Levels of apoptosis in human granulosa cells ... - Fertility and Sterility

Levels of apoptosis in human granulosa cells seem to be comparable after therapy with a gonadotropin-releasing hormone agonist or antagonist Franca Giampietro, M.D., Ph.D.,a Silvia Sancilio, Ph.D.,b Gian Mario Tiboni, M.D.,a Rosa Alba Rana, M.D.,b and Roberta Di Pietro, M.D.b a Sezione di Ostetricia e Ginecologia, Dipartimento di Medicina e Scienze dell’Invecchiamento, Facoltà di Medicina e Chirurgia, and b Sezione di Istologia, Dipartimento di Biomorfologia, Università “G. d’Annunzio” Chieti-Pescara, Chieti, Italy

Objective: To compare levels of apoptosis in granulosa cells from women treated with the gonadotropin-releasing hormone (GnRH) agonist triptorelin or the GnRH antagonist cetrorelix. Design: Randomized, prospective study. Setting: University hospital. Patient(s): Thirty-two women undergoing assisted reproduction techniques after ovulation induction with recombinant follicle-stimulating hormone (FSH) plus GnRH agonist or antagonist. Intervention(s): Granulosa cells were isolated from follicular aspirates after oocyte removal. Main Outcome Measure(s): Apoptosis was assessed with Annexin V binding assay, terminal deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) assay, flow cytometric analysis of DNA, and ultrastructural analysis of cell morphology in transmission electron microscopy. Serum and follicular hormonal levels were also determined. Result(s): Annexin V binding and TUNEL assays revealed comparable percentages of apoptosis in the two groups under investigation. Analysis of DNA histograms revealed a similar cell cycle distribution in the two groups. Ultrastructural analysis only occasionally displayed patterns of chromatin margination in apoptotic cells. The mean concentrations of all the follicular fluid steroid hormones evaluated (E2, T, and P) were significantly lower in the GnRH antagonist–treated group. Conclusion(s): Therapy with a GnRH agonist or antagonist is associated with comparable levels of apoptosis in granulosa cells. (Fertil Steril威 2006;85:412–9. ©2006 by American Society for Reproductive Medicine.) Key Words: Apoptosis, human granulosa cells, GnRH agonist, GnRH antagonist, assisted reproduction techniques

Apoptosis, or programmed cell death, is the physiologic process of cell deletion and plays a critical role in normal ovarian physiology (1, 2). During mammalian folliculogenesis, numerous follicles start to develop, but only one or two reach maturation and ovulation, whereas the majority undergoes atresia by means of an apoptotic process that initiates in granulosa cells (GCs) (3–5). There is increasing evidence that ovarian apoptosis is significantly modulated by the hormonal milieu resulting from the interplay of many factors that are locally produced and transported by plasma (6, 7). A hormonal-mediated influence is supported by the evidence that, in the rat, ovarian apoptosis can be triggered by gonadotropinreleasing hormone (GnRH) and its analogues (4, 8) and androgens (9) but blocked or inhibited by estrogen (9), FSH, LH, and hCG (10). Controlled ovarian hyperstimulation is achieved by pharmacologic manipulation of folliculogenesis. The major pharmacologic tools used include gonadotropins and GnRH agonists and antagonists. Gonadotropin-releasing hormone agonists Received March 22, 2005; revised and accepted August 2, 2005. Reprint requests: Gian Mario Tiboni, M.D., Sezione di Ostetricia e Ginecologia, Dipartimento di Medicina e Scienze dell’Invecchiamento, Università “G. d’Annunzio”, Via dei Vestini 17, 66013 Chieti, Italy (FAX: ⫹39-0871-540037; E-mail: [email protected]).

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have been introduced in assisted reproduction techniques (ART) to induce pituitary desensitization to gonadotrope hormones by means of a down-regulation of GnRH receptors exposed on cell membrane. Conversely, GnRH antagonists achieve a rapid decline in gonadotropin secretion by the competitive blockade of the GnRH receptors. In addition to the effects of GnRH and its analogues on the pituitary– gonadal axis, possible extrapituitary effects have been postulated (11, 12). The evidence that specific GnRH receptors are present in rat and human GCs offers biological support of this hypothesis (13, 14). A proapoptotic effect of GnRH agonists has been documented by several lines of research showing the ability of these agents [1] to increase the incidence of apoptosis in porcine and human cultured GCs (15, 16), [2] to decrease in vitro human luteinized GC proliferation (17), [3] to reduce the stability of the BCL-XL protein in rat follicles, resulting in an imbalance in favor of proapoptotic protein levels (18), and [4] to act in vivo as ovarian atretogenic factors (4). By contrast, there is limited information concerning the effects of GnRH antagonists on the ovarian apoptotic process in animal models (19, 20), and no data have been published regarding possible actions of GnRH antagonists on ovarian apoptosis in humans. Remarkably, the incidence of GC ap-

