| | The effect of mifepristone on apoptosis and caspase-3 activation in human ovarian luteinized granulosa cellsReceived 24 May 2007; received in revised form 17 April 2008; accepted 4 July 2008. published online 29 August 2008. Abstract ObjectiveTo investigate the effect of mifepristone, an oral contraceptive, on apoptosis in human ovarian luteinized granulosa cells. Study designHuman ovarian luteinized granulosa cells were treated in vitro with 1.25, 2.5, 5.0, 10.0, or 20.0 μM of mifepristone. Nuclear morphology, apoptosis ratio, and level of caspase-3 expression were determined with immunofluorescence microscopy, the terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay, and flow cytometry. ConclusionFrom these results, we propose that mifepristone induces human ovarian luteinized granulosa cells to undergo apoptosis by activating caspase-3. 1. Introduction  Mifepristone is a progesterone receptor antagonist which is used as a medical method to terminate pregnancy. Some studies have shown that mifepristone can also be used as an emergency postcoital contraceptive [1], [2]. Mifepristone has a number of advantages over other methods of contraception. Compared to the standard Yuzpe regimen, mifepristone has fewer side effects and is equal in efficacy to the levonorgestrel regimen [3], [4]. However, the mechanism by which mifepristone functions as a contraceptive has not been fully elucidated. Applying low doses of mifepristone in the follicular phase of the menstrual cycle inhibits follicular development and ovulation at a serum mifepristone concentration of 2.5μM [5]. By taking 2 or 5mg of mifepristone daily, ovulation can be routinely suppressed [6], [7]. This provides a novel contraceptive method in which the risk of taking estrogen-containing combined oral contraceptives (COC) can be avoided, e.g., venous thromboembolism. Although mifepristone has been extensively studied for its potential use as a contraceptive drug, further investigations are required to make it more effective, safe, and acceptable for large scale clinical applications and acceptance. Greater than 99% of follicles undergo a degenerative process in mammalian ovaries, known as “atresia,” and only a few follicles ovulate during ovarian follicular development [8]. In the past, studies have investigated the molecular mechanism of selective follicular atresia in mammalian ovaries, and have reported that follicular selection depends largely on granulosa cell apoptosis [9], [10], [11]. Granulosa cells are known to contribute to maturation of oocytes, and most of the growth factors exert their action via granulosa cells. It has been established that granulosa cell death during follicular atresia and luteolysis results from apoptosis [12]. However, the precise mechanistic pathways of granulosa cell apoptosis have not yet been defined. Caspases, a family of cysteine proteases with aspartate substrate specificity, are produced in cells as inactive zymogens. Caspase-3 conversion of cytoplasmic DNase into an active form results in characteristic inter-nucleosomal clearance of DNA [13]. This is functionally required for normal execution of apoptotic cell death in granulosa cells, causing specific changes in cell surface and nuclear morphologic features characteristic of apoptosis [14], [15]. The existence of such endonuclease activity in differentiated granulosa cells has previously been demonstrated in animals. However, to our knowledge, it has not yet been studied in humans. In this study, we tested whether mifepristone can induce apoptosis in in vitro cultured human ovarian luteinized granulosa cells and whether this is via caspase-3 activation. Nuclear morphology, the apoptosis ratio, and caspase-3 expression in granulosa cells were determined with immunofluorescence microscopy, TUNEL, and flow cytometry. 2. Materials and methods 2.1. Granulosa cell isolation and culture Primary luteinized granulosa cells were obtained from women undergoing IVF (in vitro fertilization) at the Women's Hospital at the School of Medicine of Zhejiang University (Hangzhou, China). Patients received a GnRH analog in combination with FSH, followed by administration of hCG. 