Application of dead end‐knockout zebrafish as recipients of germ cell transplantation

Abstract

Germ cell transplantation is a promising technology for the propagation of endangered or valuable fishes. In this technique, sterile male and female recipient fish are injected with donor germ cells so they can produce viable gametes derived from the donor cells. The dead end (dnd) gene is involved in the migration of primordial germ cells; therefore, dnd-knockout zebrafish are expected to be germ-cell-free, making them suitable recipients for germ cell transplantation. dnd mutants were produced by microinjecting 2 nl of 10 ng/μl cRNAs encoding zinc finger nucleases against dnd into the blastodisc of zebrafish embryos before the cell- cleavage stage. One of the resulting founder males was mated with a wild-type female, and produced heterozygous mutants in the F1 generation. Mating of these F1 mutants produced an F2 generation with approximately 25% of the clutch being homozygous mutant (dnd-knockout) male, and lacking germ cells (as confirmed by expression analyses of vasa). The resulting dnd-knockout zebrafish males were tested for suitability as germ cell transplantation recipients by intraperitoneal transplantation of testicular cells prepared from vasa-gfp zebrafish. GFP-positive germ cells incorporated into the germ-cell-free gonads of the dnd-knockout recipients matured into functional sperm. Progeny tests revealed that the sperm from these dnd-knockout recipients were derived entirely from donor cells. Thus, we demonstrated that homozygous dnd mutants became germ-cell-free males that are able to nurse donor-derived germ cells.

Hybrid Sterility in Fish Caused by Mitotic Arrest of Primordial Germ Cells

“…zebrafish, ablation of PGCs by knockdown or knockout studies for the dead end (dnd) gene promotes the masculinization of genetically female fish (Ciruna et al 2002;Slanchev et al 2005;Dai et al 2015;Li et al 2017). Further, during the embryonic stage of zebrafish, a certain number of PGCs must be present in the genital ridges for the subsequent ovarian development in females to successfully proceed (Tzung et al 2015).…”

“…In marine fish, the production of triploids by preventing second polar body exclusion using heat or pressure shocks is technically more difficult than in freshwater fish because of their small egg sizes, unstable egg quality, and high mortality at early stages (Felip et al 1997;Kaji et al 1999). Recently, germ cell-less fish have been obtained by dnd-knockdown and -knockout studies in several freshwater fishes, such as zebrafish (Weidinger et al 2013;Li et al 2017), loach Misgurnus anguillicaudatus (Fujimoto et al 2010), goldfish Carassius auratus (Goto et al 2012), sterlet Acipenser ruthenus (Linhartová et al 2015), and salmon species (Wargelius et al 2016;Yoshizaki et al 2016); however, microinjection experiments of antisense morpholinos or reagents for genome editing to marine fish embryos also require high levels of skill and advanced equipment. Since hybridization can be achieved only by artificial insemination and the usual rearing of the resulting hybrid embryos, hybrid fish showing the germ cell-less phenotype would be suitable recipients for germ cell transplantation, not only in sciaenids but also in other marine fishes.…”

Biotechnology applied to fish reproduction: tools for conservation

“…After those findings, spermatogonial transplantation in larvae was performed many other times involving species, such as the Nibe Croaker Nibea mitsukurii (Takeuchi et al 2009;Yoshikawa et al 2016), the yellowtail Seriola quinqueradiata (Morita et al 2012), the Nile tilapia Oreochromis niloticus (Farlora et al 2014), and the zebrafish D. rerio (Li et al 2017), among others. However, since germline transmission rate to offspring was considered low because of niche competition between donor and endogenous germ cells, subsequent studies commonly used sterile hosts, such as Okutsu et al (2007) that produced trout fry from triploid sterile salmon.…”

“…One of these genes, encoding a RNA-binding protein crucial for migration and survival of PGCs-the Dead end (dnd) gene-is effectively knocked down by injection of the chemically modified Morpholino oligonucleotide antisense (MO) into the eggs, as tested in the sterlet sturgeon Acispenser ruthenus (Linhartová et al 2015). Many other fish were also proven to be fully sterile when dnd gene is suppressed by MO, such as zebrafish (Ciruna et al 2002;Li et al 2017) and rainbow trout Oncorhynchus mykiss (Yoshizaki et al 2016). Although effective, as shown by Saito and colleagues that used zebrafish morphants as host (Saito et al 2008), this technique cannot be applied at large-scale because the embryos must be individually injected, as well as resulting in higher percentage of males.…”

Enrichment of transplantable germ cells in salmonids using a novel monoclonal antibody by magnetic‐activated cell sorting

“…We previously succeeded in establishing a germ cell transplantation system in various teleosts (Farlora et al, ; Hamasaki et al, ; Li, Fujii, Naito, & Yoshizaki, ; Morita et al, ; Morita et al, ; Octavera & Yoshizaki, ; Okutsu, Shikina, Kanno, Takeuchi, & Yoshizaki, ; Okutsu, Suzuki, Takeuchi, Takeuchi, & Yoshizaki, ; Seki et al, ; Takeuchi, Yoshizaki, & Takeuchi, ; Yoshikawa et al, ; Yoshizaki & Yazawa, ). In this transplantation system, A spermatogonia (ASGs) or oogonia isolated from donor gonads were transplanted into the peritoneal cavity of newly hatched larvae that did not yet have a mature immune system.…”