目的:探讨原癌基因c-erbB2在原始卵泡启动生长中表达变化及可能的作用。方法:选用2日龄SD大鼠卵巢在Waymouth培养体系中进行体外培养,用原位杂交、RT-PCR和免疫组化方法检测c-erbB2mRNA和蛋白在原始卵泡启动生长中及在表皮生长因子(EGF)作用下的表达情况,用Westernblot方法同步测定卵泡活化生长的重要标志物——增殖细胞核抗原(PCNA)和磷酸化细胞外信号调节激酶1/2(p-ERK1/2)的表达情况,并分析p-ERK1/2与c-erbB2mRNA表达变化的相关关系。结果:伴随原始卵泡启动生长过程,PCNA表达逐渐增加,EGF能促进原始卵泡的增殖和分化;原始卵泡中有c-erbB2mRNA及蛋白的表达,且随原始卵泡的启动生长及在EGF作用下表达增强;RT-PCR结果显示,c-erbB2mRNA表达在2日龄大鼠卵巢培养8d后与培养0d相比显著增加(0.297±0.018vs0.178±0.011,P〈0.05),并在EGF作用下进一步增强;p-ERK1/2含量的变化与c-erbB2mRNA表达的变化呈显著的正相关关系(rs=0.900,P〈0.05)。结论:c-erbB2在原始卵泡启动生长中起重要促进作用,并为介导EGF促进原始卵泡启动生长的关键信号分子;ERK-MAPK信号通路可能在介导c-erbB2调控原始卵泡生长中起作用。
Objective:To study the expression and possible roles of proto-oncogene c-erbB2 during the initiation growth of primordial follicles.Methods:Ovaries were collected from 2-day-old SD rats and cultured in the Waymouth culture system.In-situ hybridization,RT-PCR and immunohistochemistry were performed to assess the expressions of c-erbB2 mRNA and protein during the initiation growth of primordial follicles and after the effect of EGF.Western blot was used to observe the PCNA,p-ERK1/2 contents and correlation analysis was used to study the correlation relationship between contents of p-ERK1/2 and expressions of c-erbB2 mRNA at the same time of the primordial follicles growth.Results:PCNA protein levels appeared to be more intense during the initiation growth of primordial follicles,EGF could promote the proliferation and differentiation of the primordial follicles.c-erbB2 mRNA existed in the oocytes endochylema and ErbB2 existed in the oocytes membran,the expressions of c-erbB2 mRNA and ErbB2 appeared to be more intense when primordial follicles were cultured for 8 d than cultured for 0 d in the Waymouth culture system and were further increased with 50 ng/ml EGF for 4 d and 8 d.The same results were observed by RT-PCR,too.p-ERK1/2 protein levels were consistent with the changes of c-erbB2 mRNA and protein.Furthermore,Spearman rank correlation analysis showed there was a significant positive correlation relationship between the changes of p-ERK1/2 and the changes of c-erbB2 mRNA during the primordial follicles growth and after the effect of EGF(rs=0.900,P0.05).Conclusion:It was suggested that proto-oncogene c-erbB2 may be play an important role during the initiation growth of primordial follicles with EGF,and it is indirectly suggested that c-erbB2 promotes the development of the primordial follicles via ERK-MAPK signal transduction.