Production of germ cell‐deficient salmonids by dead end gene knockdown, and their use as recipients for germ cell transplantation

Abstract

We previously established a spermatogonial transplantation model in fish using triploid recipients. Although triploid salmonids are sterile, they carry a limited number of immature triploid germ cells that potentially compete with the donor-derived germ cells for their niche. We therefore assessed the biological characteristics of germ cell-deficient gonads in rainbow trout for their suitability as recipients for germ cell transplantation in this study. Antisense morpholino oligonucleotides against the dead end gene were microinjected into the fertilized eggs of rainbow trout to eliminate endogenous germ cells, leaving only their supporting cells. Unlike similar approaches performed in zebrafish and medaka, these germ cell-deficient rainbow trout did not show a male-biased sex ratio. Approximately 30,000 spermatogonia were then transplanted into the body cavities of both germ cell-deficient and control recipients. The donor-derived germ cells showed significantly higher proliferation in the gonads of germ cell-deficient recipients than those in the gonads of the control recipients. Finally, the applicability of the germ cell-deficient recipients for xenogeneic transplantation was evaluated by transplanting rainbow trout spermatogonia into germ cell-deficient masu salmon recipients. The resulting recipient salmon matured normally and produced trout gametes, and early survival of the resulting trout offspring was as high as that of the control offspring. Thus, dead end-knockdown salmonids appear to be ideal recipients for the intraperitoneal transplantation of spermatogonia.

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

“…Phenotypes obtained in knockout studies are not always consistent with those obtained in knockdown studies (Kok et al, ; Rossi et al, ). In the case of dnd , however, our knockout phenotype was similar to those previously reported for dnd ‐knockdown zebrafish (Saito et al, ) and rainbow trout (Yoshizaki et al, ). The dnd ‐knockdown treatment eliminated all endogenous germ cells during early embryogenesis, and the resulting fish exhibited no expression of the dnd gene from any remaining somatic cells.…”

“…Although phenotypes of the dnd‐ knockout and ‐knockdown zebrafish were almost identical, there are two potential advantages of dnd ‐knockouts over individuals produced by knockdown treatment. First, dnd‐ knockouts can completely wipe out the endogenous germ cells, whereas dnd knockdown fish sometimes possessed small numbers of endogenous germ cells (Yoshizaki et al, ) that can proliferate and contribute to fecundity. This minor population could be a disadvantage since somatic resources would not be fully allocated to the production of donor‐derived germ cells.…”

“…Similar results were obtained by xenotransplantation of PGCs into dnd ‐knockdown zebrafish (Saito, Goto‐Kazeto, Arai, & Yamaha, ). In previous research, we obtained germ‐cell‐deficient masu salmon by injecting dnd antisense morpholino oligonucleotide into 1‐cell‐stage embryos, and then transplanting rainbow trout germ cells, resulting in dnd ‐knockdown salmon that produced 100% rainbow trout offspring (Yoshizaki et al, ). Unfortunately, the sterility phenotype of dnd ‐knockdown recipients was never passed to the next generation, making the knockdown approach labor‐intensive since fertilized eggs need to be injected individually to produce each knockdown fish.…”

“…Indeed, only a small portion of the F1 offspring from these recipients was genetically derived from the donors (Okutsu et al, 2006). Based on these outcomes, we explored the possibility of creating surrogate recipients that only produce donor-derived offspring (Okutsu et al, 2007;Yoshizaki et al, 2016). We were able to produce recipients that produced only donor-derived trout gametes by transplanting spermatogonia into sterile, triploid masu salmon (Okutsu et al, 2007).…”

bmp15l, figla, smc1bl, and larp6l are preferentially expressed in germ cells in Atlantic salmon (Salmo salar L.)

“…In model organisms like zebrafish (Danio rerio), sterility can be induced by a number of methods that eliminate the germ cells in adults or the primordial germ cells of embryos (reviewed by Wong and Zohar, 2015b); similar approaches produced germ cell-free non-model fish. Indeed, knocking out or knocking down the dead end (dnd) gene in embryos yielded sterile adult salmon (Wargelius et al, 2016), sterlet (Acipenser ruthenus) (Linhartova et al, 2015), and rainbow trout (Yoshizaki et al, 2016). Such functional studies also provided valuable information on the complex biology of germ cell formation and identified factors that could be temporarily blocked to achieve sterility.…”