Growth Hormone Size Variants: Changes in the Pituitary During Development of the Chicken (44464) CARLOS ARA ´ MBURO,* ,1 MARICELA LUNA,* MARTHA CARRANZA,* MARISA REYES,* HILDA MARTI ´ NEZ-CORIA,* AND COLIN G. SCANES² ,2 *Centro de Neurobiologı ´a, Campus UNAM-UAQ Juriquilla, Universidad Nacional Auto ´noma de Me ´xico, Quere ´taro, Me ´xico; and ²Department of Animal Science, Rutgers-The State University of New Jersey, New Brunswick, New Jersey 08903 Abstract. There is considerable evidence for the existence of structural variants of growth hormone (GH). The chicken is a useful model for investigating GH heteroge- neity as both size and charge immunoreactive-(ir) variants have been observed in the pituitary and plasma. The present study examined the size distribution of ir-GH in the pituitary gland of chicken, from late embryogenesis through adulthood. Pituitaries were homogenized in the presence of protease inhibitor, and the GH size variants were separated by SDS-PAGE, transferred by Western blotting, immunostained with a specific antiserum to chicken GH, and quantitated by chemiluminescence followed by laser densitometry (chemiluminescent assay). Under nonreducing conditions ir-GH bands of 15, 22, 25, 44, 50, 66, 80, 98, 105 and >110 kDa were observed. Both the relative proportion of the GH size variants and the total pituitary content varied with developmental stage and age. The proportion of the 15-kDa fragment was greatest in the embryonic stage, and then it decreased. The proportion of the monomeric 22-kDa form was lowest at 18 days of embryogenesis (dE) and highest at 20 dE. In contrast, the high MW forms (≥66 kDa) were lowest in embryos, and they increased (P < 0.05) after hatching. The 22-, 44-, 66-, and 80-kDa forms were assayed for activity by radio- receptor assay following isolation by semipreparative SDS-PAGE. Only the 22-kDa GH variant showed radioreceptor activity. Under reducing conditions for SDS-PAGE, ir- GH bands of 13, 15, 18, 23, 26, 36, 39, 44, 48, 59 and 72 kDa were oberved, but most of the high MW form disappeared. There was a concomitant increase in the proportion of the monomeric band and of several submonomeric forms. The present data indicate that the expression, processing, and/or release of some if not all size variants are un- der some differential control during growth and development of the chicken. [P.S.E.B.M. 2000, Vol 223] T here is considerable evidence for the existence of structural variants of GH. This may be due to gene duplication (e.g., human GH-N and GH-V forms) (1) or alternative splicing of the mRNA as in the case of 20 kDa human GH (1) and also perhaps eel GHI and GHII (2). Multiple forms of GH are also due to post-translational modification of GH due to deamidation, proteolytic cleav- age, cleavage and reduction, glycosylation, phosphoryla- tion, and aggregation (1, 3, 4). Some of these post- translationally modified forms exhibit differences in biological activity (e.g., human (5), bovine (6), chicken (7)). The chicken appears to be a useful model for the investi- gation of GH variants as both charge and size ir-variants (including oligomers) have been observed in the pituitary gland (7–9) as have glycosylated (10, 11), phosphorylated (12, 13), and cleaved forms (14). One isomer has been isolated that presumably correponds to the nontransformed monomer (15). This monomeric variant exhibits a range of biological activities including increasing the rate of both IGF-I secretion by hepatocyte in vitro and lipolysis in vitro This is a paper of the Journal Series of the New Jersey Agricultural Experiment Station (project 06140) supported by the Hatch Act and State Funds (C.G.S.) and also by grants from The Rockefeller Foundation, The Fogarty International Center (NIH), DGAPA-UNAM (Me ´xico, PAPIIT IN206196), and CONACYT(3455-PN) to C.A. 1 To whom requests for reprints should be addressed at Centro de Neurobiologı ´a, Campus UNAM-UAQ Juriquilla, Universidad Nacional Auto ´ noma de Me ´xico, Apdo. Postal 1–1141, Quere ´taro, Qro., 76001, Mexico. E-mail: aramburo@servidor. unam.mx 2 Present address: College of Agriculture, Iowa State University, Ames, IA 50011. Received March 25, 1999. [P.S.E.B.M. 2000, Vol 223] Accepted July 30, 1999. 0037-9727/00/2231-0067$14.00/0 Copyright © 2000 by the Society for Experimental Biology and Medicine ONTOGENY OF GH VARIANTS 67