Loss of Inhibin Advances Follicle Activation and Female Puberty Onset but Blocks Oocyte Maturation in Zebrafish

Abstract

Inhibin was first characterized in mammals as a gonadal dimeric protein that inhibited pituitary follicle-stimulating hormone (FSH) secretion. As in mammals, inhibin-specific α subunit (INHA/Inha/inha) has also been characterized in teleosts; however, its functions and physiological importance in fish reproduction remain unknown. Using CRISPR/Cas9 method, we generated an inha-deficient zebrafish line and analyzed its reproductive performance. As expected, pituitary expression of fshb increased significantly in both young and adult inha mutant. The expression of lhb also increased in the mutant, but only in sexually mature adults. Interestingly, the expression of activin βA (inhbaa) increased significantly in both ovary and testis of inha mutant and the expression of ovarian aromatase (cyp19a1a) also increased dramatically in the mutant ovary. The juvenile female mutant showed clear signs of early follicle activation or precocious puberty onset. However, the adult female mutant was infertile with follicles arrested at full-grown stage without final oocyte maturation and ovulation. Although follicle growth was overall normal in the mutant, the size and distribution of yolk granules in oocytes were distinct and some follicles showed granulosa cell hypertrophy. In contrast to females, inha null males showed normal spermatogenesis and fertility. As reported in mammals, we also found sporadic tumor formation in inha mutants. Taken together, our study not only confirmed some conserved roles of inhibin across vertebrates such as inhibition of FSH biosynthesis and tumor formation, but also revealed novel aspects of inhibin functions such as disruption of folliculogenesis and female infertility but no obvious involvement in spermatogenesis in fish.

Disruption of Epidermal Growth Factor Receptor but Not EGF Blocks Follicle Activation in Zebrafish Ovary

“…Our previous studies proposed that EGF promotion of oocyte maturation in zebrafish was likely mediated by activins in follicles ( Pang and Ge, 2002 ), and that EGF significantly stimulated expression of activin subunits ( inhbaa , inhbab , and inhbb ) ( Pang and Ge, 2002 ; Wang and Ge, 2004a ; Tse and Ge, 2010 ; Chung and Ge, 2012 ) but suppressed that of activin binding protein follistatin ( fsta ) ( Wang and Ge, 2004a ) in cultured follicle cells. Our recent study showed that the loss of inhibin ( inha ), an antagonist of activin, advanced follicle development by promoting follicle activation or PG–PV transition, leading to precocious puberty ( Lu et al, 2020 ). This is opposite to the disruption of egfra , which blocked PG–PV transition as reported in this study.…”

“…This was further evidenced in the present study by novel phenotypes shown by egfra and inha double mutant. Although the loss of inhibin ( inha−/− ) alone accelerated follicle activation or PG-PV transition ( Lu et al, 2020 ), it could not rescue the phenotype of PG-PV blockade shown by the egfra mutant. Instead, egfra and inha double mutant induced a novel phenotype not shown by single mutants, viz .…”

“…The AB strain zebrafish and inhibin null mutant ( inha −/−) used in this study were maintained in the ZebTEC Multilinking Rack zebrafish system (Tecniplast, Buguggiate, Italy) at 28°C with a lighting scheme of 14-h (8:00 am–10:00 pm) light and 10-h dark. The inha mutant (umo19 with ZFIN) was recently created in our laboratory ( Lu et al, 2020 ). The fish were handled according to the guidelines and protocols approved by the Research Ethics Panel of the University of Macau.…”

Homozygous Mutation of gsdf Causes Infertility in Female Nile Tilapia (Oreochromis niloticus)

“…More evidence proves that the TGF-β signaling genes, besides Amh , gsdf and Amhr2 discussed above, are critical for regulating gonadotropin signaling pathways in vertebrates. The other two TGF-β subfamily members, Activin and Inhibin, antagonize each other to regulate pituitary fshb expression and Fsh secretion in vertebrates ( 51 ). Inhibin contains a unique inhibin-specific α subunit (Inha) and shares a common β subunit (Inhba and Inhbb) with Activin to form Inhibin A, or Inhibin B. Activins are homodimers of either Inhba or Inhbb or their heterodimers.…”

“…Inhibin contains a unique inhibin-specific α subunit (Inha) and shares a common β subunit (Inhba and Inhbb) with Activin to form Inhibin A, or Inhibin B. Activins are homodimers of either Inhba or Inhbb or their heterodimers. In mammals, Activin stimulates, and Inhibin inhibits the Fsh section ( 51 , 52 ).…”