The Localization And Functional Analysis Of Bex4 In Mice Sertoli Cells

Objective:The brain expressed X-linked（Bex） gene family are comprised of six proteins including Bex1, Bex2, Bex3, Bex4, Bex5 and Bex6. Recent studies suggest that Bex genes play roles in the development and function of the nervous system. Bex4, also named Bex L1 or Tceal7, is highly expressed in heart, skeletal muscle, liver and kidney. Our preliminary experiments showed that Bex4 also was present in testis. Therefore, this study aims to investigate the expression pattern and cellular localization of Bex4 in mouse testis, and explore the possible roles of Bex4 in the regulation of Sertoli cells. Methods:1. Immunohistochemistry and immunofluorescence methods were employed to identify the expression of Bex4 in mouse testis.2. The mice were randomly divided into groups containing 6 mice. The mice received 3.5 mg/kg Cd Cl₂, administered via intraperitoneal injection, and were sacrificed 5d thereafter. Subsequently, testis were removed for protein extraction or morphological analysis. The effects of Cd Cl₂ treatment on the expression levels of Bex4 in the testes of mice were determined using Western blotting. In vitro, TM4 Sertoli cells were treated with Cd Cl₂ for 24 h or 48 h. Subsequently, Western blotting was employed to investigate the protein levels of Bex4 in TM4 sertoli cells with Cd Cl₂ treatment.3. To investigate the role of Bex4 in TM4 Sertoli cells, TM4 Sertoli cells were transfected with Bex4 lentiviruses to upregulate the expression of Bex4. Then protein expression levels of Bex4 in TM4 Sertoli cells were detected by Western blotting. The cell cycle was analyzed by FACS. Additionally, Real-time PCR technique was used to detect the effects of Bex4 on specific genes of Sertoli cells. Results:1. The results of immunohistochemistry and immunofluorescence revealed that Bex4 was present in the Sertoli cells, Leydig cells and germ cells of mouse testis. Further study indicated that Bex4 protein was localized within the cytoplasm of TM4 Sertoli cells.2. To confirm the relationship between Bex4 and male reproductive function, a Cd Cl₂-induced mice model was established. Western blotting results indicated that treatment of mouse testis with Cd Cl₂ resulted in a significant increase in Bex4 expression. Furthermore, Bex4 expression was also significantly increased in Cd Cl₂-treated Sertoli cells in vitro,, which suggested that an increase in Bex4 expression is assisoated with Cd Cl₂-induced damage of male reproductive function. Additionally, Western blotting analyses suggested that the p38 signaling pathway was involved in the effect of CdCl₂ on Bex4 expression.3. Bex4-overexpression TM4 Sertoli cell model was established successfully. MTT showed that the proliferation of TM4 Sertoli cells had been suppressed by Bex4 overexpression. Flow cytometry indicated that Bex4 overexpression inhibited cell cycle progression occurred in the G1 phase in TM4 Sertoli cells. Real-time PCR results demonstrated that Bex4 downregulated several specific genes of Sertoli cells, such as DHH, Rhox5, ABP, Inhibin and clusterin.Conclusions:The study demonstrats that Bex4 is expressed in mouse testis, and is localized within the cytoplasm of Sertoli cells. In addition, Bex4 shows a significant increase in Cd Cl₂-treated mice or TM4 Sertoli cells. The p38 signaling pathway is involved in the effect of Cd Cl₂ on Bex4 expression in Sertoli cells. Bex4 overexpression is associated with proliferation, differentiation and functions of Sertoli cells. Our study demonstrated that Bex4 may be one of the targets of the reproductive toxicity of Cd Cl₂. The increase in the expression of Bex4 may affect the function of Sertoli cells, which in turn, results in testicular damge.