mM Tris and 0.84% NH4Cl, pH 7.4, to lyse red blood cells (15 min shaking at 37 C). Several washings in PBS achieved elimination of the debris. Cells were plated in DMEM (DMEM/Ham’s F-12, 1:1), supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), and 5% fetal calf serum (FCS). Attachment of the granulosa cells to the bottom of the dishes (35- or 60-mm plastic dishes, NUNC brand products, Roskilde, Denmark) was already achieved after 24 h at 37 C. Medium was removed, and cells were washed five times with PBS (1 ml for 35-mm plastic dish and 4 ml for 60-mm plastic dish) to remove remaining red and nonadherent white blood cells (lymphocyte) as well as cell debris. Costaining of the cells on the culture dish with DAPI for general nuclear DNA and with anti-StAR antibodies revealed that 92.42 ± 2.86% of the total population (random counting in six cultures) was positive in staining of mitochondria StAR (typical for steroidogenic cells) and under a phase microscope showed typical morphology of granulosa cells (e.g. lipid droplets; see Fig. 1Go). The rest of the cells demonstrated fibroblast- or monocyte-like morphology with negative staining for StAR. Cells were cultured for additional 48 h in 5% FCS and washed every 24 h with PBS (twice) to remove any minor contamination of nonadherent red and white blood cells. The 3 d of cell culture before stimulation with LH were essential to release the cells from refractoriness to LH/CG stimulation (5). Cells were washed on d 4 of culture with PBS and incubated in serum-free medium containing the various stimulants.



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  		Figure 1. Staining of primary culture of human granulosa cells with DAPI and anti-StAR antibodies. Monolayers of cells were fixed with 3% paraformaldehyde 24 h after plating and doubly stained with DAPI and anti-StAR antibodies, followed by FITC-conjugated goat antirabbit second antibodies. A, Combined fluorescent and phase microscopy showing healthy cultured granulosa cells, where the DAPI staining delineates the nuclei and the granulated cytoplasm, which is typical for accumulation of lipid droplets (arrowheads). B, Fluorescent microscopy of the same field showing mitochondrial stained with anti-StAR antibodies (arrows). Bar, 10 µm.

 
Biochemical assay

Progesterone released to the culture medium was determined by RIA at the end of stimulation (20, 37). The extent 100 nM Dex cross-reaction with the progesterone RIA was less than 0.01%. The intra- and interassay variations did not exceed 7.5%. Protein was quantified by the Bradford method (38).

Western blot analysis

Cells were washed with cold PBS and harvested with a rubber policemen using lysis buffer containing 50 mM HEPES (pH 7.2), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 30 mM NaF, 30 mM sodium pyrophosphate, 1 mM orthovanadate, and 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 5 µg/ml aprotinin. Lysates were boiled in sample buffer for 10 min. Samples containing 50 µg protein were separated by 12% SDS-PAGE (to detect Bcl-2 and CX-43). The blots were then blocked using 5% milk powder in PBS plus 0.05% Tween 20 and reacted (overnight at 4 C) with antihuman Bcl-2 or CX-43 antibody, followed by 1-h incubation at room temperature with goat antirabbit IgG conjugated to horseradish peroxidase. The detection of the specific protein bands was carried out using enhanced chemiluminescence (10, 39).

Phase contrast and immunofluorescence microscopy

Phase contrast and fluorescence microscopy of cells labeled with anti-CX- 43 antibodies and DAPI or with FITC-labeled phalloidin were carried out as described previously (32, 36, 39, 40). Fixed and permeabilized cells were incubated with 0.16 µg/ml FITC-labeled phalloidin or with 0.5 µg/ml DAPI for 30 min at 24 C. Cells were gently washed with PBS (three times), mounted in Mowiol (Aldrich Chemical Co., Milwaukee, WI), and analyzed under a fluorescence microscope. Microscopic examination of the specimens was carried out using a Zeiss Axioskop microscope (Carl Zeiss, Inc., Oberkochen, Germany) in both phase and fluorescent modes.

Measurement of apoptosis

For quantifying apoptotic events, photographs of 5–10 random fields of each treated culture were taken through a x40 objective lens. Apoptotic and total cell nuclei were counted on x400 magnified images (194–238 nuclei/treatment), and the percentages of the apoptotic nuclei in the total nuclei in each treatment were calculated. Data from three independent different experiments were combined and expressed as the mean ± SEM. This method of in situ DNA staining allows visualization of chromatin condensation and fragmentation as typical patterns of apoptosis (5, 32).

Analysis of the mean size of gap junctions between human granulosa cells in the different treatments

The size of the junctions (long axis) was measured in photographs of labeled cultures with polyclonal rabbit anti-CX-43 antibodies, followed by goat antirabbit IgG conjugated to FITC (see Fig. 4Go). Images taken at x1000 magnification were enlarged to a magnification of x2620. Junctions smaller than 0.15 µm and appearing as round dots inside the cells were not scored and were assumed to contain mainly fragmented internalized gap junctions. For each treatment 60 junctions between 20 cells were scored randomly.



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  		Figure 4. Incidence of apoptosis in human granulosa cells stimulated by LH, FK, and Dex. Apoptotic incidences were scored on fluorescent images of cultured cells stained with DAPI (A) or propidium iodide (B). A, Counts of normal and apoptotic nuclei were made on 5–10 microscopic fields of each treatment at x400 magnifications. B, Sub-G1 cells, essentially apoptotic cells, were scored by FACS analysis of propidium iodide-labeled cells (5000 cells/treatment). Cultures were treated at identical conditions as in Fig. 2Go. Data are the mean ± SD of three independent measurements. Essentially similar results were obtained from pools of six patients (see Materials and Methods). a < b and c, b > e, and c > f, P < 0.01; a' > b', a' < c', d' and c' > f', and d' > g', P < 0.05.