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optosis has been recently related to ART outcome (21, 22). In view of the possibility that GC apoptosis might represent an important issue in clinical situations, the aim of this study was to compare levels of apoptosis in GCs from women undergoing ART treated with either the GnRH agonist triptorelin or the GnRH antagonist cetrorelix. A multiparametric analysis of apoptotic patterns was performed with Annexin V binding assay, terminal deoxynucleotidyl transferase (TdT)mediated nick-end labeling (TUNEL) assay, flow cytometric analysis of DNA, and ultrastructural analysis of cell morphology by transmission electron microscopy.

Hormonal Assays Peripheral venous blood was taken from each patient on the day of hCG administration. The sera were stored at ⫺20°C until assayed for LH, E2, and P levels. After isolation of the oocytes, the clear follicular fluid from each patient was pooled and immediately centrifuged for 10 minutes. Follicular fluid supernatant was stored at ⫺20°C until used for hormonal assays, including measurement of E2, testosterone (T), and progesterone (P) levels. Quantification of hormonal concentrations was performed with a commercially available immunoassay kit (DiaSorin, Stillwater, MN).

MATERIALS AND METHODS Patients A total of 32 women with primary infertility undergoing ART were included in the study. All the patients met the following inclusion criteria: age ⬍38 years; regular menstrual cycles; normal basal serum follicle-stimulating hormone [FSH] (ⱕ10 IU/L), luteinizing hormone [LH] (ⱕ10 IU/L), and estradiol [E2] (ⱕ60 pg/mL) levels; body mass index ⬍30 kg/m2; normal prolactin [PRL] (⬍25 ng/mL), thyroid-stimulating hormone [TSH] (range, 0.3– 4 mU/L), free thyroxin (Ft)3 (range, 2.2–5.5 pg/mL), and Ft4 (range, 7.8 –19.4 pg/mL) levels; and a normal karyotype. Because endometriosis has been reported to be associated with an increased incidence of apoptosis in GCs (23, 24), patients with clinical evidence of this disease were excluded from this study. Patients underwent controlled ovarian hyperstimulation and were randomly assigned to receive either a GnRH agonist (group 1, n ⫽ 15) or a GnRH antagonist (group 2, n ⫽ 17). The study was approved by the institutional review board of the University “G. d’Annunzio” of ChietiPescara. No financial support was received.

GC Isolation After isolation of the oocytes, all follicular fluids from each patient were pooled in a tube so as to have sufficient material for analysis, and the tube was left standing for 5 minutes to allow the GCs to sediment. The GCs were then transferred to a tube containing 5 mL of Ham’s F-10 medium (Sigma Aldrich, Milano, Italy) and gently resuspended. After centrifugation on a density gradient (Ficoll-Hypaque; Sigma Aldrich) at 3,000 rpm for 10 minutes, GCs were purified from red blood cells and washed with 5 mL of Ham’s F-10 medium. To prevent cell aggregation, GCs were treated with hyaluronidase solution (0.1% vol/wt in Ham’s F-10) for 15 minutes, mechanically dispersed with repeated aspiration and expiration through a fine Pasteur glass pipette, and resuspended in phosphate-buffered saline (PBS) without calcium and magnesium. The entire procedure was completed within 1 hour after follicle aspiration to prevent cell death.

Stimulation Protocols Women belonging to group 1 received triptorelin (Decapeptyl; IPSEN Laboratories, Milan, Italy) at a daily dose of 0.1 mg, starting on day 21 of the previous cycle and continuing up to the day of human chorionic gondatropin (hCG) administration. Women belonging to group 2 received cetrorelix (Cetrotide; Serono Laboratories, Geneva, Switzerland) at a dose of 0.25 mg per day (multiple dose protocol) starting from the day when the dominant follicle reached a mean diameter of ⱖ14 mm and continuing until the day of hCG administration. This flexible protocol of GnRH antagonist administration was recently found effective in preventing a premature LH surge (25). Recombinant FSH (Gonal-F, Serono Laboratories; Puregon, Organon Laboratories, Oss, The Netherlands) was given at a daily dose of 150 –300 IU, starting on the second day of the menstrual cycle. The dose was then adjusted according to the patient’s individual ovarian response. In both groups, hCG (10,000 IU IM; Profasi; Serono Laboratories) was administered when at least three follicles reached a mean diameter of 18 mm, and oocyte retrieval was performed 36 hours later. Fertility and Sterility姞

