Function Study Of Hnrnph1 In Mouse Spermatogenesis

Spermatogenesis is the production of sperm from the primordial germ cells. It's a kind of cleavage and differentiation process involves a number of complex developmental events, including successive mitotic, meiotic and post-meiotic phases. This process which takes place in the seminiferous tubules of male testis is precisely regulated by a lot of external and internal factors. Once the balance around is destroyed, spermatogenesis dysfunction or even infertility may happen. The proteins associated with spermatogenesis are expressed differently during multiple stages. They together play a temporal-spatial control role in spermatogenesis. Searching for these factors and further functional study are very crucial not only to the elucidation of molecular mechanism involving in spermatogenesis but also to the finding of new contraceptive methods.It has been known that spermatogenesis needs a relative low temperature. It will cause reversible seminiferous dysfunction when the ambient temperature becomes higher. Therefore, the heat-treated testes may be one of the suitable models for us to do functional study of spermatogenesis related proteins. Previously, we utilized two-dimensional gel electrophoresis (2-DE) as well as mass spectrometry (MS) to isolate and identify a serial of proteins with altered expression in both mouse and human testes at different times after heat exposure. One protein called Hnrnph1 whose homologous protein in human is HNRNPH1 attracted our attention. It is one kind of RNA binding protein with altered expression in both experiments. Consistently, the protein expressed lower when spermatogenesis was destroyed so badly by heat exposure. And it grows higher when spermatogenesis process recovers. Hnrnph1, which is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) H protein family, is likely important for the processing of RNAs. It suggested that Hnrnph1 may have some relationship with regulation of genes in testes. In this research, we focused on the function of Hnrnph1 during spermatogenesis.Firstly, we verified the expression of Hnrnph1 in testes by western blot. Then, we collected mouse testes from postpartum 0 day, 1 week, 2 week, 3 week and 4 week. With the method of immunohistochemistry, we examined the dynamic expression changes of above-mentioned puberal testes. Meanwhile, we did the same thing to both mouse and human adult testes. It was showed that Hnrnph1 expressed in the nucleus of earlier spermatogenic cells before the stage of spermatid. It also expressed in the cytoplasm of somatic cells in testes such as Leydig cells and Sertoli cells. The results were coincident by immunofluorescence.Secondly, we utilized the GC2-spd cell line as a model to study the function of Hnrnph1 in vitro as follows:(1)We checked the translational level of Hnrnph1 in the cell line by western blot and immunofluorescence.(2)Subsequently, we checked the dependability between the expression level of Hnrnph1 and apopotosis.We imitated the heat treatment to the cell line. After we gave the 42℃heat exposure for 3 hours, we put the cells back in the 37℃incubator. We set a time course of 0h, 3h, 6h, 24h after heat exposure, and made the non-heat treatment group as control ones. When the cells were digested from culture flask, we put everyone into two parts. One was extracted into cell protein and examined the expression of Hnrnph1 by western blot, while the other was labeled with AV-FITC and PI and checked by the flow cytometry. From that, we discovered that the apoptosis rate of the cells from 24h was the highest compared with the other four groups (P<0.05), while its expression of Hnrnph1 was the lowest (P<0.05). Given to the relevance of Hnrnph1 with apoptosis, we supposed it may be concerned with spermatogenesis dysfunction.(3)Then we knocked down the expression of Hnrnph1 in GC2-spd by shRNA. We used the same method to check the apoptosis rate between RNAi group and negative control one. When Hnrnph1 was downregulated, the apoptosis rate was significantly higher than control (P<0.01). Meanwhile, the apoptosis rate in RNAi group from 24h was notably higher than control (P<0.01). It gave us a hint that down regulation of Hnrnph1 could promote apoptosis in GC2-spd. In other words, Hnrnph1 could be an anti-apoptosis factor in GC2-spd.(4)To find the potential anti-apoptosis molecular pathway of Hnrnph1, we utilized the proteomic approach mentioned above to isolate and identify altered expressed proteins between two cell lines. Compared with the control, we identified 72 protein spots with altered expression. 27 of the total spots decreased, while 45 remained increased. They were corresponding to 66 proteins. According to existing reference, 31 of the total 66 proteins were involved in the control of cell proliferation and apoptosis. Clic4/Gmfb/Hspa5/Park7/Cfl1/Sod2/Eef2/Numa1 were likely involved in mitochondrial apoptotic pathway, while Arl3/Calu/Des/Dstn/ENO1/Fkbp52/Glo1/ Naca/Pkm2/Pgk1/Psat1/Rpsa/Set/Tubb were involved in proliferation. We expected to find the downstream factors regulated by Hnrnph1, so as to illustrate the mechanism during spermatogenesis well.To conclude, we guessed that Hnrnph1 may be one of protective proteins to inhibit apoptosis during spermatogenesis. It may function as a direct or indirect factor together with multiple proteins to restrain the apoptosis of spermatogenic cells.