title: Development, validation, and utilization of a novel antibody specific to the type III chicken gonadotropin-releasing hormone receptor.
abstract: Two gonadotropin-releasing hormone receptors (GnRH-Rs) have been characterized in chickens to date: cGnRH-R-I and cGnRH-R-III, with cGnRH-R-III being the predominant pituitary form. The purpose of the present study was to first validate a novel antibody for the specific detection of cGnRH-R-III and second, using this antibody, detect changes in cGnRH-R-III protein levels in the pituitary gland of male and female chickens during a reproductive cycle. The localization of cGnRH-R-III within the anterior pituitary gland was also determined. Western blotting of pituitary extracts and transiently transfected COS-7 cell lysates revealed that our antibody is highly specific to cGnRH-R-III protein. Similarly, when used in immunocytochemistry, this antibody specifically detects cells expressing cGnRH-R-III and not cGnRH-R-I. Western blot analyses of chicken pituitary gland homogenates show that cGnRH-R-III protein levels are significantly greater in sexually mature birds than in immature birds or birds at the end of a reproductive cycle (P < 0.0001). A similar pattern was observed for both males and females. Additionally, the antibody was able to detect cGnRH-R-III in cells along the periphery of the cephalic and caudal lobes of the anterior pituitary where the cells containing the gonadotropins are located. In summary, we successfully validated a novel antibody to cGnRH-R-III and showed levels of cGnRH-R-III protein in the pituitary fluctuate with respect to the reproductive status in both male and female chickens.
authors.affiliation: Department of Animal and Poultry Science, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada.
authors.forename: H O
authors.initials: HO
authors.lastname: McFarlane
authors.forename: N T
authors.initials: NT
authors.lastname: Joseph
authors.forename: S R
authors.initials: SR
authors.lastname: Maddineni
authors.forename: R
authors.initials: R
authors.lastname: Ramachandran
authors.forename: G Y
authors.initials: GY
authors.lastname: Bédécarrats
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D030161:Avian Proteins / Q000032:analysis / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods*; D019556:COS Cells; D002645:Chickens; D002522:Chlorocebus aethiops; D005260:Female; D008297:Male; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000378:metabolism; D011966:Receptors, LHRH / Q000032:analysis / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D000906:Antibodies; D030161:Avian Proteins; C434455:GnRH receptor 3; D011966:Receptors, LHRH