The establishment of novel technologies for the long-term storage of fish genetic resources has become critical as aquatic species around the world are experiencing rapid declines due to overexploitation and global climate change. This project will examine an innovative area of reproductive stem cell biology to investigate optimal conditions for vitrification of spermatogonial stem cells from Rainbow trout (Oncorhynchus mykiss). Vitrification involves ultra-rapid cooling that results in instant solidification of the cell without crystallization. Development of a vitrification protocol is desirable since vitrification is fast and the process avoids trauma to cells by eliminating intracellular ice formation and instead creates a glass-like (vitreous) state, resulting in higher cell survival. This study will be conducted at the Tokyo University of Marine Sciences and Technology in Tokyo, Japan in collaboration with and under the supervision of Dr. Goro Yoshizaki, a renowned expert in fish reproductive stem cell biology. The proposed study will provide insight into fundamental aspects of stem cell cryobiology and could lead to real-world application of an inexpensive, economical method for preserving fish genetic resources for the conservation of endangered fish and aquaculture purposes.
The proposed experiments for this project will use Gfp-vasa+ transgenic rainbow trout to 1) determine the effects of the vitrification process on spermatogonial stem cell survival and 2) investigate the functionality of vitrified spermatogonial stem cells. Stem cell survival will be quantified by using flow cytometry, which will separate cell populations into viable or non-viable based on fluorescence patterns. A germ cell transplantation assay will be utilized to determine if the viable vitrified stem cells retain their functionality potential to colonize and proliferate in recipients. This research will improve understanding of the complex effects of cryopreservation and vitrification on stem cell function while providing the information necessary to successfully implement stem cell research as a novel artificial reproductive technology method to maximize genetic diversity for the conservation of threatened aquatic species. This NSF EAPSI award is funded in collaboration with the Japan Society for the Promotion of Science (JSPS).
