The Effect Of Busulfan On Production Of Testicular Germ Cells, Sexual Behaviour And Sterility Of Male Mice

Background:Busulfan is a kind of alkylating agent, also known as myleran, named as:1, 4-Butanediol dimethyl sulfonate. Though it is widely used in the chemical treatment of chronic myelocytic leukaemia and pretreatment of haematopoietic stem cell transplantation in patients, systemic studies of the effect of busulfan on male's production of germ cells, sexual behaviour and sterility are very few. Busulfan is also usually used to make recipient animal models before spermatogonial transplantation, but the optimal dosage of busulfan is not clear.Objective:To investigate the effect of busulfan on production of spermatogenic cells, sexual behaviour and sterility of male mice; to investigate the optimal dose of busulfan for depleting testicular germ cells of recipient mice before spermatogonial transplantation.Methods:A total of 221 male BALB/c mice aged 4-6 weeks received different doses of busulfan (10-50mg/kg body weight) by intraperitoneal (i.p.) injection, and the livability was examined. Then the body weight of the mice, testicular mass and histomorphological changes of testes affected by different doses of busulfan were examined throughout the experimental period. Flow cytometry was also used to examine the effect of different doses of busulfan on the percentage of haploid cells (spermatids and spermatozoa) in testes. Optimal dose of busulfan for depleting recipient germ cells before spermatogonial transplantation was determined by statistical analysis of detailed quantitative data mentioned above. After all or most of the seminiferous tubules became normal, the male mice were caged with the homogenic estrous virginal females in couples. The sexual behaviors of the males suffered by different doses of busulfan including mounting, intromission and ejaculation in 15 minutes immediately after the meeting were examined. The females were separated from the males after mating or after 5 days if no mating took place, and the childbirth rate of the females was also examined.Results:The livability of the mice treated by 10 to 20mg/kg busulfan was 100%.30mg/kg busulfan led to a little decreased livability (93.5%), which had no statistical difference compared with the control group.40 and 50mg/kg busulfan led to obviously decreased livabilities, which were 13.3% and 0% respectively. Owing to the unacceptable high mortality, the mice treated with 40-50mg/kg busulfan were omitted from further studies.10-30mg/kg busulfan had no obvious effect on the body weights of the mice, but did harm to the production of the testicular germ cells. At the end of week 4 after the administration of 10-30mg/kg busulfan, we found that the higher doses treated, the more testicular mass lost, and the lower percentage of normal seminiferous tubules and haploid cell in testes remained. Then we traced these parameters in 8 to 48 weeks after the busulfan treatment, and found that the higher doses of busulfan administrated, the longer time needed for the parameters to become normal, including testicular mass, percentage of normal seminiferous tubules and haploid cells in testes. The statistical analysis of the detailed quantitative data mentioned above indicated that 30mg/kg busulfan can create sufficient niches by depleting maximal endogenous spermatogenic cells, and maintain the microenvironments for a sufficient long time so that colonization of donor-derived spermatogonial stem cells can take place. At week 24 after the administration of 20-30mg/kg busulfan, the male BALB/c mice showed lower incidence rate of mounting and the coupled females appeared lower childbirth rate compared with the control group.Conclusion:1. Doses of busulfan no more than 30mg/kg had no obvious effect on the livability of the mice.40-50mg/kg busulfan led to a clearly decreased survival rate, among which 50mg/kg was 100% lethal. 2. The higher doses of busulfan administered, the more damage on production of testicular germ cells, and the longer time needed for the self-recovery of the spermatogenic ability.3. Busulfan can inhibit the sexual behaviour of the male mice and lead to sterility.4. 30mg/kg was the optimal dose of busulfan for depleting recipient germ cells before spermatogonial transplantation.