The Functions And Mechanisms Of CK1α Regulating Mouse Spermatogenesis

Spermatogonial stem cells(SSCs)have the ability to self-renew and differentiate into spermatogonial progenitor cells,which ultimately produce large numbers of sperm.The balance between self-renewal and differentiation of SSCs is a key component in maintaining mammalian spermatogenesis.SSCs are regulated by a network of external hormones as well as internal genes and proteins.It has been demonstrated that casein kinase(CK)consists of two different kinases,CK1 and CK2.In mammals,there are seven CK1 isoforms(α,β,γ1,γ2,γ3,δ and ε),of which CK1αis encoded by Csnk1a1.CK1α,a serine/threonine(Ser/Thr)protein kinase that is widely distributed in organisms,is highly expressed in the reproductive system and regulates ovarian and follicular development,but CK1α function in the male reproductive system is unknown.In this study,conditional Csnk1a1 KO mouse model in spermatogonia was established using Stra8cre mice to investigate the regulatory function of CK1α on mouse spermatogenesis and development and related mechanisms.The obtained results of the study are as follows:1.CK1α is highly expressed in mouse testisTo investigate CK1α expression in mouse testis,the expressions of CK1α mRNA and protein and the cell types of CK1α protein expression during C57BL/6 mouse testis development were detected using real-time PCR,western blot,enrichment assays of spermatogonia and somatic cells,and double immunofluorescence staining techniques.The results showed that Csnk1a1 mRNA expression was significantly higher in mouse testis than in other organs;Csnk1a1 mRNA and CK1α protein expression remained relatively low from P1 to P17,and significantly increased at P21 and persisted until P60;CK1α protein was expressed in Sertoli cells,spermatocytes,P/D spermatocytes,spermatogonial progenitor cells,and SSCs.The expression of CK1α protein was significantly higher in spermatogonia than in somatic cells(p<0.01).2.CK1α conditional knockout mice in spermatogonia is infertileTo investigate CK1α function in mouse spermatogenesis,CK1α conditional knockout mice in spermatogonia was established using Stra8cre mice and Csnk1a1flox mice.Then the effects of CK1α KO on mouse fertility,testicular CK1α KO efficiency and histomorphology were analyzed by fertility assay,real-time PCR,western blot,immunofluorescence staining,and histomorphology.The results showed that CK1αKO had no significant effect on mouse development,but severely hindered testicular growth;Csnk1a1 mRNA and CK1α protein expression in P7 cKO testes were significantly decreased by 51.3%(p<0.0001)and 62.6%(p<0.001)respectively.In addition,germ cells were significantly reduced in cKO mouse testes,there was no sperm in the epididymis,and adult male cKO mice were infertile.3.CK1α conditional KO leads to a reduction in the number of spermatogonial progenitor cellsTo further investigate the effects of CK1α in the development of spermatogenesis in mice,the effects of CK1α KO on the expression of marker molecules related to spermatogenesis process as well as cell proliferation and apoptosis were examined using real-time PCR,western blot and immunofluorescence staining techniques.The results showed that the spermatogonial marker Plzf mRNA level decreased significantly by 69%(p<0.001)and 73%(p<0.01),PLZF protein expression level decreased by 79%and 70%in P7 and P14 cKO testes respectively;The spermatogonial progenitor marker Sox3 mRNA level decreased by 81%(p<0.01)and 87%(p<0.0001)and Sox3 protein expression level decreased by 92%and 83%.The number of SOX3+spermatogonial progenitor cells and PLZF+ spermatogonia was significantly reduced,However,CK1α KO had no significant effect on the proliferative capacity and apoptosis of GFRα1+SSCs.4.CK1α conditional KO activates the MDM2/p53 signaling pathwayTo investigate the molecular mechanism of CK1α regulation of mouse spermatogonial progenitor cell,the signaling molecules associated with CK1αregulation of spermatogonial progenitor cell development were analyzed using RNA sequence,western blot,real-time PCR,immunofluorescence staining,and immunoprecipitation techniques.The results showed that CK1α KO increased the expression of MDM2 protein to 1.5-fold(p<0.05)and p53 protein to 2.3-fold(p<0.001)in P7 mouse testes,and p53 protein was expressed in mouse spermatogonia nuclei.This suggests that CK1α affects spermatogenesis and development through the interaction with MDM2 and p53.5.CK1α regulates spermatogonial progenitor cell development through the p53/Sox3 signaling pathwayBased on the above findings,we used western blot,immunofluorescence staining,RNA sequence,dual luciferase assay,chromatin immunoprecipitation,in situ hybridization to demonstrate whether CK1α regulates spermatogonial progenitor cell development through the p53/Sox3 signaling pathway.The results showed that p53 binds to the Sox3 gene promoter to inhibit Sox3 transcriptional expression;p53 inhibition rescued the reduced phenotype of spermatogonial progenitor cells in cKO mice.These results suggest that CK1α regulates spermatogenesis and development through the p53/Sox3 signaling pathway.In conclusion,this study demonstrates that CK1α is one of the key regulators of spermatogonial progenitor cell maintenance.The findings have important theoretical and practical implications for the understanding of spermatogonial progenitor cell maintenance and spermatogenesis mechanism.