Studies On The In Vitro Maturation And Meiosis Regulation Of Buffalo Oocytes

1.The role of protein kinase C(PKC)in the buffalo oocyte maturation was investigated.Addition of PMA(a PKC activator)to the maturation medium inhibited the spontaneous maturation of buffalo oocytes and this inhibitory action could not be overcome by addition of FSH.Addition of Calphostin C(a PKC inhibitor)to the maturation medium stimulated meiotic resumption of buffalo oocytes and also overcame the meiosis inhibitory action of milrinone(a phosphodiesterase inhibitor),The decrease of PKC activity at Metaphase I may be essential in progressing from MⅠto anaphase and MⅡof buffalo oocytes.2.Dynamic change of the expression of PKC gene and its relation to the maturation of buffalo ooctyes were expored.Buffalo oocytes were collected at 0,4,8,12,16,20 and 24h of in vitro maturation,and the relative level of PKC mRNA transcript in buffalo oocytes at the different maturation stages was determined using the real-time PCR.The expression level of PKC gene increased with GVBD during the first 12 h of in vitro maturation,and then decreased with the extrusion of PBI during the last 12 h of in vitro maturation, indicating that PKC may play a vital role during buffalo oocyte maturation.3.Effects of PKC activation during the maturation of buffalo oocytes on their subsequent fertilization and embryonic development were examined.Buffalo oocytes were randomly allocated into five groups and matured in TCM-199 supplemented with 1 mg/ml PVA(negative control);5%fetal calf serum (positive control);1.62×10^-6mol/L PMA and 1mg/ml PVA(treatment); 1.62×10^-6mol/L 4a-PDD and 1mg/ml PVA(PMA control)for 24h.The PKC protein of buffalo oocytes at the end of maturation was determined by SDS-PAGE and Western blotting.The PKC protein was detected in the oocytes matured in the medium without PMA but not in the oocytes matured in the medium with PMA,indicating that long treatment with PMA may destroy the PKC protein stored in buffalo oocytes.Addition of PMA to the maturation medium resulted in more oocytes forming two pronuclei and less oocytes cleaved after in vitro fertilization.Addition of Calphostin C at lower concentration(less than 10^-7mol/L)during the maturation of 12 to 24 h did not affect the cleavage rate of oocytes but decreased the cleavage rate at high concentration(beyond 1.26×10^-6mol/L).4.Effects of milrinone on the in vitro maturation of buffalo oocytes were investigated.Nuclear and cytoplasmic maturation of buffalo oocytes were evalulated according to the proportion of oocytes appearing GVBD and embyonic development after in vitro fertilization.The spontaneous maturation of buffalo oocytes could be inhibited by milrinone at a dose-dependent manner and this inhibitory action was relative stable for a certain period and can be overcome partly by addition of FSH at late stage of in vitro maturation.The maturation inhibitory action of milrinone was reversible,as buffalo oocytes can resume and complete their final maturation when milrinone was removed from the maturation medium.