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 thrice. The follicular fluid was centrifuged at 300×g for 5min to separate granulosa cells from red blood cells. The supernatant was discarded and the pellet resuspended in 10ml of PBS. The cells were dispersed using 0.25% trypsin-EDTA (Gibco-BRL, Grand Island, NY, USA). The suspension was layered onto 10ml of a 50% Percoll solution and centrifuged at 300×g for 30min to sediment the red blood cells. The isolated granulosa cells were aspirated from the interface, washed, and resuspended in PBS. The resulting pellet was resuspended in 10mm Tris and 0.84% NH4Cl (pH 7.4), to lyse red blood cells (15min of shaking at 37°C). Several washings in PBS achieved elimination of the debris. Cells were counted on a hemacytometer. Viable cells (2×105 per well) were plated in 6-well plates with RPMI-1640, supplemented with penicillin (100IU/ml), streptomycin (100μg/ml), and 10% fetal calf serum (FCS). Attachment of the granulosa cells to the bottom of the dishes (Falcon, Heidelberg, Germany) was already achieved after 24h at 37°C in a humidified atmosphere of 5% CO2 and air. The medium was removed and the cells were washed five times with PBS to remove the remaining red and non-adherent white blood cells (lymphocytes), as well as cell debris. In each experiment, the cells were plated at an equal density for each treatment group. The culture media were changed 24h after plating to either fresh serum-supplemented media that contained vehicle or treatments. Granulosa cells were exposed to varying concentrations of mifepristone (1.25, 2.5, 5.0, 10.0, and 20.0μM) and the cells not treated with mifepristone served as controls. In our experiments, ethanol, as the carrier molecule for the mifepristone, was in the same concentration in the controls and all treatments. The cells were collected 24h later. For the TUNEL experiments, granulosa cells were grown on glass coverslips. Informed consent was obtained from each woman undergoing an IVF procedure. The study was approved by the Ethics Committee of the Women's Hospital at the School of Medicine of Zhejiang University. 2.2. DAPI staining After treatment with mifepristone, granulosa cells were washed with 1× PBS, fixed with 70% ethanol for 20min at room temperature, washed again with 1× PBS, smeared on a glass slide, and air-dried. Cell nuclei were stained with 0.1μg/ml 4,6-diamino-2-phenylindole (DAPI; Sigma, St. Louis, MO, USA) in PBS containing 0.1% Tween 20 (Sigma). The slides were washed three times in PBS, air-dried, and observed under a phase fluorescence microscope (Olympus Optical Co., Tokyo, Japan). The total number of cell nuclei and the number of apoptotic cell nuclei were counted in five randomly chosen fields on each treated or control coverslips per experiment. 2.3. TUNEL labeling TUNEL was performed on granulosa cells, cultured as described above on glass coverslips, and fixed for 10min in 4% neutral buffered formalin using an ApopTag peroxidase in situ cell apoptosis detection kit (Boster Biological Technology Co., Wuhan, China), according to the manufacturer's instructions. Granulosa cells were treated with proteinase K for 5min at room temperature. Then, the cells were treated with 3% H2O2 for 5min and labeled with biotin-dUTP by incubation with reaction buffer containing terminal deoxynucleotidyl transferase enzyme for 30min at 37°C. The slides were further incubated with streptavidin–horseradish peroxidase conjugate to detect biotinylated nucleotides for 30min at room temperature. Diaminobenzidine reacted with the labeled samples to generate an insoluble colored substrate at the site of DNA fragmentation. Negative control slides were processed in the same manner except the labeling enzyme was omitted. Finally, slides were counterstained with methylene green and photographed via a differential interference contrast microscope (Nikon Microphot-FX, Tokyo, Japan) that creates a clear distinction between nuclear and cytoplasmic components of the cell. TUNEL labeling was then analyzed microscopically by a technician unaware of the study group. Using a 10×10 reticule and 20× objective, the total number of cell nuclei and the number of TUNEL-positive cell nuclei were counted in five randomly chosen fields on each treated or control coverslips. 