Studies on the growth of the fetal guinea pig. The effects of nutritional manipulation on prenatal growth and plasma somatomedin activity and insulin-like growth factor concentrations

In guinea pigs between days 41-46 of pregnancy prenatal growth has been manipulated by alteration of nutritional state. Three methods were used. Uterine artery ligation at day 30 of pregnancy depressed fetal growth rate by greater than 50% and was associated with falls in plasma insulin, IGF-1, cortisol, thyroid hormone, glucose, acetate and free fatty acid concentrations and rises in that of IGF-2, glucagon and amino acids. Fetal plasma was inhibitory to sulphate incorporation into pig costal cartilage. Complete food withdrawal from pregnant guinea pigs for 2 days at days 43-44 of pregnancy caused mild fetal growth retardation and similar changes in plasma constituents, except in that plasma IGF-2 concentrations were now depressed and plasma was not inhibitory to sulphate incorporation into pig costal cartilage. Production of hypoglycaemia by 4-times-daily maternal injections of glucose between days 41-46 of pregnancy accelerated fetal growth rate. It also elevated fetal plasma concentrations of insulin, IGF-1, IGF-2, sulphation-promoting activity, thyroid hormones, glucose and free fatty acids and depressed that of glucagon and amino acids. Fetal growth rate during the experimental period showed a good correlation with plasma glucose, insulin and IGF-1 and, to a certain extent, with sulphation-promoting activity. It did not correlate closely with fetal plasma IGF-2 concentration. Hepatic glycogen concentrations showed a good correlation with plasma IGF-2 levels.     

Studies on the growth of the fetal sheep. Effects of surgical reduction in placental size, or experimental manipulation of uterine blood flow on plasma sulphation promoting activity and on the concentration of insulin-like growth factors I and II

The effect of long- and short-term manipulations of uterine blood flow on fetal plasma levels of IGF-I and -II have been studied in sheep at days 125-139 of pregnancy and compared with those in near term rats and guinea pig. The primary objective is to show that both long- and short-term reduction of uterine blood flow is associated with increase in the fetal plasma concentration of IGF-II while that of IGF-I falls. In the pregnant sheep long-term depression of utero-placental blood flow was caused by surgical reduction in placental mass (carunclectomy) prior to conception. This reduced fetal weight to 2.42 +/- 0.49 kg (SD) compared with 3.41 +/- 0.46 in controls; the respective values for uterine blood flow being 1694 +/- 558 and 913 +/- 324 ml/min respectively. This was associated with a fall in fetal plasma IGF-I concentration from 22.6 +/- 3.4 ng/ml to 14.9 +/- 1.31 ng/ml and a rise in IGF-II from 1952 +/- 284 ng/ml to 3360 +/- 914 ng/ml respectively. Similar changes in the plasma concentrations of IGF peptides were observed in fetal rats and guinea pigs in response to uterine artery ligation. Short-term reduction (60 min) of the uterine blood flow was caused either by compression of the common uterine artery to depress flow from 1491 +/- 375 to 648 +/- 216 ml/min or through intraarterial infusion of adrenaline at 35 ug/min to lower flow from 1628 +/- 339 to 1195 +/- 128 ml/min. Such falls in uterine blood flow had no significant effect on fetal plasma IGF-I levels but increased IGF-II levels by 30 to 60%.     

Studies on the growth of the fetal guinea pig. The effects of ligation of the uterine artery on organ growth and development

The effects of reduced maternal placental blood flow on the growth and development of the fetal guinea pig have been studied by unilateral ligation of the uterine artery at day 30 of pregnancy. Fetal guinea pigs were investigated about 20 or 30 days later. In about one-third of cases fetal death occurred, in another third fetuses less than 60% of normal weight were observed and in the remainder all fetuses were in the normal weight range. In the growth retarded fetuses prenatal growth occurred at about 50% of the rate in control. There was no postnatal 'catch up' as growth still remained lower than in controls. Restricted fetal growth affected particularly development of the visceral tissues in which case size declined in proportion to body weight. Brain and adrenal by comparison were less affected as their contribution to total body weight increased, but even so in the severely retarded fetuses the mass of both fell. The responses of the liver were in general consistent with a delay in the pattern of development. Thus DNA, RNA, protein and haematopoietic cell content changes occurred later than normal. In contrast an enhanced deposition of glycogen was apparent in the liver of the growth-retarded fetus. The results indicate some of the ways in which nutritional deprivation of the fetuses leads to reprogramming of growth and maturation of selected fetal tissues to allow non-essential changes to await more favourable times.     

