The IGF system is one of the most important endocrine and paracrine growth factor systems that regulate fetal and placental growth. We hypothesized that intrauterine growth restriction (IUGR) in guinea pigs is mediated by the altered expression of IGFs and/or IGF binding protein (BP) mRNAs in tissues and is related to growth of specific tissues. IUGR was induced by unilateral uterine artery ligation on day 30 of gestation, and fetal plasma, amniotic fluid and tissue samples were collected at 55–57 days (term about 68 days) from paired IUGR and control fetuses (n=6). Western ligand blotting and immunoblotting were used to compare IGFBP levels in plasma and amniotic fluid. Total RNA was extracted from placenta and fetal tissues, and the relative abundance of IGF-II and IGFBP-1–6 mRNA was determined by Northern blotting, using species-specific probes where available. IUGR fetuses had decreased (P<0.01, by Student’s t-test) placental weight and body weight with an increase in the brain:liver weight ratio. The principal IGFBPs in fetal plasma migrated at 40–35, 30 and 25 kDa and were identified as IGFBP-3, -2 and -4 respectively. IUGR was associated with elevated plasma IGFBP-2 and IGFBP-4 and reduced IGFBP-3 levels. IGFBPs were detected at low levels in amniotic fluid of control fetuses but at higher levels in IUGR fetuses. In IUGR placentae, there was a small increase in IGFBP-4 mRNA (P<0.05). IGFBP-2 mRNA increased (P<0.001) in liver of IUGR fetuses. IGF-II and IGFBP mRNA expression did not change in fetal muscle. The results are consistent with reduced IGF action, directly or through inhibition by IGFBPs, particularly by circulating and tissue IGFBP-2, as a potential causal factor in decreased growth of the placenta and certain fetal tissues.

ABSTRACT

Plasma levels of ACTH and cortisol in fetal sheep increase progressively during late pregnancy, providing the stimulus for birth. However, little information is available concerning either sources of pro-opiomelanocortin (POMC, the precursor to ACTH) or changes in POMC gene expression, which may be responsible for the elevated fetal plasma ACTH concentrations. We therefore studied the relative amount of POMC mRNA in fetal sheep hypothalami, anterior pituitaries and adrenals at discrete times of pregnancy between day 60 and term (approximately 145 days) and from newborn lambs. Total RNA from these tissues was analysed by Northern blot hybridization using a human POMC DNA probe, and the amount of POMC mRNA was expressed relative to the signal obtained for 18S ribosomal RNA. A single 1·2 kb transcript was detected by day 60 in the anterior pituitary, and its relative amount did not change significantly until after days 125–130. Pituitary POMC mRNA levels increased significantly at days 138–143, remained elevated at term and increased further in newborn lambs. In contrast, POMC mRNA was undetectable in hypothalami and adrenal glands of fetuses at all ages.

The results suggested that the prepartum rise in plasma ACTH concentrations in fetal sheep is due to increased POMC biosynthesis in the fetal pituitary. The increase in POMC mRNA occurs at a time when fetal plasma cortisol concentrations are elevated, indicating that the negative feedback effects of circulating glucocorticoids on the fetal hypothalamicpituitary axis may be obscured by other mechanisms that increase pituitary POMC mRNA accumulation during the last week of gestation.

Journal of Endocrinology (1991) 131, 483–489

Abstract

We have investigated the patterns of expression and cellular localization of polypeptides and mRNAs encoding IGF-I and IGF-II in intact bovine oviduct and two bovine oviductal primary cultures (monolayers and vesicles) which are utilized for supporting development in vitro. IGF-I and IGF-II polypeptides were localized by immunocytochemistry in intact oviduct and in both primary cultures for an 8-day culture interval, but IGF-II polypeptide displayed a more restricted distribution in day 8 monolayer cultures. IGF-I and IGF-II mRNAs were localized in both oviductal cell cultures as assessed by in situ hybridization. We were unable to detect IGF-I and IGF-II mRNAs in intact oviduct by in situ hybridization; however, transcripts encoding IGF-I and IGF-II mRNAs were detected in intact oviduct cell preparations and all primary culture samples by reverse transcription-PCR methods. The origin and phenotypic stability of these cultures was assessed by immunostaining with antibodies raised against vimentin (mesenchymal cell marker) and cytokeratin (epithelial cell marker). Over the culture period, the proportion of vimentin-immunoreactive cells increased in the monolayer cultures but remained at a low level in the vesicle cultures which were predominantly composed of cytokeratin-positive cells. The results suggest that oviductal cell co-culture may facilitate early mammalian development, in part, by the establishment of paracrine growth factor circuits.

