Genetic analysis of activin/inhibin β subunits in zebrafish development and reproduction

Activin and inhibin are both dimeric proteins sharing the same beta subunits that belong to the TGF-beta 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 beta subunits (beta Aa, inhbaa; beta Ab, inhbab; and beta B, inhbb) in zebrafish using CRISPR/Cas9. The loss of beta Aa/b but not beta B 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 beta A mutant together with aromatase (cyp19a1a) in the ovary. The single mutant of beta Aa/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 beta Aa/b mutant males showed normal spermatogenesis and fertility. As for activin beta B 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. Author summary Activin and inhibin are important growth factors that play essential roles in animal development and reproduction. Activin and inhibin share the same beta subunits, but they are antagonistic in function. Using gene editing technology, we studied the functional roles and importance of all three beta subunits (beta As: inhbaa and inhbab; beta B: inhbb) in zebrafish development and reproduction. Our results showed that the loss of all three subunits resulted in early death in zebrafish larvae, indicating functional importance of these molecules in development. Further analysis demonstrated essential roles of the beta subunits especially beta As (inhbaa and inhbab) in controlling female follicle development in the ovary. The loss of beta As especially inhbaa showed delayed follicle activation and ovarian fibrosis. Without inhbaa gene, the ovary deteriorated progressively after 6 months and the formation of eggs stopped completely after 18 months. In summary, the present study provided comprehensive evidence for the relative importance of each beta subunit in zebrafish reproduction.