Screening Small Molecules To Maintain Spermatogonial Stem Cells Self-Renewal In Vitro And Mechanistic Studies

The germ cells not only carry the genetic information for the reproduction of the species,but also bear the important responsibility for the evolution of the species through mechanisms such as the association and crossover of the meiotic process.Therefore,germ cells,as the spark that transmits the continuation and evolution of species in the vast river of history,play a key role not only for individual species,and for the continuation of the whole species and even the development of civilization.However,the incidence of infertility has been increasing in recent years,which is not only closely related to the defects of germ cells,but has also become a major challenge to people’s health.According to the World Health Organization,nearly 15%of couples around the world suffer from infertility,of which 30%-50%is caused by male factors.Data show that the incidence of infertility in China is 12.5%-15%,and the number of patients is more than 40 million,which means that every 8 couples of childbearing age have 1 couple facing infertility problems,which is a huge number.As the basis of spermatogenesis in adult males,spermatogonial stem cells play a critical role in maintaining the spermatogonial pool through self-renewal and initiating meiotic differentiation into mature spermatozoa,and the long-term maintenance of spermatogonial stem cells in vitro is dependent on their self-renewal properties.Therefore,the study of cell fate regulation of spermatogonial stem cells and the establishment of an in vitro germ cell differentiation system to research the developmental regulatory mechanisms of spermatogonial stem cells can help to establish relevant disease models,study the pathogenesis,and carry out drug screening,which will promote the research of reproduction and infertility as well as the treatment of the disease.However,a long-term in vitro culture system for human spermatogonial stem cells has not been reported,because the traditional mouse spermatogonial stem cell culture system cannot be applied to humans,and there is an urgent need to find new targets to promote the self-renewal of spermatogonial stem cells;in addition,the limited availability of human testicular tissues,and the lack of a way to obtain spermatogonial stem cells induced by human pluripotent stem cells have greatly limited the progress of screening for the human spermatogonial stem cell culture system.Therefore,the aim of this study was to 1)conduct a screening based on mouse spermatogonial stem cells to identify targets that can promote spermatogonial stem cell self-renewal differently from the traditional culture method,and to analyze the mechanism and assess the effectiveness of their application to primate spermatogonial stem cells;and 2)optimize the in vitro culture system for human pluripotent stem cell-induced germ cell development.We conducted a small molecule screening to identify the small molecule compounds,lovastatin and SM-8#,that can maintain the self-renewal of mouse spermatogonial stem cells without relying on GDNF cytokines.Among them,we found that SM-8#inhibits the expression of differentiated genes by reducing the binding of H4R3me2s histone modification at the promoter of these genes through the inhibition of Prmt5 enzyme activation site,thus achieving the maintenance of self-renewal of mouse spermatogonial stem cells in vitro.More importantly,SM-8#effectively promotes the proliferation of undifferentiated spermatogonia in vivo and maintains the cellular identity of primate spermatogonia for maintaining self-renewal in vitro.In addition,we optimized the existing in vitro culture system for human pluripotent stem cell-induced germ cell development to be closer to the in vivo developmental time and to obtain the DDX4-positive late primordial germ cell-like cellular stage more quickly.In summary,this study found that Prmt5 regulates the cell identity of spermatogonial stem cells and reveals its mechanism of action,which lays the foundation for the establishment of an in vitro culture system for primate spermatogonial stem cells;in addition,it also provides a good research platform for better realizing the acquisition of spermatogonial stem cell-like cells from human pluripotent stem cells induced in vitro.The above studies not only broaden our understanding of spermatogonial stem cell fate regulation,but also provide a research platform for applications in reproductive development and regenerative medicine.