T3 Reglute The Expression Of Connexin43 And Maturation Of Seminiferous Epithelium Induced With Flutamide In SD Rat

Objective To establish the animal model of cryptorchidism induced with flutamide in gestation period of SD rat. The purpose is to investigate flutamide inpact on lumen of seminiferous tubule, maturation of Sertoli cell, expression of connexin43 in testis, and T3 regulate expression of connexin43 , maturation of Sertoli cell.Methods First, we established SD rat cryptorchidism model by subcutaneous injection flutamide(25mg/kg·d)in gestation 12-21day, And in postnatal 1-13 day, injected T3(15μg/kg·d). Second, we investigated the protein distribution and mRNA expression of connexin43 in testis on PD13, PD 15, PD17 and PD20 by immunohistochemistry and real-time PCR. Third, we observed lumen index, diameter, area of seminiferous tubule and distribution of Gonocytes on PD10 by HE stain. Fourth, we analyzed the SGP-2 mRNA expression of Sertoli cells on PD13, PD 15 and PD17 by real-time PCR. Fifth, we counted Sertoli cells which incorporated BrdU on PD13, PD 15 and PD17 by immunohistochemistry to evaluation labeling index.Results①The connexin43 mRNA expression in flutamide-induced testis was significantly lower than control group on PD13 and PD15(P﹤0.05). However, there was no significance different between flutamide-induced and control group on PD17(P﹥0.05). The expression of connexin43 mRNA in flutamide-induced undescended testis was significant lower than control and flutamide-induced descended testis group on PD20(P﹤0.01), but there was no significance different between flutamide-induced descended testis and control group on PD20(P﹥0.05). After treated by T3, the expression of connexin43 mRNA in flutamide-induced undescended testes treated by T3 was significant higher than that without T3 group (P﹤0.05), but there is not significance different with normal testis (P﹥0.05) .②The connexin43 protein expression in flutamide-induced testis group was lower than control group on PD13 and PD15 (Fig 1-5A,B and 1-6A,B) . In normal testis on PD17 and PD20, there was no connexin43 protein location in spermatogonial cell layer of seminiferous tubule, but in testis of flutamide-induced group on PD17 and PD20, the connexin43 protein could been observed in spermatogonial cell layer (Fig 1-6C,D and 1-7A,B,C). After treated by T3, in seminiferous tubule, the connexin43 protein expression in testis flutamide-induced on PD13 was significantly increased, and without any significantly different with control group(Fig 1-5C) . On PD20 , the connexin43 protein in flutamide-induced descended testis treated by T3 was located in right area of seminiferous tubule , but in flutamide-induced undescended testis treated by T3, the connexin43 protein could also been observed in spermatogonial cell layer of seminiferous tubule (Fig 1-7D,E).③With time from PD13 to PD17, both the sertoli cell labeling index of BrdU in control and flutamide-induced group were decreasing. The sertoli cell labeling index in flutamide-induced testis on PD13, PD15 and PD17 was higher than control (P﹤0.01,Fig 2-7). After treated by T3, the sertoli cell labeling index in flutamide-induced testis on PD13 was significance lower than which without treated by T3 and control group(P﹤0.01).The SGP-2 mRNA expression in flutamide-induced testis was no statistical significance with control group on PD13(P﹥0.05) ; But on PD15 and PD17, the SGP-2 mRNA expression in flutamide-induced testis was lower than control group(P﹤0.01). After treated by T3, the SGP-2 mRNA expression in flutamide-induced testis on PD13 was significance higher than which without treated by T3 and control group (P﹤0.01).Conclusions①After induced by flutamide in gestation period of SD rat,the connexin43 mRNA expressed down-reglutiong and protein located abnormally,T3 may be up-reglute expression of connexin43 and maintaining it in a normal level.②After induced by flutamide in gestation period of SD rat, the function maturation of sertoli cells may be delayed, T3 maybe have function to promote maturation of sertoli cells.③After induced by flutamide in gestation period of SD rat, the formation of lumen of seminiferous tubule was delayed and gonocytes always remained in centre of seminiferous tubule, T3 maybe have function to promote formation of lumen of seminiferous tubule.