Modified technique for spermatogonial stem cell transplantation into the seminiferous tubules in mouse model

This study aimed to develop a modified technique for spermatogonial stem cell (SSC) transplantation with the aid of an operating microscope in an infertile mouse model. Male neonatal C57BL/6 (B6) mice served as SSC donors. SSCs labeled with the PKH26-GL marker were detected by flow cytometry to verify purity. Adult B6 males were rendered infertile by busulfan treatment as the recipient. One month later the SSC suspension was delivered into recipient seminiferous tubules by manual microinjection under microscope with 100x magnification. This was compared to the conventional mechanical micromanipulator method via efferent ducts, rete testis, and seminiferous tubules, respectively. The volume injected and time required in the different procedures were compared. The recipient accepted manual microinjection via seminiferous tubules was subjected to histology, confocal laser scanning microscopy, and real-time fluorescent PCR at different checkpoints after transplantation. Positive controls received neither busulfan treatment nor transplantation. Negative controls were injected with an equal amount of transplant medium. The results showed that manual microinjection took 10 minutes per testis for the complete delivery of 50 µl of the SSC suspension, which was significantly less time-consuming and delivered a larger volume of SSC suspension than other methods. Transplanted SSCs demonstrated the earliest transference and colonization in recipient testes 7 days after transplantation. The newly generated germ cell layers appeared to be intact during spermatogenesis 90-days post-transplantation. This manual injection technique under microscope provides an alternative method to deliver the SSCs into the recipient seminiferous tubules.