The Effects Of Roscovitine On Granulosa Cell Culture And Oocyte In Vitro Maturation Of The Yak

Granulosa cells(GC)are inherent to the process of oocyte maturation.The decrease in the developmental competence of in vitro-matured(IVM)oocytes may be due to asynchronous nuclear and cytoplasmic maturation,leading to insufficient cytoplasmic maturity.Roscovitine is a selective inhibitor of the cyclin-dependent kinases and an oocyte meiosis inhibitor,which could synchronize nuclear and cytoplasmic maturation and in hence improve matured oocyte quality.Therefore,the objective of this study were to assess the effects of roscovitine on GC function and oocyte IVM of the yak and their regulatory mechanisms.1.The effects of roscovitine on yak GC cultureYak GCs were cultured in culture medium containing different concentrations of roscovitine(0,6.25,12.5,25,50 and 100 μM)for different time(3,6 and 9 h),and then cultured in culture medium without roscovitine.GC proliferation,cells cycle,progesterone(P4)secretion,and the expression of genes related to cell proliferation,apoptosis and hormone synthesis were detected.The results showed:(1)The GC proliferation in 6 h+12.5 μM group was significantly higher than that of the other combinations.Therefore,6 h+12.5 μM was used as pre-treatment for treatment in the subsequent experiments.(2)The proliferation in the treatment was significantly higher than in the control at 24 hand 48 h(P<0.01).(3)At 24 h of culture,P4 concentration in treatment was significantly lower than that in control(P<0.05).(4)After roscovitine pre-treatment,the percentage of GC at G0/G1 stage were significantly higher in treatment than those in the control(P<0.05),but after further cultured for 24 h in medium without roscovitine,the cells at G0/G1 stage were significantly less than those in the control(P<0.05).(5)q PCR results showed that the BCL-2/BAX ratio was not significant difference between treatment and control(P>0.05)at the end of pretreatment,but the BCL-2 /BAX ratio in treatment was significantly higher than that in control(P<0.05)at 24 h in medium without roscovitine.(6)The relative expression levels of HSD3B1,STAR,CDK5 and PCNA in treatment were significantly lower than those in control(P<0.01)at the end of treatment,but their expression levels in the treatment were significantly higher(P<0.01)at 24 h in medium without roscovitine.The relative expression of P53 in treatment was significantly higher than that in control(P<0.01)at 6 h,but there was no significant difference at 24 h between the two groups(P>0.05).2.The effects of roscovitine on in vitro maturation of yak oocytesYak cumulus-oocyte complexes(COCs)were culture in IVM medium containing different concentrations of roscovitine(0,6.25,12.5,25,50 μM)for 6 h,and then cultured in IVM medium without roscovitine for 18 h.The expansion of cumulus cells(CEI),and reactive oxygen species(ROS),mitochondria,transzonal projections(TZPs),apoptosis and developing-related genes of oocytes were determined.The results showed that CEI was high in 12.5 μM roscovitine group,and this was used as the treatment concentration for the subsequent research.Compared with control,ROS content of oocytes in treatment was significantly decreased(P<0.05),and the percentage of oocytes with homogeneous mitochondrial distribution in treatment was significantly higher(P<0.05).The fluorescence intensity of TZPs of yak oocytes in treatment was significantly higher than that in control(P< 0.05).The expression levels of SOD2,BCL-2 /BAX,GDF9 and EGFR in treatment were significantly higher than those in control(P<0.01).In conclusion,pre-treatment with 12.5 μM roscovitine for 6 h followed by conventional cell culture could improve yak GC proliferation and function.Pretreatment with 12.5 μM roscovitine for 6 h followed by conventional IVM could improve yak oocyte quality after IVM.