Expression And Correlations Of Piwil2 Protein And Stat3 Protein And Bcl-2 Protein In Testis Of Male Infertitle Mice

Objective: We established the mouse infertility models,and detected the expression of Piwil2 protein,Stat3 and Bcl-2 proteins in the testis of male sterile mice.We explored the potential location and quantity between the three expressions;and further elucidated the role of Piwil2 protein in the development of infertility and related mechanisms.Methods: Sixty male Kunming mice were randomly divided into experimental group and control group,with 30 mice in each group.The experimental group of mice were used the tripterygium glycosides to estabilish the infertitle mice.Tripterygium wilfordii suspension was perfused daily at a dose of 0.001 ml/g,according to the body weight of the mice,once a day for 28 days.The mice in the control group were given the same dose of normal saline for intragastric administration,frequency and duration.Time is the same as the experimental group.After modeled,the male mice of two groups were mated with female mice to observe the pregnancy status of the female mice.After one week,the male mice were sacrificed,and taken out the testis tissues and epididymis of the two groups of mice.We counted the numeber of sperms in the epididymis of the mice of two groups.We useed Immunohistochemical staining and Western blotting to detect the expression of Piwil2,Stat3 and Bcl-2 proteins in the testis.And used quantitative PCR to examine the expression levels of m RNA of the three proteins in testicular tissues.We compaired the test results of the experimental group and the control group,and figured out the difference in protein expression in two groups;and observed the correlation of the three protein expressions.Results: 1.H.E staining results showed that the testis tissue structure of the control group was intact.The thickness of the spermatogenic epithelium was uniform.The proliferation and development of spermatogonial cells,primary and secondary spermatocytes and Leydig cells were normal(Fig.3.1a).Compared with the testis tissue of the control group,the structure of the mouse was significantly destroyed,and the spermatogenic epithelium was significantly thin.The distribution of seminiferous tubules was sparse compared to the control group,and the proliferation of spermatogonia,primary and secondary spermatocytes and testicular stromal cells was reduced,dysplasia,and sperm cells were clearly absent(Fig.3.1b).After mating with female mice,the pregnancy rate of female mice decreased significantly.The epididymis count of mice in the experimental group was significantly reduced,indicating that the infertitle mice model were successfully modeled.2.Immunohistochemical staining(IHC)showed that Piwil2 protein was mainly expressed in the cytoplasm of spermatogonia and primary and secondary spermatocytes,and also expressed in the Leydig cells.Bcl-2 protein was mainly expressed in cytoplasm and nucleus of spermatogonia,primary and secondary spermatocytes,and sperm cells;Stat3 protein is mainly expressed in the cytoplasm of Leydig cells(Fig.3.2).The staining scores showed that the staining degree and positive cells of Piwil2,Stat3 and Bcl-2 proteins in the experimental group were significantly lower than those in the control group(P < 0.01).The results of IHC staining showed that Piwil2 protein was in each spermatogenesis in the control group.Stable higher expression in the tubules(including spermatogonia,primary and secondary spermatocytes)and a few Leydig cells in the control group than in the experimental group.The expression level of Piwil2 protein in spermatogonia and primary and secondary spermatocytes,and the Leydig cells the experimental group were significantly lower than the control group(P < 0.001)(Table 3.2).The Bcl-2 and Stat3 proteins in the same control group were all stably expressed,and the expression level of the experimental group was significantly lower than the control group(P < 0.001)(Table 3.2).3.Western Blot results showed that the protein expression of the experimental group was higher than the control group.The decrease of Piwil2 protein was over 60%,and Stat3 and Bcl-2 protein were over 90%.The protein expression levels of the two groups were significantly different.(Piwil2 protein P < 0.05,Stat3 protein P < 0.05,Bcl-2 protein P < 0.01).4.Quantitative PCR results showed that the relative expression levels of the three protein m RNAs in the experimental group were significantly lower than the control group.The m RNA expression of Bcl-2 protein decreased more obviously.The difference of two groups was statistically significant(Piwil2 protein P < 0.01,Stat3 protein P < 0.001,Bcl-2 protein P < 0.05)Conclusion: The results of this study showed that the expression of Piwil2,Stat3 and Bcl-2 proteins were significantly decreased in spermatogonial cells,primary spermatocytes,secondary spermatocytes,sperm cells and Leydig cells of testicular tissues of male sterile mice.And there is a correlation between the expression levels of the three proteins in the seminiferous tubules(including spermatogonia,primary spermatocytes,secondary spermatocytes,sperm cells)and Leydig cells.Through the detection of the expression and location of Piwil2,Stat3 and Bcl-2 proteins,three proteins can be used to regulate mouse sperm production.The deletion of three proteins is essential for the occurrence of mice infertility.There is a potential correlation between the three protein interactions.