3. Results 3.1. Morphologic characteristics of granulosa cells treated with mifepristone In apoptotic cells, the nucleus is typically condensed and forms buds, frequently with a crescent shape of condensed chromatin, and the fragments of cells containing condensed chromatin referred to as apoptosis bodies which is considered as the morphologic hallmark of apoptosis. Without mifepristone treatment as a negative control (Fig. 1A), few cells showed morphologic characteristics of apoptosis. When treated with 1.25 or 2.5μM of mifepristone (Fig. 1B and C), a few cells showed evidence of apoptosis (arrow). When treated with 5.0μM of mifepristone, a greater number of cells were found to bear characteristic morphologic features of apoptosis (arrow; Fig. 1D). Finally, when the dose of mifepristone reached 10.0–20.0μM, cells were found to contain more circular bodies in their nuclei. A typical mass of circular bodies, nuclear membrane shrinkage, and shrunken nuclei uniformly stained by light green fluorescence (arrow) were indicative of a late stage of apoptosis (Fig. 1E and F). The rectangles on the top right corner of Fig. 1A–F depict the replicative enlarged views of the granulosa cells. The percentage of apoptotic cells for the cell groups treated with 0.0, 1.25, 2.5, 5.0, 10.0, and 20.0μM of mifepristone was 4.00±0.63%, 12.16±1.16%, 14.33±1.03%, 21.00±2.82%, 31.16±2.13%, and 48.33±5.85%, respectively, as shown in Fig. 2. A significant difference was found between the mifepristone-treated and control groups (P<0.05). Significant differences were also seen between groups with different doses of mifepristone (P<0.05). 3.2. Induction of DNA fragmentation in granulosa cells by mifepristone Detection of the DNA fragmentation has been a principal method for identification of apoptotic cells in atretic follicles, and in human granulosa cells during luteal regression. In the control group (Fig. 3A), positive staining rarely occurred. The number of positive staining cells increased in the group treated with 1.25 or 2.5μM of mifepristone (Fig. 3B and C). In the groups treated with 5.0 and 10.0μM of mifepristone, more abundant positive staining cells were observed (Fig. 3D and E). In the group treated with 20.0μM of mifepristone (Fig. 3F), the majority of cells had positive staining. Fig. 3G shows a negative control that lacked positive staining. Human lymphocyte cells were used as a positive control (Fig. 3H). Therefore, as shown in Fig. 4, following the dose increase of mifepristone, the density of apoptotic granulosa cells increased. A quantitative analysis of the TUNEL staining for each group is as follows: control, 5.00±0.27%; 1.25μM mifepristone treatment, 11.67±0.59%; 2.5μM mifepristone, 12.18±0.99%; 5.0μM mifepristone, 18.52±0.53% and 10.0μM mifepristone, 26.64±1.51% and 20.0μM mifepristone, 49.96±2.13%. A significant difference was found between mifepristone-treated and control groups (P<0.05). Significant differences also existed between groups with different drug doses (P<0.05), except those with treated with 1.25 and 2.5μM of mifepristone. 3.3. Induction of caspase-3 activity in granulosa cells by mifepristone To determine whether caspase-3 antigen mediates apoptosis of granulosa cells, flow cytometry was performed to assess the average fluorescence intensity of active caspase-3 levels in the human ovarian luteinized granulosa cells. A significant increase in the caspase-3 activity levels for mifepristone-treated cells was observed (Fig. 5). The average fluorescent intensity of caspase-3 for cell groups treated with 0.0, 1.25, 2.5, 5.0, 10.0, and 20.0μM of mifepristone was 2.3±0.15%, 17.09±0.36%, 20.05±0.4%, 29.86±2.05%, 36.11±1.62%, and 46.03±1.86%, respectively, as shown in Fig. 5. 4. Discussion Ovarian follicles of all growth stages, from primary to large antral follicles, undergo atresia [12], [16]. Moreover, this process occurs throughout life, from the fetal period to the onset of reproductive senescence. Initial studies from rat, avian, and porcine models have documented a role of cellular apoptosis in the loss of granulosa cells during follicular atresia [10], [11]. Apoptosis is a cellular mechanism involved in ovarian follicular atresia and luteal regression. In the early stage of follicular development, atresia is initiated by oocyte apoptosis, followed by the death of granulosa cells [17]. Atresia of maturing and mature follicles was first demarcated by scattered granulosa cell apoptosis [18], [19]. As atresia progresses in these follicles, the number of dying granulosa cells increases dramatically, and large masses of apoptotic bodies are shed into the antral space [20], [21]. Apoptosis is characterized by specific changes in cell surface and nuclear morphologic features, and is caused mostly by caspase-3 [22], [23]. In the current study, human