Isolation and Characterization of Rabbit Spermatogonial Stem Cells as a Promising Animal Genetic Resource for Biodiversity Protection.

The continual spermatogenesis throughout adulthood is ensured by a rare and unique cell group named spermatogonial stem cells (SSCs), which undergo self-renewal and/or differentiate into sperm cells. SSCs also become a promising genetic source for the protection of animal biodiversity. However, the isolation and culture of SSCs in vitro is still a big challenge and poorly explored in rabbits. The main objective of this study was to isolate, culture, and deeply characterize SSCs obtained from rabbit testes. Briefly, rabbit testicular tissue was mechanically and enzymatically dissociated, and obtained testicular somatic and germ cells were cultured for a short term in culture media supplemented with specific molecular factors maintaining SSC self-renewal and proliferation (GDNF, GFRα-1, FGF2, etc.). Immunofluorescent and PCR techniques were used for molecular profiling of cultured SSCs, while TEM analysis revealed their ultrastructure. After a few weeks, round and grape-like SSC colonies emerged, growing on the feeder cell layer. Rabbit SSCs showed positive staining for DBA, GFRA1, PLZF, RET, PGP9.5, DAZL, and DDX4. Increased expression of additional SSC markers was noticed using RT-qPCR and dd PCR (RET, PLZF, PGP9.5, DAZL, DDX4, CDH1, CD9, CD14, CD90, c-kit, ALDH, SSEA-4, SALL4, OCT4, and SOX2), while ultrastructure typical for primitive undifferentiated cells was observed under TEM. In conclusion, we successfully established a method for rabbit SSC isolation, culture, and phenotyping, which might facilitate their collection for further cryopreservation. However, the self-renewal, proliferative, and differentiation capacities of cultured SSCs still need to be confirmed through an in vivo SSC transplantation experiment.