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  • Author: S C Butterwith x
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C Goddard, R Johnson, H J Gilhooley, J O Gardner, A Gray, R S Wilkie, and S C Butterwith

ABSTRACT

The increase in muscle weight in neonatal animals is a consequence of increased protein accretion and DNA content. GH increases protein accretion but direct effects of GH on myogenic cell proliferation have not been demonstrated. Sex-linked dwarfism in the chick is caused by mutation or deletion in the GH receptor gene and has provided a useful model to study the physiological consequences of GH insensitivity. This study determined the consequences of GH receptor gene mutation on muscle cell proliferation in vivo. Northern and Southern blotting and PCR analysis revealed restriction fragment length polymorphism patterns and a 1·7kb deletion of the intracellular domain of the GH receptor gene in commercial dwarf broiler chicks, similar to the Connecticut strain in which there is a dysfunctional GH receptor. Cell proliferation was measured in muscle sections from normal and dwarf chicks after incorporation of 5-bromo-2′-deoxyuridine (BrdU; 25 mg/kg) in vivo at 2, 5 and 13 days of age. Incorporation of BrdU into nuclei was measured in frozen sections, counterstained with propidium iodide to estimate the total number of nuclei by quantitative image analysis, and the labelling index was calculated. Paraffin-embedded sections of breast muscle were stained using an anti-human IGF-I polyclonal antibody. Expression of IGF-I mRNA in muscle from each genotype at 5 days of age was measured by RNAse protection assay.

The labelling index was similar in 2-day-old chicks from both genotypes (normal, 20·14 ± 2·39%; dwarf, 19·79 ± 5·83%). By day 5 the labelling index had decreased but was significantly higher (P<0·02) in normal (12·53 ± 3·36%) compared with the dwarf (6·25 ± 1·39%). By 13 days of age, there was a further decrease in labelling index but no difference between the groups (normal, 4·92 ± 1·28%; dwarf, 4·96 ± 1·51%). IGF-I mRNA was expressed and IGF-I peptide was identified in muscle sections but there was no difference between genotypes. The results show that cell division in breast muscle in vivo is high in neonatal chicks but it declines with increasing age. The absence of a functional GH receptor in the dwarf is associated with a greater decline in DNA synthesis and suggests that GH may directly affect a proportion of cells, since there was no difference in IGF-I mRNA or peptide.