 
Fluorescence-activated cell sorting (FACS) analysis of DNA content

Cells were released from the culture dish by trypsinization after removing the culture medium, which also contained some floating cells. Floating and trypsinized cells from each treatment were collected and combined to ensure complete recovery of the cell population. Cells were washed with cold PBS and fixed in cold methanol (-20 C) for 1 h. Subsequently, cells were centrifuged, resuspended in 0.5 ml cold PBS, and stained for 15 min with 50 µg/ml propidium iodide in the presence of ribonuclease A (100 µg/ml). Cells were analyzed by FACS. Five thousand events from the gated sub population of each treatment were recorded separately (10, 39).

Statistical analysis

All experiments were performed in triplicate culture plates obtained from two pools, each one of three women. All values are expressed as the mean ± SD (n = 6). Analysis of progesterone production and densitometer tracing of Western blot autoradiograms (mean ± SD) were performed using ANOVA, followed by Fisher’s probable least squares differences multiple comparison. Differences between treatment groups were considered statistically significant at P < 0.05.

Results

To examine the rate of apoptosis and its possible modulation by glucocorticoids, we cultured freshly isolated granulosa cells, from patients who had undergone gonadotropin stimulation before retrieval of oocytes, on plastic dishes as monolayers for 3 d at 37 C and subsequently for 24 h with no additions or with Dex (100 nM), LH (1 IU/ml), FK (20 µM), or FK plus Dex (100 nM). Apoptosis was quantified by two independent methods: DNA staining by DAPI and FACS analysis, followed by propidium iodide labeling. Moreover, progesterone release to the medium was measured in parallel in the same cultures. Progesterone production was 31% higher in the presence of serum compared with that in serum-deprived cultures (Fig. 2Go). LH and FK increased progesterone production by 115% and 50%, respectively (P < 0.05), whereas Dex further increased progesterone production by 56% and 37%, respectively (P < 0.05). In contrast, no elevation in progesterone production was found in cultures treated with Dex alone.



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  		Figure 2. Progesterone production in LH-, FK-, and Dex-treated primary cultures of human granulosa cells. Progesterone release to the medium during 24 h at 37 C was measured by RIA. All treatments except for 5% FCS were in serum-free medium. Data are the mean ± SD of triplicate plates. Essentially similar results were obtained from pools of six patients (see Materials and Methods). hLH, 3 IU/ml; FK, 20 µM; Dex, 100 nM. A < b–d; c < f and d < g, P < 0.05.

 
Apoptosis incidence was 10–13%, measured in parallel by DAPI staining (Figs. 3Go and 4AGo) and FACS analysis after labeling the cells with propidium iodide (Fig. 4BGo). Serum deprivation increased apoptosis by 53% (Fig. 4BGo). LH and FK caused a pronounced increase in apoptosis (by 100% and 269% compared with serum-deprived control in Fig. 4AGo, respectively; P < 0.05). Dex attenuated the number of apoptotic events to 76% of that in serum-deprived cultures and suppressed LH and FK stimulation of apoptosis (to 60% of that in LH-stimulated cells and to 24% of that in FK-stimulated cells; Fig. 4BGo). There was a close correlation between apoptotic incidence measured by DAPI labeling and FACS analysis (Fig. 4Go, compare A and B).



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  		Figure 3. DNA staining of primary human granulosa cells cultures treated with LH, FK, and Dex. Aldehyde-fixed cells were stained with DAPI. Cultures were either nonstimulated (A) or stimulated with Dex (B), LH (C), LH plus Dex (D), FK (E), or FK plus Dex (F) under identical conditions as in Fig. 2Go. Arrows indicate apoptotic cell nuclei, which appear smaller and revealed, in general, more highly condensed chromatin compared with normal nuclei.

 
Staining of the cells with specific antibodies to CX-43 revealed intact gap junctions between nonstimulated cells in the size of 1.3 ± 0.16% (mean ± SEM; n = 60; Fig. 5Go, A and A', and Fig. 6Go). In contrast, in FK-treated cells the intensity of staining was clearly elevated. However, fragmented gap junctions (fluorescent bright dots and diffused staining) were abundant in these cultures (Fig. 5Go, B and 5B'Go). The size of the junctions was reduced to 0.82 ± 0.11% ( P < 0.05). Treatment of the cells with Dex suppressed at least part of the junction breakdown and revealed a high incidence of intact gap junctions (mean, 1.05 ± 0.11%; Fig. 5Go, C and 5C'Go). Cells treated with LH demonstrated a high incidence of medium-sized gap junctions (1.6 ± 0.18%; Fig. 5Go, D and 5D'). In contrast, cells treated with hLH plus Dex showed many gap junctions of variable size; the largest ones reached 6–7 µm (Fig. 5Go, E and E'; mean, 2.23 ± 0.28%). Because the cultures were doubly stained with DAPI and CX-43 antibodies, the appearance of gap junction protein could be compared individually in apoptotic and nonapoptotic cells within the same cultures (Fig. 5Go). Staining with anti CX-43 antibody clearly demonstrated that apoptotic cells showed much lower and more diffused and fragmented staining compared with nonapoptotic cells, suggesting a clear breakdown of gap junctions during apoptosis.



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  		Figure 5. Visualization of gap junctions between cultured human granulosa cells treated with FK, LH, and Dex. Gap junction protein was visualized in cultures doubly stained with DAPI (left panel) and anti-CX-43 antibody (right panel) and in nontreated cells (A and A') or cells treated with FK (B and B'), FK plus Dex (C and C'), LH (D and D'), and LH plus Dex (E and E'). Cultures were treated at identical conditions as in Fig. 2Go. Left panel, Cells were visualized in both phase and fluorescent mode (for DNA). Right panel, The same fields visualized with the fluorescent mode for the detection of gap junction protein CX-43. White long arrows point at apoptotic nuclei; black short arrows point at gap junctions.

