Possible Roles Of Proto-oncogene C-erbB₂ During The Initiation Growth Of Rat Primordial Follicles

ObjectiveTo establish the culture system of rat primordial follicles in vitro and study the possible roles of proto-oncogene c-erbB2 during the initiation growth of primordial follicles.Methods(1) Ovaries were collected from 2-day-old SD rats and cultured in the Waymouth and the DMEM culture systems; the morphous and the quantity of every stage follicles of ovaries cultured for 0d,4d and 8d were observed by histological sections stained with hematoxylin; the expressions of PCNA in the ovaries were examined by immunohistochemistry and western blot.(2) 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.(3) Western blot was used to observe the p-ERK1/2 contents during the initiation growth of primordial follicles 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(1) Compared with the DMEM culture system according to the results of the morphous and the quantity of every stage follicles of ovaries, the expressions of PCNA in the ovaries,the Waymouth culture system is a more ideal model to study the initiation growth of primordial follicles. 50ng/ml EGF could further promote the initiation growth of primordial follicles in the Waymouth culture system.(2) In-situ hybridization showed c-erbB2 mRNA existed in the oocytes endochylema, the expressions of c-erbB2 mRNA appeared to be more intense when primordial follicles were cultured for 8d than cultured for 0d in the Waymouth culture system and were further increased with 50ng/ml EGF for 4d and 8d. The same results were observed by RT-PCR,too. Immunohistochemistry showed ErbB2 existed in the oocytes membrane, the expressions of ErbB2 appeared to be more intense when primordial follicles were cultured for 8d than cultured for 0d in the Waymouth culture system and were further increased with 50ng/ml EGF for 4d and 8d.(3) Western blot analysis showed that p-ERK1/2 protein levels were increased when primordial follicles were cultured for 4d and 8d or supplyment with 50ng/ml EGF. These changes 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,P<0.05).Conclusion(1) The culture system of primordial follicles in vitro was successfully established in this study.(2) C-erbB2 mRNA and protein existed in the primordial follicles, the expressions increased as the growth of the primordial follicles and after the effect of EGF, it was suggested proto-oncogene c-erbB2 maybe play an important role during the initiation growth of primordial follicles.(3) There was a significant positive correlation relationship between the contents of p-ERK1/2 and the expressions of c-erbB2 mRNA during the primordial follicles growth, and it was indirectly suggested that c-erbB2 promotes the development of the primordial follicles via ERK-MAPK signal transduction.