ABSTRACT

The ontogenesis of the pulsatility of GH in the ovine fetus was determined by obtaining blood samples at 20-min intervals for 3-h periods from fetuses (n = 33) at various stages of development (76–147 days gestation), and in neonatal life (n = 19). A significant increase (P <0·01) in the GH mean, nadir and maximum, and pulse height was observed between the ages of 100 and 130–139 days of gestation. An analysis of the difference in the mean, maximum and nadir concentrations between 100 and 139 days of gestation revealed that males had higher GH levels than females (P <0·05).

There was a significant fall in plasma GH concentrations from 140 days of gestation to term, but before the onset of active labour. There was a more rapid fall in the circulating levels of fetal GH directly following birth. Immediately before birth fetal GH levels were still relatively high, but within 60 min of birth they had fallen by more than 80%.

It is suggested that these changes in the pulsatile pattern of GH release are a consequence of both maturational changes in the hypothalamic-pituitary unit and the effects of pregnancy-related factors on GH release. The sexually dimorphic nature of GH release in the adult is also observed in the sheep fetus during late gestation.

J. Endocr. (1986) 109, 307–312

ABSTRACT

A specific radioimmunoassay for insulin-like growth factor I (IGF-I) has been used to study the distribution of circulating IGF-I on its carrier proteins in sheep. Serum or plasma samples obtained from fetal, neonatal and adult sheep were chromatographed at neutral pH on Sephadex G-200 columns. IGF-I eluted primarily in the 20 000–100 000 dalton region and to a lesser extent in the 20 000–100 000 dalton region in the adult. In the fetus, IGF-I eluted primarily in the 20 000–50 000 dalton region. Studies in the neonatal lamb showed IGF-I activity to fluctuate between different size binding proteins but by 7 days after birth the pattern was similar to that of the adult. When labelled IGF-I and IGF-II were added to serum which was chromatographed over Sephadex G-200 at a neutral pH, binding was displayed in the 50 000– 100 000 dalton region for both adult and fetus. However in the fetus, but not in the adult, binding of ¹²⁵I-labelled IGF-II was also found at > 200 000 daltons. These studies demonstrate that the distribution of IGF-binding proteins varies between the adult and the fetal lamb. The appearance of the 150 000 dalton binding protein is coincidental with the appearance of the somatogenic receptor in the infant liver and is consistent with the GH-dependent nature of this form of binding protein in other species. The marked developmental changes in the circulating form of IGF-I in the perinatal lamb may be of functional significance and lead to altered biological availability of circulating IGF-I.

J. Endocr. (1986) 109, 333–338

Specific radioligand assays for the two somatomedins, insulin-like growth factor (IGF)-I and IGF-II, have been used to study the ontogeny of somatomedin secretion in the perinatal lamb. Plasma samples were obtained from sheep fetuses from 54 days of gestation to term (147 days) and from neonatal lambs. All samples were first extracted in acid-ethanol to remove the somatomedin-binding proteins. Concentrations of IGF-I measured by radioimmunoassay were lower (P <0·01) in the fetus than in the adult sheep (0·96 ± 0·17 (s.d.) units/ml, n = 11). Fetal IGF-I values rose (P <0·01) from 0·29 ± 0·15 units/ml (n = 6) at 50–80 days to 0·79 ± 0·18 units/ml (n=13) at 140–150 days. While values were similar 0–2 days after birth, they rose (P <0·01) to 2·4 ± 1·3 units/ml (n = 15) 3–7 days after birth. By 60 days they had fallen to adult values. In contrast, IGF-II levels measured by rat placental membrane radioreceptor assay were higher (P <0·001) in the fetus (2·71 ±1·06 units/ml, n = 18) than in the adult (1·0 ± 0·17 units/ml) and showed no gestational trend between 50 and 140 days of gestation. Longitudinal studies showed a fall (P <0·01) in IGF-II values starting several days before birth. By 12 h after birth, IGF-II concentrations were similar to the adult and showed no subsequent postnatal change. These results demonstrate that IGF-I and IGF-II are not secreted in parallel in the perinatal lamb. There are major changes in the regulation of both IGF-I and IGF-II in relationship to birth. It is suggested that the high fetal IGF-II concentrations appear to be maintained by a stimulus withdrawn before birth. The postnatal rise in IGF-I may be related to the increase in hepatic somatogenic receptors at this age.

