Growth Hormone Size Variants: Changes in the Pituitary During Development of the Chicken

Abstract

Abstract. There is considerable evidence for the existence of structural variants of growth hormone (GH). The chicken is a useful model for investigating GH heterogeneity 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 radioreceptor 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 under some differential control during growth and development of the chicken.

Non-classical Signalling of Growth Hormone in the Chick Neural Retina?

“…Retinal GH has biological activity (promoting RGC cell survival and RGC axonal growth) and its siRNA knockdown Sanders et al, 2010Sanders et al, , 2011 or immunoneutralisation (Sanders et al, 2005;) blocks these actions. However, as neither 15 kDa nor scGH are thought to bind to the classical GHR (Aramburo et al, 2000(Aramburo et al, , 2001 GH signalling in the early chick neural retina may occur through non-classical mechanisms, especially as these GH moieties are likely to be acting as autocrines rather than endocrines. The demonstration that fragments of human and mammalian GH molecules have some biological activities despite lacking the stoichiometric requirements for classical GH:GHR interactions provides evidence for the presence of some non-classical pathways in GH signalling.…”

“…In the chicken, both size and charge variants are present in the pituitary gland (Houston and Goddard, 1988;Aramburo et al, 1989;Houston et al, 1990), together with glycosylated (Berghman et al, 1988;Aramburo et al, 1989Aramburo et al, , 1990aAramburo et al, , 2004Diaz et al, 1993), phosphorylated (Aramburo et al, 1990b(Aramburo et al, , 1992, cleaved and oligomeric (Aramburo et al, 2000) forms. The abundance of these forms changes during development (Aramburo et al, 2000) and in extrapituitary tissues, tissue-specific ways (e.g. in the testis, Luna et al, 2004;Martinez-Moreno et al, 2011; in lymphoid tissue, Luna et al, 2005; in the brain, Alba et al, 2011; in the neural retina Baudet et al, 2003; in the ovarian follicle, Ahumada et al, 2012).…”

“…This moiety is thought to result from the proteolytic processing of full-length chicken GH (Aramburo et al, 2001), at position Arg 133 -Gly 134 of the 22 kDa monomer. This fragment has little affinity for the GH binding sites on chicken liver membranes (<1% of the monomer protein), as does the 1-132 sequence of recombinant chicken GH (Aramburo et al, 2000). Another submonomeric moiety, a 17 kDa variant, is the most abundant GH moiety in lymphoid (Luna et al, 2005) and reproductive (Luna et al, 2004;Martinez Moreno et al, 2011;Ahumada-Solorzano et al, 2012) tissues, but the ability of this moiety to bind to GHRs or plasma membranes or to induce biological activity is currently unknown.…”

O‐Glycosylated 24 kDa human growth hormone has a mucin‐like biantennary disialylated tetrasaccharide attached at Thr‐60

“…Glycosylated chicken GH has a longer half‐life than non‐glycosylated GH 84, 86, stimulates deiodination of thyroid hormone in the chick embryo 78, has a lower affinity than non‐glycosylated GH for liver receptors 87, and stimulates Nb2 cell proliferation 87. Moreover, the release of glycosylated GH from pituitary explants and primary cell cultures is up‐regulated by GH releasing hormone 84, and its levels in the pituitary gland fluctuate during development 87, 88 as do its levels in tissues of the immune system 89. In pigs, the levels of glycosylated porcine GH increased in the pituitary during progression of fetal life 90, and obese pigs had less glycosylated GH than lean pigs 80.…”