Study On The Mechanisms Of Mir-194-5p Regulating Apoptosis And Autophagy In Spermatogenesis Through Stra8 & Study On The Role Of Testis-specific Protein 33 (Tex33) In Mouse Spermatogenesis

Chapter Ⅰ Study on the mechanisms of miR-194-5p regulating apoptosis and autophagy in spermatogenesis through Stra8Objective:To explore the molecular mechanisms of miR-194-5p regulating cell apoptosis and autophagy during spermatogenesis via Stra8Methods:1.We synthesized the precursor gene sequence of miR-194-5p and constructed the recombinant lentivirus plasmid GV369-miR-194-5p.HEK293T cell was used to package plasmids into lentivirus,and the lentivirus was used to infect testicular teratoma cells(F9 cells),after puromycin selection,F9 cell line with stable overexpression of miR-194-5p was obtained,then the proportion of overexpression cells was analyzed by flow cytometry,the expression level of miR-194-5p after overexpression was determined by qRT-PCR,while the expression level of after overexpression was determined by qRT-PCR and Western Blot.2.Flow cytometry was used to detect the apoptosis level after miR-194-5p overexpression.Pre-constructed RNA-Seq was analyzed to screen out the different expressed molecules related with cell apoptosis in Stra8 knockout mice.qRT-PCR and Western Blot were used to detect the expression trends of these molecules in miR-194-5p overexpression F9 cells and Stra8 HOM mice model,and the signaling pathway regulating apoptosis was further revealed.3.MDC staining was used to detect the autophagy level after miR-194-5p overexpression.The expression trends of autophagy molecules in miR-194-5p overexpression F9 cells and Stra8 HOM mice model were detected by qRT-PCR and Western Blot.Results:1.Sequencing showed that the recombinant lentivirus plasmid GV369-miR-194-5p was correct and without mutation,miR-194-5p lentivirus was successfully packaged by HEK293T cells and then infected F9 cells.After selection,F9 cell line with stable over expression of miR-194-5p was obtained.Flow cytometry analysis revealed a high proportion of overexpression cells.qRT-PCR and Western Blot showed that the expression level of miR-194-5p increased,while the mRNA and protein expression level of Stra8 decreased in the miR-194-5p overexpression F9 cell lines.2.Flow cytometry analysis showed that the apoptosis level of miR-194-5p overexpression F9 cells increased,as compared with the control group.The expression level of Bc12 decreased in the miR-194-5p overexpression F9 cells,while the expression levels of JNK,p-JNK,Bax,Bak,Cytochrome C,caspase9 and Caspase3 increased,compared with control F9 cells.This kind of expression trend was also verified by comparing the testes of Stra8 HOM mice with WT mice.3.MDC staining showed that the autophagy level of miR-194-5p overexpression F9 cells increased,as compared with the control group.The expression level of P62,which is known as autophagy substrate molecule,decreased in the miR-194-5p overexpression F9 cells,while the expression levels of autophagy-associated molecules Ulkl,Atg5,Vps18,Nrldl,and autophagy molecular marker LC3 increased,and LC3-Ⅱ/Ⅰ ratio raised after miR-194-5p overexpression.Moreover,the expression level of p62 decreased,while the expression level of LC3 increased,and the ratio of LC3-Ⅱ/Ⅰ elevated in testes of Stra8 HOM mice compared with WT mice,which were consistent with the expression trend in miR-194-5p overexpression F9 cells.Conclusion:miR-194-5p may promote the cell apoptosis of F9 cells and spermatogenic cells through JNK mitochondrial apoptosis signaling pathway via repressing Stra8,and may promote the cell autophagy of F9 cells and spermatogenic cells through inhibiting the expression of Stra8.Chapter Ⅱ Study on the role of testis-specific protein 33(Tex33)in mouse spermatogenesisObjective:To investigate the expression patterns of Tex33 and its roles in spermatogenesis.Methods:1.Preparation and identification of Tex33 polyclonal antibody:PCR was used to amplify Tex33 open reading frame,then the prokaryotic expression plasmid pET-30a-Tex33 was constructed.Tex33 prokaryotic protein was induced by IPTG,and then purified by Ni-NTA affinity chromatography.After regaining activity through gradient urea,Tex33 purified protein was used to immunize the male New Zealand white rabbits,and then Tex33 polyclonal antibody was obtained.ELISA was used to detect the titer of polyclonal antibody,and Western Blot was used to determine the specificity and validity of antibody.2.RT-PCR and Western Blot were