ovarian luteinized granulosa cells isolated from patients undergoing IVF were treated with different doses of mifepristone. Fluorescence microscopy revealed morphologic evidence of apoptosis in these cells, including nuclear membrane shrinkage and formation of apoptotic bodies. We found that the numbers of apoptotic cells were increased in the groups treated with different doses of mifepristone. In the control group, few granulosa cells showed the morphologic changes characteristic of apoptosis. This result demonstrated that human ovarian luteinized granulosa cells can be induced to undergo apoptosis by mifepristone. Detection of the DNA fragmentation by TUNEL assay has been used as a principal method to identify and quantify the apoptotic cells in atretic follicles, during luteal regression, and in human graulosa cells. In this study, the TUNEL assay showed that there is significant difference of apoptotic ratio between cells treated with or without mifepristone, and the difference remains significant among the cells treated with different doses of mifepristone, except for the two groups exposed to 1.25 and 2.5μM of mifepristone. Moreover, the apoptotic ratio increased as the mifepristone concentration increased. These results suggest that mifepristone can induce granulosa cells to undergo apoptosis in a dose-dependent manner. Expression of caspase-3 has been found in ovarian leukocytes and in follicular cells of atretic follicles [24], [25]. Robbles [26] showed that healthy granulosa cells possess the inactive (pro-) form of the enzyme almost exclusively, whereas apoptotic granulosa cells process the procaspase-3 to the active enzyme. In addition, apoptotic DNA degradation occurs during both follicular atresia and luteal regression, and cleavage of poly-(ADP-ribose)-polymerase (PARP) is found during luteal regression [27]. The existence of such endonuclease activity in differentiated granulosa cells has previously been demonstrated in animals [28]. Apoptosis is characterized by specific changes in cell surface and nuclear morphologic features caused mostly by caspase-3 [15]. After initial formation of the antrum, activation of caspase-3 is a normal physiologic process of the follicle during atresia and luteinization [24], [29]. In the present study, we examined the existence of active caspase-3 in human luteinized granulosa cells obtained during IVF to determine whether the activation of caspase-3 plays a role in the occurrence of apoptosis treated with mifepristone. In the mifepristone-treated cells, flow cytometry showed an increase in the active caspase-3 levels in a dose-dependent manner. The fluorescent intensity of caspase-3 was significantly different between control and treatment groups. All apoptotic granulosa cells expressed active caspase-3. These results suggest that the luteinized granulosa cells treated with mifepristone undergo apoptosis through a caspase-3 involved mechanism. Previous studies have suggested that in vitro mifepristone inhibits proliferation and induces apoptosis in prostate cancer [30], cervical carcinoma [31], and in some endometrial cancer cell lines [32]. Similar studies have shown similar effects of mifepristone on non-neoplastic cells. Specifically, increased apoptosis and decreased proliferation have been demonstrated in non-neoplastic endometrial stromal and endometrial epithelial cells in culture, as well as in the endometrium of E2-treated, ovariectomized monkeys [33], [34]. Li et al. [35] suggested that mifepristone inhibited cell growth by arresting cell cycle progression at the S phase, induced apoptosis through caspase-3 activation, and modulated apoptosis regulatory gene BAX in Ishikawa cells. Immunofluorescent double labeling of Ishikawa cells in the absence or presence of mifepristone revealed that BAX protein expression increased and translocated from cytosol to mitochondria. Recent published data showing that mifepristone-associated apoptosis of EM42 endometrial cells and granulosa cells demonstrated nuclear factor kB activation suggested a mitochondria-mediated pathway [36], [37]. 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Women's Hospital, School of Medicine, Zhejiang University, 310006 Hangzhou, Zhejiang, China  Corresponding author.
PII: S0301-2115(08)00299-6 doi:10.1016/j.ejogrb.2008.07.031 © 2008 Elsevier Ireland Ltd. All rights reserved. | |
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