Biomedical subjects
Publications and source records attributed to V K Han.
Classical gene targeting has identified many genes important for fetal and placental development. Null mutation of these genes may lead to fetal growth restriction, malformation or embryonic death. Growth restriction of epigenetic basis can predispose to adult-onset diseases. The mechanisms underlying this process, termed 'fetal programming', are beginning to be understood.
BACKGROUND: Disorders of kidney development represent a major cause of renal failure and end-stage renal disease in the pediatric population. To understand further the prenatal pathogenesis of obstructive renal dysplasia, a fetal monkey model was developed using ultrasound-guided techniques. METHODS: Ureteropelvic obstruction (N = 13) was induced during the early or late second trimester by the injection of purified guluronic alginate spheres. All fetuses were monitored sonographically, and then fetal tissues were removed at varying time points during the second and third trimesters. RESULTS: There was no evidence of oligohydramnios during the course of gestation, and the obstructed kidneys were typically progressively smaller than the contralateral (nonobstructed) kidneys when monitored sonographically over time. Obstructed kidneys displayed most features of renal dysplasia, including numerous cortical cysts of various sizes derived predominantly from collecting ducts and glomeruli. Mesenchymal changes included expansion of both the cortical and medullary interstitium, as well as mesenchymal-myocyte transformation, expressed as pericystic and peritubular fibromuscular collar formation. An important feature of this model was the disruption of normal glomerular development and architecture, associated with significant podocyte apoptosis, evident as early as the prevascularized S-shaped nephron. As in other models, collecting duct cell apoptosis was apparent, particularly in areas of cyst formation and cellular atrophy. CONCLUSIONS: These results demonstrate the importance of this nonhuman primate model for exploring the pathophysiology of congenital obstructive uropathy and highlight the potential role of podocyte injury in determining long-term renal function associated with this condition.
The IGF system is a key modulator of somatic fetal growth. Studies with human fetal tissues have shown a specific spatial and temporal pattern of expression of IGF and IGF binding protein (IGFBP) mRNAs, but have been limited to defined periods during gestation (i.e. 8-20 wk gestation) because of tissue availability. To fully assess the role of these peptides in the primate growth process, a longitudinal study was conducted that focused on the expression of IGF-II and IGFBP-1 and IGFBP-3 genes in the rhesus monkey (Macaca mulatta). Liver, kidney, brain, and lung were collected from rhesus monkey fetuses approximately every 2 wk from 65 (early second trimester) through 150 d gestation (term 165 +/- 10 d) (n = 50), then processed for in situ hybridization using radiolabeled human cDNAs. IGF-II mRNA was abundantly expressed in fetal kidney (maturing glomerulus, supporting mesenchyme, cells of the developing nephrons), liver (hepatocytes), cerebral cortex (choroid plexus, capillaries), and lung (blood vessels, connective tissues, lamina propria, cartilage framework). IGFBP-1 was expressed only in the hepatocytes and IGFBP-3 mRNA was modestly expressed within the kidney (developing nephrons, collecting system mesenchyme), and liver (hepatocytes). These studies have shown that (1) IGF-II, IGFBP-1, and IGFBP-3 are expressed in specific cell types of the fetal monkey indicating a paracrine/autocrine role during development; (2) changes in IGF-II and IGFBP mRNA expression occur with advancing gestation; and (3) fetal monkey tissues express IGF-II and IGFBPs in a similar manner when compared with the human fetus.
