Abstract
Zebrafish is one of the most commonly used model organisms in biomedical, developmental and genetic research. The production of several thousands of transgenic lines is leading to difficulties in maintaining valuable genetic resources as cryopreservation protocols for eggs and embryos are not yet developed. In this study, we utilized testis cryopreservation (through both slow-rate freezing and vitrification) and spermatogonia transplantation as effective methods for long-term storage and line reconstitution in zebrafish. During freezing, utilization of 1.3 M of dimethyl sulfoxide (Me2SO) displayed the highest spermatogonia viability (~60%), while sugar and protein supplementation had no effects. Needle-immersed vitrification also yielded high spermatogonia viability rates (~50%). Both optimal slow-rate freezing and vitrification protocols proved to be reproducible in six tested zebrafish lines after displaying viability rates of >50% in all lines. Both fresh and cryopreserved spermatogonia retained their ability to colonize the recipient gonads after intraperitoneal transplantation of vasa::egfp and actb:yfp spermatogonia into wild-type AB recipient larvae. Colonization rate was significantly higher in dnd-morpholino sterilized recipients than in non-sterilized recipients. Lastly, wild-type recipients produced donor-derived sperm and donor-derived offspring through natural spawning. The method demonstrated in this study can be used for long-term storage of valuable zebrafish genetic resources and for reconstitution of whole zebrafish lines which will greatly improve the current preservation practices.
Highlights
Zebrafish Danio rerio is a well-established, and one of the most widely used vertebrate model organisms in biomedical, developmental and genetic research[1] mostly due to its small size, ease of culture, high fecundity, transparent embryos, 70% homology between the zebrafish and human genomes, as well as good annotation of the genome[2,3]
As SSC transplantation and surrogate production technology coupled with cryopreservation offer new possibilities in the conservation of valuable genetic resources, in the present study we have successfully developed methods for cryopreservation of zebrafish spermatogonia and demonstrated the reproducibility of the presented protocols in several zebrafish lines
The protocols developed in this study can be applied to the reconstitution of whole zebrafish lines since testes of donor individuals can be crypreserved for theoretically indefinite periods, and subsequently transplanted into sterile individuals producing donor-derived gametes and offspring
Summary
Zebrafish Danio rerio is a well-established, and one of the most widely used vertebrate model organisms in biomedical, developmental and genetic research[1] mostly due to its small size, ease of culture, high fecundity, transparent embryos, 70% homology between the zebrafish and human genomes, as well as good annotation of the genome[2,3]. These are the baseline cells of spermatogenesis with a specific function to both self-renew and differentiate into later-stage germ cells and into spermatozoa[11] Their ability to colonize recipient gonads after intraperitoneal transplantation, proliferate and differentiate into functional gametes (either spermatozoa or eggs depending on the sex of the recipient) after cryopreservation and to produce donor-derived offspring by recipients has been demonstrated by several studies[12,13,14]. An additional challenge of the SSC transplantation strategy is the visualization of donor-derived germ cells within the recipient gonads To this end, most studies utilized transgenic strains in which vasa-positive cells are fluorescently labeled[19,20], enabling the visualization of fluorescently-labelled donor cells within recipient gonads
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