Preliminary Establishment Of A Mouse Model For Spermatogonial Stem Cell Transplants

In the adult male, spermatogonial stem cell continuously undergoes self-renewal and differentiation, which is the basis of spermatogenesis. Spermatogonial stem cell transplants are the gold standard of identification of spermatogonial stem cell, and provide the possibility for the treatment of male infertility. The objective of this study was to build a model of mouse testis, and lay a good technical basis for subsequent study for large animals.Chapter1:Optimal conditions of busulfan treatment for recipients were investigated. Physiological parameters (relative testis weights, the rate of hollow seminiferous tubule, and Mortality of mice) were measured as the evaluation standards for the effect of busulfan treatment on Od,3d,14d,21d,28d,35d,42d. The result showed that the relative testis weights for10mg·kg-1group were very low6w after treatment. The relative testis weights were low4w after treatment for the15mg·kg-1group, which was significantly different compared to3w after treatment, but there was no difference compared to5w and6w after treatments. The relative testis weights of all groups of treatments for the intraperitoneal injection were low4w after treatment, and there was no significant difference compared with5w and6w after treatment. Following single injections of busulfan, the cells in the seminiferous tubule disintegrated and died gradually. The rate of hollow seminiferous tubule of10mg/kg group was100%6w after treatment. The rate of hollow seminiferous tubule for the15mg·kg-1group was100%4w after treatment, and there was significant difference compared with3w after treatment. There was no sign of recovery in the two weeks. The rate of hollow seminiferous tubule of all groups with intraperitoneal injection was100%4w after treatment, and there was no significant difference compared with5w and6w groups. Histological sections revealed that nuclei of the cells had become pyknotic and had then undergone karorrhexis and karyolysis in the process of cell death. Although there was no significant difference in mortality among the groups, the mortality rate raised as the increase of the dose. The dose of40mg·kg-1was the LD50for animal survival. When compared with40mg·kg-1group, the mortality rate for15mg· kg-1group reduced by25%. In conclusion, for the safety of survival of the donor cells after transplantation, the optimum time for the spermatogonial transplants would be5w after injection with the dose of15mg·kg-1even though the relative testis weights were low4w after treatment.Chapter2:Transplant of spermatogonial stem cell and identification of sperm were studied. After the recipients were prepared according to the first chapter, spermatogonial transplants were operated and detected by microsatellite DNA technique after3mon. The selection of spermatogonial stem cells for spermatogonial transplants was based on different adherent rate and the cells were transplanted into seminiferous tubule through efferent tubes. Three months after transplantation, the mice were killed, and testises were removed for histological sections, and tails and epididymis were removed for detection by microsatellite DNA technique. The results showed that there was no sperm from the donor cells.In summary,5w after injection with the dose of15mg·kg-1is the optimum time for spermatogonial transplants. And transplantation technique was mastered. But the reason why the cells from the donor did not proliferate and differentiate in the seminiferous tubule of recipient mice needs further research.