070 Production of donor-derived offspring by allogenic transplantation of cryopreserved spermatogonia in medaka

Although numerous inbred lines and endangered wild populations of medaka exist, the only method currently available for preserving the resources is to rear live individuals, as protocols for cryopreservation of fish eggs or embryos have not yet been successfully established due to their large size. However, rearing live individuals is always associated with the risk of mutation due to mobile elements, and the loss of parent fish through infectious disease or accidents related to rearing conditions and equipment. Our group developed a novel method named surrogate broodstock technology. By transplanting donor germ cells into recipient fish of another species, the donor germ cells mature into sperm or oocytes in recipient gonads (Takeuchi et al., 2004; Okutsu et al., 2006; Yoshizaki et al., 2011). More importantly, the germ cells cryoporeserved can be differentiated into functional oocytes (Okutsu et al., 2007; Lee et al., 2013). In this study, we aimed to cryopreserve spermatogonia that can be differentiated into both sperm and eggs. When medaka spermatogonia were dehydrated with hypertonic solutions at 25°C, the survival was decreased in 0.5 M sucrose solution. However, survival rates were improved, when dehydration was carried out at 0°C. Toxicity assay of cryoprotectant (−10%, 25°C) revealed that propylene glycol (PG) was the least toxic, while ethylene glycol (EG) proved less toxic than glycerol and Me2SO. Based on these results, vitrification solutions containing ficoll and sucrose were based with either PG or EG and the testes were vitrified after exposure to them at 0°C. Although survival of cells treated with PG was low, survival rate of 44.9% was obtained using vitrification solution containing EG. Thus, vitrification method of medaka whole testis includes spermatogonia was established. When vitrified spermatogonia from vasa-GFP transgenic strain (body color: orange) were transplanted into larvae of non-transgenic strain (body color: black), female recipients produced oocytes showing green fluorescence. In their gonads, however, there were recipient-derived oocytes together with donor-derived green fluorescent oocytes. Since recipients producing gametes derived only from transplanted donor germ cells are desirable, infertile triploids were used as the recipients in the next experiment. Triploid medaka was prepared by placing fertilized eggs at 41°C water for 5 min (Naruse et al., 1985). When cryopreserved spermatogonia were transplanted into triploi