The presence of a peptide closely related to porcine NPY has been demonstrated in the goldfish brain and pituitary by means of radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). The RIA data demonstrate that displacement curves of brain extracts are parallel to a porcine NPY standard and that in HPLC a compound present in brain extracts is co-eluted with porcine NPY. The distribution of this NPY-like factor within the central nervous system was studied by radioimmunoassay and immunohistochemistry. The results indicated that NPY has a widespread distribution with the highest concentrations being found in the telencephalon and diencephalon. In the pituitary gland, NPY immunoreactive terminals characterized at the electron microscope level were found in the different lobes and, in particular, in close association with the gonadotrophin (GTH) secreting cells. Using anin vitro perifusion system, it was shown that NPY causes a dose dependent increase of GTH release from anterior lobe fragments.These data indicate for the first time in teleosts that NPY is present and widely distributed in the brain and pituitary, and that among other putative functions, could be implicated in the multihormonal release of GTH from the pituitary.
An enzyme-linked immunosorbent assay (ELISA) for goldfish gonadotropin (GTH) was developed with the intent of devising a simple, reliable and nonradioisotopic assay for the measurement of GTH in goldfish biological samples. In this assay, soluble GTH of the standards or samples competes with carp GTH (cGTH) immobilized on a solid support (96-well microplate) for the fixation on antibodies to the beta-subunit of carp gonadotropin. The immobilized antigen-antibody complexes are then revealed by the peroxidase-antiperoxidase (PAP) technique. After revelation of the peroxidase activity, the absorbance value of each well is measured with a microplate reader. The cGTH concentration used for coating the wells is 2 ng/ml and the final dilution of the specific antibody is 1:80,000. The assay can be performed within 24 h and can be used over a range of 0.125-4 ng/ml. At about 50% binding, the intra- and interassay coefficients of variation are 5% and 9% respectively. The displacement curves generated by goldfish plasma or pituitary perifusion fractions were strictly parallel to the standard cGTH. In addition, the stimulation by salmon gonadotropin-releasing hormone of pituitary fractions perifused in vitro caused an immediate increase in the GTH measured in the collected fractions, strongly reinforcing the assumption that this assay indeed measures GTH.
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