Journal of Endocrinology (1996) 149, 41–53

ABSTRACT

Insulin is a major regulatory hormone for optimal tissue growth and function in utero. Its continued availability to the growing fetus depends on increasing islet cell mass. The purpose of the study was to examine the interactions between nutrient availability and insulin-like growth factor (IGF) release and action during DNA synthesis by isolated fetal rat islets of Langerhans. Specifically, we wished to determine (a) whether the availability of glucose or total amino acids altered the release of endogenous IGF-I or -II, (b) if both IGF-I and -II were effective mitogens for pancreatic β-cells, (c) whether islets released IGF-binding proteins (IGFBPs) and their possible regulation by nutrient availability and (d) how IGFBPs might regulate the ability of IGFs to alter islet DNA synthesis. Islets of Langerhans were isolated from fetal rat pancreata on day 22 of gestation by collagenase digestion. Islets enriched in β-cells following a 5-day preincubation regime were maintained in various concentrations of glucose (1·4–16·7 mmol/l) or amino acids (×1–×3 total concentrations), with or without exogenous IGF-I, -II, IGFBP-1 or IGFBP-2. The release of insulin and endogenous IGF-I and -II were each determined by radioimmunoassay, and IGFBP release characterized by Western ligand blot analysis. DNA synthesis was measured by the incorporation of [³H]thymidine. Isolated islets demonstrated an increased release of insulin in response to increasing amounts of both glucose and amino acids, demonstrating functional viability. Both classes of nutrients also increased the DNA synthetic rate of islets. Islets released almost twice as much IGF-II (0·22 ± 0·08 nmol/l, mean ± s.e.m., n=4) as IGF-I (0·14 ± 0·03 nmol/l) in cultures containing 8·7 mmol glucose/1 and × 1 amino acids. Lesser or greater concentrations of glucose did not alter the release of either IGF, but the release of IGF-II was significantly increased (0·53 ± 0·08 nmol/l, P<0·01) in the presence of × 2 amino acids. Exogenous IGF-I was fivefold more active in stimulating DNA synthesis by islets (half maximal concentration (ED₅₀) 1·6 ± 0·4 nmol/l, n = 3) than was IGF-II (ED₅₀ 8·1 ± 0·6 nmol/l), regardless of glucose concentration. Isolated islets released four species of IGFBP with molecular sizes of approximately 19, 25, 35 and 46 kDa respectively. The 35 kDa form was identified by Western immunoblot as IGFBP-2. Increasing the glucose concentration between 1·4 mmol/l and 16·7 mmol/l caused a dose-related increase in the release of the 19, 25 and 35 kDa IGFBP species. Increasing amino acid concentrations from × 1 to × 2 concentrations increased the relative amounts of all IGFBP species, but greater concentrations were inhibitory. Exogenous IGFBP-1 and BP-2 synergized with sub-effective concentrations of IGF-I or -II to increase DNA synthetic rate. The results show that isolated fetal rat islets release more IGF-II than IGF-I, but that IGF-I is a more potent stimulus to DNA synthesis. The ability of glucose to increase islet DNA synthesis was not accompanied by altered release of endogenous IGFs, but did result in increased release of IGFBPs. Increasing the concentration of total amino acids increased the release of both IGF-II and IGFBPs. Since exogenous IGFBPs were able to potentiate the mitogenic actions of IGFs, it is likely that nutrients, IGFs and IGFBPs interact to promote islet cell hyperplasia in late gestation.

Journal of Endocrinology (1993) 138, 401–412

ABSTRACT

The presence of insulin-like growth factors (IGFs) in blood is regulated by their association with specific IGF-binding proteins (IGFBPs). In turn, the level of IGFBPs in the blood is likely to depend on a dynamic equilibrium between peptide production and clearance to extravascular tissues or organ-specific degradation. Since circulating IGFBPs may largely derive from liver we have employed partial hepatectomy in the rat to study the clearance rate of endogenous IGFBPs from blood once a major site of production is removed. Adult male rats were partially hepatectomized and serum and the remaining liver removed between 30 min and 7 days after surgery. Ligand blot analysis revealed two major species of IGFBP, of 28–30 kDa and 40–44 kDa in sera from control rats or sham-operated rats respectively. The larger species corresponded in size to rat IGFBP-3, but the smaller form was not recognized by antisera against rat IGFBP-1, bovine IGFBP-2 or human IGFBP-5 following Western immunoblot. Following hepatectomy, the levels of both IGFBP forms in the serum declined within 30 min and were barely detectable after 3 h or 6 h. They began to increase again in serum 24 h following surgery. The reduction in IGFBPs following hepatectomy was not primarily due to degradation by specific proteases in serum. Circulating levels of insulin were increased fivefold 3 h after hepatectomy but subsequently returned to control values. The rise in insulin was accompanied by a significant (P < 0·05) reduction in circulating IGF-I after 3 h which persisted at 24 h. Glucose levels in serum showed a transient but non-significant reduction between 90 min and 6 h after hepatectomy. Total RNA was extracted from remnant liver and subjected to Northern blot hybridization with ³²P-labelled cDNAs encoding rat IGFBP-1, -2 or -3. Messenger RNA encoding IGFBP-1 was barely detectable in liver from control or sham-operated animals, but increased within 30 min of partial hepatectomy and peaked at 3 h. It subsequently declined and was again barely detectable after 24 h. No expression of IGFBP-2 or -3 mRNAs was found by Northern blot analysis in the liver of control animals or following partial hepatectomy. These results suggest that both IGF-I and IGFBPs in rat serum decreased rapidly following partial hepatectomy, and that this was due largely to the rapid clearance of the peptide and its binding proteins once the major source of production was removed. A rapid induction of IGFBP-1 in the remaining liver may be unrelated to the circulating IGFBPs since immunoreactive IGFBP-1 was not detected in rat serum.

Journal of Endocrinology (1993) 137, 271–280