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...

A sensitive radioimmunoassay (RIA) for the determination of inhibin in peripheral plasma and tissue homogenates of different species has been developed using antisera to partially purified bovine follicular fluid (bFF) inhibin and 125I-labelled bFF 32 kDa inhibin. Antisera were produced by immunization of rabbits with partially purified bFF inhibin prepared by immunoaffinity chromatography. Increasing doses of a high titre antiserum could neutralize the suppressing effect of bFF, porcine follicular fluid and rat ovarian homogenate on FSH secretion from rat anterior pituitary cells in culture. Sensitivity of the assay was 3.1 ng International Research Standard of porcine inhibin per tube. Parallel inhibition curves were obtained for inhibin preparations from female and male animals of ten species, i.e. cattle, goats, sheep, cats, dogs, monkeys, pigs, horses, rats and man. Inhibin subunits and related proteins cross-reacted minimally with the antiserum used in the study. Plasma concentrations of inhibin in adult male and female rats were measured by the RIA before and at various times after gonadectomy. Inhibin levels in peripheral plasma before gonadectomy were significantly higher in adult female than in adult male rats. Inhibin levels decreased abruptly after gonadectomy in both sexes and they correlated negatively with plasma concentrations of FSH. This inhibin RIA will facilitate studies of the physiology of inhibin in various species of animals.

No abstract

No abstract

No abstract