Studies on the growth of the fetal guinea pig. Changes in plasma hormone concentration during normal and abnormal growth

The endocrine changes associated with fetal growth retardation caused by unilateral uterine artery ligation of guinea pigs at day 30 of pregnancy were studied. Plasma hormone levels in fetuses that, about 20 or 30 days later, were 35-50% of normal size were measured by radioimmunoassay. The small fetuses were severely hypoglycaemic and hypoinsulinaemic; both showing close correlation and relationship to the degree of growth retardation. Plasma thyroid and cortisol and concentrations were much lower than normal and that glucagon and androstenedione were much higher. Plasma growth hormone level appeared to be unaffected by growth retardation. The developmental changes in glucagon and thyroid hormone concentrations were consistent with a delay in the timing of prenatal events in growth-retarded fetuses. However the late cortisol rise, although somewhat blunted, still occurred at 58-60 in the small fetal guinea pigs.     

Early treatment of the pregnant guinea pig with IGFs promotes placental transport and nutrient partitioning near term

Appropriate partitioning of nutrients between the mother and conceptus is a major determinant of pregnancy success, with placental transfer playing a key role. Insulin-like growth factors (IGFs) increase in the maternal circulation during early pregnancy and are predictive of fetal and placental growth. We have previously shown in the guinea pig that increasing maternal IGF abundance in early to midpregnancy enhances fetal growth and viability near term. We now show that this treatment promotes placental transport to the fetus, fetal substrate utilization, and nutrient partitioning near term. Pregnant guinea pigs were infused with IGF-I, IGF-II (both 1 mg.kg-1.day-1) or vehicle subcutaneously from days 20-38 of pregnancy (term=69 days). Tissue uptake and placental transfer of the nonmetabolizable radio analogs [3H]methyl-D-glucose (MG) and [14C]aminoisobutyric acid (AIB) in vivo was measured on day 62. Early pregnancy exposure to elevated maternal IGF-I increased placental MG uptake by>70% (P=0.004), whereas each IGF increased fetal plasma MG concentrations by 40-50% (P<0.012). Both IGFs increased fetal tissue MG uptake (P<0.048), whereas IGF-I also increased AIB uptake by visceral organs (P=0.046). In the mother, earlier exposure to either IGF increased AIB uptake by visceral organs (P<0.014), whereas IGF-I also enhanced uptake of AIB by muscle (P=0.044) and MG uptake by visceral organs (P=0.016) and muscle (P=0.046). In conclusion, exogenous maternal IGFs in early pregnancy sustainedly increase maternal substrate utilization, placental transport of MG to the fetus, and fetal utilization of substrates near term. This was consistent with the previously observed increase in fetal growth and survival following IGF treatment.     