Determination of GC Apoptosis Annexin V–Propidium Oxide Detection in Flow Cytometry. Early apoptotic cells are recognizable through reversible binding of Annexin V to phosphatidyl serine, a membrane phospholipid that is exposed at the beginning of the apoptotic process (26). Purified GCs were assessed for apoptosis, in parallel with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-treated Jurkat T cells used as positive controls, as previously described (27, 28). A commercial kit (human Annexin V–fluorescein isothiocyanate [FITC] Kit; Bender MedSystem, Vienna, Austria) was used according to the manufacturer’s instructions. Briefly, the cells were gently resuspended in binding buffer and incubated for 10 minutes at room temperature in the dark with Annexin V-FITC. Samples were then washed and supravitally stained with propidium iodide (PI, 50 ␮g/mL), a membrane-impermeable stain that allows the discrimination between membranealtered necrotic (bright) and apoptotic (dim) cells. Analyses were performed with an EPICS Coulter flow cytometer with the FL3 detector in a log mode using Expo 32 analysis software (Beckman Coulter Inc., Brea, CA). For each sample, 10,000 –20,000 events were collected. Vital cells were Annexin V⫺/PI⫺, early apoptotic cells were Annexin V⫹/ PI⫺, and necrotic cells were Annexin V⫹/PI⫹. 413

Immunofluorescent Staining of DNA Strand Breaks (TUNEL). To visualize on a per-cell basis possible DNA damage caused by endogenous endonuclease activation, we performed, on the same samples, in situ TUNEL assay, which detects single or double DNA strand breaks by means of labeled nucleotides polymerized to free 3=-hydroxyl termini in a reaction catalyzed by TdT (29). For TUNEL assay, cells were cytocentrifuged, fixed in paraformaldehyde (4% vol/vol in PBS pH 7.4) for 30 minutes at room temperature and incubated in a permeabilizing solution (0.1% Triton X-100, 0.1% sodium citrate) for 2 minutes on ice. Deoxyribonucleic acid strand breaks were identified with an in situ cell death detection kit (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s instructions. Slides were counterstained with 4=,6-diamidino-2-phenylindole (DAPI) (Vector laboratories, Burlingame, CA), mounted in glycerol, and observed with a light microscope (Leica, Wetzlar, Germany) equipped with a videocamera (CoolSNAP; Photometrics, Tucson, AZ) for acquiring digital images. The extent of DNA fragmentation was quantified through direct visual counting of green fluorescent labeled nuclei at ⫻40 magnification. Five slides per sample were examined, and apoptotic cells were scored out of a total of 100 cells. Positive control samples consisted of GCs treated with deoxyribonuclease I at 2–5 mg/mL for 10 minutes at room temperature. Flow Cytometric Analysis of DNA Samples containing 2–5 ⫻ 105 cells were harvested through centrifugation at 200 ⫻ g for 10 minutes at 4°C, fixed with 70% cold ethanol for at least 1 hour at 4°C, and treated as detailed elsewhere (28). Analysis of PI fluorescence was performed with an EPICS Coulter flow cytometer with the FL3 detector in log mode using Expo 32 analysis software. A total of 10,000 –20,000 events were collected for each sample. Multicycle software (Multicycle; Phoenix Flow Systems, San Diego, CA) was used for cell cycle phase analysis. Quantitative evaluation of apoptosis was assessed by calculating the subdiploid (⬍2n) DNA content as described (28, 30) and expressed as percentage of apoptotic vs. nonapoptotic cells, regardless of the specific cell cycle phase.

Transmission Electron Microscopy For ultrastructural analysis, samples were prefixed in 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.6, for 60 minutes at 4°C, and postfixed in 1% osmium tetroxide for 60 minutes at 4°C. Samples were then dehydrated in alcohol at progressively higher concentrations and embedded in Spurr resin. Ultrathin sections were cut with an ultramicrotome (Reichert, Depew, NY), mounted on 300-mesh nickel grids (Electron Microscopy Sciences, Fort Washington, WA), and photographed with an electron microscope (Zeiss EM-109; Jena, Germany). 414

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Statistical Analysis Data are expressed as mean ⫾ SEM. Student’s t-test was used for statistical comparisons. Statistical significance was defined as P⬍.05. RESULTS Patients The difference in mean patient age for the two groups (32 ⫾ 1.22 years and 33.06 ⫾ 0.89 years in groups 1 and 2, respectively) was not statistically significant (P⬎.05). The differences in the distribution of the causes of infertility were also not statistically significant (P⬎.05): male factor 60%, tubal disease 20%, and unexplained 20% in group 1; male factor 82%, tubal disease 6%, and unexplained 18% in group 2. Determination of GC Apoptosis A representative result obtained with Annexin V–PI detection in flow cytometry is shown in Figure 1. No significant differences were found with respect to the mean percentage of Annexin V⫹/PI⫺cells (group 1 ⫽ 7.87 ⫾ 1.27, group 2 ⫽ 9.92 ⫾ 0.98; P⬎.05). Power analysis for Student’s t-test for

