原癌基因 c-src 通过 PI3K、PKC 和 MAPK 信号通路参与原始卵泡的激活。
The proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways.
机构信息
Medical Experimental Teaching Department, Nanchang University, Nanchang, 330006, China.
出版信息
Reprod Biol Endocrinol. 2012 Aug 20;10:58. doi: 10.1186/1477-7827-10-58.
BACKGROUND
C-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.
METHODS
Ovaries 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.
RESULTS
The 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.
CONCLUSIONS
C-src plays an important role in regulating primordial follicle activation and growth via the PI3K-PKC- ERK1/2 pathway.
背景
C-src 是一种进化上保守的原癌基因，调节细胞增殖、分化和凋亡。在我们之前的研究中，我们报道另一个原癌基因 c-erbB2 在原始卵泡激活和发育中发挥重要作用。我们还发现 c-src 在哺乳动物卵巢中表达，但它在原始卵泡激活中的功能尚不清楚。本研究的目的是研究 c-src 在原始卵泡生长过程中的作用和机制。
方法
将 2 日龄大鼠的卵巢在体外培养 8 天。设计了三种靶向 c-src 和一种阴性对照 siRNA，并用于本研究。通过 PCR、Western blot 和原始卵泡发育评估慢病毒 c-src siRNA 的沉默效率及其对原始卵泡起始的影响。使用 PCR 方法和免疫组织化学染色检测原始卵泡生长中 c-src mRNA 和蛋白的表达。此外，使用 MAPK 抑制剂 PD98059、PKC 抑制剂 Calphostin 和 PI3K 抑制剂 LY294002 来探索 c-src 在原始卵泡发生中的可能信号通路。
结果
结果表明，Src 蛋白分布在原始卵泡的卵质膜和颗粒细胞膜中，c-src 的表达水平随着原始卵泡的生长而增加。靶向 c-src 的慢病毒 siRNA 对 c-src mRNA 和蛋白表达具有沉默作用。用 c-src siRNA 转染大鼠卵巢 8 天后，冷冻卵巢切片中的 GFP 荧光在荧光显微镜下清晰可见，其相对表达水平比对照组高 5 倍。此外，靶向 c-src 的慢病毒 siRNA 将其相对表达水平降低了 1.96 倍。我们还发现，c-src 表达被敲低后，培养的原始卵泡的发育完全被抑制。此外，我们的研究表明，Calphostin、PD98059 或 LY294002 处理抑制了卵泡发生起始，但它们都没有下调 c-src 的表达。相反，c-src siRNA 转染后，卵泡中 p-PKC、p-ERK1/2 和 p-PI3K 的表达水平明显降低。相应地，Calphostin 和 LY294002 处理均导致卵泡中 p-PKC 水平降低，但 PD98059 组未见变化。最后，LY294002 处理降低了卵泡中 p-PI3K 的表达水平，但 PD98059 和 Calphostin 组未见变化。
结论
C-src 通过 PI3K-PKC-ERK1/2 通路在调节原始卵泡激活和生长中发挥重要作用。
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