Somatomedin-C/insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues

Somatomedin-C or insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II) have been implicated in the regulation of fetal growth and development. In the present study 32P-labeled complementary DNA probes encoding human and mouse Sm-C/IGF-I and human IGF-II were used in Northern blot hybridizations to analyse rat Sm-C/IGF-I and IGF-II mRNAs in poly(A+) RNAs from intestine, liver, lung, and brain of adult rats and fetal rats between day 14 and 17 of gestation. In fetal rats, all four tissues contained a major mRNA of 1.7 kilobases (kb) that hybridized with the human Sm-C/IGF-I cDNA and mRNAs of 7.5, 4.7, 1.7, and 1.2 kb that hybridized with the mouse Sm-C/IGF-I cDNA. Adult rat intestine, liver, and lung also contained these mRNAs but Sm-C/IGF-I mRNAs were not detected in adult rat brain. These findings provide direct support for prior observations that multiple tissues in the fetus synthesize immunoreactive Sm-C/IGF-I and imply a role for Sm-C/IGF-I in fetal development as well as postnatally. The abundance of a 7.5-kb Sm-C/IGF-I mRNA in poly(A+) RNAs from adult rat liver was 10-50-fold higher than in other adult rat tissues which provides further evidence that in the adult rat the liver is a major site of Sm-C/IGF-I synthesis and source of circulating Sm-C/IGF-I. Multiple IGF-II mRNAs of estimated sizes 4.7, 3.9, 2.2, 1.75, and 1.2 kb were observed in fetal rat intestine, liver, lung, and brain. The 4.7- and 3.9-kb mRNAs were the major hybridizing IGF-II mRNAs in all fetal tissues. Higher abundance of IGF-II mRNAs in rat fetal tissues compared with adult tissues supports prior hypotheses, based on serum IGF-II concentrations, that IGF-II is predominantly a fetal somatomedin. IGF-II mRNAs are present, however, in some poly(A+) RNAs from adult rat tissues. The brain was the only tissue in the adult rat where the 4.7- and 3.9-kb IGF-II mRNAs were consistently detected. Some samples of adult rat intestine contained the 4.7- and 3.9-kb IGF-II mRNAs and some samples of adult liver and lung contained the 4.7-kb mRNA. These findings suggest that a role for IGF-II in the adult rat, particularly in the central nervous system, cannot be excluded.     

Characterization of fetal growth by repeated ultrasound measurements in the wild guinea pig (Cavia aperea)

Fetal growth during pregnancy has previously been studied in the domesticated guinea pig (Cavia aperea f. porcellus) after dissecting pregnant females, but there are no studies describing the fetal growth in their wild progenitor, the wild guinea pig (C aperea). In this study, 50 pregnancies of wild guinea pig sows were investigated using modern ultrasound technique. The two most common fetal growth parameters (biparietal diameter [BPD] and crown-rump-length [CRL]) and uterine position were measured. Data revealed similar fetal growth patterns in the wild guinea pig and domesticated guinea pig in the investigated gestation period, although they differ in reproductive milestones such as gestation length (average duration of pregnancy 68 days), average birth weight, and litter mass. In this study, pregnancy lasted on average 60.2 days with a variance of less than a day (0.96 days). The measured fetal growth parameters are strongly correlated with each (R = 0.91; P < 0.001) other and with gestational age (BPD regression equation y = 0.04x − 0.29; P < 0.001 and CRL regression equation y = 0.17x − 2.21; P < 0.01). Furthermore, fetuses in the most frequent uterine positions did not differ in their growth parameters and were not influenced by the mother ID. Our results imply that ultrasound measurement of a single fetal growth parameter is sufficient to reliably estimate gestational age in the wild guinea pig.     

The effects of maternal undernutrition on maternal and fetal serum insulin-like growth factors, thyroid hormones and cortisol in the guinea pig.

The insulin-like growth factors (IGF-I and -II) are potential mediators of the effects of maternal undernutrition on fetal growth and muscle development. The effects of a 40% reduction in maternal feed intake on serum levels of the IGFs, the thyroid hormones and cortisol, were investigated for the last two trimesters (day 25 to birth). This level of undernutrition is known to cause a 35% reduction in fetal and placental weights, and a 20-25% reduction in muscle fibre number. Maternal IGF-I level was greater than non-pregnant levels on day 25 gestation, in both control and restricted dams, and declined with gestational age. The increase in IGF-I level in the 40% restricted group was approximately two-thirds that of control animals. Fetal serum IGF-I was also reduced in undernourished fetuses throughout gestation. Maternal IGF-II did not change with gestational age and was unaffected by undernutrition. Fetal IGF-II reached a peak at day 55 of gestation, this peak was greatly diminished by maternal feed restriction. Both IGF-I and IGF-II tended to be related to fetal, placental and muscle weights at day 65 of gestation. Thyroid hormone concentration declined in maternal serum and increased in fetal serum with increasing gestational age. Levels were not significantly affected by undernutrition. Both triiodothyronine (T3) and thyroxine (T4) were correlated with IGF-I in maternal serum (P < 0.05), but not in fetal serum. Cortisol levels were elevated by undernutrition in both maternal and fetal serum, and increased with gestational age. Cortisol was inversely correlated with serum IGF-I in both maternal and fetal serum. Maternal serum IGF-I may mediate the effects of undernutrition on fetal growth by affecting the growth and establishment of the feto-placental unit in mid-gestation. Fetal IGF-I may mediate the effects on muscle growth, whereas IGF-II seems to be related to hepatic glycogen deposition. Cortisol may play a role via its effect on the IGFs, but the thyroid hormones are unlikely to be important until the late gestation/early postnatal period.     

