ISOLATION, CHARACTERIZATION, AND RADIOIMMUNOASSAY OF ATLANTIC HALIBUTSOMATOLACTIN AND PLASMA-LEVELS DURING STRESS AND REPRODUCTION IN FLATFISH

Somatolactin (SL), a recently identified teleost pituitary hormone whi ch is a member of the growth hormone/ prolactin family, was isolated f rom pituitary tissue of Atlantic halibut (Hippoglossus hippoglossus). Pituitary proteins were extracted in ammonium bicarbonate (pH 7.8), fr actionated using gel filtration chromatography, and purified using rev ersed-phase high-performance liquid chromatography. Halibut SL was ide ntified on the basis of molecular size (determined by gel electrophore sis and mass spectroscopy), cross-reactivity of the putative hormone w ith antisera to cod SL, and N-terminal amino acid sequence. Polyclonal antibodies to purified halibut SL were raised in rabbits, and a radio immunoassay (RIA) was developed for measurement of plasma concentratio ns of SL using purified halibut SL as a standard. The RIA was tested i n several flatfish species including Pacific halibut (Hippoglossus ste nolepis), English sole (Pleuronectes vetulus), and rock sole (Lepidops etta 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 Atl antic halibut SL standard curve, indicating that the assay is valid fo r a range of flatfish species. Using halibut SL antiserum, SL was loca lized in the pars intermedia of English sole pituitary, where it has b een identified in previously examined teleost species. The RIA was use d to measure plasma levels of SL in Atlantic halibut and English sole during reproductive development, and in English sole subjected to vari ous types of environmental stressors, including handling and crowding. In both sole and halibut, plasma SL concentrations remained relativel y constant throughout gonadal development, but dropped during or follo wing ovulation. Plasma SL levels in English sole tended to increase in response to acute stress, in parallel with plasma cortisol levels. (C ) 1997 Academic Press.