Human Chorionic Gonadotropin Mediated Generation of Reactive Oxygen Species Is Sufficient to Induce Meiotic Exit but Not Apoptosis in Rat Oocytes

Abstract Generation of reactive oxygen species (ROS) is associated with final stages of follicular development and ovulation in mammals. The human chorionic gonadotropin (hCG) mimics the action of luteinizing hormone and triggers follicular development and ovulation. However, it remains unclear whether hCG induces generation of ROS, if yes, whether hCG-mediated increased level of ROS could induce meiotic exit and/or apoptosis in rat oocytes. For this purpose, cumulus–oocyte complexes (COCs) were collected from ovary of experimental rats injected with 20 IU pregnant mare's serum gonadotropin for 48 h followed by 20 IU hCG for 0, 7, 14, and 21 h. The morphological changes in COCs, meiotic status of oocyte, total ROS, hydrogen peroxide (H2O2), inducible nitric oxide synthase (iNOS), nitric oxide (NO), Bax, Bcl-2, cytochrome c, telomerase reverse transcriptase (TERT) expression levels, and DNA fragmentation were analyzed in COCs. Our data suggest that hCG surge increased total ROS as well as H2O2 levels but decreased iNOS expression and total NO level in oocytes. The hCG-mediated increased level of ROS was sufficient to induce meiotic cell cycle resumption in majority of oocytes as evidenced by meiotic exit from diplotene as well as metaphase-II (M-II) arrest and their meiotic status. However, increase of ROS level due to hCG surge was not sufficient to trigger Bax and cytochrome c expression levels and DNA fragmentation in COCs. In addition, increased TERT activity was observed in oocytes collected 21 h post-hCG surge showing onset of oocyte aging. Taken together, these results suggest that hCG induces generation of ROS sufficient to trigger meiotic exit from diplotene, as well as M-II arrest, but not good enough to induce apoptosis in rat oocytes.


Introduction
Ovary is a metabolically active organ that generates excess amount of reactive oxygen species (ROS) during final stages of follicular development, maturation, and ovulation. [1][2][3][4] During this period, oocyte achieves meiotic competency within the antral follicle just before ovulation in mammals. [5][6][7][8] The meiotic competency starts when oocyte resumes meiosis from diplotene stage. 1,9 The diplotene stage is morphologically identified by the presence of germinal vesicle (GV) and nucleolus in the oocyte cytoplasm. [5][6][7][8][9][10] The diplotene stage may last for several months to several years in various mammals. 1,2,[11][12][13] The cell cycle arrest for such a long period could be due to several factors either released from encircling granulosa cells or by oocyte itself. 5,10 Granulosa cells play important role in the synthesis and secretion of various signal molecules required for the maintenance of meiotic arrest at diplotene stage. 2,6,[11][12][13][14][15] Acquisition of meiotic competency in oocytes starts with resumption from diplotene arrest, 5,13,[16][17][18] progress through metaphase-I (M-I), and ends with the achievement of metaphase-II (M-II) stage by extruding first polar body (PBI) in most of the mammalian species. 6,11,12,[19][20][21][22][23][24][25] Pituitary gonadotropin(s) surge induces follicular development, maturation, and ovulation. 26,27 The increased metabolic activity during follicular development or increased inflammation during follicular rupture at the time of ovulation may cause the generation of ROS. 3,4 The increased level of ROS has been observed during luteinizing hormone (LH)induced ovarian stimulation protocol. 4,28 The increase in level of ROS associates with meiotic resumption, fertilization, and improved reproductive outcome in human. 29,30 In contrast, excess amount of ROS could induce susceptibility towards oocyte apoptosis. [31][32][33][34][35][36][37][38] Human chorionic gonadotropin (hCG) has commonly been used in ovarian stimulation protocol to induce final maturation and ovulation in several mammalian species during assisted reproduction. 28,39 Due to structural and biological similarities with LH, hCG binds to LH receptors. 39 It is proposed that hCG surge generates ROS due to increased cell proliferation during final stages of folliculogenesis and mediate meiotic resumption. 2,4 Another source of ROS could be the inflammation generated due to follicular rupture during ovarian stimulation protocol. This notion is supported by the observations that the depletion of ROS impairs ovulation. 3,4 Pituitary gonadotropins modulate expressions of various nitric oxide synthase (NOS) isoforms. 40 Both pregnant mare's serum gonadotropin (PMSG) and hCG have been shown to influence inducible nitric oxide synthase (iNOS) expressions in rat ovary. 41 Recent studies from our laboratory suggest that decreased level of nitric oxide (NO) is associated with meiotic exit from diplotene, as well as M-II arrest, 12,17,22 while high level of NO induces cell cycle arrest and apoptosis. 31,33 After ovulation, oocyte experiences aging process, 42-44 which can be determined by telomerase reverse transcriptase (TERT) activity. 45 The increased TERT activity has recently been reported during postovulatory egg aging in rat. 25 Oxidative stress may result in shortening of telomere length. 45 Recent studies from our laboratory suggest that increased oxidative stress reduces Bcl-2 expression and increased TERT activity. 25,[36][37][38] However, it remains unclear whether hCG-mediated generation of ROS triggers meiotic resumption. If yes, whether, its amount is sufficient to induce apoptosis. Therefore, present study was aimed to find out the impact of hCG surge on ROS, hydrogen peroxide (H 2 O 2 ), iNOS, NO, Bax, Bcl-2, cytochrome c, TERT expression, and DNA fragmentation in rat cumulus-oocyte complexes (COCs).

