Successful transplantation of spermatogonial stem cells into the seminiferous tubules of busulfan-treated mice.

Spermatogonia (SSCs) in the testis transmit genetic information to the next generation. Successful SSC transplantation into infertile men is an advanced therapeutic application in reproductive biology research. In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology­immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. IMH analysis showed different expression levels of Zbtb16 in both populations. Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene and the down-regulated expression of DAZL, VASA, and Zbtb16 during SSCs differentiation of (P < 0.05). Flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. We performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm. Data analysis confirmed that zbtb16 is expressed in the undifferentiated germ cells located on the basal membrane of seminiferous tubules and SSCs in vitro. Also, spermatogenesis was resumed, and fertility improved after transplantation of undifferentiated cells into busulfan-treated mice; thus, improvements in vitro SSCs transplantation, isolation and culture would be helpful in future clinical treatments to solve the reproductive problems of families influenced by infertility.