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Possible roles of proto-oncogene c-src on the initiation growth of rat primordial follicles

【摘要】:正Objective:Primordial follicle initiation and growth are the vital center of animal reproduction,but the mechanisms are unknown.The aim of this article is to explore the expression,distribution of c-src mRNA and protein in the ovaries as primordial follicle development and the role and mechanisms of c-src on the growth of primordial follicle.Methods:Ovaries were collected from 2-day-old rats,and cultured in Waymouth culture system; the morphous and the quantity of every stage follicles of ovaries cultured for 0 d,4 d and 8 d were observed by histological sections stained with hematoxylin.In situ hybridization,RT-PCR,western blot and immunohistochemistry were performed to assess the expressions of c-src mRNA and protein during the initiation growth of primordial follicles.RNA interference was used to observe the effect of proto-oncogene c-src on the initiation of primordial follicles;and inhibitors such as MAPK inhibitor(PD98059),PKC inhibitor(calphostin),PI-3K inhibitor(LY294002) were used to investigate the upstream or downstream relationship with c-src in the initiation of primordial follicle growth.Results:Immunohistochemical technique,insitu hybridization and RT-PCR showed that c-src mRNA and protein existed in primordial follicles,their expression levels increased as the primordial follicles growth,c-src mRNA and proteins distributed in oocyte cytoplasm and granulosa cells.RNA interfering showed that the development of primordial follicles were remarkablely suppressed after c-src siRNA transfection and corresponding,c-src mRNA and protein,the levels of p-PKC,p-ERK1/2 and p-PI3K in follicles were significantly decreased.The initiation of primordial follicles delayed obviously,but c-src mRNA and protein showed no significant change after PD98059,LY294002 and calphostin effect;p-ERK1/2 level in follicles decreased after PD98059,calphostin and LY294002 effect;p-PKC level in follicles decreased after calphostin and LY294002 effect but no change in PD98059; p-PI3K level in follicles decreased after LY294002 effect,but no change in PD98059 and calphostin group. Conclusion:The culture system of primordial follicles in vitro was successfully established in this study.c-src mRNA and protein existed in the primordial follicles and the expressions increased as the growth of the primordial follicles,c-src pay important role in regulating primordial follicle initiation and growth through p-PKC,p-ERK1/2 and p-PI3K signaling pathway and the upstream and downstream relationship among them may be c-src→p-PI3K→p-PKC→p-ERK1/2.

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