remove all IGFBP from plasma, particularly the lower molecular mass IGFBP-1, IGFBP-2 and IGFBP-4. These residual binding proteins confound assays for IGF.
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Ontogenic and nutritional changes in circulating insulin-like growth factor (IGF)-I, IGF-II and IGF-binding proteins in growing ewe and ram lambs K L Gatford, K J Quinn1,2, P E Walton1,2, P A Grant1,3, B J Hosking, A R Egan and P C Owens1,3 Department of Agriculture and Resource Management, University of Melbourne, Parkville, Victoria 3052, Australia, 1The Cooperative Research Centre for Tissue Growth and Repair, PO Box 10065 Gouger Street, Adelaide, South Australia 5000, Australia, 2CSIRO Division of Human Nutrition, PO Box 10041 Gouger Street, Adelaide, South Australia 5000, Australia, and 3Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide, South Australia 5005, Australia (Requests for offprints should be addressed to K L Gatford who is now at Department of Physiology, University of Adelaide, Adelaide, South Australia 5005, Australia) (B J Hosking is now at Better Blend Stockfeeds Pty Ltd, PO Box 21, Oakey, Queensland 4401, Australia) (P E Walton is now at Diagnostic Systems Laboratories Inc., 445 Medical Center Blvd, Webster, Texas 77598–4217, USA)
Abstract The ontogeny of the IGF endocrine system was investigated in 15 young lambs before and after weaning at 62 days of age. Before weaning, plasma IGF-I concentrations were higher in rams than ewes, and plasma concentrations of IGF-II and IGF-binding protein-3 (IGFBP-3) also tended to be higher in rams than in ewes. Feed intake of ewes and rams was restricted after weaning to remove sex differences in feed intake. Plasma concentrations of IGF-I and IGFBP-3 did not differ between
Introduction The somatotropic axis plays a major role in regulation of growth in mammals. Growth hormone (GH) is secreted from the anterior pituitary of the sheep in a pulsatile fashion (Davis et al. 1977). In well-nourished sheep, exogenous GH stimulates synthesis of insulin-like growth factor-I (IGF-I) and its major circulating binding protein, IGFBP-3, resulting in increased circulating IGF-I and IGFBP-3 concentrations (Hodgkinson et al. 1991, Hua et al. 1993, Pell et al. 1993). IGF-I acts as both an endocrine and paracrine growth factor, and appears to mediate many of the growth-promoting actions of GH (Salmon & Daughaday 1957, Cohick & Clemmons 1993, Jones & Clemmons 1995). IGFBPs increase the half-life of circulating IGF, and also modulate IGF actions on target tissues (Baxter 1993, Jones & Clemmons 1995). In sheep the half-life of IGF-I in plasma is increased from 10 min in the free form to 545 min when it is bound to IGFBP-3 in the 150 kDa complex (Davis et al. 1989). IGFBP-3 is the major carrier of IGF-I in adult sheep plasma, whilst in the fetal sheep IGFBP-3, IGFBP-2 and a soluble form of the IGF-II receptor each appear to carry about one-third Journal of Endocrinology (1997) 155, 47–54 0022–0795/97/0155–0047 $08.00/0
rams and ewes at 100 days of age, but plasma IGF-II was higher in rams than in ewes at this time. Since circulating concentrations of GH were higher in rams than in ewes at 100 days of age, this implies that the restricted feed intake blocked the IGF-I and IGFBP-3 responses to GH. We conclude that sex differences in circulating IGF-I and IGFBP-3 concentrations in the growing lamb alter with age, and are not present when nutrition is restricted. Journal of Endocrinology (1997) 155, 47–54
of circulating IGF (Hodgkinson et al. 1989b, Gallaher et al. 1992). The ontogeny of the IGF–IGFBP system in the growing lamb is poorly understood at present. Early studies of plasma IGF used acid–ethanol techniques for separation of IGF from IGFBP before assay (Roberts et al. 1990, Medrano & Bradford 1991). This technique does not remove all IGFBP from plasma, particularly the lower molecular mass IGFBP-1, IGFBP-2 and IGFBP-4. These residual binding proteins confound assays for IGF (Mesiano et al. 1988, Breier et al. 1991). This is particularly a problem in ontogenic and nutritional studies, since the proportions of high and low molecular mass IGFBP in blood change with development and nutrition. We have therefore used size exclusion chromatography of plasma under acidic conditions to dissociate IGF from IGFBP to ensure complete removal of IGFBP from plasma extracts before assay for IGF. In this study we have characterised the ontogeny of IGF and IGFBP from birth to weaning in male and female sheep. In the rat, patterns of GH concentrations, as well as mean GH concentration, are important in determining growth and hormonal responses ( Jansson et al. 1982, Clark
? 1997 Journal of Endocrinology Ltd Printed in Great Britain
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K L GATFORD
and others
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IGF and IGFBP in the growing lamb