FIGURE 1 Results of flow cytometric analysis of granulose cells of one representative experiment (GnRHagonist-treated women). Early apoptotic cell populations (Annexin V⫹/PI⫺) can be discriminated from vital (Annexin V⫺/PI⫺) or necrotic cells (Annexin⫹/PI⫹) according to their fluorescence emission. Cells in quadrant D4 are apoptotic. Quadrants D1 and D2 represent dead and damaged cells, respectively, and vital cells are situated in quadrant D3.

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GnRH agonist/antagonist and apoptosis

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FIGURE 2 Phase contrast and fluorescent micrographs showing granulosa cell apoptotic (A) and normal (N) nuclei assayed with the terminal deoxynucleotidyl transferase (TdT)-mediated nickend labeling (TUNEL) technique. Original magnification, ⫻40.

independent data using the values obtained in this study (setting ␣ to 0.05, difference of the means ⫽ 2, SD of all groups ⫽ 1.5, and dimension of the two groups of 15 and 17) yielded 95.4% power. Analysis of DNA strand breaks detected with TUNEL revealed a comparable percentage of apoptotic cells in the two groups under investigation (percentage of fluorescent nuclei: 18.00 ⫾ 3.87 in group 1 vs. 20.50 ⫾ 6.56 in group 2, P⬎.05) (Fig. 2). Flow cytometry analysis of cell cycle profile did not show a statistically significant difference in the distribution of cell cycle phases in the two groups and failed to reveal the presence of a subdiploid peak (Fig. 3). The absence of a population of GCs containing subdiploid levels of DNA was confirmed by DAPI nuclear counterstaining, which did not detect any typical micronuclei (Fig. 3). Transmission electron microscopy showed a low incidence of typical hallmarks of apoptotic cell death independent of the treatment. In fact, the greater portion of GCs appeared as typical large cells with round and eccentric nuclei, with the presence of clearly visible nucleoli, well-developed Golgi apparatus and smooth endoplasmic reticulum, mitochondria with complex tubular cristae, and typical osmiophilic droplets scattered throughout the cytoplasm (Fig. 4). Only occasional nuclei with aspects of chromatin condensation and margination were observed, together with patterns of phagocytic removal (Fig. 4).

FIGURE 3 Deoxyribonucleic acid histogram of granulose cells (GCs) stained with PI and analyzed with flow cytometry. The inset shows a representative field of GC nuclei counterstained with 6-diamino-2phenylindole (DAPI) and observed with fluorescent microscopy. No evidence of typical micronuclei was observed.

Giampietro. GnRH agonist/antagonist and apoptosis. Fertil Steril 2006. Giampietro. GnRH agonist/antagonist and apoptosis. Fertil Steril 2006.

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FIGURE 4 (A) Electron micrograph showing a typical granulose cell (GC) with a large, round, and eccentric nucleus (Nu) with the presence of dispersed chromatin and distinctly visible nucleoli (white arrows). Note abundant osmiophilic lipid droplets scattered throughout the cytoplasm (black arrows). Well-preserved mitochondria (M) with complex tubular cristae are often located adjacent to electron-dense droplets. Original magnification, ⫻7,000; ⫻50,000 in inset. (B) Apoptotic GC with the aspect of peripheral chromatin condensation and margination. Original magnification, ⫻4,000. (C) A normal GC which is removing by phagocytosis a highly condensed nucleus (black arrow) of an apoptotic cell. Original magnification, ⫻4,000.

