Identification of the second gonadotropin-releasing hormone in chicken hypothalamus: Evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species (reproduction/neuropeptide) KAORU MIYAMOTO*, YOSHIHISA HASEGAWA*, MITSuo NOMURA*, MASAO Since the structural determination of porcine hypothalamic luteinizing hormone (LH)-releasing hormone (LHRH) in 1971 (1), the concept has widely been acepted that gonadotropin secretion is controlled by a sole gonadotropin-releasing hormone (GnRH), such as mammalian LHRH. On the other hand, we have demonstrated that gonadotropin secretion is not controlled by LHRH under certain physiological and experimental conditions (2). However, there has been no structural evidence that two or more than two GnRHs coexist and function in any vertebrate.The present paper represents the structural demonstration of the existence of two different GnRHs at least in avian species.Recently, we (3, 4) and King and Millar (5-7) have independently isolated chicken LHRH and determined its structure as [Gln8]LHRH. We have purified chicken LHRH by monitoring its biopotency towards rat anterior pituitary cells. During the course of the purification, another gonadotropin-releasing activity was found in the chicken hypothalamic extract, which was well separated from that due to chicken LHRH on ion-exchange chromatography.The present paper describes the isolation and structural determination of the second GnRH in chicken hypothalamic extract (designated chicken GnRH-II).Chicken GnRH-II has been purified by successive gel filtration, ion-exchange HPLC and reverse-phase HPLC. A pure peptide has been isolated in a yield of 7 pg (6 nmol), starting from 10,000 chicken hypothalamic fragments.The amino acid composition of the peptide indicated a decapeptide structure consisting of SerGlxPro1Gly2Tyrl-His2Trp2. By amino acid analyses and terminal analyses of enzymatic fragments derived from the purified decapeptide, the complete structure of chicken GnRH-II has been determined to be: pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly- NH2.The present paper also describes the gonadotropin-releasing activity of chicken GnRH-II compared with that of mammalian LHRH. MATERIALS AND METHODSAssay of Gonadotropin-Releasing Activity. Gonadotropinreleasing activity was estimated on the rat anterior pituitary cell culture system as reported (3).Anterior pituitaries were obtained from female Holzman rats (180-to 220-g body weight), agitated in trypsin solution (0.25%), and finally dispersed with 1% Viokase (GIBCO) solution. Cells were collected and washed three times with Dulbecco's modified Eagle's medium (DME medium) containing gentamycin (100 ,ug/ml), fungizone (25 ttg/ml), 5% horse serum, 5% human cord serum, 2.5% bovine calf serum, 0.1% glutamine, and 1% nonessential amino acids (GIBCO). Cells were resuspended in an appropriate volume of the medium described above, and 0.2-ml portions (containing 5 x 104 cells) were put into 96 multi-well tissue culture plates (Nunc,. Th...

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Four peptides having SRIF-like bioactivity when assayed by a rat anterior pituitary cell culture system have been isolated from chicken hypothalamic extracts. These four peptides have been purified from 2,000-5,000 chicken hypothalami in a yield of 2.2 nmol to 16 nmol. The isolated SRIFs have been characterized by amino acid analysis. One consists of 28 amino acids which are identical with mammalian SRIF-28 except that one Met residue is replaced by a Leu residue. Three other SRIF-like substances with low mol wt have also been isolated in a pure state. One is identical with mammalian SRIF-14, as judged by chromatographic comparison on HPLC and its amino acid composition as well as the specific bioactivity on rat pituitary cells. The amino acid composition and specific bioactivity of another one is identical with that of mammalian SRIF-14, but it is different from mammalian SRIF-14 in its elution position on HPLC. The third SRIF-like substance consists of 16 amino acids and is less potent than mammalian SRIF-14. It is concluded that several SRIF-like peptides exist and that SRIF-28, with one amino acid substitution from Met to Leu, and SRIF-14, identical with the mammalian one, are the major components in chicken hypothalamus.