To better understand the role of the insulin-like growth factors (IGF-I and -II) and their binding proteins (IGFBPs 1-6) in placental development and function, it is important to review similarities and differences between species in expression of the respective mRNAs. In human placenta, IGF-II mRNA is expressed in chorionic mesoderm and first trimester villous cytotrophoblast, but not in syncytiotrophoblast. In contrast, in rhesus monkey placenta, IGF-II mRNA is expressed in syncytiotrophoblast but not in chorionic mesoderm. IGFBP-3 mRNA is present in the chorionic mesoderm of placental villi from both these species and may modulate IGF-II action through a paracrine mechanism. In rodent placentae, IGF-II mRNA is expressed both in fetal mesoderm and in the trophoblast of the placental labyrinth. In guinea pig, where IGFBP-5 mRNA is expressed in the marginal and interlobular syncytium and IGF-II mRNA in the labyrinth, interaction between IGF-II and IGFBP-5 mRNA may be involved in vascularization of the placenta by fetal vessels. In sheep placenta, IGF-II mRNA is expressed, not in the trophoblast layer, but in the fetal mesoderm immediately adjacent to it. In the basal plate of human, rhesus monkey and baboon placentae, extravillous trophoblasts express IGF-II mRNA and uterine decidual cells IGFBP 1-6 mRNAs. The inference is that there is interaction between IGF-II and IGFBPs at the maternal-fetal interface of the primate placenta during trophoblast invasion and decidualization. IGFBP-1 expressed by the decidua may also interact with alpha(5)beta(1)integrin expressed by the extravillous trophoblast. The placentae of rodents are also of the invasive type. Glycogen cells of the mouse placenta are analogous with human extravillous trophoblast and express IGF-II mRNA. However, expression of IGFBP mRNAs in the mouse, as in the guinea pig, is confined to non-decidualized endometrium and myometrium. IGF-II mRNA is strongly expressed by trophoblasts invading uterine vessels in human and guinea pig placentae. Interactions probably occur between IGF-II expressed by these trophoblasts and IGFBPs expressed in the vessel walls. However, it is possible that IGFBPs expressed by maternal vessels are associated with processes that are independent of trophoblast invasion. Thus, IGFBP-3 mRNA is highly expressed in the maternal blood vessels of the non-deciduate sheep placenta. Findings to date highlight the diversity in the expression of the IGF system among placentae of man and different laboratory animals, and even between closely related species. Comparative studies will continue to be required to understand the functional role of IGFs and IGFBPs in each species.
Insulin-like growth factor-II (IGF-II) from the invading extravillous cytotrophoblasts (EVTs) and insulin-like growth factor binding proteins (IGFBPs) from the maternal decidua interact at the feto-maternal interface and regulate implantation and placentation. To determine whether a local stimulus from the fetus is important in the regulation of IGFBP gene expression in the human decidua, we compared the expression of IGFBP genes in intra- and extrauterine (tubal) pregnancies. The expression of IGF-II and IGFBP-1 to IGFBP-6 mRNAs was determined by in-situ hybridization in the Fallopian tubes of extrauterine pregnancies and concurrent decidua (n = 6), and in the placentae and Fallopian tubes of intrauterine pregnancies (n = 6). All six IGFBP mRNAs were identified in the decidualized endometrium and decidualized Fallopian tubes of intra- and extrauterine pregnancies, with IGFBP-1 mRNA being the predominant mRNA. IGFBP-4 was the second most predominant mRNA and was slightly more abundant in the decidua of extrauterine pregnancies than of intrauterine pregnancies. IGF-II mRNA was expressed mainly in cells of fetal origin. The fact that the IGFBP mRNAs were expressed similarly in both intra- and extrauterine pregnancies indicates that the local physical stimulus from an implanting fetus is not necessary to induce or maintain decidual IGFBP gene expression.
cDNAs for ovine surfactant-associated protein (SP) A, SP-B, and SP-C have been cloned and shown to possess strong similarity to cDNAs for surfactant apoproteins in other species. These reagents were employed to examine the effect of fetal hypoxia on the induction of surfactant apoprotein expression in the fetal lamb. Postnatal lung function is dependent on adequate growth and maturation during fetal development. Insulin-like growth factor (IGF) I and IGF-II, which are present in all fetal tissues studied, possess potent mitogenic and proliferative actions, and their effects can be modulated by IGF-specific binding proteins (IGFBPs). Hypoxia can lead to increases in circulating cortisol and catecholamines that can influence lung maturation. Therefore, the effects of mild hypoxia in chronically catheterized fetal lambs at gestational days 126-130 and 134-136 (term 145 days) on the expression of pulmonary surfactant apoproteins and IGFBPs were examined. Mild hypoxia for 48 h resulted in an increase in plasma cortisol that was more pronounced at later gestation, and in these animals, there was a twofold increase in SP-A mRNA. SP-B mRNA levels also increased twofold, but this was not significant. SP-C mRNA was not altered. No significant changes in apoprotein mRNA were observed with the younger fetuses. However, these younger animals selectively exhibited reduced IGFBP-5 mRNA levels. IGF-I mRNA was also reduced at 126-130 days, although this conclusion is tentative due to low abundance. IGF-II levels were not affected at either gestational age. We conclude that these data suggest that mild prolonged fetal hypoxia produces alterations that could affect fetal cellular differentiation early in gestation and can induce changes consistent with lung maturation closer to term.
