Cryopreservation of male and female gonial cells by vitrification in the critically endangered cyprinid honmoroko Gnathopogon caerulescens
We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively.
KeywordsCryopreservation Endangered cyprinid Oogonia Spermatogonia Vitrification
The authors thank Syuichi Shimomura (Kusatsu Honmoroko Seisan Kumiai), Tetsuo Yamamoto (Yamasho Honmoroko Youshoku Koubou), and Taemon Yamamoto (Yamamoto Yougyojo) for providing honmoroko G. caerulescens and carp serum. We also thank Takefumi Yamamoto and Yasuhiro Mori (Shiga University of Medical Science) for their excellent technical supports.
This work was funded in part by the Grant-in-Aid for challenging Exploratory Research (23651248 and 15 K14440 to T.T.) and the Scientific Research on Priority Area (21028021 to T.T.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. This study was also supported by the NIG Collaborative Research Program (2010-A41, 2011-A36, 2012-A28, 2013-A47, 2014-A45, and 2016-A36 to T.T.), Ritsumeikan Global Innovation Research Organization (R-GIRO) program (to T.T.), Center of Innovation Trial Program from Japan Science and Technology Agency, JST (to T.T.), and Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan (to S.H.).
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