Longitudinal data for intrauterine levels of fetal IGF-I and IGF-II

OBJECTIVE: An increasing body of evidence supports a major role for the insulin-like growth factors (IGFs) in the control of human fetal growth. Individual data at various times of pregnancy suggest that IGF-I and IGF-II levels remain stable up to the 33rd week of pregnancy. Thereafter, both increase to reach values 2-3 times higher at term. In order to provide an accurate reflection of fetal IGFs in utero, we sampled fetal blood from the umbilical cord by cordocentesis. METHODS: We measured IGF-I and IGF-II in 12 fetuses longitudinally for up to 5 times between the 21st week of gestation and delivery. RESULTS: All patients showed a progressive increase in IGF-I and IGF-II levels. Data determined during different time intervals (before 29th, 29th to 32nd, after 32nd week) were compared and the main increase was found after the 32nd week. The median for IGF-I before the 29th week was 33.5 ng/ml (range 19-40.5) and increased to 41 ng/ml (32-59) between the 29th to 32nd and further to 54.1 ng/ml (range 17-70) thereafter. During the same time interval, the median for IGF-II increased from 217 ng/ml (86-326) to 349 ng/ml (227-467). In 7 patients, cord blood after delivery was available. For IGF-II a further increase was consistently found after birth (from 282 ng/ml (175-511) to 393 ng/ml (297-513)), whereas only 2 fetuses showed an increase in IGF-I. CONCLUSION: We conclude that in human fetuses, IGF-I and IGF-II levels increase longitudinally throughout pregnancy. Therefore, they may become important markers of healthy fetal development.     

Isocaloric maternal low-protein diet alters IGF-I,IGFBPs, and hepatocyte proliferation in the fetal rat

We investigated the effect of an isocaloric maternal low-protein diet during pregnancy in rats on the proliferative capacity of cultured fetal hepatocytes. The potential roles of these changes on the IGF-IGF-binding protein (IGFBP) axis, and the role of insulin and glucocorticoids in liver growth retardation, were also evaluated. Pregnant Wistar rats were fed a control (C) diet (20% protein) or a low-protein (LP) diet (8%) throughout gestation. In primary culture, the DNA synthesis of hepatocytes derived from LP fetuses was decreased by approximately 30% compared with control hepatocytes (P < 0.05). In parallel, in vivo moderate protein restriction in the dam reduced the fetal liver weight and IGF-I level in fetal plasma (P < 0.01) and augmented the abundance of 29- to 32-kDa IGFBPs in fetal plasma (P < 0.01) and fetal liver (P < 0.01). By contrast, the abundance of IGF-II mRNA in liver of LP fetuses was unaffected by the LP diet. In vitro, the LP-derived hepatocytes produced less IGF-I (P < 0.01) and more 29- to 32-kDa IGFBPs (P < 0.01) than hepatocytes derived from control fetuses. These alterations still appeared after 3-4 days of culture, indicating some persistence in programming. Dexamethasone treatment of control-derived hepatocytes decreased cell proliferation (54 +/- 2.3%, P < 0.01) and stimulated 29- to 32-kDa IGFBPs, whereas insulin promoted fetal hepatocyte growth (127 +/- 5.5%, P < 0.01) and inhibited 29- to 32-kDa IGFBPs. These results show that liver growth and cell proliferation in association with IGF-I and IGFBP levels are affected in utero by fetal undernutrition. It also suggests that glucocorticoids and insulin may modulate these effects.     

