Induction of germ cell-deficiency in grass puffer by dead end 1 gene knockdown for use as a recipient in surrogate production of tiger puffer

The surrogate broodstock technique produces donor-derived gametes via transplantation of germ cells into surrogate fish. The technique is a promising approach for improving the establishment and management of aquaculture strains with desirable genetic traits. The tiger puffer (Takifugu rubripes) is one of the most popular aquaculture fishes in Japan. However, brood stocks have large body sizes and require a long time to reach sexual maturity. The current study established a sterilization technique using gene knockdown in the grass puffer (T. alboplumbeus), which has a small body size and matures in half the time taken by the tiger puffer, and assessed the possibility of using germ cell-deficient grass puffers as recipients to efficiently produce donor-derived tiger puffer gametes. Dead end 1, which has two transcribed variants, was identified in the grass puffer. Morphants resulting from the microinjection of antisense morpholino oligonucleotides against the transcribed variants, into fertilized eggs, showed germ cell deficiency. Germ cell-deficient morphants exhibited sexual dimorphism with respect to their morphological and gene expression patterns. Testicular germ cells prepared from the testes of tiger puffer were intraperitoneally transplanted into morphant hatchlings. These recipients matured normally and produced functional donor-derived gametes separately from endogenous gametes. Thus, the use of germ cell-deficient recipients that produce only donor-derived gametes improves the efficiency of surrogate production and may accelerate the breeding process in tiger puffer aquaculture.