 


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  		Figure 6. Size of the gap junction in primary human granulosa cells. Granulosa cells were cultured as monolayers for 3 d and subsequently for 24 h with no stimulation or with LH (1 IU/ml), FK (20 µM), FK plus Dex (100 nM) or LH plus Dex. Data are the mean ± SEM length of 60 junctions in each treatment. a is significantly different from b–f, P < 0.05.

 
Quantitation by Western blot of CX-43 revealed a higher content of the protein in LH-, FK-, and Dex-treated cells compared with control cultures (140%, 273%, and 140%, respectively). However, cells treated with LH plus Dex or FK plus Dex did not show any greater increase in CX-43 content (Fig. 7Go). The morphology of the cells in the different treatment was greatly changed. Although control cells were widely spread on the culture dish, cells treated with LH were partially rounded up with long cytoplasmic extensions, and cells treated with FK were further rounded up. Dex treatment led to spreading of the cells similarly to that in the control cultures (Figs. 5Go and 8Go). Staining of cellular actin with FITC-conjugated phalloidin demonstrated dramatic changes in actin filament organization with the different treatments, in line with the changes in cell shape (Fig. 8Go). In nonstimulated cultures, healthy cells that were well spread on the culture dish showed a well developed network of actin filaments. In contrast, apoptotic cells (in the same cultures) were shrunken and showed a diffuse pattern of actin, which was highly concentrated in the apoptotic blebs (Fig. 8Go, A and A'). In Dex-treated cultures (Fig. 8BGo) most of the cells were well spread, demonstrating well developed actin filaments network, whereas the apoptotic shrunken cells demonstrated a diffuse pattern of actin. Cells treated with LH and FK (not shown) were more rounded up than control and Dex-treated cells, with a very fine network of actin. Cells that were completely rounded up and apoptotic cells demonstrated a diffuse pattern of actin (Fig. 8CGo). Cotreatment of LH- or FK-stimulated cells with Dex prevented to a large extent the depolymerization of actin and the rounding of cells (Fig. 8DGo).



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  		Figure 7. CX-43 in cultured human granulosa cells treated with FK, LH, and Dex. Cell lysates of the different groups were prepared for Western blot using specific antibodies to CX-43. CONT, Nonstimulated cells. hLH, 1 IU/ml; FK, 20 µM; Dex, 100 nM. Cultures were treated under identical conditions as in Fig. 2Go. Upper part, CX-43 appearance in blots as triple bands at 43 kDa. The two upper bands probably represent phosphorylated CX-43 protein. Data are the mean ± SD of three independent measurements of the density of the 43-kDa labeled protein (total of three bands) in the different treatments. a < b–f, P < 0.05.

 


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  		Figure 8. The actin cytoskeleton in cultured human granulosa cells. Nonstimulated cultures (A and A') and cells stimulated with Dex (100 nM; B), hLH (1 IU/ml; C), and Dex plus LH (D) were compared. Cells were doubly stained for actin (with FITC-labeled phalloidin) and DNA (with DAPI). Arrows point to well developed actin filaments in healthy cells. Solid arrowheads point to diffuse actin in rounded cells, and open arrowheads point to diffuse actin in apoptotic cells, which is highly concentrated in apoptotic blebs. A–D, Fluorescent microscopy after staining the cultures with FITC-conjugated phalloidin. A', The same field as in A after staining of cell DNA with DAPI (the open arrowheads in A' point to the apoptotic nuclei).

 
Because Bcl-2 protein was demonstrated as a survival and antiapoptotic factor, we detected the intracellular levels of this protein by Western blots. Bcl-2 as a 27-kDa protein was clearly visible in all cell cultures (Fig. 9Go). Densitometric tracing analysis revealed the reduction of Bcl-2 protein in LH- and FK-treated cells (to 77% and 83% compared with control, respectively; P < 0.05). In contrast, Bcl-2 was clearly elevated in Dex-treated cultures by 92% compared with the control (P < 0.05). These significant levels of Bcl-2 did not decline in cultures cotreated with Dex plus LH or Dex plus FK.



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  		Figure 9. Modulation of Bcl-2 expression in cultured human granulosa cells treated with LH, FK, and Dex by Western blot. Human granulosa cells were incubated under the different conditions indicated in Fig. 2Go. Bcl-2 protein was visualized as 27-kDa protein (upper panel). Densitometer analysis of three independent measurements is presented in the lower panel. Data are the mean ± SD (n = 3). Essentially similar results were obtained from pools of six patients. a > b and c, a–c < d–f, P < 0.05.

 
Discussion

Glucocorticoid receptors are expressed in granulosa cells, which implies the potential for direct action in the ovary (41, 42, 43). Glucocorticoids have been found to interfere with luteolysis, possibly by blocking the synthesis of prostaglandin F2{alpha}, but the entire mechanism of their action is not fully understood (44, 45, 46). In the present work we demonstrate that expression of Bcl-2, integrity of gap junction, and the actin cytoskeleton may play important roles in controlling apoptosis in human lutein-granulosa cells. Moreover, Dex could prevent apoptosis induced by LH/cAMP through modulation of these parameters and stabilization of the actin cytoskeleton.

