Genetic Analysis of Activin/Inhibin β Subunits in Zebrafish Development and Reproduction

Activin and inhibin are both dimeric proteins sharing the same β subunits that belong to the TGF-β superfamily. They are well known for stimulating and inhibiting pituitary FSH secretion, respectively, in mammals. In addition, activin also acts as a mesoderm-inducing factor in frogs. However, their functions in development and reproduction of other species are poorly defined. In this study, we disrupted all three activin/inhibin β subunits (βAa, inhbaa ; βAb, inhbab ; and βB, inhbb ) in zebrafish using CRISPR/Cas9. The loss of βAa/b but not βB led to a high mortality rate in the post-hatching stage. Surprisingly, the expression of fshb but not lhb in the pituitary increased in the female βA mutant together with aromatase ( cyp19a1a ) in the ovary. The single mutant of βAa/b showed normal folliculogenesis in young females; however, their double mutant ( inhbaa−/− ; inhbab−/− ) showed delayed follicle activation, granulosa cell hypertrophy, stromal cell accumulation and tissue fibrosis. The ovary of inhbaa−/− deteriorated progressively after 180 dpf with reduced fecundity and the folliculogenesis ceased completely around 540 dpf. In addition, tumor- or cyst-like tissues started to appear in the inhbaa−/− ovary after about one year. In contrast to females, activin βAa/b mutant males showed normal spermatogenesis and fertility. As for activin βB subunit, the inhbb−/− mutant exhibited normal folliculogenesis, spermatogenesis and fertility in both sexes; however, the fecundity of mutant females decreased dramatically at 270 dpf with accumulation of early follicles. In summary, the activin-inhibin system plays an indispensable role in fish reproduction, in particular folliculogenesis and ovarian homeostasis. ### Competing Interest Statement The authors have declared no competing interest.