Stem cells and spermatogenic lineage development in the primate testis

[摘要] Stem cells in the primate testis, Adark and Apale, have been designated as 'reserve” and 'active” stem cells, respectively. It is not clear if Adark participate in steady-state spermatogenesis; little is known about how these descriptions correlate with molecular markers or clone size and there are knowledge gaps on the potential use of primate stem cells for fertility preservation/restoration. We show that Adark are mitotically active, slow-cycling, label-retaining cells during steady-state spermatogenesis, with evidence of self-renewal and differentiation. We found that UTF1, ENO2 and UCHL1 are markers of Adark and Apale spermatogonia during development and they mark undifferentiated spermatogonia in adult Rhesus Testis. We also demonstrate that undifferentiated spermatogonia (UTF1+/cKIT- cells) exist more frequently as clones of 1 or 2, less frequently as clones of 3 or 4, clones greater than 4 are rare. cKIT+ clones of 1 (~20%) and 2 (~15%) cells were also observed. Our novel approach to staging seminiferous epithelium through EDU staining revealed that cKIT+ clones of 1 or 2 cells are not stage dependent. Our data indicate that primate spermatogonia undergo both symmetric (undifferentiated) and asymmetric (differentiating) divisions during steady-state spermatogenesis. For cell-based application of primate stem cell, we show that the concentration of xenotranplantable stem cells was higher in neonatal testis. Using ITGA6, stem and progenitor population were enriched by 18-fold (FACS) and 9-fold (MACS) while stem cell activity was enriched by 5-fold (MACS).We established foundational primate SSC culture conditions. ITGA6-positive cells produced colonies with 'grape-like” appearance in culture. ITGA6-positive cells retain phenotype of stem and progenitor cells up to 14 days in culture. For tissue-based application of primate stem cell, we grafted fresh and frozen-thawed prepubertal testicular tissues in the subcutis of the back and scrotal skin. Volume of testicular tissue grafts increased in graft sites. Although weight of recovered tissue grafts from the scrotal area were higher than from the back, spermatogenesis was confirmed in grafts with spermatids/sperm cells in more than 70% of tissue cross-section. Millions of sperm were recovered per graft and sperm were competent to fertilize oocytes, resulting in hatching blastocyst in vitro and pregnancy following embryo transfer.

[发布机构] the University of Pittsburgh