Gonocytes are primitive germ cells that reside in neonatal testis and are believed to be progenitor-type stem cells that differentiate into spermatogonial stem cells. Because of their self-renewal ability, gonocytes may be one of the targets for cryopreservation of genetic resources in domestic animals and in endangered species. However, there are only a few reports regarding the preservation of gonocytes and spermatogonial stem cells isolated from the testis. In this experiment, porcine gonocytes were used as a model for preservation of genetic resources. Porcine testes were collected at 2–6 days after birth. They were divided into the 5 experimental groups for storage: (1) DMEM/F12 medium, (2) DMEM/F12 + 15 mm HEPES, (3) PBS, (4) PBS + 15 mm HEPES, and (5) Liquid-Free, and stored at 4�C for 24 h. The testes were minced by scissors and digested with 2-step enzyme treatments. The gonocytes were isolated by Percoll density gradients and recovered from the fraction between 50 and 60%. The viability of cells was assessed using trypan blue dye exclusion. To determine optimum cryopreservation conditions for gonocytes, 10% DMSO, 10% glycerol, and 0.07 mm sucrose were used as cryoprotectants. The isolated gonocytes were suspended in DMEM/F12 + 10% FBS containing cryoprotectant at 4�C, kept at –80�C overnight, and finally immersed in liquid nitrogen. After freezing and thawing of gonocytes, cells were examined for viability and then cultured in DMEM/F12 + 10% FBS in 5% CO2, 95% air at 37�C in humidified atmosphere. Identification of gonocytes was performed using a specific marker of gonocytes, a lectin Dolichos biflorus agglutinin (DBA; Goel et al. 2007 Biol. Reprod. 77, 127–137). The gonocytes were recovered from testes at the purity level of around 70%. Cell viability in average after storage of testes at 4�C was significantly higher in DMEM/F12 + HEPES (95.3%) and PBS + HEPES (89.8%) than in DMEM/F12 (73.9%), PBS (79.7%), and Liquid-Free (72.2%) (P < 0.05; ANOVA). The addition of HEPES in storage medium seemed to be effective for improving cell viability. The use of 10% DMSO and 0.07 mm sucrose as cryoprotectants supported high cell viability (74.4%) of gonocytes after freezing and thawing. The addition of glycerol had an adverse effect on cell viability after freezing (18.3%). When cells were cultured, gonocytes started to form colonies after 3 days and continued to proliferate for at least 7 days in culture. These colonies showed DBA affinity and maintained their nature as gonocytes. The viability of gonocytes can be maintained in the testis at 4�C for at least 24 h and after freezing and thawing. The stored gonocytes successfully proliferated in culture for at least 7 days. In conclusion, these results may provide useful information for short-term storage of primitive germ cells and preservation of genetic resources in domestic animals and in endangered species. It may also have implications for assisted reproductive technology in humans.
Restoration of Genetic Resources from Ehime Native Chicken via Transferred Primordial Germ Cells (PGCs)