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Hormonal Assays Follicular fluid aspirated from women treated with the GnRH antagonist showed lower levels of E2 in comparison with women treated with the GnRH agonist (Table 1). A comparable trend was noted in serum (P⬍.001) (Table 1). Also, follicular fluid P and T mean concentrations were significantly lower in the patients from group 2 when compared with those from group 1 (P⬍.05) (Table 1). As shown in Table 1, no significant differences (P⬎.05) were found in serum P and LH concentrations between the two groups. DISCUSSION Recent studies suggest a relationship between GC apoptosis and ART outcome, but data are controversial (21, 22). The incidence of apoptotic bodies has been regarded as a predictive marker of ART outcome (21), and the incidence of apoptosis in aspirated GCs has been postulated to reflect the competency of the oocyte in terms of pregnancy rate (21, 22). Conversely, Piquette et al. (5) found that GC apoptosis is not predictive of the result of ART procedures (5), and Clavero et al. (31) failed to find a relationship between the percentage of apoptosis and oocyte maturity and fertilizability by intracytoplasmic sperm injection. In an attempt to provide further insight into the issue of GC apoptosis, in the present study we compared for the first time the levels of apoptosis in GCs from women undergoing ovarian stimulation achieved with protocols using either a GnRH agonist (triptorelin) or a GnRH antagonist (cetrorelix). The results of this study indicate that the observable levels of apoptosis in the two groups investigated are comparable. In our study, GC apoptosis was detected with current methods, offering the possibility of a multiparametric analysis of several cellular attributes, including asymmetry of the plasma membrane, DNA fragmentation, cell cycle position, and ultrastructure. These approaches permitted the evaluation and discrimination between early and late phases of apoptosis and the acquisition of information concerning the plasma membrane integrity of the dying GCs. Measuring apoptosis in flow cytometry has the advantage of increasing outcome precision in determining apoptosis, allowing the analysis of far more GCs in a short time and the detection of early phases of apoptosis. In fact, the externalization of phosphatidyl serine at the cell plasma membrane level, a very early phenomenon during apoptosis, can be detected by virtue of its affinity for Annexin V with fluorescently labeled Annexin V in flow cytometry. The levels of apoptosis found by us seem to be higher than those found by other investigators (21). This inconsistency could be explained by differing sensitivities of the assays used for characterizing apoptosis. In fact, whereas Nakahara et al. (21) measured the percentage of GC apoptosis by using fluorescence microscopy, evidencing only apoptotic bodies, which are indicative only of the late stages of apoptosis, in the present study the cells were analyzed with flow cytometry, which has the advantage of analyzing an event that occurs earlier in the process of apoptosis. Flow cytometric analysis of DNA



TABLE 1 Serum and follicular fluid steroid hormone concentrations. Steroid hormone Serum E2 (pg/mL) Serum P (ng/mL) Serum LH (IU/mL) Follicular fluid E2 (pg/mL) Follicular fluid P (ng/mL) Follicular fluid T (ng/mL)

GnRH agonist ⴙ rFSH (n ⴝ 15)

GnRH antagonist ⴙ rFSH (n ⴝ 17)

P

4,159.0 ⫾ 623.4 1.2 ⫾ 0.1 0.6 ⫾ 0.1 3,770.6 ⫾ 502.6 8,213.3 ⫾ 446.5 5.5 ⫾ 0.8

1,656.9 ⫾ 189.5 1.0 ⫾ 0.1 1.0 ⫾ 0.1 2,386.0 ⫾ 398.6 5,306.2 ⫾ 493.4 3.0 ⫾ 0.6

.000 ns ns .037 .000 .022

Note: All values are expressed as mean ⫾ SEM. ns ⫽ not significant (P⬎.05). Giampietro. GnRH agonist/antagonist and apoptosis. Fertil Steril 2006.

was also used to examine the nuclear pattern of GCs of the two groups under investigation. The resulting absence of a typical subdiploid peak in the cell cycle profile was remarkable. This finding is coherent with the absence of apoptotic bodies, as revealed by DAPI fluorescent staining. From these results it can be inferred that, despite caspase activation (which can be responsible for DNA nick end labeling) (32, 33), the typical oligonucleosomal cleavage of DNA does not occur in this peculiar cell model, analogously with other cell systems (34 –36). The concept that GCs can display a random cleavage of DNA is also consistent with the study by Van Wezel et al. (37), who observed in GCs from healthy or atretic bovine antral follicles a pattern of randomly nicked DNA rather than the pattern of low-molecular-weight apoptotic DNA. The number of examples of DNA cleavage without apoptosis (38) or apoptosis without internucleosomal DNA fragmentation (34 –36) has added new evidence to support the use of morphologic techniques as the gold standard for the identification of apoptosis (39). In the classic mode of apoptosis, different degrees of chromatin margination and condensation are observable before the budding of the nucleus and of the cell itself into several small apoptotic bodies (39). In our study, whatever the treatment used, the great majority of the cells showed normal morphology and did not show crescent-shaped nuclei or signs of cell shrinkage, typical hallmarks of apoptotic cell death. However, it is worth noting that pyknotic and highly condensed nuclei were occasionally observed inside cells displaying a normal morphology. It is known that, in epithelia, apoptotic bodies are rapidly phagocytosed by neighboring epithelial cells, and thus free apoptotic bodies are rarely seen (39). We can hypothesize that either the apoptotic nuclei do not have the capacity to bud or, more likely, that phagocytosis is an early event in the apoptosis of human GCs, preceding any potential budding. The rapid extrusion of phosphatidyl serine at the plasma membrane documented in this study seems to support the early removal of apoptotic nuclei by means of a phagocytosis mechanism. An interrelationship between steroidogenesis and apoptosis has been suggested (40). Some of the multiple apoptotic Fertility and Sterility姞

