Gag (Mycteroperca microlepis) vitellogenin: purification, characterizationand use for enzyme-linked immunosorbent assay (ELISA) of female maturity in three species of grouper
Sa. Heppell et Cv. Sullivan, Gag (Mycteroperca microlepis) vitellogenin: purification, characterizationand use for enzyme-linked immunosorbent assay (ELISA) of female maturity in three species of grouper, FISH PHYS B, 20(4), 1999, pp. 361-374
Circulating levels of the egg yolk precursor protein, vitellogenin (VTG), c
an be used as a biochemical indicator of maturation in female fish. Here we
report on purification and partial characterization of VTG from a temperat
e marine serranid, the gag(Mycteroperca microlepis). Development of a compe
titive, enzyme-linked immunosorbent assay (ELISA) for gag VTG (gVTG) is als
o described. The gVTG was purified by DEAE-agarose anion exchange chromatog
raphy from a pooled plasma sample collected from several juvenile gag after
they were injected with 17 beta-estradiol. The protein appeared as a major
band of M(r)congruent to 183 000 after SDS-PAGE +/- Western blotting using
either a specific rabbit antiserum to gVTG or a universal monoclonal antib
ody for vertebrate VTGs. Amino acid composition analysis and N-terminal pep
tide sequencing verified that gVTG is similar in primary structure to VTG f
rom several other teleost species. The purified gVTG and its specific antis
erum were used to develop a sensitive, competitive, antibody-capture ELISA
for quantifying the protein in blood plasma from maturing females. VTG leve
ls in maturing female gag were highly correlated with oocyte growth and cir
culating testosterone and 17 beta-estradiol levels, whereas VTG was non-det
ectable in juveniles, immature females or males. Two size-based maturity sc
hedules for female gag were constructed, one utilizing detection of VTG in
their circulation as a marker of maturity and the other relying on histolog
ical evidence that their ovaries were in vitellogenic or later stages of ma
turation. The two schedules were virtually identical. The gVTG ELISA was al
so used to detect VTG in blood plasma from mature Nassau grouper (Epinephel
us striatus) and red hind (E. guttatus). As with gag, the assay was complet
ely reliable for discriminating between reproductively mature females versu
s males from these two grouper species.