used to analyze the expression profiles of Tex33 gene in mouse multiple tissues and different developmental stages of spermatogenesis;Cellular and subcellular localization of Tex33 protein in mouse testes and sperms were analyzed by immunofluorescence staining assay.3.CRISPR/cas9 gene editing technology was used to construct Tex33 knockout mice model,Western Blot and immunofluorescence staining of testes was used for mice genotyping.4.Phenotypic analysis of Tex33 HOM mice:Differences in the shapes of the testis and epididymis and testis-to-body weight ratio between Tex33 HOM mice and WT mice were observed and compared;H&E staining was used to analyze the histological structure of testes of Tex33 HOM mice;PAS staining was performed to observe the acrosome development in Tex33 HOM mice;Sperm quality of Tex33 HOM was analyzed by H&E staining and CASA system;α-Tubulin fluorescence staining was used to observe the manchette morphology of Tex33 HOM mice;H&E staining was used to analyze the ovarian morphology of Tex33 HOM mice;Cage experiments were carried out to analyze the fertility of Tex33 HOM mice.Results:1.Tex33 open reading frame was successfully amplified by RT-PCR assay.Sequencing showed that the prokaryotic expression recombinant plasmid pET-30a-Tex33 was correct and without mutation.After induction through IPTG,purification and regaining activity,Tex33 prokaryotic protein with high purity was successfully obtained.After immunizing New Zealand white rabbits for four times,Tex33 polyclonal antibody was obtained.ELISA revealed that the titer of the polyclonal antibody was 1:100 0000.Western Blot showed that the polyclonal antibody could specifically recognize Tex33 protein in testes and purified samples,and its size was consistent with prediction.2.RT-PCR analysis for mouse multiple tissues showed that three transcript variants of Tex33 gene were all testis specific,and Western Blot showed that Tex33 protein was only expressed in testes;RT-PCR also showed that Tex33 V2 expressed weakly from P21 in mice,and reached peak at P28,and remained expression into adulthood,while Tex33 V1 and V3 expressed from P28 in mice,and maintained peak into adulthood.Western Blot showed that Tex33 protein began to express from P28 and maintained high expression into adulthood.Immunofluorescence staining suggested that Tex33 protein was located on the acrosome and manchette of spermatids,and also localized on the acrosome and tail of mature sperms.3.Exon 2-4 of Tex33 gene was deleted by,CRISPR/cas9 technology.Genotype of the offsprings was identified by PCR assay.Western Blot and immunofluorescence staining showed that there was no Tex33 protein in the testes of HOM mice,suggesting that the successful construction of Tex33 knockout mice model.4.Phenotypic analysis of Tex33 HOM mice:there were no significant differences in the sizes of testis and epididymis between Tex33 HOM mice and WT mice,and the ratios of testis-to-body weight of Tex33 HOM mice showed no difference when compared with WT mice;H&E staining revealed that there was no significant difference in the histological structure of testis between Tex33 HOM mice and WT mice,and both Tex33 HOM mice and WT mice exhibited seminiferous tubules with normal diameter and spermatogenic cells;PAS staining was used to analyze spermatogenic stages,and the result showed that there was no significant difference in the acrosome development of Tex33 HOM mice;H&E staining revealed that there were no significant differences in the sperm morphology between Tex33 HOM mice and WT mice;CASA system was performed to analyze sperm motility,and revealing that the sperm count,progressive motility and total motility of Tex33 HOM mice had no significant differences as compared with WT mice;α-Tubulin fluorescence staining showed that both Tex33 HOM mice and WT mice exhibited manchette with normal morphology;H&E staining showed that there was no significant difference in the morphology of ovary between Tex33 HOM mice and WT mice.The results of cage experiments suggested that there were no significant differences in the average litter sizes between Tex33 HOM mice and WT mice.Conclusion:Tex33 polyclonal antibody with high specificity and effectiveness was successfully prepared.Tex33 is a testis-sepcific and highly conserved gene associated with sperm acrosome and tail,while Tex33 gene knockout does not affect mouse spermatogenesis as well as fertility,it may regulate spermiogenesis with other genes,but is not required for spermatogenesis.