Dissociation, enrichment, and the in vitro formation of gonocyte colonies from cryopreserved neonatal bovine testicular tissues

Gonocytes play an important role in early development of spermatogonial stem cells and fertility preservation to acquire more high quality gonocytes in vitro for further germ cell-related research and applications, it is necessarily needed to enrich and in vitro propagate gonocytes from cryopreserved bovine testicular tissues. This study aimed to investigate the isolation, enrichment, and colony formation of gonocytes in vitro for germ cell expansion from cryopreserved neonatal bovine testicular tissues. The effects of several different in vitro culture conditions, including seeding density, temperature, serum replacement and extracellular matrices were investigated for the maintenance, proliferation and formation of gonocyte colonies in vitro. Frozen/thawed two-week-old neonatal bovine testicular tissues were digested and gonocytes were enriched using a Percoll density gradient. Cell viability was accessed by trypan blue staining and cell apoptosis was evaluated by TUNEL assays. Gonocytes were identified and confirmed by immunofluorescence with the PGP9.5 germ cell marker and the OCT4 pluripotency marker while Sertoli cells were stained with vimentin. We found that neonatal bovine gonocytes were efficiently enriched by a 30%–40% Percoll density gradient (p < 0.05). No significant differences were detected between neonatal bovine testicular cells cultured at 34 °C or 37 °C. The formation of gonocyte colonies was observed in culture medium supplemented with knockout serum replacement (KSR), but not fetal bovine serum (FBS), at a seeding density higher than 5.0 × 104 cells/well. A greater number of gonocyte colonies were observed in culture plates coated with laminin (38.00 ± 6.24/well) and Matrigel (38.67 ± 3.78/well) when compared to plates coated with collagen IV and fibronectin (p < 0.05). In conclusion, bovine neonatal gonocytes were able to be efficiently isolated, enriched and maintained in gonocyte colonies in vitro; the development of this protocol provides vital information for the clinical translation of this technology and the future restoration of human fertility.