Effect of a high maternal dietary intake during mid-gestation on components of the utero-placental insulin-like growth factor (IGF) system in adolescent sheep with retarded placental development

The aim of the present study was to investigate the effects of administering a high plane diet during early to mid-gestation on the uterine and placental insulin-like growth factor (IGF) system and on systemic IGF-I concentrations in pregnant adolescent ewes with restricted placental growth. Embryos recovered from superovulated ewes inseminated by a single sire were transferred in singleton to the uterus of adolescent recipients. After transfer ewes were offered a high (H) or moderate (M) amount of a complete diet calculated to promote rapid or normal maternal growth rates, respectively. Five ewes from each group were switched from either M to H or H to M diets at day 52 of gestation. Maternal and fetal blood samples and placental tissues were collected from all animals at day 104. Ewes on the high plane diet from mid-gestation (HH, MH groups) had restricted placental mass (P < 0.01) and tended to have smaller fetuses. This was associated with increased maternal plasma IGF-I concentrations (P < 0.001). The pattern of expression of components of the IGF system in the uterus and placenta was studied by in situ hybridization. IGF-I mRNA concentrations were below the limit of detection. IGF-II mRNA expression was high in the fetal mesoderm and present in maternal stroma, but was not influenced by nutritional treatment. In contrast, IGF binding protein 1 (IGFBP-1) mRNA expression was higher (P < 0.05) and IGFBP-3 mRNA expression was lower (P < 0.05) in the endometrial glands of ewes in HH and MH groups. In the fetal trophoblast, IGFBP-3 mRNA expression was higher in the MH group. Type 1 IGF receptor expression was increased (P < 0. 01) in the luminal epithelium of the HM group and IGFBP-2 mRNA expression was highest in the placentome capsule of ewes in the HH group. Together, these results indicate that reprogramming of the uterine and placental IGF axis by maternal nutrition could contribute to placental growth retardation in growing adolescent sheep.     

Insulin-like growth factors and binding proteins in the fetal rat: Alterations during maternal starvation and effects in fetal brain cell culture

Maternal malnutrition adversely affects fetal body and brain growth during late gestation. We utilized a fetal brain cell culture model to examine whether alternations in circulating factors may contribute to reduce brain growth during maternal starvation; we then used specific immunoassay and western blotting techniques, and purified peptides to investigate the potential role that altered levels of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) may play in impaired growth during maternal nutritional restriction.Fetal, body, liver, and brain weight were reduced after 72 hr maternal starvation, and plasma from starved fetuses were less potent than fed fetal plasma in stimulating brain cell growth. Circulating levels of IGF-I were reduced in starved compared to fed fetuses, while levels of IGF-II were similar in both groups. In contrast, [¹²⁵I]-IGF-I binding assay demonstrated an increase in the availability of plasma IGFBPs following starvation. Western ligand blotting and densitometry indicated that levels of 32 Kd IGFBPs were 2-fold higher in starved compared to fed fetal plasma. Immunoblotting and immunoprecipitation with antiserum against rat IGFBP-1 confirmed that heightened levels of immunoreactive IGFBP-1 accounted for the increase in 32 Kd IGFBPs in starved plasma. Levels of 34 Kd BPs, representing IGFBP-2, were unaffected by starvation. Reconstitution experiments in cell culture showed that IGF-I promoted fetal brain cell growth, and that when they were supplemented with IGF-I, the growth promoting activity of starved fetal plasma was restored to fed levels. These changes were measured using MTT to assess mitochondrial reductase activity. Conversely, addition of physiological amounts of rat IGFBP-1 inhibited the effects of fed fetal plasma on brain cell growth, and bioactivity was reduced even further with higher concentrations of IGFBP-1. Based on these results, we conclude that reciprocal changes in circulating levels of IGFBP-1 (increased) and IGF-I (decreased) may combine to reduce the availability of IGF-I to this tissue and limit fetal brain cell growth when maternal nutrition is impaired.     

