Purification and partial characterization of vitellogenin from shorthead redhorse (Moxostoma macrolepidotum) and copper redhorse (Moxostoma hubbsi) and detection in plasma and mucus with a heterologous antibody

Abstract

Vitellogenin (VTG), the egg yolk precursor protein, was purified from plasma of estradiol-3-benzoate (E2B)-treated male shorthead redhorse (Moxostoma macrolepidotum) and immature copper redhorse (Moxostoma hubbsi) by a two-step chromatographic procedure without precipitation. Intact VTGs appeared as dimers with apparent molecular masses, determined by gel filtration, of approximately 425 kDa (copper redhorse) and approximately 450 kDa (shorthead redhorse). In native polyacrylamide gel electrophoresis (PAGE), dimeric redhorse VTGs appeared as a 520 kDa band. Both VTGs were reduced to a single monomer of approximately 150 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions, indicating that monomers are not linked by disulfide bonds in the dimer form. The purified proteins were characterized as phospholipoglycoproteins. Isoelectric focusing of both VTGs revealed components with isoelectric points ranging from 5.3 to 6.0, suggesting charge heterogeneity. The amino acid composition of both VTGs contains a high proportion of nonpolar amino acids and was similar to those of other teleosts. An antibody developed against carp (Cyprinus carpio) VTG showed cross-reactivity with VTG from both redhorse species. Using this antibody, VTG was detected in plasma and surface mucus of E2B-treated redhorse. This is the most extensive report on purification and characterization of vitellogenin from catostomidid species.

A proteomic view on the developmental transfer of homologous 30 kDa lipoproteins from peripheral fat body to perivisceral fat body via hemolymph in silkworm, Bombyx mori

“…Considering the homology of 30 kDa lipoproteins to M. sexta vitellogenin, a female-specific lipo-glyco-carenoprotein, it was not surprising that they showed cross-reactivity with its monoclonal antibody (see Additional file 4) [55,58]. After 1D-PAGE separation of PVFB, the response of a protein at ~29 kDa was noted in Western blot.…”

“…These dyes target hydrophobic compounds like neutral triglycerides, lipids and fatty acids, but it remains unclear to what extent sequence stretches of hydrophobic amino acids respond to the dye. For redhorse vitellogenin poor staining was observed after SDS-PAGE suggesting that lipid content was reduced by the procedure [58], that free lipids were removed and that only covalently bound lipidoic moieties were present.…”

Use of Vitellogenin as Biomarker Indicator in Sex Identification of Giant Grouper (Epinephelus lanceolatus)

“…Maltis and Roy, demonstrated shorthead redhorse and copper redhorse Vtg was successfully detected by using Western blot technique [29]. They used carp anti-Vtg polyclonal antibody to recognize the purified redhorse Vtg.…”

Investigating the Utility of Measuring 11α‐Ketotestosterone and Vitellogenin in Surface Mucus as an Alternative to Plasma Samples in Assessments of the Reproductive Axis of White Bass

“…Significant relationships between mucus and plasma samples for both VTG OD and 11-KT are also reported for other species. Mucus and plasma concentrations of VTG have been measured in Shorthead Redhorse Moxostoma macrolepidotum, Copper Redhorse M. hubbsi (Maltais and Roy 2009), Atlantic Salmon Salmo salar (Meucci and Arukwe 2005), Cichlasoma dimerus (Moncaut et al 2003), and Striped Bass (Kishida et al 1992) through Western-blot and gel electrophoresis. Our use of commercially available VTG ELISA TABLE 1.…”

“…Surface mucus is a potential alternative sampling method for measuring many steroids and VTG and would allow for sampling a wide range of fish sizes when making assessments of the hypothalamicpituitary-gonadal (HPG) axis of fishes. Surface mucus of numerous freshwater (Moncaut et al 2003;Maltais and Roy 2009) and marine species (Schultz et al 2005) have been assayed successfully for 11α-ketotestosterone (11-KT) and VTG. An important feature when characterizing the HPG axis of hatchery fish is that the sampling methodology should employ a minimum amount of handling stress.…”