Knock out of a major vitellogenin receptor gene with eight ligand binding repeats in medaka (Oryzias latipes) using the CRISPR/Cas9 system

Abstract

Recent studies of vitellogenesis engendered a novel model of teleost yolk formation in which multiple yolk precursors, vitellogenins (Vtgs), and their receptors (Vtgrs) interact to ensure proper yolk composition for embryonic development and larval growth. As a step toward verification of this concept, we examined the role of one candidate Vtgr, termed low-density lipoprotein receptor relative with eight ligand-binding repeat (Lr8), in the medaka, a representative teleost and established laboratory model. A homozygous lr8 knock out (lr8-KO) medaka was produced to perform reverse-genetic functional analyses. In ovaries of wild type (WT) medaka, Western blotting detected a putative Lr8 protein band at ~130 kDa, while immunohistochemistry detected the putative Lr8 signal at the periphery of the oocyte underneath the zona radiata. These signals disappeared in ovaries of the lr8-KO group. Offspring of lr8-KO medaka exhibited decreased survival rate compared to WT fish, but KO of lr8 was not 100% lethal. There was no significant difference in total yolk protein content or size of eggs between WT and lr8-KO fish. However, LC-MS/MS analyses revealed a remarkable decrease in the relative abundance of yolk proteins derived from VtgAb in lr8-KO eggs, in conjunction with a compensatory increase in proteins derived from VtgAa1. These findings strongly support the conclusion that Lr8 is an important receptor for VtgAb in medaka. The disruption of proper yolk composition by lr8-KO is possibly one cause of the low offspring survival.