Study On The Regulatory Mechanism Of PLZF+ Spermatogonia Pool In Mouse Testis

Androgen plays a key role in spermatogenesis,but the mechanism by which androgen acts on testicular germ cells and regulates their fate remains unclear.The transcriptional repressor Promyelocytic Leukemia Zinc Finger(PLZF)is essential for maintaining undifferentiated spermatogonial cell populations(also known as spermatogonial progenitor cells,SPCs),but the specific regulatory mechanisms remain largely unclear,especially the potential relationship between androgen signaling pathway and PLZF is still a blank.This study first analyzed the regulatory relationship between the androgen and the fate of the PLZF+SPCs population.Based on immunohistochemistry results,we confirmed the lack of endogenous AR in testicular germ cells from onset of spermatogenesis,indicating that androgen is not required for initiation of SPCs differentiation.Subsequently,total cells were isolated from 5dpp(day post partum)mouse testes to establish a somatic cells-spermatogonia co-culture system,and androgen DHT and/or the androgen antagonist bicalutamide was added to disturb androgen signal in this system,and the expression of PLZF was found to be negatively related to androgen signaling only when Sertoli cells existed.This result indicates that Sertoli cells is required for spermatogenesis during androgen signal stimulation.Then,ChIP-seq assay was performed in purified primary Sertoli cells to screen the downstream target genes of AR and Wilms Tumor' 1(WT1),and gene silencing and overexpression were used to verify the results from ChIP-seq assays in Sertoli cells.The results showed that Gata2 was identified as the target gene of AR,and the?1-integrin was a target gene of WT1 in the Sertoli cells.The androgen signal negatively regulates the ?1-integrin on the Sertoli cells via GATA2 and WT1,while the ?1-integrin on the Sertoli cells interacts with E-cadherin on the SPCs to regulate the fate of the SPCs.Subsequently,we specifically overexpressed androgen receptors in germ cells using transgenic mouse model,to analyze the impact of continuous overexpression of endogenous androgen receptors in germline on spermatogenesis.The DDX4-Cre male mice were mated with R26hARQ9floxed/+female mice to obtain R26hARQ9floxed/+:DDX4-Cre germline AR overexpressing offspring.Immunohistochemical results showed that the size of PLZF+SPCs population decreased at various stages of mouse development compared to R26hARQ9floxed/+controls,and most of seminiferous tubules in testes of adult R26hARQ9floxed/+:DDX4-Cre male mice became empty:the germ cells were lost,but only the supporting cells adhered to the basement membrane,indicating that specific overexpression of AR in germline induced loss of SPCs.In summary,this study reveals an indirect regulatory pathway that AR in Sertoli cells regulated the fate of undifferentiated spermatogonial cells,and overexpression of AR in germline causes the blockage of spermatogenesis due to loss of SPCs,demonstrating that AR promotes the differentiation of PLZF+ spermatogonia pool.This study provides theoretical support for the in vitro differentiation of SPCs and its molecular mechanism of interaction with microenvironment,and also offers a new idea for the maintenance of fecundity in male animals.