Depletion of endogenous germ cells in tree shrews in preparation for spermatogonial transplantation

To achieve successful spermatogonial transplantation, endogenous germ cells must be depleted in recipient animals to allow donor germ cells to colonize efficiently. Busulfan is commonly used for the depletion of endogenous germ cells in recipient males. However, the optimal dose of busulfan is species-specific, and the optimal dose in tree shrews is yet to be determined. The current study aimed to determine the optimal dose of busulfan for effective suppression of endogenous spermatogenesis in tree shrews. Different doses (15, 20, 25, 30, 35, 40 and 45 mg/kg) of busulfan were injected into tree shrews intraperitoneally. Survival rates of the different treatment groups were calculated at 2 weeks and body weights were measured at 4, 6, 8, 10 and 28 weeks post-busulfan treatment. The testes were also removed and weighed at 4, 6, 8, 10 and 28 weeks post-treatment, and the cross and longitude diameters of the testes and diameters of the seminiferous tubules were measured and histologically evaluated. It was observed that there were no significant differences in the survival rates between the 15-35 mg/kg treatment groups and the control group (P>0.05), while the survival rate of the 40 mg/kg treatment group significantly decreased relative to the control group (P<0.05) and the survival rate of the 45 mg/kg treatment group was 0% (P<0.05 vs. control). In addition, the weight and diameters of the testes, diameters of the seminiferous tubules and proportion of normal type tubules in the 40 mg/kg group significantly decreased over 4-10 weeks relative to the control group (P<0.05), though gradually recovered with time. At 28 weeks, the recovery was significant relative to 4 weeks (P<0.05). Similarly, histological analysis indicted that recovery of abnormal tubules was delayed in the 40 mg/kg group relative to the lower dose groups as the 40 mg/kg dose tree shrews had more tubules with no spermatogenesis compared with the lower dose group at the same time points. These data indicate that a busulfan dose of 40 mg/kg is optimal for the depletion of endogenous germ cells in tree shrews. This dose led to maximum suppression of endogenous spermatogenesis while maintaining an acceptable survival rate of >50% of the lethal dose of busulfan for tree shrews.