Materials and Methods
Chemicals and preparation of culture medium Chemicals used in present study were purchased from Sigma Chemical Co., St. Louis, MO, and culture media from HiMedia Laboratories, Mumbai, India unless otherwise specified. Culture medium (AL094A, M-199) was prepared as per the company manual protocol. The sodium bicarbonate (0.035% w/v) was added, and the pH was adjusted to 7.2 -0.05. The osmolarity was checked (290 -5 m Osmol) and then medium was supplemented with antibiotics, that is, L-glutamine, penicillin, and streptomycin (GPS; 1 lL/mL; Cat. No. A007). The culture medium was stored at 4°C and discarded if not used within 15 days.

Collection of COCs
Experimental immature female rats (Rattus norvegicus) of Charles-Foster strain (22-24 days old; 45 -5 g body weight) were housed in light-controlled room with food and water in ad libitum. For the first series of experiments, rats were subjected to a single subcutaneous injection of 20 IU PMSG for 48 h and 20 IU hCG for 0, 7, 14, and 21 h. Thereafter, rats were euthanized, and ovary along with fallopian tube was collected in 35-mm Petri dish containing 2 mL of prewarmed culture medium-199 (M-199). The ovary was punctured using a 26-gauge needle attached to 1 mL of tuberculin syringe under a stereomicroscope (Nikon type 104, Japan) for the collection of diplotene, as well as exit from diplotene arrest (EDA) COCs, while M-II and exit from metaphase-II (EM-II) stages of COCs were collected from the ampulla of fallopian tube. A group of 12-14 COCs were collected using microtubing attached with disposable glass micropipette (Clay Adams, NJ) and few of them were partially denuded by manual pipetting using hyaluronidase (0.01%). COCs and denuded oocytes were transferred on slide and fixed with 4% buffered formaldehyde. In the second series of experiment, COCs were collected from ovary, as well as ampulla of fallopian tube, as described for first series of experiments. The COCs were denuded and immediately used for the quantitative analysis of H 2 O 2 and total NO levels. In the present study, three independent experiments were conducted using total 60 experimental animals to collect COCs and denuded oocytes sufficient for morphological, biochemical, and immunofluorescence studies. Determination of meiotic status of oocytes The COCs were partially denuded by repeated manual pipetting several times and after washing with M-199; meiotic status of oocytes was confirmed by Hoechst-33342 using fluorescence microscope (Model, Ni-U; Nikon Eclipse Tokyo, Japan). For this purpose, *12-14 denuded oocytes collected from each group of animals were washed twice with phosphate-buffered saline (PBS) and then incubated (10 min) with 10 lg/mL of Hoechst-33342 in PBS. Thereafter, denuded oocytes were washed 10 times with PBS and then checked for their meiotic status under fluorescence microscope at 350 nm (400· magnification). Three independent experiments were conducted to confirm the meiotic stage, and representative photographs are shown in the Results section.

