Isolation, characterization, and radioimmunoassay of Atlantic halibut somatolactin and plasma levels during stress and reproduction in flatfish.
Somatolactin (SL), a recently identified teleost pituitary hormone which is a member of the growth hormone/prolactin family, was isolated from pituitary tissue of Atlantic halibut (Hippoglossus hippoglossus). Pituitary proteins were extracted in ammonium bicarbonate (pH 7.8), fractionated using gel filtration chromatography, and purified using reversed-phase high-performance liquid chromatography. Halibut SL was identified on the basis of molecular size (determined by gel electrophoresis and mass spectroscopy), cross-reactivity of the putative hormone with antisera to cod SL, and N-terminal amino acid sequence. Polyclonal antibodies to purified halibut SL were raised in rabbits, and a radioimmunoassay (RIA) was developed for measurement of plasma concentrations of SL using purified halibut SL as a standard. The RIA was tested in several flatfish species including Pacific halibut (Hippoglossus stenolepis), English sole (Pleuronectes vetulus), and rock sole (Lepidopsetta bilineata). The assay was specific for SL as indicated by absence of cross-reactivity with Atlantic halibut growth hormone, prolactin, and GTH alpha subunit. Dilutions of plasma and pituitary extracts from Pacific halibut, English sole, and rock sole were parallel to the Atlantic halibut SL standard curve, indicating that the assay is valid for a range of flatfish species. Using halibut SL antiserum, SL was localized in the pars intermedia of English sole pituitary, where it has been identified in previously examined teleost species. The RIA was used to measure plasma levels of SL in Atlantic halibut and English sole during reproductive development, and in English sole subjected to various types of environmental stressors, including handling and crowding. In both sole and halibut, plasma SL concentrations remained relatively constant throughout gonadal development, but dropped during or following ovulation. Plasma SL levels in English sole tended to increase in response to acute stress, in parallel with plasma cortisol levels.
Johnson
L L
LL
Northwest Fisheries Science Center, NMFS, Seattle, Washington, 98112, USA.
Norberg
B
B
Willis
M L
ML
Zebroski
H
H
Swanson
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D029941:Fish Proteins; D005399:Fishes; D005412:Flatfishes; D006023:Glycoproteins / Q000032:analysis / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D006854:Hydrocortisone / Q000097:blood / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D010902:Pituitary Gland / Q000033:anatomy & histology / Q000737:chemistry* / Q000276:immunology; D010907:Pituitary Hormones / Q000032:analysis / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D012098:Reproduction / Q000276:immunology / Q000502:physiology*; D013312:Stress, Physiological / Q000097:blood* / Q000276:immunology / Q000378:metabolism; D013997:Time Factors
D029941:Fish Proteins; D006023:Glycoproteins; D007106:Immune Sera; D010907:Pituitary Hormones; C064448:somatolactin protein, fish; D006854:Hydrocortisone