| Background:Spermatogonia stem cells (SSCs), the origin of spermatogenesis, are a cluster of adult stem cells which have the potential of both self-renewal and differentiation. To uncover the mechanism behind SSCs proliferation and differentiation is a prerequisite to the progress in SSCs culturing and transplantation and regulation in spermatogenesis. The extremely low content of SSCs in germ cells and the concurrence of all stages of spermatogenesis in seminiferous tubules constitute two main obstacles to this mechanism research in vivo. Busulfan, an alkylating agent, can kill germ cells in a manner of dose and species dependant. If busulfan evanishes most SSCs, doubtless, SSCs' self-renewal will be strengthened and their differentiation suspended in the initial of spermatogenesis, from which synchronization of SSCs proliferation and differentiation could be achieved.Objective:To establish a model of spermatogenesis recovery and achieve the relative synchronization of the SSCs proliferation and differentiation in male Kunming mice, and base on that, to detect the difference in gene expression patterns of mouse testis during SSCs proliferation and differentiation and probe preliminarily into the molecular regulation mechanism in SSCs proliferation and differentiation.Methods:Adult male Kunming mice were randomly treated with busulfan injection intraperitoneally at different doses or types (A:single dose of 10mg/kg; B: single dose of 20mg/kg; C:two doses of lOmg/kg,24 days apart; D:two doses of 15mg/kg,24 days apart). Mice that received single injection of 50% DMSO (10ml/kg) were used as control. Testes were dissected out 1w,2w,3w,4w,6w and 8w after treatment. Testes of mice in control group were all taken out one week after the treatment. The expression of Ki-67 in germ cells was detected by immunohistochemistry in two doses of busulfan treatment groups and control group. A mice model of spermatogenesis regeneration was reestablished with the suitable busulfan dose determined by the aforementioned study. A 36k Mouse Genome Array was used to detect the differential gene expression profiles between stages of SSCs proliferation and differentiation. Bioinformatics analysis was conducted in GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway to describe the potential roles that may play in regulation of spermatogonial stem cells behavior. Oct4 and Thy-1 expression patterns in spermatogonia were detected by immunohistochemistry.Results:The damage effects in seminiferous epithelium by two doses of 10mg/kg or 15mg/kg busulfan injection were between single dose of 10mg/kg and 20mg/kg. Two weeks after the second injection, only fewer dispersed spermatogonia (mainly As type) survived in close contact with the basal portions of adjacent Sertoli cells in group C and D, vacuolization of Sertoli cells appeared in some seminiferous tubulues in group D. In week 3, the number of As spermatogonia increased. In week 4, differentiated spermatogonia and spermatocytes appeared. In week 6-8, spermatogenesis regeneration took place and morphology of seminiferous epithelium got back to normal gradually in group C, while diameter of seminiferous tubulues and thickness of seminiferous epithelium in group D were significantly lower than control group. In week 1 and 2, the Ki-67 positive rates of spermatogonia in group C and D were extremely lower than control group. They rebounded rapidly to apex in week 3, descended in week 4 and restored to the control level in week 6 and 8. In week 2 and 3, Ki-67 positive rates of spermatogonia in group C were significantly higher than group D.911 differential expression genes were identified by gene arrays in mice testes, consisting of 608 up-regulated and 303 down-regulated in SSCs proliferation stage vs. SSCs differentiation stage. The differential expression genes were classified by their biological process, molecular function and cellular component, respectively. Alterations with statistical significance (P<0.05) appeared in 84 KEGG signal pathways, including Notch and Wnt signaling pathways which have been proved to be important for stem cell maintenance.56 differential expression genes were selected as genes related to stem cells, among which 40 genes were up-regulated, including some stem cell biomarkers (such as Cd9, Stra8, Itgbl, Oct4 and Thy1) and some growth factors (such as Fgf2, Pdgfa and Csfl). Oct4 antigen was expressed in spermatogonia located in the basal portions. The positive rate of Oct4 in spermatogonia in SSCs proliferation stage was higher than that in SSCs differentiation stage.Conclusions:24 days apart two doses of 10mg/kg busulfan treatment could establish an ideal mice model of spermatogenesis recovery. Relative synchronization of spermatogenesis could be achieved in this model. After the administration, the 3rd week was mainly proliferation stage of spermatogonial stem cells, the 4th week was mainly differentiation stage, and week 6 to 8 was stage of spermatogenesis regeneration. The regulation of SSCs proliferation and differentiation involved in many genes expressed differentially locating in various signal pathways. This study provided a basis for the elucidation of the molecular mechanism behind self-renewal and differentiation of spermatogonial stem cells in vivo.
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