stimuli in the ovarian follicle have been found to negate steroidogenesis (41), and some enhance steroidogenesis (42). Furthermore, stimuli for apoptosis or cell survival can be endocrine, paracrine, or autocrine factors (43). In fact, androgens enhance ovarian GC apoptosis (9), E2 has been attributed a role in GC proliferation (9), and P might play a part in GC survival, preventing apoptosis by inhibiting the cell oxidation pathway (44, 45). Therefore, we also evaluated follicular fluid hormonal profiles, to provide further insight into crosstalk between steroidogenesis and apoptosis. Currently, there is no consensus as to the effects of GnRH agonists on human ovarian steroidogenesis (46, 47), and little is known concerning the possible direct effects of GnRH antagonists (48). Our results are in agreement with those from the recent study by Garcia-Velasco et al. (49), who found a significantly lower follicular fluid E2 concentration and a trend toward lower P and T levels in GnRH antagonist–treated patients. In the present study, despite the significant action exerted by a GnRH antagonist on ovarian steroidogenesis, levels of GC apoptosis in the two groups were comparable, suggesting that no correlation exists between steroid levels in follicular fluid and GC apoptosis. Early studies demonstrated that hypophysectomy in female rats causes a progressive increase in GC apoptosis in spite of a dramatic increase in P release from the ovarian follicle and a parallel drop in follicular fluid androstenedione, T, and E2 concentrations (50). Recent studies on immortalized rat and human steroidogenic GC lines demonstrated enhanced P production, despite the parallel occurrence of apoptosis (40). Another study on steroidogenic production from human GCs failed to find any correlation between GC apoptosis and steroid levels in follicular fluid (31). These observations suggest that apoptosis and steroidogenesis can coexist in the same cell population, at least temporarily, and that different, still unidentified, mechanisms are presumably involved in the regulation of the two processes. In conclusion, the results of our study indicate that, under the stimulation protocols used, [1] comparable levels of apoptosis are observable in GCs from women exposed to a GnRH agonist or antagonist, and [2] in agreement with 417

previous observations, GnRH antagonist therapy in women undergoing ART has a significant effect on ovarian follicular steroidogenesis. The clinical significance of these findings remains to be determined.

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Acknowledgment: The authors thank Domenico Bosco for the electron micrographs. 21.

REFERENCES 1. Tilly JL. Apoptosis and ovarian function. Rev Reprod 1996;1:162–72. 2. Palumbo A, Yeh Y. Apoptosis as a basic mechanism in the ovarian cycle: follicular atresia and luteal regression. J Soc Gynecol Invest 1995;2:565–73. 3. Tilly JL, Kowalski KIN, Johnson AL, Such AD. Involvement of apoptosis in ovarian follicles atresia and postovulatory regression. Endocrinology 1991;129:2799 – 801. 4. Billig H, Furuta I, Hsueh AJ. Gonadotropin-releasing hormone directly induces apoptotic cell death in the rat ovary: biochemical and in situ detection of deoxyribonucleic acid fragmentation in granulosa cells. Endocrinology 1994;134:245–52. 5. Piquette GN, Tilly JL, Prichard LE, Simon C, Polan ML. Detection of apoptosis in human and rat ovarian follicles. J Soc Gynecol Invest 1994;1:297–301. 6. Chun SY, Eisenhauer KM, Minami S, Hsueh AJ. Growth factors in ovarian follicle atresia. Semin Reprod Endocrinol 1996;14:197–202. 7. Andreu C, Parborell F, Vanzulli S, Chemes H, Tesone M. Regulation of follicular luteinization by gonadotropin-releasing hormone agonist: relationship between steroidogenesis and apoptosis. Mol Reprod Dev 1998;51:287–94. 8. Knecht M, Amsterdam A, Catt KJ. Inhibition of granulose cell differentiation by gonadotropin-releasing hormone. Endocrinology 1982; 110:865–72. 9. Billig H, Furuta I, Hsueh AJ. Estrogens inhibit and androgens enhance ovarian granulose cell apoptosis. Endocrinology 1993;133:2204 –12. 10. Chun SY, Billing H, Tilly JL, Furuta I, Tsafriri A, Hsueh AJ. Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insuline-like growth factor I. Endocrinology 1994;135:1845–53. 11. Hsueh AJ, Erickson GF. Extrapituitary action of gonadotropin-releasing hormone: direct inhibition of ovarian steroidogenesis. Science 1979; 204:854 –5. 12. Fraser HM, Bramley TA, Miller WR, Sharpe RM. Extra pituitary actions of LHRH analogs in tissues of the human female and investigation of the existence and function of LHRH-like peptides. Prog Clin Biol Res 1986;225:29 –54. 13. Latouche J, Crumeyrolle-Arias M, Jordan D, Kopp N, AugendreFerrante B, Cedard L, et al. GnRH receptors in human granulosa cells: anatomical localization and characterization by autoradiographic study. Endocrinology 1989;125:1739 – 41. 14. Ortmann O, Weiss JM, Diedrich K. Ovarian actions of GnRH antagonists. Hum Reprod 2001;16:608 –11. 15. Zhao S, Saito H, Wang X, Saito T, Kaneko T, Hiroi M. Effects of gonadotropin-releasing hormone agonist on the incidence of apoptosis in porcine and human granulosa cells. Gynecol Obstet Invest 2000;49:52– 6. 16. Takekida S, Deguchi J, Samoto T, Matsuo H, Maruo T. Comparative analysis of the effects of gonadotropin-releasing hormone agonist on the proliferative activity, apoptosis, and steroidogenesis in cultured porcine granulose cells at varying stages of follicular growth. Endocrine 2000;12:61–7. 17. Matsubara H, Ikuta K, Ozaki Y, Suzuki Y, Suzuki N, Sato T, et al. Gonadotropins and cytokines affect luteal function through control of apoptosis in human luteinized granulosa cells. J Clin Endocrinol Metab 2000;85:1620 – 6. 18. Parborell F, Pecci A, Gonzalez O, Vitale A, Tesone M. Effects of a

