Abstract

Obtaining somatic cells from preserved organs or tissues is useful for the conservation and regeneration of genetic resources by nuclear transfer (NT). Bovine cells for NT have been obtained from cooled carcasses stored at 0°C for several days (Arat et al. 2005 Reprod. Fert. Dev. 17, 164 abst) and from fetal skin tissue cryopreserved with DMSO (Fahrudin et al. 2001 J. Vet. Med. Sci. 63, 1151–1154). However, frozen storage of organs or tissues without cryoprotectants was considered to be quite inappropriate for obtaining viable cells. We report here that viable donor cells for NT were obtained from bovine testicles after frozen storage without cryoprotectants. In the first experiment, we investigated whether viable cells can be recovered from frozen testicles castrated from Japanese Black bulls. The testicles were frozen at -80°C in a freezer for several days; then some were stored in liquid nitrogen for 10 months without cryoprotectants. Before thawing, the testicles were divided into 3 pieces, caput epididymis, cauda epididymis, and testis. Each piece was then put in saline at 42°C for quick thawing. Thawed tissues were minced into 5-mm pieces and incubated at 39°C for 2 h in DMEM containing 0.1% collagenase and 0.2% dispase. After filtration through a 250-µm nylon mesh filter, the filtrates were centrifuged at 250 × 4g for 5 min. Then precipitates were resuspended with MF-start^® primary culture medium (TM Cell Research Inc., Fukui, Japan) and incubated at 38.5°C under the atmosphere of 5% CO₂ in air with high humidity. After 5 days of incubation, the medium was replaced and nonadherent debris was discarded. Viable cells were obtained from the caput epididymis. These cells actively proliferated and expanded. In the next experiment, to determine whether these cells can be used for NT, the cells were electrically fused with enucleated bovine oocytes. Bovine fibroblasts taken from unfrozen ear tissue were used as controls. The NT embryos were activated by Ca-ionophore treatment, followed by treatment with cycloheximide for 6 h, and then cultured in mSOF for 168 h. NT embryos reconstructed from testicle cells did not significantly differ from NT embryos made with control cells with regard to blastocyst rates (22.1% and 20.2%), cell number of blastocysts [130 ± 43 and 121 ± 43 (mean ± SD)], and ICM ratio (21.1% and 22.6%), respectively (ANOVA). These results suggest that somatic cells derived from bovine frozen testicles can be used for nuclear transfer. Further studies are needed to examine whether viable cells can be obtained from other frozen organs or tissues.