Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido 041-8611, Japan.
Mariculture Fisheries Research Institute, Fisheries Research Department, Hokkaido Research Organization, 1-4-1 Masuura, Abashiri, Hokkaido 099-3119, Japan.
Comp Biochem Physiol A Mol Integr Physiol. 2021 Jul;257:110967. doi: 10.1016/j.cbpa.2021.110967. Epub 2021 Apr 23.
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.
最近对卵黄发生的研究产生了一个新的硬骨鱼类卵黄形成模型，其中多个卵黄前体、卵黄蛋白原（Vtgs）及其受体（Vtgrs）相互作用，以确保胚胎发育和幼虫生长所需的适当卵黄成分。作为验证这一概念的一步，我们研究了一种称为低密度脂蛋白受体相关蛋白 8 个配体结合重复（Lr8）的候选 Vtgr 在典型硬骨鱼代表之一——日本青鳉中的作用，并建立了实验室模型。通过产生 Lr8 纯合敲除（lr8-KO）日本青鳉来进行反向遗传功能分析。在野生型（WT）日本青鳉的卵巢中，Western blot 检测到约 130 kDa 的假定 Lr8 蛋白带，而免疫组织化学检测到卵母细胞周围放射区下的假定 Lr8 信号。这些信号在 lr8-KO 组的卵巢中消失。与 WT 鱼相比，lr8-KO 日本青鳉的后代存活率降低，但 KO 并不完全致死。WT 和 lr8-KO 鱼的总卵黄蛋白含量或卵大小没有显著差异。然而，LC-MS/MS 分析显示，lr8-KO 卵中的卵黄蛋白原 VtgAb 相关的卵黄蛋白相对丰度显著降低，同时 VtgAa1 相关的蛋白丰度代偿性增加。这些发现强烈支持 Lr8 是日本青鳉卵黄蛋白原 VtgAb 的重要受体的结论。由于 lr8-KO 导致卵黄组成不当，可能是后代存活率低的原因之一。
