O-38: Evaluation the Expression of Bax, Bcl-2,p53 & Survivine after Transplantation of SpermatogonialStem Cells to Cryptorchid MouseModel

Background: Disruption in spermatogenesis and infertility occurred after local testicular heating. In this study in addition to evaluation the long term effects of experimental bilateral and unilateral cryptorchidism on sperm parameters and structure of mouse testis, the genes that involved in programmed cell death after cryptorchidism and treatment methods of decreasing the germ cells in seminiferous tubules after heat exposure were examined. Materials and Methods: To induce bilateral and unilateral cryptorchidism model, immature mice (age under 2 months) were anesthetized, a small incision was made in the abdominal skin and then fat pad at the upper end of testis was sutured to peritoneum. Testes were removed after 2, 4, 6 and 8 weeks after surgery, weighted and processed for light microscopy study. Expresssion of apoptotic genes (Bax, Bcl-2, p53 and survivin) and their proteins were evaluated at different times after surgery with semiquantitative RT-PCR and immunhstochemistery methods. In next steps, Sertoli and spermatogonial cells were isolated from cryptorchid mice testes using two step enzymatic digestion and lectin immobilization. The identity of the isolated cells was confirmed by analysis of immunocytochemistry against oct-4 and vimentin. Isolated spermatogonial cells were co-cultured with Sertoli cells three weeks. Colony assay was performed by means of a light microscopy at the end of every week. Finally two treatment methods were examined. In transplantation group, cultural spermatogonial cells were marked with BrdU and with 105 concentrations transplanted into seminiferous tubules of left testis of cryptorchid mouse 3 months after descendingsurgery and then returned to scrotum. In another group, testis of mouse model returned to scrotum 2 months after ascending surgery. Eight weeks after treatment, testicular structure, ultra structure, expression of apoptoticgenes and proteins were examined. Statistical analysis performed by statistical tests using ANOVA and Tukey’s methods. Results: Spermatogenesis was arrested and the testicular weight and seminiferouse tubular diameters were significantly reduced in the bilateral undescended testis compared with unilateral undescended testis and the control mice. Spermatocytes and spermatids were the main type of germ cells undergoing apoptosis in all groups. RT-PCR data further verified the alteration of p53, Bax and Bcl-2 mRNA expression in cryptprchid testis in a time dependent manner. The expression of survivin 225 and 140 strongly decrease in bilateral groups comparing with unilateral and control groups, but survivin 332 was lower in both experimental group. These changes were remarkable in bilateral group in compare to unilateral group. Immunohistochemistry data showed that the intensity of p53 and Bax expression mainly increased in remainder cells in cryptorchid testis and rates of Bcl-2 and survivin expression mainly in bilateral group decreased. Transplantation of spermatogonial stem cells into the mouse seminiferous tubules was successful in recipients which had severe tubular degeneration after induction of cryptorchidism compared with the group that testis was descend to scrotum. After transplantation of spermatogonial stem cell in to the cryptorchid testis, germ cell colonization was shown, the number of spermatogonia, spermatocytes returned to near normal range but spermatogenesis was recoveredpartially at the late stages of spermatogenesis. Conclusion: In general, high temperature causes a decrease in all analyzed parameters except spermatogonial cell number in consequently this model is suitable for enrichment of spermatogonial stem cells. RT-PCR and immunohistochemistery methods observations suggest that multiple molecular pathways participate in the germ cell apoptosis induced by cryptorchidism. The reason of partially return of spermatozoa after transplantation maybe is the severe effects of surgery on the blood supply or innervations of the testis and also disruption in reconstruction of serous sac around the testis without adhesions, which are important for normal spermatogenesis.