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

Abstract

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.