418

Giampietro et al.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.


gonadotropin-releasing hormone agonist on rat ovarian follicle apoptosis: regulation by EGF and the expression of Bcl-2-related genes. Biol Reprod 2002;67:481– 6. Parborell F, Irusta G, Vitale A, Gonzalez O, Pecci A, Tesone M. The gonadotropin-releasing hormone antagonist antide inhibits apoptosis of preovulatory follicle cells in rat ovary. Biol Reprod 2005;72:659 – 66. Durlinger AL, Kramer P, Karels B, Grootegoed JA, Uilenbroek JT, Themmen AP. Apoptotic and proliferative changes during induced atresia of pre-ovulatory follicles in the rat. Hum Reprod 2000;15: 2504 –11. Nakahara K, Saito T, Ito M, Ohta N, Sakai N, Tezuka N, et al. Incidence of apoptotic bodies in membrane granulose of patients participating in an in vitro fertilization program. Fertil Steril 1997; 67:297–303. Oesterhuis GJE, Michgelsen HW, Lambalk CB, Schoemaker J, Vermes I. Apoptotic cell death in human granulose-lutein cells: a possible indicator of in vitro fertilization outcome. Fertil Steril 1998;70:747–9. Nakahara K, Saito H, Saito T, Ito M, Ohta N, Takahashi T, et al. Ovarian fecundity in patients with endometriosis can be estimated by the incidence of apoptotic bodies. Fertil Steril 1998;69:931–5. Toya M, Saito H, Ohta N, Saito T, Kaneko T, Hiroi M. Moderate and severe endometriosis is associated with alterations in the cell cycle of granulose cells in patients undergoing in vitro fertilization and embryo transfer. Fertile Steril 2000;73:344 –50. Escudero E, Bosch E, Crespo J, Simòn C, Remohì J, Pellicer A. Comparison of two different starting multiple dose gonadotropinreleasing hormone antagonist protocols in a selected group of in vitro fertilization-embryo transfer patients. Fertil Steril 2004;3:562– 6. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. Exposure of phosphatidyliserine on the surface of apoptotic lymphocytes triggers recognition and removal by macrophages. J Immunol 1992;148:2207–16. Di Pietro R, Secchiero P, Rana RA, Gibellini D, Visani G, Bemis K, et al. Ionizing radiation sensitizes erythroleukemic cells but not normal erythroblasts to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by selective up-regulation of TRAILR1. Blood 2001;97:2596 – 603. Zauli G, Sancilio S, Cataldi A, Sabatini N, Bosco D, Di Pietro R. PI-3K/AKT and NFKB/IKB␣ pathways are activated in Jurkat T cells in response to TRAIL treatment. J Cell Physiol 2005;202:900 –11. Ansari B, Coates PJ, Greenstein BD, Hall PA. In situ end-labelling detects DNA strand breaks in apoptosis and other physiological and pathological states. Pathology 1993;170:1– 8. Secchiero P, Zella D, Barabitskaja O, Reitz MS, Capitani S, Gallo RC, et al. Progressive and persistent downregulation of surface CXCR4 in CD(⫹) T cells infected with human herpesvirus 7. Blood 1998;92: 4521– 8. Clavero A, Castilla JA, Nunez AI, Garcia-Pena M, Maldonado V, Fonts J, et al. Apoptosis in human granulosa cells after induction of ovulation in women participating in an intracytoplasmic sperm injection program. Eur J Obstet Gynecol 2003;110:181–5. Gaido ML, Cidlowski JA. Identification, purification, and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes. J Biol Chem 1991;266:18580 –5. Zeleznik AJ, Ihrig LL, Bassett SG. Developmental expression of Ca⫹⫹/ Mg⫹⫹-dependent endonuclease in rat granulose and luteal cells. Endocrinology 1989;125:2218 –20. Zamai L, Falcieri E, Marhefka G, Vitale M. Supravital exposure to propidium iodide identifies apoptotic cells in absence of nucleosomal DNA fragmentation. Cytometry 1996;23:303–11. Vitale M, Zamai I, Falcieri E, Zauli G, Gobbi P, Santi S, et al. IMP dehydrogenase inhibitor, tiazofurin, induces apoptosis in K562 human erythroleukemia cells. Cytometry 1997;30:61– 6. Falcieri E, Bassini A, Pierpaoli S, Luchetti F, Zamai L, Vitale M, et al. Ultrastructural characterization of maturation, platelet release, and senescence of human cultured megakaryocytes. Anat Rec 2000;253: 90 –9. Van Wezel IL, Dharmarajan M, Lavranos C, Rodgers J. Evidence


