The Mechanism Of Protein 4.1R Based On Sertoli Cells In Oligozoospermia Mice Model

Infertility is a series of reproductive system diseases that seriously disturb the reproductive ability of mammals.Infertility can be caused by nutritional deficiencies,poor feeding management,seasonal causes,genetic causes(such as hybrid infertility),and diseases.Male factors account for about half of the causes of mammalian sterility.Azoospermatism,oligozoospermia and asthenospermia are main causes of male infertility.Oligozoospermia is a male infertility characterized by the significantly low sperm counts.The pathogenesis of oligozoospermia is complex and the mechanism was still unclear.Both genetic and environmental factors lead to the decrease in sperm counts.The effect of genetic factors on sperm counts is mainly reflected by the expression and regulation of the specific genes during spermiogenesis process.Therefore,it is of great theoretical and practical significance to look for specific genes about spermatogenesis and to illuminate their mechanism for accurate treatment and prognosis of oligozoospermia.Protein 4.1R is a cytoskeletal protein component encoded by the epb41 gene,which plays a role in erythrocytes and non-erythrocyte tissues.In this paper,the role and mechanism of protein 4.1R was elaborated based on Sertoli cells in oligozoospermia from the following three parts.(1)Western blotting,q-PCR and immunofluorescence were used to detect the expression characteristics of protein 4.1R in the testes of oligozoospermia C57BL/6J mice at 7-week-old induced by tripterygium glycosides.The integrity of the blood-testis barrier(BTB)was tested in oligozoospermia mice by FITC distribution,the expression detection of proteins related the BTB formation for illustrating the correlation between protein 4.1R expression and cell junction in Sertoli cells.The Wnt/β-catenin signaling pathway was detected in oligozoospermia mice for the function and mechanism of protein 4.1R in cell proliferation.Resuts from Western blotting test showed that epb41 gene expressed 135 k D and 80 k D isoforms of protein4.1R in normal C57BL/6J mice testes,named protein 4.1R-135 k D and 4.1R-80 k D.In oligozoospermia mice the expression of 135 k D isoform decreased significantly(P<0.05),and the expression of 80 k D isoform showed no significant difference compared to the normal C57BL/6J mice.The q PCR results showed that the expression of epb41 m RNA in testes of oligozoospermia mice decreased compared with the control group.Immunofluorescence test showed that the expression of protein 4.1R on the Sertoli cells membrane decreased,but the expression on various germ cells showed no significant difference compared with the control group.The integrity test of the BTB showed that the BTB impaired in oligozoospermia mice,and FITC entered the compartment from the basal compartment and dispersed in lumen of seminiferous tubules.Oligozoospermia mice showed the decreased β-catenin expression and increased E-cadherin expression compared with the control.The expression of ZO-1 was significantly decreased(P<0.05)and occludin and claudin-11 expression decreased.QPCR detection of Wnt/β-catenin signaling pathway components showed that wnt3 am RNA,gsk3βm RNA,c-mycm RNA,CTNNB1 m RNA and cdk4 m RNA expression were decreased compared with the control group.But CCND1 m RNA and cdk6 m RNA expression increased.Western blotting test of wnt/β-catenin signaling pathway components showed that the expression of protein wnt3 a and β-catenin significantly decreased,and the expression of GSK3β,c-myc and CDK4 were also decreased compared with the control group.But the expression of Cyclin D1 and CDK6 increased.(2)The recombinant plasmid including the PGL3-U6-epb41-sg RNAa-puromycin plasmid and the p Sp Cas9(BB)-epb41-sg RNAb-2A-GFP plasmid were constructed according to exon 3 of mouse epb41 gene.N2 a cells were transfected to verify the correlation between the gene sequence in exon 3 and the expression of isoforms in protein 4.1R.To explore the function of epb41 gene,the overexpressed recombinant vector LV-Epb41(43286-1)and the knockout recombinant vector LV-Epb41-sg RNA(05765-1)were constructed and transfected TM4 cells(normal mouse Sertoli cell line)and the primary Sertoli cells respectively.The expression of protein 4.1R,protein in Wnt/β-catenin signaling pathway and the protein related the BTB was detected for illuminating the roles and