Intracellular interaction between FcRY receptor and IgY in ovarian vascular endothelial cells during avian maternal IgY transfer

Maternal immunoglobulin Y (IgY) is transferred from the bloodstream to the yolk of maturing oocytes in avian species, which is crucial for the passive immunity of neonatal chicks. The receptor responsible for maternal blood IgY transfer to the yolk remains unidentified. Recently, we found that an IgY receptor, FcRY, is expressed in the vascular endothelial cells of ovarian follicles. However, there is no evidence of the direct binding of IgY to FcRY in vascular endothelial cells of avian ovarian follicles. To investigate how ovarian FcRY interacts with IgY, we performed a microscopic analysis of FcRY-IgY interactions in the primary endothelial cells isolated from quail ovarian follicles. Western blot and real-time PCR analyses showed that FcRY was expressed in the vascular endothelial cells. Microscopic analysis indicated that FcRY signals were distributed as intracellular vesicles around the center of vascular endothelial cells, suggesting that FcRY is localized to subcellular compartment of vascular endothelial cells. The percentage of QH1 (a quail endothelial marker)-positive cells was comparable to that of QH1/FcRY double-positive cells, showing that a considerable number of vascular endothelial cells express FcRY in avian ovaries. Proximity ligation assay (PLA) demonstrated that vascular endothelial FcRY binds to intracellular IgY. Importantly, the FcRY-IgY binding signal was inhibited by Pitstop 2, a selective inhibitor of clathrin-mediated endocytosis, and bafilomycin A₁, a proton pump inhibitor, suggesting that FcRY-IgY interactions depend on clathrin-mediated endocytosis and acidification of intracellular vesicles. This is the first study to demonstrate direct evidence of an FcRY-IgY interaction in primary vascular endothelial cells of an avian species, which supports a key role for FcRY in maternal IgY transfer into egg yolks. These findings open potential avenues for improving avian immunity or delivering substances into eggs through IgY-Fc fusion protein strategies.