Analysis of total ROS level
The total ROS level was analyzed using 2¢, 7¢dichlorodihydrofluorescein diacetate (H 2 DCFDA) following previous published protocol. 18 Briefly, 12-14 COCs from each group were exposed to H 2 DCFDA (10 lM) for 15 min at 37°C in CO 2 incubator (Galaxy 170 R; New Brunswick, Eppendorf AG, Hamburg, Germany, United Kingdom). Thereafter, COCs were washed five times with prewarmed PBS and then DCF fluorescence was measured at 485 nm excitation/ 520 nm emissions using fluorescence microscope (400· magnification). The denuded oocytes of corresponding COCs from three independent experiments were used for corrected total cell fluorescence (CTCF) analysis. All parameters were kept constant for each oocyte, and fluorescence intensity was analyzed using ImageJ software (version 1.44; National Institute of Health, Bethesda).

Quantitative estimation of H 2 O 2 concentration
The H 2 O 2 concentration was analyzed using Hydrogen Peroxide Assay Kit (Cat. No. K265-200) purchased from BioVision, CA. In brief, *36-42 COCs collected from each group were denuded by manual pipetting and lysed in hypotonic lysis buffer (5 mM Tris, 20 mM ethyl diamine tetraacetic acid [EDTA], 0.5% Triton X-100, pH 8). Lysates were centrifuged at 10,000 g at 4°C for 30 min, and supernatant was immediately used for quantitative estimation of H 2 O 2 following previous published protocol. 22 The optical density (OD) was determined using a microplate reader (Model: Micro Scan MS5608A; Electronics Corporation of India Limited, Hyderabad, India) set to 550 nm. Samples from three independent experiments were used for each group, and all samples were run in one assay to avoid interassay variation, and intraassay variation was 2.3%.

Quantitative estimation of total NO concentration
The total NO level was estimated using NO Assay Kit (Cat. No. KGE001) purchased from R&D Systems (MN) following previous published protocol. 17 Briefly, 36-42 COCs from each group were denuded by manual pipetting. The denuded oocytes were lysed in hypotonic lysis buffer (5 mM Tris, 20 mM EDTA, 0.5% Triton X-100, pH 8) and centrifuged at 10,000 g at 4°C for 30 min. The supernatant was stored at À20°C until all samples were collected. All samples, working standards, and reagents were thawed, brought to room temperature, and prepared following company manual protocol. The reaction diluents (50 lL) were added to the blank well and nitrate standards (50 lL) or samples to remaining wells. The nicotinamide adenine dinucleotide (reduced form) (25 lL) and then diluted nitrate reductase (25 lL) were added to each and every well. The microplate was incubated at 37°C (30 min) and then Griess reagent I and II (50 lL each) were added. The plate was incubated (10 min) at room temperature and then OD was determined using a microplate reader set at 540 nm with wavelength correction at 690 nm. Three independent samples were run in one assay to avoid interassay, and intra-assay variation was found to be 1.9%.
Detection of iNOS, Bax, Bcl-2, cytochrome c, and TERT expressions Immunofluorescence of iNOS, Bax, Bcl-2, and cytochrome c expressions were analyzed in COCs using their highly specific antibodies purchased from Santa Cruz Biotechnology (Dallas, TX), as per our published protocol with some modifications. 36 In brief, 12-14 COCs from each group were fixed with 4% buffered formaldehyde (10 min) at room temperature. Slides were washed thrice with prewarmed PBS and exposed to Triton X-100 (0.01% in PBS) for 10 min at 37°C for permeabilization. Slides were washed thrice with prewarmed PBS and then treated with sodium citrate solution (0.01 M) at 37°C (10 min) for better antigen retrieval. Slides were again washed thrice with prewarmed PBS and then incubated with blocking buffer (2.5% PBS-BSA solution) at 37°C (30 min). Thereafter, slides were exposed to 100 lL of their respective primary antibodies (NOS2 (N-20), rabbit polyclonal antibody (sc-651) raised against a peptide mapping near the N-terminus of NOS2; Bax (B-9), mouse monoclonal antibody (sc-7480) raised against amino acids 1-171 of Bax; Bcl-2 (C-9), mouse monoclonal antibody (sc-7382) raised against amino acids 1-205 of Bcl-2; cytochrome c (A-8), mouse monoclonal antibody (sc-13156) raised against amino acids 1-104 of cytochrome c; TERT (D-16), goat polyclonal antibody (sc-68720) raised against a peptide mapping within an internal region of TERT; and actin (C-2) mouse monoclonal antibody (sc-8432) specific for an epitope mapping between amino acids 350-375 at the c-terminus of actin, 1:500 dilutions in blocking buffer) at 37°C for 1 h. After 6-8 washes with PBS, slides were exposed to 100 lL of specific anti-rabbit fluorescein isothiocyanate (FITC)-labeled (sc-3839) secondary antibody for detection of iNOS, anti-mouse FITC-labeled (sc-2010) secondary antibody for detection of Bax, Bcl-2, cytochrome c, and b-actin, and anti-goat FITC-labeled (sc-2024) secondary antibody for detection of TERT expression at 37°C in humidified chamber (1:1000 dilutions in blocking buffer). After 1 h of incubation, slides were washed five times with prewarmed PBS, mounted with fluorescence mounting medium by VECTASHIELD (Vector laboratories) and then observed under fluorescence microscope, 465 nm at 100· and 400· magnification. Fluorescence intensity of b-actin was analyzed in parallel as a control to assure that all parameters were kept constant during immunofluorescence analysis. The experiment was repeated thrice to confirm the results, and the representative photographs are shown in Results section.