Cells obtained from an IVF program are highly stimulated by CG and are known to be refractory to further stimulation (3, 4, 5, 47). One week of incubation recovered their responses to LH/hCG, FSH, FK, and 8Br-cAMP (5). However, the incidence of apoptosis was quite high. Therefore, we found it optimal to use 3 d of culture, after which the response is restored to large extent, but the cells show a low incidence of apoptosis compared with 7-d cultures (5). Our observation that LH and FK (to a lesser extent) enhance steroidogenesis supports the idea that the initial steps of apoptosis do not attenuate steroidogenesis as long as the steroidogenic organelles do not collapse (16). Indeed, we demonstrated previously that breakdown of actin filaments and clustering of steroidogenic organelles enhanced steroidogenesis (reviewed in Refs.28, 29, 30). An alternative explanation is that granulosa cell populations are not homogeneous in their degree of maturation and LH receptor content (48). Therefore, a cell’s response could be either to enhance steroidogenesis or to induce apoptosis. However, we found consistency in the regulation of steroidogenesis and apoptosis in granulosa cell preparations obtained from six women who subsequently became pregnant.

The protective effect of glucocorticoids on granulosa cells is somewhat surprising, because glucocorticoids induce apoptosis in a hemopoietic cell system to block inflammation (49, 50, 51, 52). However, there are accumulating data that epithelial cells, such as in mammary gland (53) and liver, as well as gastric cancer cells derived from epithelium (54) can be protected against apoptosis by glucocorticoids. Thus, the resident cells of inflamed tissue can be protected from apoptotic signals that therefore may facilitate the healing of the inflamed tissue. This may explain the rapid healing of the follicular wall, which subsequently is luteinized (55). Our findings also may explain the importance of the enhanced conversion of cortisone to cortisol by 11ßHSD1 during and after follicular rupture (23). Our data are also in line with our recent findings that in highly steroidogenic immortalized human granulosa cells, Dex and hydrocortisone could protect against serum deprivation and p53-, cAMP-, and TNF{alpha}-induced apoptosis (20). These phenomena also involve up-regulation of Bcl-2 (20). Future studies should reveal whether other members of the Bcl-2 family, such as Bax, Bcl-xlong, and Bcl-xshort, are involved in controlling apoptosis in human granulosa-lutein cells (56). Because enhanced progesterone production by LH was not associated with reduction of the incidence of apoptosis, we believe that glucocorticoids exerted their effect via {alpha}-type glucocorticoid receptors (57) rather than progesterone receptors (18, 19). This idea is further supported by recent studies demonstrating that freshly prepared granulosa-luteal cell obtained from an IVF program undergo apoptosis in suspension after incubation with RU486, which could be blocked by progesterone, but not by glucocorticoids (58). On the other hand, it was recently demonstrated that Dex significantly decreased ovarian IL-1ß converting enzyme gene expression, which serves as a classical marker for apoptosis (59). Taken together these data suggest that the protective effect of progesterone and Dex on granulosa cell apoptosis may depend on the degree of cell maturation and the culture conditions and that these two steroid hormones probably exert their effect via their specific receptors. There is direct evidence that 11ßHSD genes are gonadotropically regulated in the rat ovary, including granulosa cells, and are consistent with a shift in glucocorticoid metabolism from inactivation (due to oxidation by 11ßHSD2) to activation (reduction by 11ßHSD1) during hCG-induced granulosa cell luteinization, which suggests the physiological role of glucocorticoids as antiapoptotic hormones during granulosa cell luteinization (60).

The results of our experiments demonstrate for the first time the importance of cell contact integrity (in particular gap junction) and the integrity of the actin cytoskeleton in maintaining lutein granulosa cells in the nonapoptotic phase. These results are in line with the observation that intact adherence junctions expressing N- or E-cadherins are important for granulosa cell viability (34, 35, 61). Because FK enhanced the expression of CX-43, we suggest that the integrity of the junction itself, rather than the concentration of its building blocks, is the important determinant in preventing apoptosis. It was earlier demonstrated that human granulosa cells in culture exhibit functional cAMP-regulated gap junctions, where 8Br-cAMP was able to increase their permeability (62). In a recent study it was demonstrated that cAMP increased apoptosis in a dose-dependent manner in human granulosa cells, which is in line with our previous observation (9, 63). It may be concluded that the change in the function and integrity of the human granulosa-luteal cell gap junction relies on the concentrations of cAMP-elevated substances as well as on the culture conditions. Breakdown of actin filaments is probably by itself not sufficient to induce apoptosis. Numerous reports indicate that enhanced steroidogenesis involves down-regulation of the actin cytoskeleton proteins, such as vinkulin, {alpha}-actinin, and tropomyosin (28, 29, 30, 64, 65, 66). However, it seems that this process is a prerequisite to granulosa cell apoptosis, because every apoptotic cell revealed in the present study demonstrated diffused, rather than filamentous, organization of actin. Moreover, it was recently demonstrated that human MCF10A mammary epithelial cells undergo apoptosis after actin depolymerization, which can be rescued by Bcl-2 (67). These observations are in line with our finding that in human granulosa cells, Dex stabilizes the actin cytoskeleton and increases the Bcl-2 intracellular level, which rescues human granulosa cells from apoptosis. The dual effects of Dex on both cytoskeleton integrity and regulation of Bcl-2 may provide novel insights into the mechanism by which glucocorticoids protect against apoptosis in ovarian follicular cells. However, the exact molecular mechanism and the cross-talk among different signaling pathways are not fully resolved.

We suggest that the new parameters revealed in the present study will be of relevance to the in vivo situation. It remains to be seen whether IVF patients who did not succeed in conceiving could be identified by their differential sensitivity to apoptotic signals when analyzed in vitro in human granulosa-lutein cells obtained from these patients. Of special interest will be women who are suffering from polycystic ovary syndrome (68), which may involve abnormal responses to apoptotic signals, or women who are hypersensitive to the high gonadotropin stimulation during IVF treatment (69, 70, 71, 72, 73, 74).

