Tracing And Characterizing The Development Of Transplanted Female Germline Stem Cells From Single Mouse

In mammals,germ cells are responsible for survival and reproduction of species.Primordial germ cells(PGCs)is the precursor cells of the female germ cells.PGCs migrate into the gonadal ridges with proliferation.Subsequently,oocytes are formed after meiosis initiation.During oogenesis,the follicle is formed and becomes a developmental unit,which consists of follicular granulose cells,thecal cells and a central oocyte.Bidirectional communications between oocytes and follicular somatic cells in follicles promotes the maturation of oocytes.In traditional view,female mammalians contain only a certain number of oocytes that arrested at the diplotene stage of meiotic prophase I,and the number exhaust with the growth of age.However,recent studies have shown that ovarian follicles are regenerated in mammalian ovary.In 2009,our group reported longterm cultured female germ line stem cells(FGSCs)isolated from neonatal and adult mouse ovaries.Subsequently,researchers isolated FGSCs from rat,human and pig.Moreover,transgenic animals were generated from FGSCs.However,there is no systematic research on the development or molecular mechanism of FGSCs post-transplantation.Stem cells have the ability to repair damaged tissue,the finding of FGSCs provides feasible method for future personalized medicine.In this study,we firstly trace the migration and development of transplanted FGSCs isolated from single mouse,then explore the molecular mechanism at transcriptome level,which provides a useful platform for future research and application of FGSCs.In this research,we isolated FGSCs from ovaries of single transgenic mouse(CAG-EGFP).The reproduction,stem cell and differentiation characteristics were identified using RT-PCR and immunofluorescence.Results showed long-term cultured FGSCs were germ cells with stem cell characteristics.Moreover,to analyze the development of FGSCs in vivo,we generated premature ovarian failure(POF)mice using chemotherapy drug or conditional gene knockout.Results showed transplanted FGSCs initiated meiosis followed migrating to the nearly cortex beneath the surface of ovary,and gradually expressed stra8,sycp3,ybx2,zp2 and zp1 and matured using immunofluorescence and single cell PCR analysis.The POF mice restored the ability of reproduction using FGSCs transplantation,and the offspring were identified using RT-PCR,southern blotting,immunofluorescence and fluorescent live image.In addition,we analyzed the folliculogenesis using single-follicle sequencing at transcriptional level.Similar functions and alternative splicings(ASs)composition of differential expression genes(DEGs)were proved during folliculogenesis between transplanted FGSCs and wild type.Further analysis,we found 98 core mRNAs and 18 core LncRNAs.More importantly,2 key LncRNAs may play important role in folliculogenesis.Taken together,we found transplanted FGSCs migrated to certain microenvironment and shared similar development characteristics with wild type.Moreover,we identified core genes in follicle development of transplanted FGSCs.This research is useful for exploring the molecular and development mechanisms of transplanted FGSCs,and also provides clues for clinical application.