DNA fragmentation analysis
The extent of DNA fragmentation was analyzed using acridine orange/ethidium bromide (AO/EtBr) staining as per our previous published protocol. 38 In brief, the 12-14 COCs from each group were fixed in 4% buffered formaldehyde (15 min) and then air-dried. Slides were washed and then exposed to nucleic acid binding dye mix and 100 lL of 1:1 mixture of AO/EtBr solutions (4 lg/mL) for 1 min at room temperature. Slides were then washed with PBS and analyzed using fluorescence microscope. The viable cells and the extent of apoptosis were recognized depending on their color due to binding of EtBr to the fragmented DNA. Normal cells with intact DNA had green fluorescence of AO, while EtBr binding to fragmented DNA changed the color from green to yellowish orange during initiation of apoptosis.
DNA fragmentation was further confirmed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, that is, TACS 2TdT in situ Apoptosis Detection Kit purchased from Trevigen, Inc., MD (4812-30-K) following company manual protocol. The 12-14 COCs from each group were transferred and fixed in 4% buffered formaldehyde (15 min). Slides were immersed in 1· PBS (10 min) and then treated with 50 lL proteinase K solution (15 min). The slides were washed with deionized water twice, 2 min each, and then immersed in 1· TdT labeling buffer (5 min). The slides were then incubated with 50 lL labeling reaction mix at 37°C in a humidity chamber for 1 h. Thereafter, slides were immersed in 1· TdT stop buffer (5 min) and then washed with 1· PBS for two times, 2 min each. Thereafter, slides were incubated with 50 lL strep-fluor solution in dark (20 min) and then again washed with 1· PBS for two times, 2 min each. After that the slides were observed under fluorescence microscope at 495 nm. The experiment was repeated thrice to confirm the results.

Statistical analysis
Data are expressed as meanstandard error of mean (SEM) of three independent experiments. All percentage data were subjected to arcsine square-root transformation before statistical analysis. Data were analyzed by one-way analysis of variance (ANOVA) using SPSS software, version 17.0 (SPSS, Inc., Chicago, IL) followed by Bonferroni post hoc analysis. A probability of p < 0.05 was considered to be statistically significant.

