Transplantation of Spermatogonial Stem Cells Suspension into Rete Testis of Azoospermia Mouse Model.

PURPOSE: The loss of spermatogonia following chemo-or radiotherapy leading to temporary or permanent infertility of the patient is a well known and unwanted side effect of many oncological therapies. MATERIALS AND METHODS: In this study, germ cells were isolated from 4 days old mouse testis cells. Busulfan treatment was used to the eliminate proliferating cells in the testis of recipient mice. The donor cells suspended in DMEM, were introduced into the rete testis of recipient mice via microinjection method. To distinguish the progeny of the transplanted donor stem cells from endogenous germ cells, BrdU-labeled cells were used. In addition, real time PCR was performed to determine expression levels of ngn3 and LIN28 (spermatogonia stem cells markers)before and after transplantation. Western blot analysis was further performed to detect an increase in - ngn3 expression after transplantation. RESULTS: Transplantations of stem cells into rete testis of the recipients was done. Our results clearly showed a significant increase in spermatozoa number in epididymal luman Spermatogonial stem cells (SSCs) did not show alkaline phosphatase activities while ngn3 and LIN28 were clearly expressed. Ngn3 and LIN28 expression were reduced after busulfan treatment compared to untreatmented mice. However, the expression of ngn3 and LIN28 increased after transplantation . BrdU-labeled testis cells were successfully transplanted into rete testis of recipient mice. These cells remained in rete testis of all recipient mice up to two months after transplantation. CONCLUSION: The present study clearly confirme that a regeneration after cytotoxic treatment was based on morphological criteria. We demonstrated the increase in stem cell numbers during regeneration and after transplantation. Transplantation of spermatogonial stem cells suspension by the injection of cells via the rete testis of recipient azoospermia model considerably enhances the efficiency of this procedure.

Isolation and culture of human spermatogonial stem cells derived from testis biopsy.

BACKGROUND: In cancer patients, chemo and radiotherapy can cause infertility by damaging spermatogenesis process. This process is based on self-renewal and differentiation of a rare population of the testicular cells called Spermatogonial Stem Cells (SSCs). Scientists have tried to isolate, enrich and culture Human spermatogonial stem cells, hoping to resolve infertility problems in cancer recovered patients in the future. METHODS: Spermatogonial stem cells were isolated and purified from human testicular biopsies sample consisting of at least 500,000 and at most 2,000,000 cells. Two enzymatic digestion steps were performed. Enriching methods, differential plating, and specific culture in serum-free medium with added growth factors: human GDNF, bFGF, EGF and LIF was performed on coated dishes. RESULTS: Human spermatogonial stem cell clusters were observed after 7 to 10 days in specific culture, then after several passages and successful expanding duration of 52 days, the cells were evaluated by three layer immunocytochemistry test (LSAB) to stain GPR125 protein as a surface marker in human spermatogonial stem cells. CONCLUSION: In current study human spermatogonial stem cell were isolated and expanded with the least manipulations in comparison with the other usual isolation methods like florescent or magnetic activated cell sorting. In contrast to the other SSCs isolation and culture methods, this system is based on the testicular biopsies against large samples, thus suggested method in this study is closer to clinical usage in the future.

The effect of human chorionic gonadotropin treatment on recipient mouse germ cell proliferation following spermatogonial stem cell transplantation of neonatal donor mice.

Spermatogonia are the male germ line stem cells whose life long expansion is needed for permanent production of spermatozoa. The present study was designed to examine the effect of hCG treatment on germ cell proliferation following stem cell transplantation in mice. Spermatogonial stem cells were isolated from neonatal mice testes and characterized by alkaline phosphatase, immunoreactivity and morphological analysis. hCG was injected into normal and cell transplanted mice. We then evaluated the testosterone levels and cell number in normal mice. After that, cyclin B1 gene expression was investigated in transplanted mice. Different doses of busulfan were injected to investigate the effects of chemotherapy on morphological criteria and preparation of recipient mice for transplantation. In this report we show proliferative potential of spermatogonial stem cells after cytotoxic treatment, transplantation efficiency by semi-quantitative RT-PCR, and hCG effect on stem cell regeneration in normal mice and following cell transplantation. The results indicate that spermatogonial stem cells can proliferate after transplantation, and the efficiency of their transplantation depends on hormonal treatment. Therefore, hormonal treatment after stem cell transplantation will be a powerful avenue for increasing the efficiency of transplantation and fertility restoration.