MAPK/PKC信号通路在原癌基因c-erbB_2调控原始卵泡启动生长中的作用

Role of MAPK and PKC signaling pathways in the regulation of c-erbB_2 in the primordial follicles onset

本研究旨在探讨原癌基因c-erbB2在原始卵泡启动生长中的调控作用,及其在信号通路中与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和蛋白激酶C(protein kinaseC,PKC)的上下游关系。实验通过脂质体介导c-erbB2siRNA转染体外培养的卵巢,用RT-PCR法检测原癌基因c-erbB2 mRNA的表达,用Western blot检测ErbB2、MAPK和PKC蛋白的表达;用MAPK及PKC通路特异性抑制剂PD98059和Calphostin加入培养液中,培养卵巢8d后检测c-erbB2 mRNA及ErbB2蛋白的表达;HE染色进行卵泡启动生长组织学观察。结果显示,c-erbB2siRNA转染卵巢后,c-erbB2 mRNA水平明显下调(P<0.01),ErbB2、MAPK和PKC蛋白的表达也明显下调(P<0.01);而在培养液中加入特异性抑制剂PD98059和Calphostin,卵巢c-erbB2 mRNA的表达和ErbB2蛋白表达均无明显变化(P>0.05);与对照组比较,培养8d后siRNA组初级卵泡和次级卵泡数明显减少(P<0.01),抑制剂组原始卵泡数增多(P<0.01),次级卵泡数明显减少(P<0.05或P<0.01)。以上结果提示,c-erbB2可能是调控原始卵泡启动生长中的蛋白激酶MAPK和PKC的上游激活物。

Our previous studies showed that the proto-oncogene c-erbB2 played an important role in primordial follicles growth. The present study was conducted to investigate the role of MAPK and PKC signaling pathways in the primordial follicle onset in neonatal rats, and the relationship between c-erbB2 and MAPK/PKC signaling pathways. Ovaries collected from 2-day-old Sprague-Dawley rats were cultured in the Waymouth culture system in vitro. Ovaries were transfected with c-erbB2 siRNA, or treated with PD98059 （50 μmol/L） or Calphostin （0.5 μmol/L） in the culture medium. RT-PCR was performed to measure the expression of c-erbB2 mRNA, and Western blot analysis was performed to measure the expression of ErbB2, MAPK and PKC protein after the neonatal rat ovaries were cultured for 8 d. The quantities of every-stage follicles of ovaries cultured for 8 d were obtained in histological section stained with hematoxylin eosin. The results showed that c-erbB2 siRNA reduced the levels of c-erbB2 mRNA （P〈0.01） and the levels of ErbB2, MAPK and PKC protein （P〈0.01） significantly. But the levels of c-erbB2 mRNA and ErbB2 protein exhibited no change （P〉0.05） in the ovaries cultured with PD98059 or Calphostin. After the ovaries were transfected with c-erbB2 siRNA or cultured with PD98059 or Calphostin for 8 d, the quantities of primary follicles and second follicles were lower than those in the control group （P〈0.05 or P〈 0.01）, but the quantity of the primordial follicles was higher than that in the control group （P〈0.01）. These results suggest that proto-oncogene c-erbB2 promotes the initiation of primordial follicle growth through the MAPK and PKC signal transduction, and c-erbB2 is possibly the upstream of PKC and MAPK signaling pathway in the regulation of primordial follicle onset.