Profiles of α-melanocyte-stimulating hormone in the Japanese flounder as revealed by a newly developed time-resolved fluoroimmunoassay and immunohistochemistry

Profiles of α-melanocyte-stimulating hormone (α-MSH) in the Japanese flounder were examined by a newly developed time-resolved fluoroimmunoassay (TR-FIA) and immunohistochemistry. A TR-FIA for α-MSH was newly developed, and its levels in the pituitary gland and plasma of Japanese flounder reared in a white or black tank for 5 months were compared. A competitive assay using two antibodies was performed among secondary antibodies in the solid phase, α-MSH antibodies, samples, and europium-labeled Des-Ac-α-MSH. The sensitivity of the assay, defined as twice the standard deviation at a zero dose, was 0.98 ng/ml (49 pg/well). The intra- and interassay coefficients of variation of the assay were 8.8% (n = 8) and 17.3% (n = 5), respectively, at about 50% binding. Cross-reactivities of Des-Ac-α-MSH and Di-Ac-α-MSH were about 100%. Cross-reactivities of adrenocorticotropic hormone, salmon gonadotropin-releasing hormone (sGnRH), and chicken GnRH-II were less than 0.2%, and that of melanin-concentrating hormone was less than 2.0% at 50% binding. Displacement curves of serially twofold-diluted hypothalamus extract, pituitary gland extract, and plasma extract of Japanese flounder with the assay buffer were parallel to the α-MSH standard curve. Moreover, displacement curves of serially twofold-diluted hypothalamus and/or pituitary gland extract of masu salmon, goldfish, red seabream, Japanese eel, tiger puffer, and barfin flounder with the assay buffer were also parallel to the α-MSH standard. In Japanese flounder, total immunoreactive (ir)-α-MSH levels in the pituitary gland were lower in the black tank, whereas those in the plasma tended to be higher in the black tank, suggesting that the synthesis and release of α-MSH are higher in the black tank. α-MSH-ir cells were detected in the pars intermedia and a small part of the pars distalis of the pituitary gland. α-MSH-ir cell bodies were located in the basal hypothalamus and α-MSH-ir fibers were distributed not only in the hypothalamus but also in the telencephalon, midbrain, cerebellum, and medulla oblongata, suggesting that α-MSH functions as a neuromodulator in the brain.