Vitellogenin purification and development of assay for vitellogenin receptor in oocyte membranes of the tilapia (Oreochromis niloticus, linnaeus 1766)

Abstract

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 32P-orthophosphate and 3H-leucine in vivo. Only one molecular form of vitellogenin was found to be induced by estrogen treatment. After iodination with Iodogen, the 1251-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°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 1251-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 (KD) of these receptors increase from the previtellogenic (9.98 x lOg/oocyte) to the vitellogenic stage (3.65 X 1012/oocyte), and remained unchanged at the preovulatory stage (3.53 X 1012/oocyte), at which time the affinity of the receptors was also highest (0.3 FM).

Identification of vitellogenin receptors in the ovary of a teleost fish, the Mediterranean sea bass (Dicentrarchus labrax)

“…Scatchard analysis of the saturation data (fig 3, inset) (Chan et al, 1991), and from 1.3 to 3.5 nM (Lancaster and Tyler, 1994) or 10 to 30 nM (Nunez Rodriguez et al, 1996) (lane 4).…”

Identification and Transcriptional Modulation of the Largemouth Bass, Micropterus salmoides, Vitellogenin Receptor During Oocyte Development by Insulin and Sex Steroids1

“…The functionality of Vtgr in fish has been examined in studies with Coho salmon [13], Nile tilapia [14], and seabass [15]; these studies focused on Vtg uptake through implementation of binding assays that demonstrated the presence of surface receptors within oocyte membranes. Furthermore, the existence of a new system of multiple Vtgrs that disparately bind functionally different types of Vtgs in white perch has been suggested [16].…”

Vitellogenin in tilapia (Oreochromis mossambicus): Induction of two forms by estradiol, quantification in plasma and characterization in oocyte extract

“…In teleosts, the presence of receptor-mediated endocytosis has also been demonstrated (Selman and Wallace 1982;Tyler et al 1988;Kanungo et al 1990). Recently, Chan et al (1991) have characterized a vitellogenin receptor in oocyte membranes of tilapia, Oreochromis niloticus. Vitellogenin has been isolated and characterized from a variety of fishes, including two species of tilapia, 0. aureus (Ding et al 1989) and 0. niloticus (Chan et al 1991).…”

“…Recently, Chan et al (1991) have characterized a vitellogenin receptor in oocyte membranes of tilapia, Oreochromis niloticus. Vitellogenin has been isolated and characterized from a variety of fishes, including two species of tilapia, 0. aureus (Ding et al 1989) and 0. niloticus (Chan et al 1991). In circulation, the vitellogenin molecule is known to be present mainly as a dimer with a molecular mass ranging from 300 kDa in tilapia, 0. aureus (Ding et al 1989), to 600 kDa in chum salmon, Oncorhynchus keta, and rainbow trout, Oncorhynchus mykiss (Hara 1978;Hara and Hirai 1978).…”

“…The molecular mass of the monomeric form of vitellogenin determined by SDS-PAGE under denaturing conditions ranges from 85 kDa in eels, Anguilla japonica (Hara et al 1980) to 200 kDa in Oryzias latipes (Hamazaki et al 1987). The tilapia 0. aureus is unusual in that two native forms of 300 and 500 kDa and two monomeric forms of 130 and 180 kDa have been identified (Ding et al 1989), although in the tilapia 0. niloticus only one native form of 300 kDa is present (Chan et al 1991).…”