Effects Of E₂ And FSH On The Proliferation And Apoptosis Of Granulosa/Theca Cells In Buffalo Follicles

The proliferation of follicular granulosa cells(GCs)and theca cells(TCs),which is closely related to follicular development,mainly relies on the combined action of estradiol(E2)and follicle-stimulating hormone(FSH).Due to the low reproduction rate of buffalo,the outcome of superovulation is still unsatisfactory.In order to improve the outcome of superovulation,the effects of E2 and FSH on the proliferation and apoptosis of GCs/TCs in buffalo follicles were investigated in this study.Theses works will assist us to improvethe technique of buffalo superovulation.The results are summarnzed in following.1.Effect of FSH treatment on the proliferation of buffalo follicular GCs/TCs.Different concentrations of FSH(0,2,8,14,20,26 ?g/mL)was used to treat buffalo follicular GCs/TCs for 24 h,respectively and then the proliferation rate of cell was detected.The results showed that the proliferation rate in the 14,20,26 ?g/mL FSH treated groups were significantly higher than that in the control,2,8 ?g/mL treated groups(p<0.05).Meanwhile,there was no significant difference among the 14,20,26 ?g/mL FSH treated groups(p>0.05).2.Effect of E2 and FSH treatment on the proliferation of buffalo follicular GCs/TCs.Different concentrations of E2(0,0.5,1,2,4 ng/mL)combined with FSH(14 ?g/mL)were used to treat buffalo follicular GCs/TCs for 24 h,respectively.The 0 ng/mL E2 and 0 ?g/mL FSH were set as the control group.The results showed that the proliferation rate of GCs in 1 ng/mL E2 combined with 14?g/mL FSH treated group was significantly higher than that in the control group,14 ?g/mL FSH treated group as well as 0.5 ng/mL E2 combined with 14 ?g/mL FSH treated group(p<0.05).Meanwhile,the proliferation rate of TCs in 1 ng/mL E2 combined with 14 ?g/mL FSH treated group was significantly higher than other groups(p<0.05).Therefore,0 ng/mL E2 and 0 ?g/mL FSH were set as control,the buffalo follicular GCs/TCs were treated with only 14 ?g/mL FSH or 1 ng/mL E2 combined with14 ?g/mL FSH in the following experiments.3.Effect of E2 and FSH treatment on apoptosis of buffalo follicular GCs/TCs.After 24 hours of treatment,the apoptosis rate of cells was detected.The results showed that the mortality and early apoptosis rate in the E2 combined with FSH treated group was significantly lower than the other two groups(p<0.05).Meanwhile,the survival rate were significantly higher compared to the other two groups(p<0.05).4.Effect of E2 and FSH treatment on the expression pattern of receptor genes in buffalo follicular GCs/TCs.After 24 hours of treatment,the mRNA expression pattern of receptor genes in buffalo follicular GCs/TCs were analysed.The results showed that the expression levels of FSHR and LHR in the E2 combined with FSH treated group were significantly higher than that of the other two groups.The expression level of ER-? in the E2 combined with FSH treated group was significantly lower than the control group in GCs(p<0.05),while there was no significant difference between the other two groups(p>0.05).Meanwhile,the expression level of ER-?,in the E2 combined with FSH treated group was significantly lower than the other two groups in TCs(p<0.05).5.Effect of E2 and FSH on the expression pattern of proliferation related genes in buffalo follicular GCs/TCs.After 24 hours of treatment,the expression pattern of proliferation related genes in buffalo follicular GCs/TCs were also analysed.The results showed that the expression levels of PCNA,Cyclin D1 and Cyclin D2 in the E2 combined with FSH treated group were significantly higher than that in the other two groups(p<0.05).6.Effect of E2 and FSH treatment on the expression pattern of apoptosis related genes in buffalo follicular GCs/TCs.After 24 hours of treatment,the expression pattern of apoptosis related genes in buffalo follicular GCs/TCs were analysed.The results showed that both in buffalo follicular GCs/TCs,the expression level of Bcl-2 in the E2 combined with FSH treated group was significantly higher than the other two groups.At the same time,the expression levels of Bax,Caspase-3 and Caspase-9 in the E2 combined with FSH treated group were significantly lower compared to the control group(p<0.05).The above results indicated that the proliferation can be promoted,and the apoptosis can be decreased after the treatment of 14 ?g/mL FSH or lng/mLE2 combined with 14 ?g/mL FSH in GCs/TCs.Moreover,the combined action of E2 and FSH is better than FSH treatment.The E2 combined with FSH treatment can improve the response ability of FSH and LH in buffalo follicular GCs/TCs by increasing the expression level of hormone receptors genes like FSHR and LHR,decreasing the expression of ER-?.Therefore,the expression of proliferation genes like PCNA,Cyclin D1,Cyclin D2,and apoptosis inhibiting gene Bcl-2 are up-regualted.Meanwhilethe expression of genes such as Bax,Caspase-3,Caspase-9 are down-regulated.Ultimately the proliferation is promoted and the apoptosis is inhibited in buffalo follicular GCs/TCs.In summary,the efficacy of buffalo superovulation may hopefully improve by the combined injection with proper concentration of E2 and FSH injection.