72 PRESERVATION OF PORCINE GONOCYTES AT 4°C AND IN LIQUID NITROGEN – A PRESERVATION MODEL OF GENETIC RESOURCES IN DOMESTIC ANIMALS AND IN ENDANGERED SPECIES

Abstract

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.