Preliminary Study On The In Vitro Maturation And Apoptosis Of Buffalo Oocytes

To improve the in vitro maturation (IVM) system for buffalo oocytes, factors affecting the IVM, cortical granules (CGs) distribution during IVM and in vitro fertilization (IVF), and the apoposis of oocyte and granulose cells were investigated in this study.Effects of EGF and IGF-I on the nuclear and cytoplasmic maturation of buffalo oocytes was investigated according to the proportion of oocytes extruding PB1 and the monolayer distribution of CGs beneath the ovum membrane. There were no significant difference in the maturation rate among control group and the groups with EGF 10 20 30ng/mL, but addition of 50 ng/mL EGF to the maturation medium resulted in significantly more oocytes extruded PB1 in comparison with other groups (55.4% vs 32.3%, P<0.05). The distribution pattern of CGs changed gradually from middle to cortex as the concentration of EGF increased to 50 ng/mL, and then changed gradually from cortex to middle as the concentration of EGF increased to 100 ng/mL; There were no significant difference in the maturation rate of oocytes between control group and IGF-I 10 ng/mL group, but the maturation of buffalo oocyte was improved significantly by addition of 30 ng/mL IGF-I to the maturation medium (P<0.05). The distribution pattern of CGs changed gradually from middle to cortex as the concentration of IGF-I increased to 30 ng/mL, and then changed gradually from cortex to middle as the concentration of IGF-I increased to either 50 ng/mL or 100 ng/mL; Significant more oocytes matured when they were cultured in the maturation medium containing 20 ng/mL EGF and 30 ng/mL IGF-I in comparison with them cultured in either 30 ng/mL IGF-I or 20 ng/mL EGF group (P<0.05). These results indicate that EGF and IGF- I in appropriate concentration can improve IVM of buffalo oocyte in a cooperative manner.Changes of buffalo oocyte CGs during IVM and IVF were investigated by laser scanning microscope (LSM) with FITC-LCA label. The CGs was located in the central of plasma in 70.1% of oocytes before IVM, and CGs was moved to the cortex in 22.1% of oocytes after IVM of 16 h. Finally, CGs in 59.8% of oocytes was located in cortex after IVM of 24 h, the distribution pattern of CGs changed gradually from cluster to dispersion. When buffalo oocytes matured in different time of IVM were used for IVF, 12.9% of oocytes matured for 12h were exocytosis of CGs at 3 h after IVF, 48.5 % of oocytes matured for 20h released CGs at 1.5 h' after IVF, 97.1% andl00% of oocytes matured for 24 h were CGs exocytosis at 1.5 and 3 h after IVF. Meanwhile the CGs distribution of buffalo oocytes was also detected after chemical activation. Chemical activation can induce the exocytosis of CGs. There were only 10.0% of oocytes matured for 12 h with CGs exocytosis at 3 h after activation. No oocytes matured for 20 h had CGs exocytosis in 0.5 h after activation, and only 33.3% oocytes had CGs exocytosis at 1.5 h after activation, which was less than IVF significantly. At 1.5 h after activation, 96.7% of oocytes matured for 24 h had CGs exocytosis. These results indicated that the distribution pattern of CGs changes gradually from middle to cortex as the maturation time increased, the speed of CGs exocytosis after IVF and chemical activation is related to the maturity of oocytes, and the speed of CGs exocytosis in chemical activation is slower than in IVF.Effects of EGF and IGF-I on the apoptosis of buffalo oocytes during IVM were investigated in this study. Addition of EGF at the concentration tested (10, 20, 30, 50, 100 ng/mL) resulted in a decrease in the proportion of buffalo oocytes with apoptosis. The apoptosis (10.9%) of buffalo oocyte was decreased significantly by addition of 20 ng/mL EGF (P<0.05). The apoptosis was decreased in the maturation as the concentration of EGF increased, and the apoptosis (6.2%) in 50 ng/mL EGF group was lower significantly than other groups(control and 10ng/mL). Addition of 10, 30, 50 and 100 ng/mL IGF-I to the maturation medium resulted in a significantly decrease in the proportion of oocytes with apoptosis (8.2%, 7.6%, 10.6%, 15.4% vs 29.4%, P<0.05). Significantly less oocytes apoptosis when they were cultured in the maturation medium containing 20 ng/mL EGF and 30 ng/mL IGF-I in comparison with them cultured in either 30 ng/mL IGF- I or 20 ng/mL EGF group (P<0.05). Thematuration rate of oocytes was 7.8%, 50.9% and 61.4% respectively at 12, 20, 24h of FVM in the mature medium containing 20 ng/mL EGF+30 ng/mL IGF- I, and the apoptosis rate was 1.7%, 4.4% and 4.7% respectively. The maturity of buffalo oocyte was improved significantly (P<0.05), but the rate of apoptosis was not significantly (P>0.05) different. These results indicate that EGF and IGF- I in an appropriate concentration can decrease the apoptosis of buffalo oocytes.The relationship between the developmental ability of buffalo oocytes and the apoptosis of granulosa cells was investigated in this study. The apoptosis rate of oocytes increased as the maturation of oocytes progressed. As the scoring and developmental capacity of the oocyte decreased, the apoptosis rate of granulosa cells was increased. These results indicate that there is closely relationship between the developmental capacity of the oocyte and apoptosis of granulosa cells. The apoptosis rate of granulose cells can be used as an index for predicting the developmental potential of oocytes.