Production of fertile zebrafish (Danio rerio) possessing germ cells (gametes) originated from primordial germ cells recovered from vitrified embryos.
This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos.
Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. ytaka@vetmed.hokudai.ac.jp
Shogo
S
Higaki
Yoshiki
Y
Eto
Yutaka
Y
Kawakami
Etsuro
E
Yamaha
Noriko
N
Kagawa
Masashige
M
Kuwayama
Masashi
M
Nagano
Seiji
S
Katagiri
Yoshiyuki
Y
Takahashi
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002469:Cell Separation; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D015925:Cryopreservation; D046149:Embryo Culture Techniques; D004625:Embryo, Nonmammalian; D005260:Female; D005298:Fertility / Q000502:physiology*; D005854:Germ Cells / Q000166:cytology / Q000502:physiology*; D008297:Male; D015017:Yolk Sac / Q000166:cytology; D015027:Zebrafish / Q000196:embryology* / Q000502:physiology