GENERAL AND COMPARATIVE ENDOCRINOLOGY 79, 83-88 (1990) Development and Application of Homologous Radioimmunoassay for Newt Prolactin KOUHEIMATSUDA,KAZUTOSHI YAMAMOTO,AND SAKAE KIKUYAMA~ Department of Biology, School of Education, Waseda University, Shinjuku-ku, Tokyo 169, Japan Accepted August 29, 1989 A specific and sensitive homologous radioimmunoassay (RIA) for newt (Cynops pyrrho- gaster) prolactin (PRL) was developed. PRL isolated from newt pituitary glands was used for generating antiserum in a rabbit, for radioiodination, and for the standard. Several dilutions of plasma and pituitary homogenate of newts yielded dose-response curves which were parallel to the standard curve. Plasma from hypophysectomized newts showed the least amount of cross-reaction. Pituitary homogenates of other species of urodeles such as Ambystoma mexicanum and Onychodactylus japonicus gave inhibition curves which were parallel to the standard curve. Purified PRLs of anurans such as Rana catesbeiana and Bufo japonicus gave inhibition curves which did not parallel the standard. Bovine PRL and ovine PRL showed no inhibition of binding even at relatively high doses in this RIA. The RIA was applied to the determination of plasma and pituitary PRL levels in the adult newts treated with dopamine agonist (bromocriptine) and/or antagonist (pimozide). Pimozide enhanced PRL levels and bromocriptine antagonized it, while pituitary PRL levels were not appre- ciably changed. 8 1990 AC&~~C PM. IIIC. A possible involvement of prolactin (PRL) in various biological phenomena in urodeles has been reported (see Nicoll and Bern, 1972; Ensor, 1978; Kikuyama, 1988). However, there is no information about the plasma and pituitary PRL levels in urode- les. Employing purified anuran PRLs (Ya- mamoto and Kikuyama, 1981; Yamamoto et al., 1986) and antisera against them, we have developed radioimmunoassay (RIA) systems (Yamamoto and Kikuyama, 1982; Yamamoto et al., 1989) which are applica- ble to the measurement of PRLs of Rana and Bufo species (Kuhn et al., 1985; Nii- numa et al., 1986; Andersen et al., 1989; Ishii et al., 1989) but not to the measure- ment urodele PRLs (Yamamoto and Kikuyama, 1982). Recently we have obtained a highly puri- fied urodele PRL from the pituitary glands ’ To whom all correspondence should be addressed. of newts, Cynops pyrrhogaster and raised an antiserum against it (Matsuda et al., 1990). In this paper, the development and application of RIA for newt PRL will be described. MATERIALS AND METHODS Newt PRL. PRL for the production of antiserum, radioiodination, and use as a reference standard was purified from pituitary glands of newts, C. pyrrhog- aster, by extraction of acetone-dried powder with acid acetone and high-performance liquid chromatography on anion exchange, gel futration, and reverse-phase columns as described previously (Matsuda et al., 1990). Production of antiserum. Antiserum was raised in a mature female albino rabbit by the lymph nodes injec- tion technique (Goudie et al., 1966). Details for immu- nization have been described elsewhere (Matsuda et al., 1990). Radioimmunoassay. Radioiodination of newt PRL with Na’? (Carrier free; The Radiochemical Centre Amersham, England) was carried out at room temper- ature according to the modified lactoperoxidase method (Sakai et al., 1975). The antiserum against newt PRL exhibited an ability to bind the radioligand 83 0016-6480190 $1.50 Copyright 0 1990 by Academic Press, Inc. All iigllts of repmduction in any foml reserved.