The Function And Mechanism Research Of RNA Binding Protein HnRNP H1 In Spermatogenesis

Part 1 The expression pattern of hnRNP H1 in mice[Purpose] Conservative analysis of the amino acid sequence of hnRNP H1 in different species,in the meantime,the expression levels and cellular localizations of hnRNP H1 in different mouse tissues were detected.[Methods] Multi-alignment and phylogenetic analyses of the amino sequence of hnRNP H1 by bioinformatics were performed to explore the conservation of hnRNPH1 among different species,including humans,mice,rats,dogs and chickens.RT-q PCR,Western Blot,and immunofluorescence were performed to explore the expression pattern of hnRNP H1 in different tissues of mice and their cellular localization in seminiferous tubules.[Results] hnRNP H1 is conserved in different species.The amino acid homology between human and mouse is 99.78%(only one amino acid is different).hnRNP H1 is highly expressed in nucleus of germ cells and somatic cells in mouse testes.[Conclusions] hnRNP H1 shows high conservation in different species.hnRNP H1 is localized in the nucleus in germ cells and sertoli cells.Part 2 Function of hnRNP H1 in mouse male germ cells[Purpose] To elucidate the physiological role of hnRNP H1 in male germ cells developments.[Methods]Germline specific knockout mice were generated using the Cre-Loxp system and Stra8-Cre transgenic mice.The physiological role of hnRNP H1 in spermatogenesis was clarified by various phenotypic analysis methods such as morphological detection and fertility test.[Results] Germline specific knockout mice were successfully generated.After conditional knockout of hnRNP H1 in mouse germ cells,the testes became significantly smaller and spermatogenesis was disrupted.[Conclusions] hnRNP H1 in male germ cells play a critical role in spermatogenesis.Part3 Fuction of hnRNP H1 in mouse sertoli cells[Purpose] To elucidate the physiological role and underlying mechanism of hnRNP H1 in Sertoli cells during spermatogenesis.[Methods] Sertoli cells specific knockout mice were generated using the Cre-Loxp system and Amh-Cre transgenic mice.Fertility statistics,immunofluorescence,apoptosis detection,and Transmission Electron Microscope was performed to observe the ultrastureture and explore the functions of hnRNP H1 in sertoli cells and spermatogenesis.[Results] Sertoli cells specific knockout mice were successfully generated.Conditional mutation of hnRNP H1 in Sertoli cells causes testis size reduction and complete male infertility,which characterized by lacing mature sperm in the epididymis,blood-testis barrier disruption,and increase of the levels of autophagy in Sertoli cells.hnRNP H1 can bind to several cell junction-related genes,and the expression of them was abnormal in knockout mice.[Conclusions] hnRNP H1 in Sertoli cells plays an important role in the regulation of spermatogenesis.Specific knockout of hnRNP H1 led to abnormal expression of cell junction-related genes,which in turn caused disruption in the blood-testis barrier and biological dysfunction of Sertoli cells.The specific knockout of hnRNP H1 eventually leads to the microenvironment of seminiferous alteration tubules and male infertility.