IGF availability during bone development in foetal sheep

Abstract

Skeletal growth is achieved through endochondral ossification at the growth plate and intramembranous ossification at the bone surface. Evidence suggests that the insulin-like growth factors, IGF-I and IGF-II, are major factors involved in these processes. The objective of this thesis was to identify the potential role IGFs play in the growth and development of foetal sheep long bones. To achieve this, the localisation of IGF-I, IGF-II and IGF BP proteins was determined by immunohistochemistry in long bones isolated from foetal sheep between 50 and 140 days gestation. IGF-I and IGF-II binding sites were localised in bone by radioligand binding, and further characterised using an affinity cross-linking assay. The local production of IGF-I, IGF-II and IGF BP-2, -4 and -5 was analysed by in situ hybridisation and Northern blot hybridisation. To investigate whether circulating IGF-I could contribute to the pool of IGF-I in bone, labelled IGF-I was infused into the circulation of foetal sheep in utero and the localisation of labelled IGF-I was analysed in long bones. The results demonstrated that IGF-I, IGF-II and IGF BP proteins were similarly localised during foetal development in many areas of bone. In the hypertrophic zone of the growth plate, the presence of IGF-I, IGF-II and IGF BPs in chondrocytes coincided with specific cellular binding of IGF-I and IGF-II, which was found to be to the Type I and Type II IGF receptors, respectively. Additional binding of IGF-I and IGF-II was found in cartilage matrix, which was likely to be to extracellular matrix proteins. In bone, IGF-I, IGF-II and IGF BPs were not only found in cells from the bone cell lineage, but were also detected in bone matrix. IGF-II appeared to be locally produced during foetal development in the growth plate and diaphysis, from multiple IGF-II transcripts sized between 1.0 and 6.4 kb. At the cellular level, IGF-II mRNA was abundant in those cells where IGF-II peptide was located. Although IGF-I mRNA (7.4 kb) and IGF-I peptide were also co-localised, levels of IGF-I mRNA were barely detectable in foetal bone at any age. There was no evidence of local production of IGF BP-2, -4 or -5 in the growth plate. Labelled IGF-I derived from the circulation was available to a limited number of cells in bone, located at the boundary between the growth plate and metaphysis, and within vascular canals embedded in the growth plate. Overall, results from this study demonstrate that IGF components are widely and similarly distributed in long bones during foetal sheep development. However, the availability of IGF-I and IGF-II to cells in bone appear to be different. While the local production of IGF-II was likely to produce effects on many cells in bone, neither the level of IGF-I expression nor the presence of circulating IGF-I was likely to contribute sufficient levels of IGF-I to enable cells in most areas of bone to respond to IGF-I actions. Sufficient levels of IGF-I appeared to be available in only one area, located at the boundary between the growth plate and bone. This area has recently been identified as an important regulatory region for endochondral ossification.