ABSTRACT

This report describes essential requirements for the validation of a radioimmunoassay (RIA) for insulin-like growth factor-I (IGF-I) and presents solutions to some problems and pitfalls commonly observed. The preparation of IGF-I to be used as radioligand or standard has to be selected carefully since some IGF-I preparations are contaminated with variants which demonstrate different potencies for different antisera used in the RIA.

Accurate assessment of IGF-I levels in blood plasma requires an efficient extraction method for the IGF-binding proteins (IGFBPs). Extraction methods to remove the influence of IGFBPs in the RIA were compared using blood plasma of considerable differences in IGF-I/IGFBP ratios. Acidification of plasma before column chromatography on Sephadex G-75 (G75) is generally considered to be the most reliable extraction method, but it is very time-consuming. The acid–ethanol extraction (AE) of plasma is not valid in many situations. Non-parallel displacement to the IGF-I standard was observed with AE-extracted plasma samples in the RIA. In addition, a comparison of IGF-I values obtained in the RIA after AE or G75 extraction of fetal ovine plasma has shown no significant correlation.

We report an extraction technique based on a modified AE extraction followed by cryo-precipitation (AEC). AEC extraction on blood plasma reduced residual IGFBPs to a level that did not interfere in the assay. Furthermore, AEC-extracted plasma samples showed parallel displacement in the RIA to highly purified preparations of authentic IGF-I. We observed high correlations, with a slope close to unity, of IGF-I values obtained in the RIA using the AEC or G75 extraction for plasma from different species including adult and fetal sheep, rat, mouse and man. The AEC extraction provides a rapid and simple alternative to G75 extraction for blood plasma from a variety of species provided that high-affinity antisera are used for the RIA.

Journal of Endocrinology (1991) 128, 347–357

ABSTRACT

The binding of bovine GH (bGH) to hepatic membranes obtained from steers on either high (3% dry matter of body weight per day) or low (1% dry matter of body weight per day) planes of nutrition with or without an oestradiol-17β implant was studied (n = 5 per group). Binding studies were performed on both crude membrane homogenates and on 100 000 g microsomal membrane fractions; identical results were obtained using both preparations. In all four groups of animals, linear Scatchard plots were obtained, but following pretreatment of the membranes with MgCl₂ to remove endogenously bound hormone, curvilinear plots were obtained in the groups on the high plane of nutrition. Analysis of these curves suggested the presence of a high- and low-affinity binding site, the high-affinity site being fully occupied in the absence of MgCl₂ pretreatment.

The specific binding of bGH in MgCl₂-pretreated crude membranes was greater (P < 0·01) in well-fed steers (14·8 ± 1·6%) than in poorly fed steers (9·8 ± 0·9%). Scatchard analysis showed this to be due to the presence of a high-affinity site (dissociation constant (K _d) = 11·6 ± 3·3 pmol/l) in the well-fed animals only. In addition, there was an increase (P < 0·01) in the affinity, but not in the capacity, of the low-affinity site (K _d = 106·4 ± 22·8 pmol/l in well-fed steers and 197·0 ± 23·8 pmol/l in poorly fed steers).

