Polyploid Formation Through Disrupting Mitotic Sister Chromatid Separation of Spermatogonia based on <i>espl1</i> Heterozygous Knockout in Fish

Polyploidy is a major driver of speciation and evolutionary changes in plants and animals. Production of unreduced gametes is considered as a main pathway for polyploid formation. However, the precise molecular mechanisms underlying unreduced gamete production, particularly those arising from mitotic defects of spermatogonia (SG)/oogonia, remain poorly understood. Here, a heterozygous espl1 (extra spindle pole bodies like 1) knockout line was generated in diploid loach (Misgurnus anguillicaudatus) by CRISPR/Cas9 technology. Interestingly, we found about 90% diploids and 10% triploids in the progeny of espl1+/- males and wild-type (WT) diploid females. espl1+/- male could produce 1n sperms, along with a certain volume of unreduced sperms (2n). All offsprings of espl1+/-♀ and espl1+/+♂ were diploid, indicating that espl1+/- female produced normal ploidy eggs. Heterozygous espl1 deficiency impaired mitotic sister chromatid separation of some SG, resulting in their chromosome number doubling, thus causing the unreduced sperm production. The triploid loach (espl1+/+/-) from espl1+/+♀ mating with espl1+/-♂ could produce triploid sperms, which gave tetraploid heterozygotes by fertilization with haploid eggs. Resultant tetraploids yielded all-triploid progeny, when mated with WT diploid males. This study was extended to model fish zebrafish, where heterozygous espl1 knockout zebrafish produced about 5% unreduced diploid sperms. Here, we showed that the heterozygous loss of espl1 was enough to induce spermatogonial mitotic sister chromatid separation defects, causing the production of unreduced sperms. Notably, our results provide new strategies for the aquaculture-oriented polyploid breeding.