There is increasing evidence that ovarian cell death and, in particular, granulosa-lutein cell apoptosis can be controlled by multifactors and can serve as an important criteria for ovarian function in health and disease. It was recently demonstrated that in addition to gonadotropic hormones, TGFß1, macrophage colony-stimulating factor (21), N-cadherin (33), basement membrane materials (32, 75), and activation of MAPK cascade, probably by growth factors such as fibroblast growth factor (16, 76), may be involved in the support of luteal function via suppression of apoptosis. On the other hand, in addition to prostaglandin F2{alpha}, saturated fatty acids, such as stearic palmitic acid (77), dioxin (78), and geldanamycin (79), can cause apoptosis in such cells, making them an attractive sensitive model for the analysis of the effects of environmental pollutants and internal effectors on female reproductive health.

Acknowledgments

We thank Drs. A. M. Kaye and J. Taplick for helpful discussion, Dr. K. Tajima for graphic assistance, Dr. A. Mayerhofer (Anatomical Institute, Technical University of Munich, Munich, Germany) and Dr. J. F. Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA) for the generous gifts of anti-CX-43 and StAR antibodies, respectively. We thank the IVF unit at Sheba Medical Center, headed by Dr. S. Dor, and Dr. S. Kees for collecting and transporting human follicular fluids aspirated during oocyte retrieval.

Footnotes

This work was supported by grants from the Yad Avraham Center for Cancer Research at the Levine Center of Applied Research at the Weizmann Institute of Science.

A.A. is incumbent of the Joyce and Ben B. Eisenberg Professorial Chair of Molecular Endocrinology and Cancer Research.

Abbreviations: 8Br-cAMP, 8-Bromo-cAMP; CX-43, connexin 43; DAPI, 4',6-diamido-2-phenylindole hydrochloride; Dex, dexamethasone; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; FK, forskolin; 11ßHSD, 11ß-hydroxysteroid dehydrogenase; IVF, in vitro fertilization; hLH, human LH; StAR, steroidogenic acute regulatory protein.

Received December 10, 2001.

Accepted April 4, 2002.