Results
Changes in morphological and oocyte meiotic status during progression of meiotic cell cycle As shown in Figure 1 initiation of extrusion of second polar body (PBII), a morphological sign of EM-II arrest (pink arrow; Fig. 1D). The various meiotic stages such as diplotene arrest (Fig. 1E), EDA (Fig. 1F), M-II arrest (Fig. 1G), and EM-II arrest (Fig. 1H) were further confirmed using Hoechst staining. As shown in Figure 1I, majority of follicular oocytes were arrested at diplotene stage after 48 h of PMSG+hCG (0 h) surge. In contrast, hCG surge for 7 h triggered meiotic resumption from diplotene arrest in majority (68.2% -1.5%) of oocytes, while 14 h post-hCG surge induced M-II stage in most of the freshly ovulated oocytes (96.2% -0.8%). In addition, 21 h post-hCG surge showed meiotic exit from M-II arrest as evidenced by the initiation of extrusion of PBII in majority of oocytes (87.4% -2.9%) (one-way ANOVA, F = 579.03, p < 0.001; Fig. 1I). Figure 2 shows fluorescence intensity of total ROS level in COCs and their corresponding denuded oocyte and H 2 O 2 concentration in denuded oocytes. Generation of ROS has been observed as evidenced by increase of ROS specific fluorescence intensity in denuded oocytes (Fig. 2B2, B4) of COCs collected after 7 h (Fig. 2A2)  hCG surge decreases iNOS expression and total NO level As shown in Figure 3A, a decrease ( p < 0.05) of iNOS expression was noticed in oocyte collected after 7 h (Fig. 3A2)  hCG surge did not alter Bax, Bcl-2, as well as cytochrome c expression levels As shown in Figure 4, hCG surge did not modulate Bax (Fig. 4A1-A4), Bcl-2 ( Fig. 4C1-C4), as well as cytochrome c expressions (Fig. 4E1-E4), as evidenced by their unaltered immunofluorescence intensity during the entire study. Peripheral granulosa cells of 21 h post-hCG surge showed little reduced Bcl-2 expression and increased Bax expression, but it was not significantly altered Increase of TERT expression associates with postovulatory oocyte aging As shown in Figure 5, hCG surge increased ( p < 0.01) immunofluorescence intensity of TERT in oocyte that showed meiotic resumption from M-II arrest after 21 h post-hCG surge (Fig. 5A4) compared to other groups (Fig. 5A1-A3) showing either diplotene or M-II arrest. The CTCF analysis (Fig. 5B) of the denuded oocyte ( Fig. 5A1-A4, in box) of corresponding COCs further strengthens our observations (one-way ANOVA; F = 22.12; p < 0.001).

Increase of ROS and H 2 O 2 levels associates with meiotic exit from diplotene, as well as M-II arrest
hCG surge did not induce DNA fragmentation As shown in Figure 6, hCG surge did not induce DNA fragmentation in any group of oocytes in the present study as evidenced by TUNEL fluor negative staining ( Fig. 6A1-A4) and background green fluorescence of AO ( Fig. 6C1-C4). However, peripheral granulosa cells underwent apoptosis as evidenced by TUNEL fluor positive green fluorescence (Fig. 6A4, yellow arrows). The CTCF analysis (Fig. 6B) of the denuded oocyte ( Fig. 6A1-A4, in box) further confirms our observations.
For all the immunofluorescence studies, b-actin was analyzed as a control in parallel. As shown in Figure 7, b-actin did not alter during the entire period of hCG surge (Fig. 7A1-A4). The CTCF analysis of denuded oocyte of corresponding COCs further strengthens our observations (Fig. 7B).

