Characterization Of Female Mouse Germline Stem Cells And The Effect Of Rapamycin On Their Proliferation

There are many kinds of stem cells existing in the tissues and organs of mammal animals. Recently, the researchers have found the existence of female germline stem cells(FGSCs) have been identified within several species, but their origins and physiological functions are currently unknown, and lack the convincing evidences. Zou et al isolated mouse FGSCs with germline specificity and proliferative ability from the immature and adult mouse ovary in 2009(Zou et al., 2009). But there is no evidence demonstrating those cells could involve in follicle formation or oogenesis in later life. Although the research of FGSCs is not deeply enough, exploring the mechanism of proliferation and differentiation and physiological activities of eggs will contribute to our understanding of diseases like premature ovarian failure and infertility. Rapamycin, as an inhibitor of mTOR pathway, can inhibit the development of primordial follicles, and reduce the consumption of primordial follicles, and thus contribute to the maintenance of the oocyte pool and ovarian fertility.The current study mainly focused on the inhibited effect of rapamycin on primordial follicle activation, whereas little research is about rapamycin on proliferation and differentiation and multipotency of mouse ovaries and FGSCs. With the continuous exploration of the mechanism of rapamycin in ovary, it will bring a new prospect in premature ovarian aging.In this experiment, we digested ovarian surface cells, and got the cells with multipotency and germline specificity, and passaged through seven generations; the one month old female mice were intraperitoneal injected rapamycin every other day for 30 days, and we found the rapamycin had a positive effect on the proliferation of mouse ovaries and FGSCs, and negative effect on differentiation and multipotency of them by quantitative reverse transcription-PCR(qRT-PCR)and immunofluorescence examination.1. Cultivation, Isolation and Characterization of mouse FGSCs.As we know, the FGSCs express pluripotent maker gene Oct4, and the locations are on and inside the oogonial surface epithelium. So we aimed at separating the FGSCs of Oct4 promoter-GFP transgenic mouse with two features above. Finally, we got the cells that stably expressed Oct4 and could passage for seven times. The clones of FGSCs showed the weak alkaline phosphatase(AP) positive and had multidirectional growth potentials and maintain their proliferative ability. The mouse FGSCs we got shared some similar abilities with Spermatogonial stem cells(SSCs) and Embryonic stem cells(ESCs).2. The effect of rapamycin on the mouse ovaries and FGSCsIn the mouse ovary, it has been reported that rapamycin can inhibit the activation of primordial follicles as the inhibiter of mTOR pathway.In this experiment, we attempted to explore the effect of rapamycin on the proliferation and differentiation and multipotency of mouse ovaries and FGSCs. We used the method that intraperitoneal injection of rapamycin to one-month-old female mice every other day for 30 days. By qRT-PCR and immunofluorescence examination of the whole ovary and isolated FGSCs, we could conform rapamyin not only played a up-regulated role on the proliferation of mouse ovaries and FGSCs, but also inhibited the differentiation and multipotency of them.