References

  1. Polan ML, Laufer N, Dlugi AM, Tarlatzis BC, Haseltine FP, DeCherney AH, Behrman HR 1984 Human chorionic gonadotropin and prolactin modulation of early luteal function and luteinizing hormone receptor-binding activity in cultured human granulosa-luteal cells. J Clin Endocrinol Metab 59:773–779[Abstract/Free Full Text]
  2. Veldhuis JD, Klase PA, Sandow BA, Kolp LA 1983 Progesterone secretion by highly differentiated human granulosa cells isolated from preovulatory Graafian follicles induced by exogenous gonadotropins and human chorionic gonadotropin. J Clin Endocrinol Metab 57:87–93[Abstract/Free Full Text]
  3. Dennefors BL, Hamberger L, Nilsson L 1983 Influence of human chorionic gonadotropin in vivo on steroid formation and gonadotropin responsiveness of isolated human preovulatory follicular cells. Fertil Steril 39:56–61[Medline]
  4. de los Santos MJ, Tarin JJ, Gomez E, Remohi J, Pellicer A 1993 Daily measurements and in-vitro effects of human chorionic gonadotrophin in the early luteal phase. Hum Reprod 8:2047–2051[Abstract/Free Full Text]
  5. Breckwoldt M, Selvaraj N, Aharoni D, Barash A, Segal I, Insler V, Amsterdam A 1996 Expression of Ad4-BP/cytochrome P450 side chain cleavage enzyme and induction of cell death in long-term cultures of human granulosa cells. Mol Hum Reprod 2:391–400[Abstract/Free Full Text]
  6. Yuan W, Giudice LC 1997 Programmed cell death in human ovary is a function of follicle and corpus luteum status. J Clin Endocrinol Metab 82:3148–3155[Abstract/Free Full Text]
  7. Izawa M, Nguyen PH, Kim HH, Yeh J 1998 Expression of the apoptosis-related genes, caspase-1, caspase-3, DNA fragmentation factor, and apoptotic protease activating factor-1, in human granulosa cells. Fertil Steril 70:549–552[CrossRef][Medline]
  8. Matikainen T, Perez GI, Zheng TS, Kluzak TR, Rueda BR, Flavell RA, Tilly JL 2001 Caspase-3 gene knockout defines cell lineage specificity for programmed cell death signaling in the ovary. Endocrinology 142:2468–2480[Abstract/Free Full Text]
  9. Zwain IH, Amato P 2001 cAMP-induced apoptosis in granulosa cells is associated with up-regulation of P53 and bax and down-regulation of clusterin. Endocr Res 27:233–249[CrossRef][Medline]
  10. Hosokawa K, Aharoni D, Dantes A, Shaulian E, Schere-Levy C, Atzmon R, Kotsuji F, Oren M, Vlodavsky I, Amsterdam A 1998 Modulation of Mdm2 expression and p53-induced apoptosis in immortalized human ovarian granulosa cells. Endocrinology 139:4688–700[Abstract/Free Full Text]
  11. Keren-Tal I, Suh BS, Dantes A, Lindner S, Oren M, Amsterdam A 1995 Involvement of p53 expression in cAMP-mediated apoptosis in immortalized granulosa cells. Exp Cell Res 218:283–295[CrossRef][Medline]
  12. Rotmensch S, Dor J, Furman A, Rudak E, Mashiach S, Amsterdam A 1986 Ultrastructural characterization of human granulosa cells in stimulated cycles: correlation with oocyte fertilizability. Fertil Steril 45:671–679[Medline]
  13. Oosterhuis GJ, Michgelsen HW, Lambalk CB, Schoemaker J, Vermes I 1998 Apoptotic cell death in human granulosa-lutein cells: a possible indicator of in vitro fertilization outcome. Fertil Steril 70:747–749[CrossRef][Medline]
  14. Chun SY, Eisenhauer KM, Minami S, Billig H, Perlas E, Hsueh AJ 1996 Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor. Endocrinology 137:1447–1456[Abstract]
  15. Tilly JL, Tilly KI, Kenton ML, Johnson AL 1995 Expression of members of the bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-xlong messenger ribonucleic acid levels. Endocrinology 136:232–241[Abstract]
  16. Aharoni D, Dantes A, Oren M, Amsterdam A 1995 cAMP-mediated signals as determinants for apoptosis in primary granulosa cells. Exp Cell Res 218:271–282[CrossRef][Medline]
  17. Zhao S, Saito H, Wang X, Saito T, Kaneko T, Hiroi M 2000 Effects of gonadotropin-releasing hormone agonist on the incidence of apoptosis in porcine and human granulosa cells. Gynecol Obstet Invest 49:52–56[CrossRef][Medline]
  18. Makrigiannakis A, Coukos G, Christofidou-Solomidou M, Montas S, Coutifaris C 2000 Progesterone is an autocrine/paracrine regulator of human granulosa cell survival in vitro. Ann NY Acad Sci 900:16–25[Medline]
  19. Svensson EC, Markstrom E, Andersson M, Billig H 2000 Progesterone receptor-mediated inhibition of apoptosis in granulosa cells isolated from rats treated with human chorionic gonadotropin. Biol Reprod 63:1457–1464[Abstract/Free Full Text]
  20. Sasson R, Tajima K, Amsterdam A 2001 Glucocorticoids protect against apoptosis induced by serum deprivation, cyclic adenosine 3',5'-monophosphate and p53 activation in immortalized human granulosa cells: involvement of Bcl-2. Endocrinology 142:802–811[Abstract/Free Full Text]
  21. Matsubara H, Ikuta K, Ozaki Y, Suzuki Y, Suzuki N, Sato T, Suzumori K 2000 Gonadotropins and cytokines affect luteal function through control of apoptosis in human luteinized granulosa cells. J Clin Endocrinol Metab 85:1620–1626[Abstract/Free Full Text]
  22. Rodger FE, Fraser HM, Duncan WC, Illingworth PJ 1995 Immunolocalization of bcl-2 in the human corpus luteum. Hum Reprod 10:1566–1570[Abstract/Free Full Text]
  23. Yong PY, Thong KJ, Andrew R, Walker BR, Hillier SG 2000 Development-related increase in cortisol biosynthesis by human granulosa cells. J Clin Endocrinol Metab 85:4728–4733[Abstract/Free Full Text]
  24. Amsterdam A, Sasson R 2002 The anti-inflammatory action of glucocorticoids is mediated by cell type specific regulation of apoptosis. Mol Cell Endocrinol 189:1–9[CrossRef][Medline]
  25. Anderson E, Albertini DF 1976 Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J Cell Biol 71:680–686[Abstract/Free Full Text]