et al. 1985, Isgaard et al. 1988, Maiter et al. 1988, 1992, Bick et al. 1992). In prepubertal lambs growing under pasture conditions, differences in GH concentrations and patterns are associated with gender differences in plasma concentrations of IGF-I and IGFBP-3 (Gatford et al. 1996). Plasma GH concentrations in rams are approximately twice those in ewes when feed intake is not different in ram and ewe lambs, under conditions of restricted feed intake (Gatford et al. 1997). Since IGF-I and IGFBP-3 responses to exogenous GH are blocked by fasting or undernutrition in the sheep (Hodgkinson et al. 1991, Hua et al. 1993, Pell et al. 1993), we hypothesised that concentrations of IGF and IGFBP would not differ between sexes in these animals, despite the presence of sexually dimorphic patterns of circulating GH. We have therefore also characterised circulating concentrations of IGF and IGFBP-3 in ewe and ram lambs at 100 days of age after 20 days of restricted feed intake. Materials and Methods Animal studies Management of experimental animals has been described in more detail elsewhere (Gatford et al. 1997). All experimental procedures were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and had received approval from the University of Melbourne Animal Experimentation Ethics Committee. The experiment was performed at the Mt Derrimut Field Station, Deer Park, Victoria, Australia (latitude 37 )49*S, longitude 144 )45*E). Briefly, eight ram and eight ewe (Border Leicester#Australian Merino)#Romney lambs, born between 15 August and 21 August 1993, were paired on the basis of age and litter size. Half the lambs of each gender were singletons and the remaining lambs were twins. Lambs and their mothers grazed grass–subterranean clover pastures plus hay and oat supplements until weaning. The lambs were separated from the lactating ewes at 62 days of age, and remained at pasture until their transfer to indoor single pens 6 days later. For the remainder of the experiment, lambs were fed lucerne chaff in vitro dry matter digestibility (IVDMD) 65%, total N 3·9%). Lambs were transferred to metabolism crates at 79 days of age, and for the remainder of the experiment (30&1 days, mean&...), they were restricted-fed by offering feed at 2·9&0·1% of live weight per day. The level of feed offered was calculated individually for each ewe–ram pair as a proportion of live weight based on the measured feed intake of the ewe lamb between 75 and 78 days of age. Feed intake was recorded daily and the amount of feed offered was adjusted for liveweight twice weekly throughout this restricted feeding period. Liveweights of lambs were recorded weekly until weaning and then twice weekly. One single-born ewe lamb broke a leg before measurement of feed intake and Journal of Endocrinology (1997) 155, 47–54
was replaced in the experiment by another single-born ewe lamb of similar liveweight and birthweight from the same flock. Another single-born ewe lamb failed to maintain intake after transfer to a metabolism crate, and this lamb was removed from the experiment. The ram lamb of this pair was subsequently offered the same amount of feed per unit liveweight as another single-born ewe–ram pair. The ewe and two rams in this group were treated as one pair for statistical purposes. Blood sampling Blood samples were taken by jugular venipuncture of lambs between 12 and 36 h after birth (1 day old), and then at mean ages of 16 days and 34 days and at 62 days, immediately before weaning. The post-weaning plasma sample was pooled from a series taken from all animals at 10 min intervals from 0800 h to 1800 h at 100 days of age, 21 days after commencement of the restricted feeding period, for measurement of circulating GH as described elsewhere (Gatford et al. 1997). Indwelling jugular venous catheters were inserted at least 3 days before repeated blood sampling. On the day of sampling, half of the daily ration of lucerne chaff was fed in equal portions at 30 min intervals from 0600 h to 1800 h, when the remaining feed was given. All blood samples were taken into collection tubes containing 125 IU lithium heparin (Disposable Products Pty Ltd, Adelaide, Australia). Plasma was separated by centrifugation and stored at "20 )C. Subsamples of plasma from blood taken at 2 h intervals at 100 days of age were combined for each animal and analysed for IGF and IGFBP. Measurement of circulating IGF-I , IGF-II and IGFBP-3 concentrations by RIA Plasma IGF-I, IGF-II and IGFBP-3 concentrations were analysed in all animals at each age. The IGFs were extracted from plasma by size exclusion HPLC at pH 2·5 using a modification of the original procedure (Scott & Baxter 1986) as described previously (Owens et al. 1990, Carr et al. 1995, Kind et al. 1995). Recovery of 125I-IGF-I was 96&2% (n=13). Recombinant human IGF-I and IGF-II (GroPep Pty Ltd, Adelaide, Australia) were used as standards and for preparation of tracer (Francis et al. 1989). IGF-I was measured by RIA using rabbit anti-human IGF-I (GroPep Pty Ltd) as described previously (Francis et al. 1989, Owens et al. 1990), modified according to the antibody supplier’s instructions. A similar RIA was used to measure IGF-II, using a mouse monoclonal antibody to rat IGF-II (IgG SF-2 at 1 ng/tube, a gift from Dr K Nishikawa, Kanaza Medical University, Ishakawa, Japan). The cross-reaction of IGF-II in the IGF-I RIA was 0·1), but rams tended to be heavier than ewes at weaning and at 100 days of age (P0·1). Ontogenic changes in plasma IGF-I, IGF-II and IGFBP-3 and their molar concentration ratios in ewe and ram lambs Plasma concentrations of IGF-I, IGF-II and IGFBP-3 throughout the experiment are presented in Fig. 1. Plasma IGF-I concentrations (Fig. 1a) were not affected by sex (P=0·3829), but sex–age interactions were highly significant (P
