VITELLOGENIN PURIFICATION AND DEVELOPMENT OF ASSAY FOR VITELLOGENIN RECEPTOR IN OOCYTE MEMBRANES OF THE TILAPIA (OREOCHROMIS-NILOTICUS, LINNAEUS 1766)

An assay has been successfully developed for vitellogenin receptors in isolated oocyte membranes of the tilapia (Oreochromis niloticus). Vitellogenin produced by estrogen stimulation of the male tilapia was purified by anion-exchange chromatography on DEAE-Sephacel and identified by its incorporation of P-32-orthophosphate and H-3-leucine in vivo. Only one molecular form of vitellogenin was found to be induced by estrogen treatment. After iodination with Iodogen, the I-125-vitellogenin was shown to be immunologically similar to the unlabeled protein and was used to develop an assay for vitellogenin receptors. For such assays, the optimal conditions for binding were pH 7.4, 6-h incubation at 23-degrees-C with 7.5 mM Mg2+ and 10 mM Ca2+. The magnitude of the specific binding was dependent on the amount of oocyte membranes and was saturable. The binding shows both tissue and protein specificity: the I-125-vitellogenin binds specifically to only oocyte membranes and not to those of the brain, muscles, liver, intestine, and testis; its binding was displaceable only by unlabeled vitellogenin and a yolk protein extract. Saturation studies and Scatchard analyses revealed only a single class of binding sites. These studies provide unequivocal evidence that the binding sites for vitellogenin in the tilapia oocytes are specific receptors. The number and affinity (K(D)) of these receptors increase from the previtellogenic (9.98 x 10(9)/oocyte) to the vitellogenic stage (3.65 x 10(12)/oocyte), and remained unchanged at the preovulatory stage (3.53 x 10(12)/oocyte), at which time the affinity of the receptors was also highest (0.3-mu-M).