Oestradiol treatment was associated with an increase (P < 0·01) in specific binding at both planes of nutrition, but binding was higher (P < 0·01) in well-fed (24·8 ± 2·9%) than in poorly fed (15·6 ± 3·7%) steers. Scatchard analysis after MgCl₂ pretreatment again showed a curvilinear plot at the high and a linear plot at the low nutritional plane. The effect of oestradiol was to increase (P < 0·001) the capacity of the high-affinity site from 1·87 ± 0·61 pmol/100 mg in the control well-fed group to 6·56 ± 1 ·2 pmol/100 mg. The capacity of the low-affinity site was increased (P < 0·01) from 20·1 ± 2·6 to 30·1 ± 3·2 pmol/100 mg in the well-fed group, with a similar change in the poorly fed group. Oestradiol had no effect on the apparent affinity of either binding site.

These studies demonstrate a heterogeneity of somatotrophic binding sites of hepatic membranes in steers. The presence of a high-affinity site is determined by nutritional status, whereas oestradiol primarily affects receptor capacity. Thus nutrition and oestradiol have independent and qualitatively different effects on somatotrophic binding. As the rate of weight gain correlated (P < 0·01) with the capacity of the high-affinity site, it is suggested that somatotrophic receptor modulation is a primary factor in the regulation of somatic growth in the ruminant.

J. Endocr. (1988) 116, 169–177

ABSTRACT

Plasma GH profiles and circulating concentrations of plasma insulin-like growth factors-I and -II (IGF-I and -II) were examined in 20 steers on either high (3% dry matter of body weight per day) or low (1% dry matter of body weight per day) planes of nutrition with or without an implant of oestradiol-17β. The response of plasma IGF-I and -II to a bolus injection of bovine GH (bGH) was also investigated.

Reduced feeding significantly (P <0·01) increased the mean concentration, peak height and integrated area of plasma GH. Treatment of steers with oestradiol at low nutrition significantly increased baseline GH concentrations. Treatment of steers with oestradiol at high nutrition significantly (P <0·05) increased mean, baseline, peak height, and integrated area of plasma GH. GH pulse frequency was not changed by either nutritional plane or oestradiol treatment.

Basal concentrations of plasma IGF-I were significantly (P <0·01) decreased by reduced feeding in both the oestradiol-treated and the control group. Treatment with oestradiol increased (P <0·01) basal plasma concentrations of IGF-I at both high and low levels of nutrition. After i.v. injection of bGH (0·1 mg/kg body weight), an increase in plasma IGF-I was observed only in steers at high nutrition. Basal concentrations of plasma IGF-II were not altered by nutritional manipulations but were significantly (P <0·001) increased by oestradiol treatment. After bGH infusion only steers at high nutrition showed an increase in plasma IGF-II. Significant correlations were observed between daily body weight gain and plasma concentrations of IGF-I (r= 0·91, P<0·001, n = 20) and also between the capacity of the high-affinity hepatic somatotrophic receptor and plasma IGF-I (r= 0·89, P <0·001, n= 10).

Decreased plasma concentrations of IGF-I at a low level of nutrition may abolish the growth-promoting activity of circulating GH. The increase in both GH secretion and the number of somatotrophic receptors with oestradiol treatment may represent a coordinated response of the somatotrophic axis leading to enhanced IGF-I and -II production and improved growth rate. The inferential relationships between the capacity of the high-affinity somatotrophic receptor, plasma concentrations of IGF-I and growth rates suggest that active modulation of somatotrophic receptors is an important regulatory constituent of the somatotrophic axis.

J. Endocr. (1988) 118, 243–250

ABSTRACT

The developmental pattern of plasma insulin-like growth factor-I (IGF-I) and insulin in calves subject to different patterns of weaning was investigated from birth until the age of 6 months. Fifteen male Friesian calves were fed on whole milk (10% of body weight per day) for the first 8 weeks after birth, then allocated into three balanced groups. Group 1 was weaned at 8 weeks; group 2 was weaned at 8 weeks, returned to milk-feeding at 13 weeks to be weaned again at the age of 16 weeks; group 3 was weaned at 12 weeks. After weaning the calves were fed on concentrates and lucerne hay.

