The Study On Relativity Between CTSB、CTSS Eene Expression, Apoptosis Of Sheep CCs And Developmental Capacity Of Oocytes

This research was conducted to study the cathepsin（CTSB、CTSS）transcriptionabundance through the qRT-PCR technology and detect cell apoptosis by the method ofTunel staining combined with flow cytometry in cumulus cells from adult sheep and lambduring in vitro maturation(IVM). After maturation of COCs with or without E-64（Cathepsin specific inhibitor）, we investigated the relativity among cathepsin (CTSB、CTSS) mRNA, cell apoptosis and developmental capacity of oocytes from sheep, theresults are as followed:Adding different concentrations of E-64(0μmol/L,1μmol/L,5μmol/L,10μmol/L) toin vitro maturation liquid which used to culture COCs of Adult sheep and lambs. Weobserved cathepsin transcription abundance respectively at0h,8h,16h,24h. The resultshowed that transcription (mRNA)of CTSB,CTSS expression both showed a trend ofgradual decline by the time longer;The expression of CTSB,CTSS in differentconcentrations(1μmol/L,5μmol/L,10μmol/L) E-64group lower than the control group atdifferent level, the5μmol/L,10μmol/L concentration have a good effect, while there was nosignificant difference in both two groups (P>0.05).Adding different concentrations of E-64(0μmol/L,1μmol/L,5μmol/L,10μmol/L) toin vitro maturation liquid which used to culture COCs. We observed Apoptosis at0h,8h,16h,24h in adult sheep CCs; at0h,24h in lamb CCs. It turned out that there was negative correlation betweeen accumulated cell apoptosis rates and in vitro maturation time, the new cell apoptosis rate gradually increased by in vitro maturation time longer in adult sheep,the highest apoptosis rates appeared at16h,then gradually reduced. The addition of E-64can reduced apoptosis rates in CCs and with adose-dependent manner. The concentration of5μmol/L,10μmol/L have a better inhibitory effect than0μmol/L,1μmol/L (P<0.05), while there was no significant differences between5μmol/L and10μmol/L groups (P>0.05).Comparing the CTSB, CTSS genes expression and apoptotic status between lamb andadult sheep CCs, results indicate that The cumulus cells CTSB、CTSS transcriptionabundance were all extremLy significant higher than that of adult sheep（p<0.01）， thecontrol group showed that lamb cell apoptosis rate is significantly higher than adultsheep(P<0.05),5μmol/L and10μmol/L E-64group showed extremLy significant higherthan that of adult sheep (P<0.01). We cultivate Adult sheep COCs in nutrient solution with0μmol/L,1μmol/L5μmol/L,10μmol/L E-64.After24h, COCs were fertilized. Results showed that oocytematuration rate, cleavage rate had no significant difference in the four concentrations ofE-64groups (P>0.05),but5μmol/L,10μmol/L group blastocyst rate is significantly higherthan that of control group (P <0.01), and there were no significant difference between them(P>0.05).