mechanism of protein 4.1R in Sertoli cell line on cell proliferation and junction.The results showed that protein 4.1R-135 k D no longer expressed in N2 a cells transfected with the recombinant knockout vector but protein4.1R-80 k D normally expressed.LV-Epb41-sg RNA(05765-1)lentiviral vector was designed and transfected TM4 cells and primary Sertoli cells respectively.The results showed that protein 4.1R-135 k D expression was inhibited,while protein 4.1R-80 k D normally expressed.LV-Epb41(43286-1)lentiviral vector also transfected TM4 cells and primary Sertoli cells.The results showed that the expression of protein4.1R-135 k D and 4.1R-80 k D all significantly increased compared with the control group.Compared with the control group,in TM4 cells protein 4.1R-135 k D gene knockout the expression of the BTB related proteins significantly decreased including in β-catenin,E-cadherin and ZO-1.The expression of wnt3 a,β-catenin,c-myc,GSK3β and cyclin D1 all decreased(P<0.05),CDK4 expression also decreased,but the expression of CDK6 decreased in wnt/β-catenin signaling pathway.Protein4.1R-135 k D overexpressed in TM4 cells,the expression β-catenin,ZO-1 and Occludin were increased,the expression of E-cadherin decreased with no significant differences.Wnt3 a,β-catenin,c-myc,GSK3β and Cyclin D1 expression were all increased,while CDK4 and CDK6 expression were decreased,and the differences were not significant.In the primary Sertoli cells with protein 4.1R-135 k D gene knockout,the expression of β-catenin,ZO-1 and Occludin significantly decreased;the expression of protein Wnt3 a,β-catenin and CDK4 were significantly decreased(P﹤0.05),c-myc and GSK3β expression were also decreased,and the expression of Cyclin D1 and CDK6 were increased in Wnt/β-catenin signaling pathway.Protein 4.1R-135 k D overexpressed in primary Sertoli cells,the expression of β-catenin,ZO-1 and Occludin increased,while the expression of E-cadherin decreased.The expression of Wnt3 a,β-catenin,c-myc,GSK3β,CDK4 and CDK6 were all increased,and the expression of Cyclin D1 was decreased,and the differences were not significant in Wnt/β-catenin signaling pathway.(3)PGL3-U6-epb41-sg RNAa-puromycin,p Sp Cas9(BB)-epb41-sg RNAb-2A-GFP and LV-Epb41(43286-1)were transfected into the testes of male C57BL/6J mice aged7 weeks by in vivo electroporation and lentiviral injection.The efficiency and targeting of the two transfection methods were compared,and the expression of protein 4.1R and the BTB related proteins in testes was detected by immunofluorescence for elucidating the function and mechanism of protein 4.1R on cell proliferation and junction of Sertoli cells in vivo.The test results showed that the plasmid vector was transfected into testes by electroporation,lentivirus vector transfection was acted on testes by injection.The results showed that the target protein expressed faster by the plasmid vector in vivo than lentivirus vector.The target protein by plasmid vectors expressed in the testes 3 days after transfection,and the expression of protein 4.1R decreased in Sertoli cells.By lentiviral vector injection the target protein expressed slowly,which expressed in testes 5 days after transfection.Lentiviral overexpression vector significantly increased the expression of protein 4.1R in Sertoli cells.Protein 4.1R-135 k D gene knockout in mice living testis,and the change rule of BTB-related protein expression was the same as that of oligospermia model mice.Protein 4.1R-135 k D overexpressed in vivo testis and significantly increased the expression of BTB related proteins except for E-cadherin.The conclusions were that epb41 gene expressed 135 k D(4.1R-135 k D)and 80 k D(4.1R-80 k D)isoforms of protein 4.1R in mice testes and Sertoli cells,and protein4.1R-135 k D expression was associated with reduced sperm counts.Decreased expression of 4.1R-135 k D led to decreased β-catenin expression,which resulted in abnormal expression of Wnt/β-catenin signaling pathway components,and inhibited the proliferation of Sertoli cells.The decreased expression of protein 4.1R-135 k D led to the abnormal expression of proteins related to the BTB,which resulted in abnormal cell junctions,and impaired microenvironment for spermiogenesis in adult mice testes.Therefore,the decreased expression of protein 4.1R-135 KD in Sertoli cells caused the decreased number of Sertoli cells,and the BTB was impaired.Sperm counts were significantly reduced.