Functional analysis of nuclear estrogen receptors (nERs) in zebrafish reproduction by genome editing approach.

PMID 28398516


Estrogens signal through both nuclear and membrane receptors with most reported effects being mediated via the nuclear estrogen receptors (nERs). Although much work has been reported on nERs in the zebrafish, there is a lack of direct genetic evidence for their functional roles and importance in reproduction. To address this issue, we undertook this study to disrupt all three nERs in the zebrafish, namely esr1 (ERα), esr2a (ERβII) and esr2b (ERβI), by genome-editing technology CRISPR/Cas9. Using this loss-of-function genetic approach, we successfully created three mutant zebrafish lines with each nER knocked out. In addition, we also generated all possible double and triple knockouts of the three nERs. The phenotypes of these mutants in reproduction were analyzed in all single, double and triple nER knockouts in both females and males. Surprisingly, all three single nER mutant fish lines display normal reproductive development and function in both females and males, suggesting functional redundancy among these nERs. Further analysis of double and triple knockouts showed that nERs, especially Esr2a and Esr2b, were essential for female reproduction, and loss of these two nERs led to an arrest of folliculogenesis at previtellogenic (PV, stage II) stage followed by sex reversal from female to male. In addition, the present study also revealed a unique role for Esr2a in follicle cell proliferation and trans-differentiation, follicle growth, chorion formation. Taken together, this study provides the most comprehensive genetic analysis for differential functions of esr1, esr2a and esr2b in fish reproduction.