Although numerous inbred lines and endangered wild populations of medaka exist, the only method currently available for preserving these 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. Our group developed a novel method termed 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. Importantly, the cryopreserved germ cells can be differentiated into functional oocytes. After examining cryobiological properties of medaka spermatogonia, we established a vitrification method for medaka whole testis including spermatogonia, and further established that this method can be applied for cryopreservation of zebrafish whole testis.
When vitrified spermatogonia from the olvas-GFP transgenic strain (body color: orange) were transplanted into larvae of a non-transgenic strain (body color: black), female recipients produced oocytes showing green fluorescence. In their gonads, there were recipient-derived oocytes together with donor-derived green fluorescent oocytes. Then, infertile triploids were used as the recipients in the next experiment. When cryopreserved spermatogonia were transplanted into triploid larvae, the mature recipients produced only sperm and eggs showing green fluorescence, which is the donor-derived characteristic. Moreover, the body colors of the offspring obtained by mating the recipient males and females were orange (100%, 1169/1169), suggesting that all of them were derived from cryopreserved spermatogonia. Thus, we succeeded in producing the donor-derived offspring by allogenic transplantation of cryopreserved spermatogonia in medaka. Finally, we confirmed that this system can be applied on vitrified testes for the production of an inbred line, or endangered wild populations (Tokyo-medaka). These techniques will facilitate the establishment of a stable and reliable system for preserving valuable medaka strains semi-permanently.