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Function Study of Hnrnph1 in Mouse Spermatogenesis

Author: ChenLing
Tutor: ZhouZuoMin；ShaJiaHao
School: Nanjing Medical University
Course: Human Anatomy and Embryology
Keywords: Spermatogenesis Hnrnph1/HNRNPH1 Apoptosis Thermal effect GC2-spd Flow analysis Proteomics
CLC: R321
Type: Master's thesis
Year: 2009
Downloads: 33
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Abstract

Spermatogenesis in the seminiferous tubules in the testis of continuous, complex process of cell division and differentiation, the process by the testicles inside and outside the precise control of a variety of factors. If the internal and external environment disorders, this rigorous process will be varying degrees of disorder, which led to infertility. Spermatogenesis associated protein varying degrees of expression in multiple stages of spermatogenesis, regulation of co-ordinating the entire process of spermatogenesis in the level of genes, proteins, cells, etc.. If we can grasp the function of these related factors, will help us to understand the molecular regulation of spermatogenesis, which are targeted to solve the problem of male infertility, as well as to provide new ideas for the development of male contraceptive methods in clinical. Spermatogenesis is very sensitive to the temperature of the testicles outside environment, will lead to increased local temperature testicular reversible spermatogenic failure. Therefore, the action of heat treatment of testicular help us to establish a good model for spermatogenesis associated protein. In previous work, the laboratory using two-dimensional gel electrophoresis (two-dimensional electrophoresis ,2-DE) and mass spectrometry (mass spectrometry, MS) were isolated and identified altered expression at different time points after the mouse and human testis heat protein and constructed the abundance differences significant change occurred expression profiling. These two lineages were identified to the heterogeneous nuclear ribonucleoprotein H (Heterogeneous nuclear ribonucleoprotein H, in mice for its Hnrnph1, in human highly homologous protein HNRNPH1 in). Hnrnph1 is an RNA-binding protein, belongs to the the hnRNPs superfamily subfamily hnRNP H family, the evolution of the relatively conservative play an important role in different biological events. Two protein expression shows the same trend, namely spermatogenic damage decreased expression of functional recovery of the seminiferous elevated expression of protein expression profiles in mouse and human testis hot role. This the results suggest Hnrnph1/HNRNPH1 may be involved in the regulation of spermatogenesis. In this study, we discussed the effect and mechanism of Hnrnph1 protein in spermatogenesis. We first examined the expression and cellular localization of Hnrnph1 protein in the testis. Applied Business Hnrnph1 antibody line detected by Western blot, to display Hnrnph1 specific expression of the protein in the adult mouse and human testis. Followed by application of the antibody on postpartum 0 days, one week, two weeks, three weeks, four weeks developing mouse testis, adult mouse testis and adult testis immunohistochemical detection, results showed that: in mouse testis and adult testis , Hnrnph1 mainly expressed in the nuclei of spermatocytes, also expressed in the interstitial cells and Sertoli cells cytoplasm. Mouse testis immunofluorescence result of separation of spermatogenic cells by immunofluorescence and immunohistochemistry consistent. Further, we vitro platform to the the mouse spermatocytes Department (GC2-spd) explore Hnrnph1 the functionality, mainly following: (1) First, the use of the Western blot method, GC2-spd have Hnrnph1 expression at the protein level validation . Then the immunofluorescence Hnrnph1 major expressing cell lines in spermatocytes nuclei. (2) the detection GC2-spd Hnrnph1 expression levels and apoptosis. We use the thermal effect induced apoptosis, and then detection the cells Hnrnph1 expression level, to reveal whether associated with apoptosis. GC2-spd restored to 37 ℃ for 3h after incubation at 42 ℃ heating, respectively, after rewarming 0h, 3h, 6h 24 hours cells were detected as a control group, unheated GC2-spd. Each group of cells in a part of the AV-FITC binding the PI flag flow detection of apoptosis, another part of the extraction the cell protein Western blot detection Hnrnph1 expression levels. The results showed that: the thermal effect rewarming after 24h apoptosis rate significantly higher than that in the control group (P lt; 0.05); Hnrnph1 expression was significantly lower than the control group (P lt; 0.05). The results suggest that, Hnrnph1 in GC2 apoptosis may be involved in the regulation of apoptosis process. (3) inhibition of apoptosis is detected after the expression of Hnrnph1 GC2-spd. Using RNAi technology, we will interfere with plasmid transfection GC2-spd build a stable cell line interference Hnrnph1 expression. Interference group and control group apoptosis rate of rewarming 24h cell lines apoptosis hot after rewarming 24hGC2-spd most significant in the experimental range, we detected not thermogenesis and heat. The results show: not hot role group interfere with cell lines apoptosis rate significantly higher than the unrelated group (P lt; 0.01), the action of heat group interference cell line apoptosis was significantly higher than the unrelated group (P lt; 0.01). Correlation studies, suggesting Hnrnph1 protein has anti-apoptotic effects. And this results we observed in the study of early testicular heating spermatogenic damage the protein expression was decreased, while the expression characteristics consistent with the increased expression of the protein in the spermatogenic recovery. (4) To explore the molecular pathways Hnrnph1 antagonize apoptosis. Proteomic methods, separation and identification of the GC2-SPD cell lines after the interference Hnrnph1 expression abundance of occurrence of significant changes in the protein, they may be used to describe the passage of Hnrnph1 involved in the regulation of the molecular. The results show that: the interference group compared with the control group, 72 protein spots altered expression, 27 protein spots decreased expression, elevated expression of 45 protein spots. 72 protein spots corresponding to 66 proteins have been reported in the literature, of which 31 proteins involved in the regulation of cell proliferation, apoptosis, directly involved in apoptosis signaling pathway protein 9 kinds (Clic4/Gmfb/Hspa5/Park7/Cfl1 The / Sod2/Eef2/Numa1/PICOT), where Clic4/Gmfb / Hspa5/Park7/Cfl1/Sod2/Eef2/Numa1 participate in the the classic mitochondrial apoptosis pathway. Protein involved in proliferation regulation the 14 kinds of (Arl3/Calu/Des/Dstn/ENO1/Fkbp52/Glo1/Naca/Pkm2/Pgk1/Psat1 / Rpsa / Set / Tubb,). Another 21 proteins associated with the cell cycle, or involved in cytoskeleton formation, or RNA translation related processing, as well as the related stress protection. Hnrnph1 protein by affecting the expression of these proteins, the regulation of apoptosis. In summary, in vitro, Hnrnph1 protein is a negative regulator of apoptosis by the Department of spermatocytes, combined with a series of cell lines in vitro results, we infer Hnrnph1. During spermatogenesis spermatogenic cell apoptosis in an antagonistic protection protein. Spermatogenesis spermatogenic cells through direct or indirect role in antagonizing apoptosis to promote spermatogenesis orderly synergy with many factors.

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