Radioimmunoassay for insulin-like growth factor II (IGF-II)

Insulin-like growth factor II (IGF-II) levels in human plasma were measured in physiological and pathological conditions by radioimmunoassay (RIA) with biosynthetic IGF-II. This RIA was specific for IGF-II and cross-reactivity with IGF-I was 1%. The sensitivity was 15 pg/tube with 50% displacement at 50 pg/tube. The intra- and inter-assay coefficients of variation for IGF-II were 6.3 and 9.3%, respectively. The plasma IGF-II levels in normal adults, patients with hypopituitarism and patients with active acromegaly were 589.6 +/- 15.8, 800.9 +/- 45.6 and 330.3 +/- 24.3 ng/ml, respectively. After human growth hormone (hGH) treatment in hypopituitarism, IGF-II slightly increased, but not significantly. After adenomectomy in patients with acromegaly, IGF-II significantly decreased. These data indicate that IGF-II concentrations in plasma were partially GH dependent. This GH dependency was less than that of IGF-I. IGF-II was low in patients with anorexia nervosa and with liver cirrhosis and high in patients with renal failure. In two cases with extrapancreatic tumor-associated hypoglycemia, plasma IGF-II was increased to 1123.8 and 843.5 ng/ml, and returned to normal after tumor resection. These data showed that IGF-II was partly dependent on GH and nutritional conditions and that IGF-II was the most likely cause of some cases of hypoglycemia with extrapancreatic tumor. This specific and sensitive RIA of IGF-II would be useful in evaluating its physiological and pathological role in plasma and tissue.     

Differential expression of insulin-like growth factors in the uterine epithelium and extracellular matrix during early pregnancy

We have studied the simultaneous expression of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) in the uterine epithelium and extracellular matrix during the time of trophoblast attachment and implantation. These studies reveal that IGF-I and IGF-II display different spatial and temporal patterns of expression during early pregnancy, and suggest a role for them in the process of attachment and implantation. Specifically, IGF-I is strongly expressed in the basal lamina which is the site of trophoblast invasion into the maternal stroma, and also in the apical epithelium, the site of initial trophoblast attachment. IGF-II is expressed to a lesser extent in the basal lamina, lateral plasma membranes and apical epithelium on day 3 but is only prominent apically at the time of implantation, suggesting a role in attachment.     

Insulin-like growth factor II binding and action in human fetal fibroblasts

To investigate the role of insulin-like growth factor II (IGF-II) in human prenatal growth, IGF-II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific binding of IGF-II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5-10 times the amount of IGF-I binding found in these cells. IGF-I and IGF-II caused dose-dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF-II was sevenfold less potent than IGF-I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF-II in stimulating [14C]AIB uptake approach that of IGF-I. Similar results were obtained when IGF-I and IGF-II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alpha IR-3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF-I and IGF-II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF-II is a potent metabolic and mitogenic stimulus for human fetal fibroblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF-II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.     

Heterogeneity of insulin-like growth factor-I affinity for the insulin-like growth factor-II receptor: comparison of natural, synthetic and recombinant DNA-derived insulin-like growth factor-I

Although insulin-like growth factors (IGF) I and II bind with high affinity to structurally discrete receptors, they bind with a lesser affinity to each other's receptor. We have evaluated the affinity of five different IGF-I preparations (three natural IGF-I preparations, one synthetic preparation, and one recombinant DNA-derived) for the IGF-II receptor in rat placental membranes, 18-54,SF cells and BRL-3A cells. In all tissues tested, the natural IGF-I preparations demonstrated an affinity for the IGF-II receptor which was 10-20% that of IGF-II. However, the recombinant and synthetic IGF-I preparations exhibited substantially lower affinities than natural IGF-I for this receptor, with only 10-25% reduction in (125-I)iodo IGF-II binding at peptide concentrations up to 400 ng/ml. Radioimmunoassay of the natural IGF-I preparations with an antibody directed against the unique C-peptide region of IGF-II demonstrated that contamination of IGF-I preparations with immunoreactive IGF-II could not exceed 5%. These results demonstrate that IGF-I purified from human plasma has a different affinity for the IGF-II receptor than does synthetic or recombinant IGF-I. Furthermore, these data are consistent with the hypothesis that IGF-I, itself, may be heterogeneous, and that subforms may vary in their affinities for the IGF receptors. Alternatively, IGF-I preparations which have been considered to be pure may be contaminated with small amounts of IGF-II, resulting in overestimation of the affinity of IGF-I for the type II IGF receptor.