The chemicals used to culture the oocytes and embryos in the present study were obtained from Sigma-Aldrich (St. Louis, MO, USA), whereas media (including, TCM199, FBS, formaldehyde and Triton X-100) were from Gibco; Thermo Fisher Scientific, Inc., (Waltham, MA, USA). The RNeasy^® Micro kit (cat. no. 74004) was purchased from QIAGEN (Valencia, CA, USA). SYBR Premix Ex Taq (cat. no. DRR420A) and the PrimeScript RT reagent kit with gDNA Eraser (cat. no. DRR047S) were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The One-Step Terminal Deoxynucleotidyl Transferase UTP Nick End Labeling (TUNEL) apoptosis assay kit (cat. no. C1088), the GSH and GSSG assay kits (cat. no. S0053), and the Caspase 3 activity assay kit (cat. no. C1116) were purchased from Beyotime Institute of Biotechnology (Haimen, China). All other chemicals were obtained commercially and were of reagent grade. All experiments were performed in accordance with the Guidelines for the Care and Use of Animals of the College of Animal Science and Technology, Nanjing Agricultural University (Nanjing, China) (16).

The working solutions of H₂O₂ (Sigma-Aldrich; cat. no. H3410), cystine (Sigma-Aldrich; cat. no. C7602) and cysteine (Sigma-Aldrich; cat. no. C5360) were prepared freshly prior to use. Briefly, 3.5 µl of 30% H₂O₂ was diluted in 1 ml of IVM culture media (30 mM). Subsequently, 10 and 12 µl of this solution were further diluted in 3 ml of culture media (100 and 120 µM). The 100 µM H₂O₂ solution was diluted into further concentrations of 60 and 80 µM. The cysteine stock solution (20 mM) and the cystine stock solution (100 mM) were diluted in culture media to obtain final concentrations of 100, 200 and 500 µM. Thus, four IVM culture conditions were used for the oocytes in the present study, as follows: i) control (IVM media without chemical agent), ii) H₂O₂ (IVM media with H₂O₂), iii) CC (IVM media with cystine and cysteine), and iv) H₂O₂ and CC (IVM media with H₂O₂ and CC).

Goat ovaries were collected from a slaughterhouse (Haimen goat Research Center; Haimen, Nantong, China), delivered to the laboratory within 3–4 h and washed with normal saline. The connective tissues and the attached oviducts were removed. The ovaries were transferred into Petri dishes and the 2–6 mm follicles were sectioned. Cumulus-oocyte complexes (COCs) appropriate for IVM were selected for IVM culture. The base medium for IVM comprised modified TCM199 supplemented with 10 µg/ml follicle-stimulating hormone luteinizing hormone, estrogen (all Sigma-Aldrich) and 10% FBS. This media was further supplemented with 100 µM cysteine and 100 µM cystine or 100 µM H₂O₂, according to the experimental design. The selected COCs were cultured for 20–22 h at 38.5°C under a saturated 5% CO₂ atmosphere.

Following IVM, the COCs were treated with 0.1% hyaluronidase to strip their cumulus cells by repeated pipetting through a narrow-bore pipette in culture media. Good-quality MII oocytes with a uniform cytoplasm and a well-extruded polar body were selected to assess for parthenogenetic activation.

The parthenogenetic embryos were treated with TCM 199 containing 5 µM ionomycin for 5 min, incubated in TCM 199 containing 2 mM 6-DMAP for 4 h, and washed three times in 100 µl of M16 medium. The oocytes were then transferred into 100 µl M16 droplets under mineral oil and cultured for 6 days at 38.5°C under 5% CO₂. Cleavage and development to the blastocyst stage were observed on day 2.5 (day of parthenogenetic activation=day 0.5) and day 6.5, respectively. Blastocysts, which developed on day 6.5, were fixed to examine their cell numbers. The total number of blastocysts were determined under a Nikon Eclipse TE2000 fluorescence microscope (Nikon Corporation, Tokyo, Japan) following Hoechst 33342 staining (Sigma-Aldrich; cat. no. B-2261).