  26. Amsterdam A, Josephs R, Lieberman ME, Lindner HR 1976 Organization of intramembrane particles in freeze-cleaved gap junctions of rat Graafian follicles: optical-diffraction analysis. J Cell Sci 21:93–105[Abstract]
  27. Amsterdam A, Knecht M, Catt KJ 1981 Hormonal regulation of cytodifferentiation and intercellular communication in cultured granulosa cells. Proc Natl Acad Sci USA 78:3000–3004[Abstract/Free Full Text]
  28. Amsterdam A, Rotmensch S 1987 Structure-function relationships during granulosa cell differentiation. Endocr Rev 8:309–337[Abstract/Free Full Text]
  29. Amsterdam A, Rotmensch S, Ben-Ze’ev A 1989 Coordinated regulation of morphological and biochemical differentiation in a steroidogenic cell: the granulosa cell model. Trends Biochem Sci 14:377–382[CrossRef][Medline]
  30. Amsterdam A, Plehn-Dujowich D, Suh BS 1992 Structure-function relationships during differentiation of normal and oncogene-transformed granulosa cells. Biol Reprod 46:513–522[Abstract]
  31. Amsterdam A, Rotmensch S, Furman A, Venter EA, Vlodavsky I 1989 Synergistic effect of human chorionic gonadotropin and extracellular matrix on in vitro differentiation of human granulosa cells: progesterone production and gap junction formation. Endocrinology 124:1956–1964[Abstract/Free Full Text]
  32. Aharoni D, Meiri I, Atzmon R, Vlodavsky I, Amsterdam A 1997 Differential effect of components of the extracellular matrix on differentiation and apoptosis. Curr Biol 7:43–51[CrossRef][Medline]
  33. Makrigiannakis A, Coukos G, Christofidou-Solomidou M, Gour BJ, Radice GL, Blaschuk O, Coutifaris C 1999 N-Cadherin-mediated human granulosa cell adhesion prevents apoptosis: a role in follicular atresia and luteolysis? Am J Pathol 154:1391–406[Abstract/Free Full Text]
  34. Peluso JJ, Pappalardo A, Trolice MP 1996 N-Cadherin-mediated cell contact inhibits granulosa cell apoptosis in a progesterone-independent manner. Endocrinology 137:1196–203[Abstract]
  35. Peluso JJ, Pappalardo A, Fernandez G 2001 E-Cadherin-mediated cell contact prevents apoptosis of spontaneously immortalized granulosa cells by regulating Akt kinase activity. Biol Reprod 64:1183–1190[Abstract/Free Full Text]
  36. Sommersberg B, Bulling A, Salzer U, Frohlich U, Garfield RE, Amsterdam A, Mayerhofer A 2000 Gap junction communication and connexin 43 gene expression in a rat granulosa cell line: regulation by follicle-stimulating hormone. Biol Reprod 63:1661–1668[Abstract/Free Full Text]
  37. Amsterdam A, Nimrod A, Lamprecht SA, Burstein Y, Lindner HR 1979 Internalization and degradation of receptor-bound hCG in granulosa cell cultures. Am J Physiol 236:E129–E38
  38. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  39. Hosokawa K, Dantes A, Schere-Levy C, Barash A, Yoshida Y, Kotsuji F, Vlodavsky I, Amsterdam A 1998 Induction of Ad4BP/SF-1, steroidogenic acute regulatory protein, and cytochrome P450scc enzyme system expression in newly established human granulosa cell lines. Endocrinology 139:4679–4687[Abstract/Free Full Text]
  40. Hanukoglu I, Suh BS, Himmelhoch S, Amsterdam A 1990 Induction and mitochondrial localization of cytochrome P450scc system enzymes in normal and transformed ovarian granulosa cells. J Cell Biol 111:1373–1381[Abstract/Free Full Text]
  41. Towns R, Menon KM, Brabec RK, Silverstein AM, Cohen JM, Bowen JM, Keyes PL 1999 Glucocorticoids stimulate the accumulation of lipids in the rat corpus luteum. Biol Reprod 61:416–421[Abstract/Free Full Text]
  42. Schreiber JR, Nakamura K, Erickson GF 1982 Rat ovary glucocorticoid receptor: identification and characterization. Steroids 39:569–584[CrossRef][Medline]
  43. Gaytan F, Morales C, Bellido C, Sanchez-Criado JE 2002 Selective apoptosis of luteal endothelial cells in dexamethasone-treated rats leads to ischemic necrosis of luteal tissue. Biol Reprod 66:232–240[Abstract/Free Full Text]
  44. Chatterton Jr RT, Kazer RR, Rebar RW 1991 Depletion of luteal phase serum progesterone during constant infusion of cortisol phosphate in the cynomolgus monkey. Fertil Steril 56:547–554[Medline]
  45. Flower RJ, Blackwell GJ 1979 Anti-inflammatory steroids induce biosynthesis of a phospholipase A2 inhibitor which prevents prostaglandin generation. Nature 278:456–459[CrossRef][Medline]
  46. Wang F, Riley JC, Behrman HR 1993 Immunosuppressive levels of glucocorticoid block extrauterine luteolysins in the rat. Biol Reprod 49:66–73[Abstract]
  47. Andreani CL, Pierro E, Lanzone A, , Lazzarin N, Capitanio G, Giannini P, Mancuso S 1994 Effect of gonadotropins, insulin and IGF I on granulosa luteal cells from polycystic ovaries. Mol Cell Endocrinol 106:91–97[CrossRef][Medline]
  48. Amsterdam A, Koch Y, Lieberman ME, Lindner HR 1975 Distribution of binding sites for human chorionic gonadotropin in the preovulatory follicle of the rat. J Cell Biol 67:894–900[Abstract/Free Full Text]
  49. Dowd DR, Miesfeld RL 1992 Evidence that glucocorticoid- and cyclic AMP-induced apoptotic pathways in lymphocytes share distal events. Mol Cell Biol 12:3600–3608[Abstract/Free Full Text]
  50. Sikora E, Rossini GP, Grassilli E, Bellesia E, Salomoni P, Franceschi C 1996 Interference between DNA binding activities of AP-1 and GR transcription factors in rat thymocytes undergoing dexamethasone-induced apoptosis. Acta Biochim Pol 43:721–731[Medline]
  51. Chauhan D, Pandey P, Ogata A, Teoh G, Krett N, Halgren R, Rosen S, Kufe D, Kharbanda S, Anderson K 1997 Cytochrome c-dependent and -independent induction of apoptosis in multiple myeloma cells. J Biol Chem 272:29995–29997[Abstract/Free Full Text]
  52. Schmidt M, Pauels HG, Lugering N, Lugering A, Domschke W, Kucharzik T 1999 Glucocorticoids induce apoptosis in human monocytes: potential role of IL-1ß. J Immunol 163:3484–3490[Abstract/Free Full Text]