At birth, circulating concentrations of IGF-I correlated with birth weight (r = 0·78, P< 0·001). There was a significant (P<0·001) fall in plasma IGF-I from birth (40·3 ± 2·5 μg/l) until 5 weeks (23·8± 1·3 μg/l), and then a gradual (P<0·01) rise until week 8 (35·0 ± 2·2 μg/l). Weaning (groups 1 and 2 after week 8) caused a significant (P<0·01) decrease in plasma IGF-I (20·5 ± 1·9 μg/l); thereafter plasma levels of IGF-I rose gradually (P<0·01) in animals fed on concentrates. The milk-fed calves (group 3) showed a progressive increase in plasma IGF-I with age until they were weaned at 12 weeks (51·0 ± 3·4 μg/l); IGF-I levels then decreased to be similar to group 1 (32·5 ± 2·1 μg/l). When group 2 was returned to milk-feeding, plasma IGF-I concentrations increased to 58·2 ±3·8 μg/l within 4 days and then continued to rise gradually until decreasing upon weaning. The age-related increase in the plasma concentration of IGF-I after 6 weeks was parallel in milk- and concentrate-fed calves.

Binding of ¹²⁵I-labelled bovine GH to hepatic membranes of neonatal calves was low at birth (specific binding; 1·56 ±0·29% n=3). Somatotrophic binding was apparent at 6 weeks (6·43 ±0·42%, n = 3) and increased (9·2± 1·1%, n=3) at the age of 12 weeks. Thus the early postnatal changes in plasma IGF-I may reflect the transition from GH-independent to GH-dependent IGF-I secretion.

Plasma concentrations of insulin at birth correlated with IGF-I (r = 0·54, P<0·05). However, regression analysis suggested no effect of insulin on birth weight independent of IGF-I. Plasma insulin levels rose gradually until weaning and thereafter were significantly (P<0·01) higher in the milk- than concentratefed calves.

Major changes in plasma concentrations of IGF-I related to monogastric (milk feeding) or ruminant (concentrate feeding) nutrition of the young calf are demonstrated. They may be mediated through changing plasma insulin concentrations. An age-dependent prepubertal rise in plasma IGF-I apparently independent of nutritional factors commenced with the appearance of functional somatotrophic receptors in the liver.

J. Endocr. (1988) 119, 43–50

ABSTRACT

The metabolic clearance of insulin-like growth factor-I (IGF-I) has been examined in sheep using a radioiodinated hormone preparation (¹³¹I-labelled IGF-I). Following i.v. administration, ¹³¹I-labelled IGF-I was distributed in a volume equivalent to plasma (60 ml whole blood/kg liveweight) and demonstrated a triphasic pattern of clearance with apparent half-lives (t _½) of 4·0 ± 0·4 (s.e.m.), 52·4 ± 3·4 and 792 ± 26·5 min (n = 10). No significant differences in the t _½ of the three phases were identified in fed compared with starved animals (fed, n = 4, phase 1 = 3·1 ± 0·64, phase 2 = 46 ± 5·9 and phase 3 = 756 ± 27 min; starved, n = 6, phase 1 = 4·6 ± 0·58, phase 2 = 57 ± 3·2 and phase 3 = 816 ± 38·5 min). Similarly, no significant differences in the distribution volume (fed, n = 4, 44 ± 4 ml/kg liveweight; starved, n = 6, 39 ± 2 ml/kg liveweight) or metabolic clearance rate (fed, n = 4, 2·9 ± 0·15 ml/min; starved, n = 6, 3·2 ± 0·5 ml/min) of the IGF-I were found in fed compared with starved animals. Highperformance gel filtration chromatography of sequential plasma samples following injection of ¹³¹I-labelled IGF-I revealed three clear peaks of radioactivity which demonstrated markedly different patterns of clearance. These correspond to hormone complexed to binding proteins of 150 000 and 50 000 daltons and to 'free' hormone. While radioactivity eluting in the position of free IGF-I had reduced to negligible levels by 12 min, ¹³¹I-labelled IGF-I associated with the intermediate molecular weight binding protein was still detectable 6 h after injection. In contrast, an initial increase in radioactivity associated with the 150 000 dalton binding protein was revealed. This form was cleared from the circulation very slowly, by 8 h it was the only species remaining and it was still detectable 26 h after injection. No significant differences in this pattern of bound and free hormone clearance were found in fed compared with starved animals. While there was apparently no interchange of hormone between the binding proteins in vitro the nature of the decay in vivo indicated the possibility of exchange of IGF-I between the different binding proteins.