Mature oocytes (30 oocytes per group) were collected to analyze the expression levels of genes associated with the mitochondria and apoptosis in goat oocytes. Total mRNA was extracted using the RNeasy^® Micro kit, according to the manufacturer's protocol. For the RT, total mRNA with a final volume of 20 µl (containing 0.5 mg oligo-dT, 1X RT buffer, 10 mM dithiothreitol and 10 mM dNTP) was reverse transcribed at 37°C for 50 min and at 70°C for 15 min, and the products were stored at 4°C until use. The qPCR was performed using an qPCR reagent kit with gDNA Eraser. The total reaction volume of 20 µl contained 2 µl of 5X gDNA Eraser Buffer, 1 µl of gDNA Eraser, 1 µg of total RNA, 4 µl of 5X RT Buffer, 1 µl of qPCR enzyme mix, 1 µl of RT Primer Mix, and sufficient nuclease-free H₂O. The qPCR was performed at 42°C for 2 min, 37°C for 15 min, followed by a denaturation step at 85°C for 15 sec and cooling on ice. The primer sequences for each gene are shown in Table I. The tests were performed in triplicate, and the mRNA levels in each sample were normalized to the mRNA level of GAPDH.

All transcripts were quantified by qPCR using an ABI 7300 PRISM system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The amplification products of PGC-1α, NRF-1, BAX, Bcl-2 and GAPDH were detected using SYBR green staining. The GenBank accession numbers and primer sequences used to amplify the target genes are presented in Table I.

The qPCRs were run in triplicate in a total volume of 20 µl, consisting of SYBR Premix Ex Taq, ROX Reference Dye, 200 nM of each gene-specific primer and 100 ng equivalent cDNA. The amplification conditions were as follows: DNA polymerase activation at 95°C for 30 sec, followed by 40 amplification cycles of 95°C for 5 sec and 58°C for 31 sec. At the end of the amplification cycles, melting curve analysis was performed to verify gene-specific amplification. The comparative CQ method (17) was used to quantify the relative expression levels of the target genes. For ease of comparison, the average expression level of each gene from the control group was set as one (16).

The TUNEL method was performed using a One-Step TUNEL Apoptosis Assay kit, as previously described. Briefly, the control and experimental oocytes (20 oocytes per group) undergoing shrinkage were fixed in 3.7% formaldehyde in PBS for 1 h at 18–20°C. Following fixation, the oocytes were washed in PBS and permeabilized by incubation in 0.1% (v/v) Triton X-100 for 2 min at 4°C. The oocytes were then washed twice in PBS and incubated with the TUNEL labeling media in the dark for 1 h at 37°C. Following counterstaining with 10 µg/ml Hoechst 33342 for 10 min at 37°C to label all the nuclei, the oocytes were mounted with light coverslip compression and observed under a microscope.

The GSH concentrations were measured in oocytes matured in vitro under four IVM culture conditions, according to the GSH and GSSG Assay kit. The oocytes (30 oocytes per group) were carefully denuded by repeated pipetting in 0.1% (w/v) hyaluronidase (Sigma-Aldrich) and washed several times in polyvinyl alcohol-PBS to remove any trace of materials with thiol groups. The oocytes were stored in a microtube at −20°C until they were assayed (30 oocytes per tube). All procedures complied with the manufacturer's protocol. Briefly, GSH was assayed using 5,5-dithio-bis (2-nitrobenzoic) acid (DTNB)-GSSG reductase recycling. The GSSG was measured by measuring the 5-thio-2-nitrobenzoic acid (TNB) produced from the reaction of reduced GSH with DTNB. The rate of TNB formation was measured at 412 nm using a Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific, Inc.), over 3 min. The reduced GSH concentration in the sample was calculated by subtracting the GSSG from GSH.

Caspase-3 activity was analyzed in the control and experimental oocytes using a Caspase-3 Activity kit, with Ac-DEVD-pNA as the colorimetrically-specific substrate. Briefly, 30 oocytes from each group were washed twice in PBS and then lysed in 100 µl of chilled lysis buffer (Gibco) at 4°C for 10 min. The lysate was centrifuged at 20,000 × g at 4°C for 10 min, and the supernatant was incubated for 7 h at 37°C with 10 µl of caspase-3 substrate. Substrate cleavage was measured using an Hitachi Fluorescence Spectrophotometer F-7000 (Hitachi, Ltd., Tokyo, Japan) at 405 nm and was corrected to the protein content in the lysate. The caspase-3 activity is presented as the fold increase in optical density/30 oocytes.