  53. Feng Z, Marti A, Jehn B, Altermatt HJ, Chicaiza G, Jaggi R 1995 Glucocorticoid and progesterone inhibit involution and programmed cell death in the mouse mammary gland. J Cell Biol 131:1095–103[Abstract/Free Full Text]
  54. Chang TC, Hung MW, Jiang SY, Chu JT, Chu LL, Tsai LC 1997 Dexamethasone suppresses apoptosis in a human gastric cancer cell line through modulation of bcl-x gene expression. FEBS Lett 415:11–15[CrossRef][Medline]
  55. Hillier SG 2001 Gonadotropic control of ovarian follicular growth and development. Mol Cell Endocrinol 179:39–46[CrossRef][Medline]
  56. Kugu K, Ratts VS, Piquette GN, Tilly KI, Tao XJ, Martimbeau S, Aberdeen GW, Krajewski S, Reed JC, Pepe GJ, Albrecht ED, Tilly JL 1998 Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary. Cell Death Differ 5:67–76[CrossRef][Medline]
  57. Pujols L, Mullol J, Perez M, Roca-Ferrer J, Juan M, Xaubet A, Cidlowski JA, Picado C 2001 Expression of the human glucocorticoid receptor alpha and beta isoforms in human respiratory epithelial cells and their regulation by dexamethasone. Am J Respir Cell Mol Biol 24:49–57[Abstract/Free Full Text]
  58. Svensson EC, Markstrom E, Shao R, Andersson M, Billig H 2001 Progesterone receptor antagonists Org 31710 and RU 486 increase apoptosis in human periovulatory granulosa cells. Fertil Steril 76:1225–1231[CrossRef][Medline]
  59. Irahara M, Ando M, Kol S, Adashi EY 1999 Expression and hormonal regulation of rat ovarian interleukin-1ß converting enzyme, a putative apoptotic marker: endocrine- and paracrine-dependence. J Reprod Immunol 45:67–79[CrossRef][Medline]
  60. Tetsuka M, Milne M, Simpson GE, Hillier SG 1999 Expression of 11ß-hydroxysteroid dehydrogenase, glucocorticoid receptor, and mineralocorticoid receptor genes in rat ovary. Biol Reprod 60:330–335[Abstract/Free Full Text]
  61. Peluso JJ, Pappalardo A 1994 Progesterone and cell-cell adhesion interact to regulate rat granulosa cell apoptosis. Biochem Cell Biol 72:547–551[Medline]
  62. Furger C, Cronier L, Poirot C, Pouchelet M 1996 Human granulosa cells in culture exhibit functional cyclic AMP-regulated gap junctions. Mol Hum Reprod 2:541–548[Abstract/Free Full Text]
  63. Tajima K, Hosokawa K, Yoshida Y, Dantes A, Sasson R, Kotsuji F, Amsterdam A 2002 Establishment of FSH-responsive cell lines by transfection of pre-ovulatory human granulosa cells with mutated p53 (p53val 135) and Ha-ras genes. Mol Hum Reprod 8:48–57[Abstract/Free Full Text]
  64. Ben-Ze’ev A, Baum G, Amsterdam A 1989 Regulation of tropomyosin expression in the maturing ovary and in primary granulosa cell cultures. Dev Biol 135:191–201[CrossRef][Medline]
  65. Grieshaber NA, Boitano S, Ji I, Mather JP, Ji TH 2000 Differentiation of granulosa cell line: follicle-stimulating hormone induces formation of lamellipodia and filopodia via the adenylyl cyclase/cyclic adenosine monophosphate signal. Endocrinology 141:3461–3470[Abstract/Free Full Text]
  66. Soto EA, Kliman HJ, Strauss JF, 3rd, Paavola LG 1986 Gonadotropins and cyclic adenosine 3', 5'-monophosphate (cAMP) alter the morphology of cultured human granulosa cells. Biol Reprod 34:559–569[Abstract]
  67. Martin SS, Leder P 2001 Human MCF10A mammary epithelial cells undergo apoptosis following actin depolymerization that is independent of attachment and rescued by Bcl-2. Mol Cell Biol 21:6529–6536[Abstract/Free Full Text]
  68. Homburg R, Amsterdam A 1998 Polysystic ovary syndrome–loss of the apoptotic mechanism in the ovarian follicles? J Endocrinology Invest 21:552–557
  69. Balasch J, Fabregues F, Arroyo V 1998 Peripheral arterial vasodilation hypothesis: a new insight into the pathogenesis of ovarian hyperstimulation syndrome. Hum Reprod 13:2718–2730[Abstract/Free Full Text]
  70. Beerendonk CC, van Dop PA, Braat DD, Merkus JM 1998 Ovarian hyperstimulation syndrome: facts and fallacies. Obstet Gynecol Surv 53:439–449[CrossRef][Medline]
  71. Rutkowski A, Dubinsky I 1999 Ovarian hyperstimulation syndrome: imperatives for the emergency physician. J Emerg Med 17:669–672[CrossRef][Medline]
  72. Schenker JG 1999 Clinical aspects of ovarian hyperstimulation syndrome. Eur J Obstet Gynecol Reprod Biol 85:13–20[CrossRef][Medline]
  73. Orvieto R, Ben-Rafael Z 1998 Ovarian hyperstimulation syndrome: a new insight into an old enigma. J Soc Gynecol Invest 5:110–113[Medline]
  74. Brinsden PR, Wada I, Tan SL, Balen A, Jacobs HS 1995 Diagnosis, prevention and management of ovarian hyperstimulation syndrome. Br J Obstet Gynaecol 102:767–772[Medline]
  75. Hwang DH, Kee SH, Kim K, Cheong KS, Yoo YB, Lee BL 2000 Role of reconstituted basement membrane in human granulosa cell culture. Endocr J 47:177–183[Medline]
  76. Oliver RH, Khan SM, Leung BS, Yeh J 1999 Induction of apoptosis in luteinized granulosa cells by the MAP kinase kinase (MEK) inhibitor PD98059. Biochem Biophys Res Commun 263:143–148[CrossRef][Medline]
  77. Mu YM, Yanase T, Nishi Y, Tanaka A, Saito M, Jin CH, Mukasa C, Okabe T, Nomura M, Goto K, Nawata H 2001 Saturated FFAs, palmitic acid and stearic acid, induce apoptosis in human granulosa cells. Endocrinology 142:3590–3597[Abstract/Free Full Text]
  78. Heimler I, Rawlins RG, Owen H, Hutz RJ 1998 Dioxin perturbs, in a dose- and time-dependent fashion, steroid secretion, and induces apoptosis of human luteinized granulosa cells. Endocrinology 139:4373–4379[Abstract/Free Full Text]
  79. Khan SM, Oliver RH, Dauffenbach LM, Yeh J 2000 Depletion of Raf-1 protooncogene by geldanamycin causes apoptosis in human luteinized granulosa cells. Fertil Steril 74:359–365[CrossRef][Medline]



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