Isogenic fish sperm produced by transplanting gynogenetic haploid-derived germ cells

Artificially induced haploidy is lethal in vertebrates, although it is useful for genetic screening and genome editing due to its single set of genomes. Haploid embryonic stem (ES) cell lines in mammals contribute to genetic studies and the production of gametes derived from haploid ES cells. In fish breeding, doubled haploids (DHs) induced by artificially induced gynogenesis are used to generate isogenic gametes for cloning purposes. However, gametes have not been directly differentiated from artificially induced haploid cells, even though haploid ES cell lines have been established in medaka. Here, we aimed to demonstrate that fertile haploid sperm with identical genotypes could be differentiated in germline chimeras in zebrafish, wherein primordial germ cells (PGCs) derived from an inviable haploid embryo were transplanted into sterilized recipient embryos. Haploid spermatogonia differentiated from haploid PGCs in germline chimeras, and genome doubling occurred in haploid spermatogonia with one set of chromosomes, which could not pair with counterpart homologs to form bivalents during meiosis. Subsequently, meiosis produced isogenic haploid gametes because identical elements were exchanged between chromosomes doubled from the haploid set during recombination. Consequently, haploid PGCs can survive beyond embryogenesis and potentially differentiate into fertile sperm.