Data are expressed as the mean ± standard error of the mean of triplicate samples. All percentages were subjected to arcsine square root transformation prior to statistical analysis. The data were analyzed using either Student's t-test or one-way analysis of variance followed by a multiple t-test. All statistical analysis was performed using SPSS (version 17.0; SPSS, Inc. Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

H₂O₂ induces ROS generation, causing oxidative stress. CC is usually used to improve GSH synthesis, as GSH protects cells against the destructive effects of ROS. To determine the optimal concentrations of H₂O₂ and CC, the present study evaluated goat oocyte maturation rate and parthenogenesis blastocyst formation rate. The oocytes treated with 100 µM H₂O₂ exhibited significantly decreased maturation rates and blastocyst rates, compared with the control group (52.67±1.81, vs. 71.78±2.06 and 11.98±1.27, vs. 20.29±2.06, respectively; P<0.05; Fig. 1). However, oocytes treated with 120 µM H₂O₂ exhibited marked decrease in oocyte maturation rates and blastocyst formation rates (19.34±1.43 and 2.49±1.36, respectively), and the high concentration of H₂O₂ resulted in a low survival rate of oocytes and thus, the remaining surviving oocytes were insubstantial for the completion of subsequent experiments. Thus, 100 µM H₂O₂ was considered the optimal concentration for oxidative stress. In addition, when 200 µM cysteine and 200 µM cystine were added to the IVM media, the oocyte maturation rate and blastocyst formation rate were higher, compared with those the control group (81.57±2.86, vs. 71.41±1.11% and 25.04±1.34, vs. 19.80±2.66%, respectively; P<0.05).

Culturing the goat oocytes in M199 with H₂O₂ decreased the oocyte maturation rate and blastocyst formation rate, compared with the control group (49.47±2.65% vs. 65.71±2.74 and 13.47±1.11 vs. 20.57±0.61% respectively; P<0.05; Table II). The oocyte maturation and blastocyst formation rates in the CC+H₂O₂ group were higher, compared with those in the H₂O₂ group (69.96±3.51, vs. 49.47±2.65% and 19.19±0.53, vs. 13.47±1.11%, respectively; P<0.05). The oocyte maturation rate and the blastocyst rate in the CC group were also higher, compared with those in the H₂O₂ group (78.36±2.83, vs. 49.47±2.65% and 23.06±0.59, vs. 13.47±1.11%, respectively; P<0.05). Furthermore, the mean number of cells per blastocyst in the H₂O₂ group was significantly lower, compared with those in the other three groups (P<0.05), although the embryonic cleavage rates did not differ among the four groups (P>0.05).

A TUNEL assay was used to evaluate the quality and viability of the goat oocytes cultured in M199 with stress inducers. The goat oocytes treated with H₂O₂ exhibited significantly increased DNA fragmentation, compared with the control group (121±1.89 cells/COC, vs. 7.8±1.32 cells/COC, respectively; P<0.05), as shown in Figs. 2 and 3A). The H₂O₂ media containing CC further reduced the number of apoptotic cells (36±1.13 cells/COC), similar with the CC group (25±2.10 cells/COC). These findings indicated that oxidative stress induced apoptosis during goat oocyte IVM.

The mean intracellular GSH concentrations in the MII oocytes treated with H₂O₂ were significantly lower, compared with those in the control groups (0.31±0.18, vs. 3.18±0.38 pmol/oocyte; P<0.05; Fig. 3B). The addition of CC to the media significantly increased the GSH concentrations, compared with the control and H₂O₂ groups (7.53±0.41 vs. 0.31±0.18 and 3.18±0.38 pmol/oocyte, respectively; P<0.05). In addition, the GSH concentration of the MII oocytes in the CC group was higher, compared with those of the control and H₂O₂ groups (8.47±0.46, vs. 0.31±0.18 and 3.18±0.38 pmol/oocyte, respectively; P<0.05).

To confirm whether caspase-3 is involved in the IVM of goat oocytes in H₂O₂-induced oxidative media, caspase-3 activity was measured using its specific substrate, Ac-DEVD-MCA. The activity of caspase-3 in the MII oocytes with H₂O₂ was 86.4±5.71 pmol/oocyte, which was significantly higher than the caspase-3 activities of the oocytes in the CC group and the H₂O₂+CC group (64.3±4.54 and 15±2.46 pmol/oocyte, respectively; P<0.05; Fig. 3C).

The expression levels of selected genes were determined in MII oocytes during IVM, following treatment with H₂O₂ and. or CC. The transcription levels were highly variable in all treatment groups. A comparable pattern of mRNA expression was observed for all transcripts analyzed, although certain differences were noted (Fig. 4). The expression levels of PGC-1α and NRF-1 were significantly higher in the H₂O₂ group, compared with the control group (P<0.05). Adding CC to the oxidative media significantly downregulated the expression of PGC-1α and the expression of NRF-1 (P<0.05). The gene transcription levels of BAX were significantly lower in the H₂O₂ group, compared with the control group (P<0.05). The gene expression of BCL-2 in the oocytes of the CC group was upregulated, compared with the other three groups (P<0.05). Furthermore, the BAX:BCL-2 ratio in the H₂O₂ group was higher, compared with the other three groups (P<0.05).