Mechanisms of yolk transport

Yolk is the major source of nutrients for the developing chicken embryo, but molecular details of the mechanisms for the delivery of yolk components to the embryo are still largely unknown. During oogenesis in the chicken, the main yolk components vitellogenin (VTG) and very low density lipoprotein (VLDL) are taken up into the oocytes via LDL receptor related protein LR8 (H. Bujo, M. Hermann, M. O. Kaderli, L. Jacobsen, S. Sugawara, J. Nimpf, T. Yamamoto, and W. J. Schneider (1994) EMBO J. 13, 5165-5175). In the oocyte, yolk precursors` protein moieties are partially degraded; however, this fragmentation does not abolish their binding to LR8. Our past studies have shown that stored yolk VLDL interacts with LR8 localized on the surface of the endodermal endothelial cells (EECs) of the yolk sac. Following internalization and degradation in the EECs, VLDL particles are re-synthesized in and secreted from these cells to yield embryo plasma VLDL (M. Hermann, M. G. Mahon, K. A. Lindstedt, J. Nimpf, and W. J. Schneider (2000) J. Biol. Chem. 275, 16837-16844). It is likely that members of the LDL receptor family other than LR8 also play important roles in lipid metabolism and reproduction of the chicken; we have identified LRP1, LRP2, and LRP380 as candidate proteins in the ovary and/or yolk sac. The function and regulation of these proteins in yolk-to-embryo transport are not yet known. The present study focuses on the roles of LDL receptor related proteins in the chicken yolk sac and in the EECs, and on the mechanisms for transfer of yolk components across the yolk sac into the embryonic circulation.

These studies were focused on the molecular characterization, elucidation of regulatory features, and on gaining insights into possible biological roles of two giant membrane proteins in the chicken, called LRP380 and LRP2. Yolk is the major source of nutrients for the developing chicken embryo. Our past studies have shown that stored yolk VLDL interacts with the best-characterized yolk transport receptor during follicle growth, called LR8, which is localized on the surface of the cells (EECs) surrounding the yolk sac. Following internalization and degradation in the EECs, VLDL particles are re-synthesized in and secreted from these cells into the embryo plasma. Now we focused on LRP2 and LRP380, which are members of the same family as LR8 and likely also play important roles in lipid metabolism and reproduction of the chicken. First, we investigated the levels and localization of LRP380 in the structures containing growing oocytes called follicles In different stages of follicle development, i.e., oocyte sizes, the relative levels of expression of LRP380 and LR8 during follicle growth in laying hens and in hens of the R/O strain were compared. R/O hens are unable to lay eggs due to a mutation in LR8 that disrupts its function, which is to transport yolk precursors from the hen`s blood into the growing oocytes. LRP380 levels were higher in the mutant`s follicles than in normal hens, suggesting a role of LRP380 follicle growth. LRP2 (also called megalin) is an essential mediator of protein resorption in the kidney and is the largest known member of this kind of receptors. The information on the structure of chicken LRP2 showed 73 % identity to the human protein. The analysis of LRP2 showed an interesting hormon-specific difference in expression levels in the chicken kidney and also in human cells, possibly the most important result of the project. The females` kidney, known to be an estrogen target organ in the chicken, contained significantly more LRP2 mRNA as well protein. These observations may be related to the dramatic differences in lipoprotein metabolism between roosters and laying hens. Incubation of human kidney cell lines with estrogen also results in enhanced LRP2 expression. These data suggest an important contribution of sex hormones to the regulation of kidney functions involving LRP2 action. Finally, we show that LRP2 is also present in the chicken yolk sac, the structure facilitating the transport of yolk components to the embryo, and, like LR8, is localized on the surface of EECs immediately adjacent to the yolk. The expression of LRP2 in the yolk sac, increases during the period of rapid embryo growth, indicating a function in nutrient supply of the embryo.