Discussion
Oocyte quality is one of the important factors that directly affects assisted reproductive technologies (ARTs) outcome in several mammalian species, including human. 8,46 The oocyte quality is solely dependent upon acquisition of meiotic competency in oocyte, that is, spontaneous EDA, progression through M-I to M- II, and release of PBI that results in the production of haploid female gamete. A moderate increase of ROS and decrease of NO level may be beneficial for oocyte meiotic resumption. 16,17,30 High level of ROS could also affect the oocyte quality by inducing apoptosis. [31][32][33][34][35][36][37][38] The attainment of meiotic competency involves several biochemical and molecular changes. 46,47 The successful completion of meiotic competency determines oocyte quality, which is primary requirement for successful fertilization and early embryonic development. 47,48 Oocyte meiotic competency relies on bidirectional communication between the oocyte and surrounding granulosa cells, which is regulated by various endocrine, paracrine, and autocrine factors. 48 Pituitary gonadotropin surge is the primary requirement for the induction of meiotic maturation in oocytes. 28,39 hCG has been frequently used as a surrogate for LH surge to induce ovulation in several mammalian species, 26,46 but its role in meiotic maturation remains ill understood. Data of the present study suggest that most of the oocytes (more than 90%) collected after 48 h PMSG surge were arrested at GV stage. The hCG surge for 7 h triggered meiotic resumption from diplotene stage in more than 65% of oocytes, while 14 h hCG surge not only induced granulosa cell dispersion but also pushes oocyte to reach to M-II stage as evidenced by one haploid genome in the oocyte cytoplasm and other toward polar body. The 14-h hCG surge induced PBI extrusion in more than 85% of oocytes. Furthermore, collection of COCs from ampulla of fallopian tube after 21 h post-hCG surge resulted in highly dispersed granulosa cells, reduction in number of total granulosa cells encircling oocytes, and meiotic exit from M-II arrest as evidenced by the initiation and extrusion of PBII, haploid genome in PBI area, and formation of metaphase plate in the cytoplasm of ovulated oocytes. The hCG-mediated meiotic exit from M-II arrest has been reported in rat. 20,25 Meiotic resumption from diplotene arrest could be due to hCG surge that induces final stages of folliculogenesis in mammals. A moderate increase of ROS level has been reported to induce spontaneous meiotic resumption in rat oocytes 16,21 and beneficial for fertilization, as well as reproductive outcome, after ovarian stimulation protocol. 4,28-30 Studies from our laboratory using NO donor and iNOS inhibitor suggest that the reduction in NO level results in spontaneous EDA in rat COCs cultured in vitro. 49 Our results suggest that the increase of total ROS level (almost two fold increase) was associated with meiotic exit from diplotene, as well as M-II arrest. The increase of ROS, decrease of iNOS, and thereby NO levels after 7 h of hCG surge could be due to increased cell proliferation and cellular metabolism within the follicle. After 14 h of hCG surge, follicles are ruptured, and ovulated COCs reside in the ampulla of the fallopian tube. Hence, they are disconnected from follicular mi-croenvironment that leads to a reduction of ROS, iNOS, and NO levels in ovulated COCs. Due to limited energy resources, ovulated COCs in the ampulla of the fallopian tube generate ROS, which could induce apoptosis in peripheral granulosa cells, as well as meiotic exit from M-II arrest that deteriorates oocyte quality. The quantification analysis of H 2 O 2 level further strengthens our data. Similarly increase of ROS, as well as H 2 O 2 level, has been reported to induce meiotic exit from diplotene, 16 as well as M-II arrest. 21 These data together with our previous findings suggest that hCG surge after superovulation induction generates ROS. This moderate increase of ROS level was sufficient to induce meiotic exit from diplotene, as well as M-II arrest in rat oocytes. The increased level of ROS may stimulate aging process by elevating markers of cell aging. 43,44 The exposure of increased level of ROS influences telomerase activity and subsequently the length of telomeres in human and bovine ovaries. 50,51 TERT (telomerase subunit) expression has been found to correlate closely with the telomerase activity 45 ; therefore, we analyzed TERT expression in the present study. Our results suggest that TERT expression was not altered till 14 h post-hCG surge, while its expression significantly increased in oocytes after 21 h post-hCG surge. These data corroborate with previous observations that the increased TERT expression induces meiotic cell cycle progression, as well as oocyte aging. 25 The increased level of ROS may generate oxidative stress and induce mitochondria-mediated oocyte apoptosis. [31][32][33][34][35][36][37][38] We propose that hCG surge during superovulation induction increases ROS level that may induce apoptosis in COCs. Data of the present study suggest that accumulation of ROS due to hCG surge was sufficient to induce meiotic exit but not enough to initiate ROS-mediated apoptosis. The hCG-mediated accumulation of ROS was not sufficient to alter Bax, Bcl-2, and cytochrome c levels in the oocyte cytoplasm. Furthermore, increased level of ROS due to hCG surge did not induce DNA fragmentation as evidenced by AO/Etbr, as well as TUNEL fluor negative staining in oocyte. However, peripheral granulosa cells of COCs collected after 21 h post-hCG treatment show DNA fragmentation as evidenced by TUNEL fluor positive staining.
In conclusion, data of the present study suggest that hCG-mediated increased level of ROS and decreased NO level was sufficient to induce meiotic exit from diplotene, as well as M-II arrest, but this moderate increase of ROS level was not sufficient to trigger Bax/Bcl-2 and cytochrome c expression levels and DNA fragmentation in oocytes. However, increased TERT expression was observed in oocytes collected 21 h post-hCG surge showing onset of oocyte aging. Although oocyte experiences aging process, the survival factor and DNA integrity were maintained until 21 h of hCG surge. Taken together, these results suggest that hCG induces generation of ROS sufficient to trigger meiotic exit from diplotene, as well as M-II arrest, but not good enough to induce apoptosis in rat oocytes.