Abstract
BackgroundC-src is an evolutionarily conserved proto-oncogene that regulates cell proliferation, differentiation and apoptosis. In our previous studies, we have reported that another proto-oncogene, c-erbB2, plays an important role in primordial follicle activation and development. We also found that c-src was expressed in mammalian ovaries, but its functions in primordial follicle activation remain unclear. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles.MethodsOvaries from 2-day-old rats were cultured in vitro for 8 days. Three c-src-targeting and one negative control siRNA were designed and used in the present study. PCR, Western blotting and primordial follicle development were assessed for the silencing efficiency of the lentivirus c-src siRNA and its effect on primordial follicle onset. The expression of c-src mRNA and protein in primordial follicle growth were examined using the PCR method and immunohistochemical staining. Furthermore, the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 were used to explore the possible signaling pathways of c-src in primordial folliculogenesis.ResultsThe results showed that Src protein was distributed in the ooplasmic membrane and the granulosa cell membrane in the primordial follicles, and c-src expression level increased with the growth of primordial follicle. The c-src -targeting lentivirus siRNAs had a silencing effect on c-src mRNA and protein expression. Eight days after transfection of rat ovaries with c-src siRNA, the GFP fluorescence in frozen ovarian sections was clearly discernible under a fluorescence microscope, and its relative expression level was 5-fold higher than that in the control group. Furthermore, the c-src-targeting lentivirus siRNAs lowered its relative expression level 1.96 times. We also found that the development of cultured primordial follicles was completely arrested after c-src siRNA knockdown of c-src expression. Furthermore, our studies demonstrated that folliculogenesis onset was inhibited by Calphostin, PD98059 or LY294002 treatment,but none of them down-regulated c-src expression. In contrast, the expression levels of p-PKC, p-ERK1/2 and p-PI3K in the follicles were clearly decreased by c-src siRNA transfection. Correspondingly, both Calphostin and LY294002 treatment resulted in a decrease in the p-PKC level in follicles, but no change was observed in the PD98059 group. Finally, LY294002 treatment decreased the p-PI3K expression level in the follicles, but no changes were observed in the PD98059 and Calphostin groups.ConclusionsC-src plays an important role in regulating primordial follicle activation and growth via the PI3K-PKC- ERK1/2 pathway.
Highlights
Oocytes are surrounded by somatic cells in the ovaries of newborn mammals
We have showed that the mRNA of another Proto-oncogene, c-erbB2, is expressed in the primordial follicle, and ablation of c-erbB2 in neonatal rat ovaries results in excessive inhibition of primordial follicles [12], which demonstrates that cerbB2 plays an important role in regulating primordial follicle onset
Src protein expressed in rat ovaries during primordial follicle culture The expression of c-src protein in the ovaries during primordial follicle growth was examined by immunohistochemistry analysis
Summary
Oocytes are surrounded by somatic cells in the ovaries of newborn mammals. In rats, during the first 3 days after birth, the primordial follicles are assembled and remain developmentally arrested thereafter until the primary follicles are formed later [1]. The exact factors and mechanisms that regulate folliculogenesis initiation remain elusive, the accumulated evidence suggests that the early growth stage of follicle development is not dependent on the gonadotropins but is mainly controlled by a combination of local paracrine factors within the ovaries Some factors, such as stem cell factor (SCF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), nerve growth factor (NGF), bone morphogenic protein (BMP), growth differentiation factor 9 (GDF-9) and insulin-like growth factor (IGF), promote the development of the primordial follicles. In addition to the evidence from our previous studies that c-src mRNA is expressed in mammalian ovaries, primordial follicle growth was retarded and the number of mature follicles was significantly reduced in c-src knock-out mice [13,14] Based on this finding, it is tempting to speculate that c-src might play an important role in regulating primordial follicle onset as well. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles
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