Zebrafish Nedd8 Facilitates Ovarian Development and Maintenance of Female Secondary Sexual Characteristics via Suppression of Androgen Receptor Activity


H&E staining of the ovaries of nedd8-knockout zebrafish (nedd8ˉ/ˉ) (n=12; 75%) at 4mpf (Credit:IHB)  

The research team led by Prof. XIAO Wuhan from Institute of Hydrobiology (IHB) of Chinese Academy of Sciences reported the loss of one copy of ar can partially rescue the phenotypes displayed by Neural precursor cell expressed developmentally downregulated protein 8 (nedd8)-null female zebrafish (e.g. improved oogenesis, ovarian maturation, egg fertilization rate and eliminated BTs on the pectoral fins of nedd8ˉ/ˉ female zebrafish) by CRISPR/Cas9 to generate double knockout zebrafish.    

This proves the interaction between Nedd8 and androgen receptors from a genetic perspective. The results were published online in Development.   

Nedd8 is an ubiquitin-like protein that covalently conjugates to target proteins through neddylation to regulate protein activity, stability and localization. Like ubiquitylation, neddylation involves NEDD8-activating enzyme E1 (NAE), NEDD8-conjugating enzyme E2 (usually Ubc12) and NEDD8-E3 ligase(s). However, the functions of other components of the NEDD system have not been illustrated by animal models in vivo. In particular, the function of nedd8 in vivo remains largely unknown.   

IHB researchers used CRISPR/Cas9 to knock out nedd8 in zebrafish. They found that loss of nedd8 in female zebrafish led to defects in oogenesis, disrupted oocyte maturation.   

In addition, researchers found that loss of nedd8 in female zebrafish stimulated growth of the breeding tubercles (BTs) on the pectoral fins. The BTs are normally present in males, not females. The phenotype is in contrary to what they previously observed in zebrafish with androgen receptor gene (ar) knockout.    

Notably, ar-knockout malezebrafish (arˉ/ˉ) do not develop BTs, but do exhibit some female secondary sexual characteristics. Then, they hypothesized that nedd8 might interact with androgen receptor.   

Further assays indicated that overexpressed nedd8 inhibits ar transcriptional activity in epithelioma papulosum cyprinid cells (EPC). The in vitro/in vivo neddylation assay indicated that Ar was modified by Nedd8. Then, researchers performed mutant screening by taking advantage of in vitro neddylation assays and found ar was modified by neddylation at lysine 475 and lysine 862. They thus proved lysine 862 in Ar might be targeted by neddylation specifically.   

The study not only demonstrated that Nedd8 modulates ovarian maturation and the maintenance of female secondary sexual characteristics of female zebrafish in vivo, but also indicated that androgen signaling is strictly regulated by nedd8.  

A working model of the relationship between ar neddylation and zebrafish gonadogenesis (Credit:IHB)