38.

39. 40.

41.

42.

43.

44.

for alternative pathways of granulosa cell death in healthy and slightly atretic bovine antral follicles. Endocrinology 1999;140: 2602–12. Collins RJ, Harmon BV, Gobe GC, Kerr JFR. Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. Int Radiat Biol 1992;61:451–3. Kerr JFR, Gobe GC, Winterford CM, Harmon BV. Anatomical methods of cell death. Methods Cell Biol 1995;46:1–27. Amsterdam A, Keren-Tal I, Aharoni D, Dantes A, Land-Bracha A, Rimon E, et al. Steroidogenesis and apoptosis in the mammalian ovary. Steroids 2003;68:861–7. Sasson R, Amsterdam A. 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. J Clin Endocrinol Metab 2002;87:3441–51. Aharoni D, Dantes A, Oren M, Amsterdam A. cAMP-mediated signals as determinants for apoptosis in primary granulosa cells. Exp Cell Res 1995;218:271– 82. Amsterdam A, Gold RS, Hosokawa K, Yoshida Y, Sasson R, Jung Y, et al. Crosstalk among multiple signalling pathways controlling ovarian cell death. Trends Endocrinol Metab 1999;10:255– 62. Eva CH, Svensson MD, Markstrom E, Shao R, Andersson M, Billig H.

Fertility and Sterility姞

45.

46.

47.

48.

49.

50.

Progesterone receptor antagonists ORG 31710 and RU 486 increase apoptosis in human periovulatory granulose cells. Fertil Steril 2001;76:1225–31. Sugino N, Takiguchi S, Kashida S, Takayama H, Yamagata Y, Nakamura Y, et al. Suppression of intracellular superoxide dismutase activity by antisense oligonucleotides causes inhibition of progesterone production by rat luteal cells. Biol Reprod 1999;61:1133– 8. Bussenot I, Azoulay-Barjonet C, Parinaud J. Modulation of the steroidogenesis of cultured human granulosa-lutein cells by gonadotropinreleasing hormone analogs. J Clin Endocrinol Metab 1993;76:1376 –9. Pellicer A, Mirò F. steroidogenesis in vitro of human granulosa-luteal cells pretreated in vivo with gonadotropin-releasing hormone analogs. Fertil Steril 1990;54:590 – 6. Weiss JM, Oltmanns K, Gurke EM, Polack S, Eick F, Felberbaum R, Diedrich K, et al. Actions of gonadotropin-releasing hormone antagonists on steroidogenesis in human granulose lutein cells. Eur J Endocrinol 2001;144:677– 85. Garcia-Velasco JA, Isaza V, Vidal C, Landazabal JR, Simon C, Pellicer A. Human ovarian steroid secretion in vivo: effects of GnRH agonist versus antagonist (cetrorelix). Hum Reprod 2001;16:2533–9. Braw RH, Bar-Ami S, Tsafriri A. Effect of hypophysectomy on atresia of rat preovulatory follicles. Biol Reprod 1981;25:989 –96.

419