Recipient Preparation Of Mouse Spermatogonial Stem Cells Transplantation

The transplantation of spermatogonial stem cells (SSCs) will be significant for the male fertilityrecovery, spermatogenesis mechanism research and the transgenic technology set-up. The key ofsuccessful transplantation is recipient preparation. Intraperitoneal injection busulfan is the mostcommon method in recipient preparation for operational simplicity in lots of methods such as heatshock, radial treatment and so on. However, the toxcicity and fatality rate restricts the development ofthis technology. In this study, two preparation methods including testes injection and intraperitonealinjection the medicine busulfan had been compared. The different of recipient preparation efficiencyand physiological parameters had been examined. Then, the SSCs transplantation was performed to testthe feasibility of recipient preparation by injecting busulfan to testes.At the comparative study, first, to find the optimum condition of injecting medicine from micetestes. Following bilateral testes injection of busulfan at2,3, or4mg/kg/side into4~6weeks ICR mice,we examined their sections at14,21,28, and35days after injection. The conclusion was that4mg/kg/side was the optimal dose in the recipient preparation in mice, as in this dose the germ cells inthe seminiferous tubules can Initiate apoptosis and hollow quickly. Second, the reproductive and thephysiological parameters were compared at14,21,28,35and70days after the two injection whichwere testes injection and intraperitoneal injection into the mice. After intraperitoneal injection（40mg/kg）, the death rate was35.8%, RBC, WBC, HGB, PLT in the live were significant lower than thecontrol group which injected the mice with solvent DMSO and this phenomenon not disappeared until35days, the relative testis weight initially declined14days after injection and reached the lowest28days after injection and began to recover from then on. After testes injection (bilateral testes at dose of4mg/kg/side), all the mice were live, the blood parameters were normal, the change of relative testisweight were the same as the intraperitoneal injection group. It demonstrated that it was a availablemethod to give the busulfan by the way of testes injection.During the transplantation process the donor were6~15-day C57black mice, carrying the gene ofred fluorescent protein, and the germ cells were isolated and cultured and then identified by alkalinephosphatase (AKP); Then the transplantation procedure was implemented by means of testicularefferent injection.2months after that, donor derived germ cells were founded in the basementmembrane of recipient seminiferous tubules. It demonstrated that the new recipient preparation methodcan support the donor stem cells settled and migration. The transplanted Recipients were first mated at42days and this work was done7times at42-100days. The result showed that the recipient had normalability to sire offspring.The study showed that we established a highly efficient, economical，and nonfatal method forrecipient preparation in SSCs transplantation.