Reproduction Abstracts

In mice, a technique was developed in which transplanted spermatogenic stem cells (SSCs) reconstitute complete spermatogenesis in the testes of infertile recipient. However, the dynamics of SSCs during the reconstitution process after transplantation is largely unknown. To dissect post-transplantation dynamics of SSCs, the behavioral process of donor spermatogonia was observed by in vivo live-imaging, and their fate was analyzed at a single-cell resolution. For live-imaging, singly dissociated testicular cells from UBI-EGFP mice (1×10⁶ cells/testis) microinjected into the seminiferous tubules of were filmed according to the protocol of Yoshida et al. (2007). For fate analysis, spermatogonia expressing GFRα1, a SSCs marker, were pulse-labeled with persistent GFP expression following administration of 4OH-tamoxifen to GFRα1/CreER^T2;CAG-CAT-EGFP mice, and transplantation was conducted as same way described above. The constitute numbers of cells in individual clones were scored according to the GFRα1 expression by whole-mount immunostaining of recipient tubules. The live-imaging study has revealed that a single isolated spermatogonium actively migrated with extend lobopodia while interconnected spermatogonia actively divided adjacent to the vasculature. In addition, the death of donor spermatogonia was frequently observed. Fate analysis suggested that vast majority of GFRα1+ spermatogonia that first settled on the basement membrane disappeared (probably death) within 20 days post-transplantation, while a tiny part of surviving GFRα1+ spermatogonia reconstituted colonies. Interestingly, the fates of individual pulse-labeled GFRα1+ spermatogonia were found highly variable. The average numbers of GFRα1+ spermatogoina and GFRα1⁻ spermatogenic cells in each clone were exponentially increased up to 20 days post-transplantation, and then remained constant. At present, we are trying to mathematically capture such of post-transplantation dynamics of SSCs.