Successful in vivo Transplantation of Cultured and Enriched Testicular Germ Cells of Pre-Pubertal Bucks to Busulfan-Treated Homologous Recipients

The objective of the present study was to establish a workable approach for the production of germ cell (GC)-depleted recipient goat model using intra-testicular busulfan treatment and transplantation of cultured and enriched caprine-male GC (cmGCs) into the homologous recipients under ultrasonography (USG) guidance. The evaluation of post-transplantation colonization of donor cmGCs and restoration of the normal architecture of seminiferous tubules (ST) was performed. For this, the cmGCs of pre-pubertal male goats were isolated and enriched by differential platting for culture until the third passage. Thereafter, cells were harvested and further enriched by magnetic-activated cell sorting using rabbit-anti-CD90 antibody. After confirmation of metabolic viability (MTT-assay) and cluster-forming ability (crystal violet staining) of CD90+ cmGCs, the cells were labeled with a lipophilic red-fluorescent dye (PKH26) before transplanted into the recipient male goats by injection directly into the mediastinum testes under USG guidance. The colonization and repopulation of transplanted CD90+ cmGCs into the recipient ST was observed up to 8 weeks post-transplantation. The PKH26-labeled donor cell-derived colonies were identified in enzymatically digested ST and cryosections of recipient testes. Moreover, histochemical analyses revealed the restoration of the normal architecture of ST of recipient testis after GC transplantation. Therefore, the results suggest that the reproductive competence of infertile animals can be restored through mGC therapy and thus the methodology presented herein could be useful to obtain donor mGCs-derived functional male gametes in the recipient animal testis.