Study On The Regulatory Mechanisms Of Self-renewal In Germline Stem Cells Mediated By Cadherin 1 And 22

Germline stem cells are a type of cells that have the ability to self-renew and differentiate,which plays an important role in animal reproduction.Cadherin is a Ca2+-dependent transmembrane glycoprotein involved in the regulation of the fate of germline stem cells.Its mechanism for the regulation of germline stem cells is not yet very clear.In this study,we focused on the mechanisms of self-renewal of germline stem cells by cadherin CDH1 and CDH22,respectively.Part Ⅰ:The regulation of fate of male germline stem cells by CDH1.The self-renewal and differentiation of mouse testicular spermatogonial stem cells(SSCs)are affected by a variety of factors.CDH1(also known as CDH1)has been identified as a marker expressed specifically in undifferentiated type A spermatogonia in mouse testes.The process of maintaining the undifferentiated state of spermatogonial progenitor cells(SPCs)isolated in vitro is very complicated.The transcription repressor PLZF has been identified as a key factor in the maintenance of dryness of SPCs,but the specific regulatory mechanism is not yet clear.In this study,we isolated and purified Thy-1+ cells from 5-day-old mouse testes and cultured them in vitro,we then identified their expression profiles and the results showed expression of Id4,Gfral,Plzft Cdh,Dazl,Ngn3 and c-kit.It demonstrated that the Thy-1+ cells we obtained were SPCs-enriched cell population.We demonstrated that SPCs express both CDH1,β-catenin,and PLZF by immunofluorescence assays.We then used immunoprecipitation to confirm the existence of an interaction between CDH1 andβ-catenin in mouse testis.Based on previous studies,we speculated that histone deacetylase 4(HDAC4)is a downstream target gene for β-catenin in mouse testis.So,we used siRNA to knock down β-catenin in SPCs and found that the expression of HDAC4 also decreased,confirming that β-catenin regulates the expression of HDAC4.It has been reported that HDAC4 participates in inhibiting cell differentiation through binding and cooperation with PLZF.We demonstrated the mutual binding interactions between HDAC4 and PLZF by co-immunoprecipitation.The above experimental results indicate that CDH1 regulates the sternness of SPCs by regulating β-catenin and its downstream target gene Hdac4,thereby affecting the binding activity of HDAC4 binding protein PLZF.Part II:The Preliminary Study of the Regulation of CDH22 on the Fate of Female Germline Stem Cells.Female Germline Stem Cells(FGSCs)is a germline stem cell found in the ovary of female mammals in recent years.However,little is known about the mechanisms of self-renewal and differentiation.It is important to fully understand this mechanism to maintain fecundity in female mammals.According to previous research reports,we know that CDH22 is highly expressed in the ovary of female mice,but it is not clear what role it plays for ovarian germ cells.To explore this mechanism,we used immunohistochemistry to verify that CDH22 is highly expressed in mouse ovaries at different ages(5d,10d,20d,42d).By knocking down the expression of CDH22 in female reproductive stem cells using siRNA,it was found that it is related to the proliferation of female reproductive stem cells.According to related reports,we know that the JAK-STAT signaling pathway influences the self-renewal of spermatogonial stem cells.To verify whether CDH22 and JAK-STAT signaling pathways also have similar regulation in female germline stem cells,we found that CDH22 and JAK2 also exist in female germline stem cells through co-immunoprecipitation experiments.It was initially explored that in female germline stem cells,CDH22 may affect its self-renewal through the JAK-STAT signaling pathway.In summary,we demonstrate that cadherin plays an important regulatory role for self-renewal of germline stem cells,this provides a reference for further study of germline stem cells,and also provides some new hints for the maintenance of productivity in farm animals.