J. Endocr. (1987) 115, 233–240

ABSTRACT

Three cows received injections of thyroxine (T₄; 20 mg/day), four cows GH (40 mg/day) and three cows saline (control; 10 ml/day) on days 5–8 of a 16-day experimental period during peak lactation. Milk yield increased 13% in cows given GH (from 14·6 to 16·5 kg/day) and 15% in cows given T₄ (from 15·8 to 18·2kg/day) but did not change in control cows. Injection of T₄ increased milkfat and lactose content but reduced milk protein content. Injection of GH was without effect on milk composition during the injection period but milk protein rose after injections ceased.

Injection of T₄ increased plasma concentrations of T₄ and tri-iodothyronine six- to sevenfold, with maxima occurring on day 9. Injection of GH increased the plasma concentration of GH five- to tenfold 5 h after injection. The plasma concentration of insulin-like growth factor I (IGF-I) was increased in cows given GH in both morning (08.30 h) and afternoon (14.30 h) blood samples, the difference being greatest in afternoon samples in which plasma IGF-I content increased from 3·3 to 6·8 nmol/l. Injection of T₄ reduced the plasma concentration of IGF-I in morning samples but the concentration in afternoon samples remained relatively constant throughout the 16-day experimental period. The plasma concentration of IGF-II rose in morning samples in all treatment groups to reach a maximum of 200–250 nmol/l by day 9.

The galactopoietic response to injection of GH but not T₄ was associated with an increase in plasma concentration of IGF-I. Changes in plasma concentration of IGF-II were not associated with changes in milk yield.

J. Endocr. (1987) 114, 17–24

ABSTRACT

The relationship between plasma GH profiles and circulating concentrations of insulin-like growth factor 1 (IGF-1) at three different planes of nutrition, chosen to represent a high, medium and low level of nutrition (3%, 1·8% and 1% dry matter of liveweight per day) was studied in 15 young Angus steers. All steers were maintained on 3% dry matter for 5 weeks, then on one of the three nutritional planes for 4 weeks and then all were returned to 3% dry matter for 3 weeks. Blood was sampled through jugular catheters at 15-min intervals for 25 h at the end of each phase of the study and additional samples were taken on 2 days each week.

Pulsatile release of GH occurred episodically with a diurnal increase during night and morning hours only in steers on high nutritional intakes. Reduced feeding at both the medium and the low plane abolished the diurnal rhythm and significantly increased mean plasma GH concentrations, the amplitude of GH pulses and the area under the GH profiles. Baseline concentrations of GH and pulse frequency did not change through nutritional manipulation. Upon realimentation, plasma GH concentrations decreased in both previously undernourished groups, with those fed 1% dry matter still having increased levels 10 days after refeeding. Plasma IGF-1 concentrations showed no periodicity. With nutritional deprivation, a decrease in IGF-1 concentration was observed only at negative energy balance (1% group). In this group plasma IGF-1 concentrations were progressively restored within 1 week of realimentation.

The different relationship between GH and IGF-1 release at each plane of nutrition suggests that at both medium and low levels of feed intake, tissue insensitivity to GH may exist peripherally and perhaps centrally. It is suggested that nutritional status may, through modulation of tissue sensitivity to GH, be a primary factor in determining growth and the regulation of the somatotrophic axis in the postnatal ruminant.

J. Endocr. (1986) 111, 209–215