During the rapid final stage of growth, chicken oocytes take up massive amounts of plasma components and convert them to yolk. The oocyte expresses a receptor that binds both major yolk lipoprotein precursors, vitellogenin (VTG) and very low density lipoprotein (VLDL). In the present study, in vivo transport tracing methodology, isolation of coated vesicles, ligand- and immuno-blotting, and ultrastructural immunocytochemistry were used for the analysis of receptor-mediated yolk formation. The VTG/VLDL receptor was identified in coated profiles in the oocyte periphery, in isolated coated vesicles, and within vesicular compartments both outside and inside membrane-bounded yolk storage organelles (yolk spheres). VLDL particles colocalized with the receptor, as demonstrated by ultrastructural visualization of VLDL-gold following intravenous administration, as well as by immunocytochemical analysis with antibodies to VLDL. Lipoprotein particles were shown to reach the oocyte surface by passage across the basement membrane, which possibly plays an active and selective role in yolk precursor accessibility to the oocyte surface, and through gaps between the follicular granulosa cells. Following delivery of ligands from the plasma membrane into yolk spheres, proteolytic processing of VTG and VLDL by cathepsin D appears to correlate with segregation of receptors and ligands which enter disparate sub-compartments within the yolk spheres. In small, quiescent oocytes, the VTG/VLDL receptor was localized to the central portion of the cell. At onset of the rapid growth phase, it appears that this pre-existing pool of receptors redistributes to the peripheral region, thereby initiating yolk formation. Such a redistribution mechanism would obliterate the need for de novo synthesis of receptors when the oocyte's energy expenditure is to be utilized for plasma membrane synthesis, establishment and maintenance of intracellular topography and yolk formation, and preparation for ovulation.

Upon receptor-mediated endocytosis of very-low-density lipoprotein (VLDL) and vitellogenin into growing chicken oocytes, the protein moieties of these lipoproteins are proteolytically cleaved. Unlike the complete lysosomal degradation in somatic cells, enzymatic ligand breakdown in oocytes generates a characteristic set of polypeptides, which enter yolk storage compartments for subsequent utilization by the embryo. Here, we demonstrate directly that the catalyst for the intraoocytic processing of both apolipoprotein B and vitellogenin is cathepsin D. The enzyme was purified from oocytic yolk, preovulatory follicle homogenates, and liver by affinity chromatography. When plasma VLDL and vitellogenin were incubated with the purified enzyme, fragments indistinguishable from those found in yolk were generated from both precursors under identical, mildly acidic conditions. Amino-terminal sequencing of the pure enzyme demonstrated 88% identity with mammalian cathepsin Ds over 34 residues. On the basis of this information, a full-length clone specifying chicken preprocathepsin D was isolated from a chicken follicle cDNA library by screening with a human cathepsin D probe. Whereas previous studies have demonstrated that the receptors for lipoproteins in somatic cells and oocytes, respectively, of the chicken are the products of different genes, Southern and Northern blot hybridization experiments showed that the enzymes expressed in oocytes and liver are the product of a single gene, giving rise to a 3.3-kb transcript. The primary structure of the 335-residue mature protein suggests a high degree of conservation of known crucial features of aspartyl proteases; however, the absence of the so-called processing region and of an aromatic residue in a region thought to partake in catalysis raise questions with possible evolutionary implications.

No abstract

No abstract

No abstract