PSP94 (beta microseminoprotein, beta MSP) is one of the three major proteins secreted by the normal human prostate gland. Using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blotting, PSP94 transcripts were shown in human endometrium, myometrium, ovary, breast, placenta and in the human endometrial cancer cell lines KLE and AN3 CA. Primers used in these studies were specific for human prostate PSP94, and were derived from its flanking non-coding regions. The results were confirmed by sequence analysis of two independently derived clones from normal human breast tissues and the other two from KLE cells respectively. The sequences were identical with the coding sequence of human prostate PSP94 cDNA. Using RNA from the endometrial tissues, two different transcripts of approximately 487 bp, equivalent to prostate PSP94 and approximately 381 bp, corresponding to prostate PSP57, its alternately spliced form, were amplified by RT-PCR. Human ovary, breast, placenta and endometrial cancer cell lines (KLE, AN3 CA), however, showed only the full length, approximately 487 bp, PSP94 transcript. We further demonstrated by in situ hybridization that PSP94 mRNA is expressed specifically in the glandular epithelial cells, and not in the stroma of both the human endometrial and breast tissues. Further, using image analysis of in situ hybridization data, the levels of PSP94 mRNA in the cycling endometrial tissues and in breast confirmed the differential levels of expression in the cycling endometrium (P<0.005). This study distinctly demonstrated significant expression of PSP94 mRNA in human uterine, breast and other female reproductive tissues as well in the endometrial cancer cell lines, suggesting that it may have a role in these tissues as a local autocrine paracrine factor.
Intermittent umbilical cord compression with resultant fetal hypoxia can have a negative impact on fetal growth and development. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are the most important regulators of fetal growth. In preterm (107-108 days of gestation) and near-term (128-131 days of gestation) ovine fetuses, we have determined the effect of intermittent umbilical cord occlusion (UCO) over a period of 4 days on the profile and expression of IGFs and IGFBPs. In experimental group animals (preterm n=7; near term n=7) UCOs were carried out by complete inflation of an occluder cuff (duration 90 s) every 30 min for 3-5 h each day, while control fetuses (preterm n=7; near term n=7) received no UCOs. Ewes were euthanized at the end of day 4, and fetal heart, lung, kidney, liver, skeletal muscle and placenta were collected. During UCOs, PO(2! ) fell (by approximately 13 mmHg), pH fell (by approximately 0.05) and PCO(2) increased (by approximately 7 mmHg), and changed to a similar extent in both preterm and near-term groups. In both preterm and near-term groups, there was no difference in fetal body or organ weight between UCO and control fetuses. No significant changes were observed in plasma IGF-I and -II concentrations or IGFBP-1, -2, -3 or -4 levels throughout the 4-day study at either gestational age. In the preterm group UCO fetuses, IGF-II mRNA (1.2-6.0 kb) levels were lower in fetal lung (33%, P<0.05), heart (54%, P<0.01) and skeletal muscle (29%, P<0.05), but there were no differences in IGF-I mRNA levels (7.3 kb); IGFBP-2 mRNA (1.5 kb) levels were lower in the right lobe of the liver (42%, P<0.05) and kidney (22%, P<0.01), but hig! her in the heart (72%, P<0.01), while IGFBP-4 (2.4 kb) levels were lower in skeletal muscle (21%, P<0.01). In the near-term group UCO fetuses, IGFBP-2 mRNA levels were greater in the placenta (39%, P<0.05). Thus, intermittent UCO as studied has a greater effect on the expression of genes encoding certain peptides of the fetal IGF system in selected tissues in preterm fetuses than that in near-term fetuses. Altered IGFBP-2 mRNA levels with reduced IGF-II mRNA levels in selected tissues may mediate changes in growth and/or differentiation that might become apparent if the length of the UCO study were extended.
To determine the temporal and spatial distribution of insulin-like growth factor (IGF) and its family of binding proteins (IGFBPs), guinea-pig yolk sac and chorioallantoic placentae were collected at 15, 20, 25, 29, 44-45, 55 and 65-66 days of gestation. Messenger RNAs for IGF I, IGF II and IGFBP 1-6 were identified in tissue sections by in situ hybridization, using 35S-cRNA probes. Epithelial and mesenchymal cell types were identified by immunohistochemistry for cytokeratin and vimentin, respectively. At 15 days of gestation, IGF-II mRNA was expressed in ectoplacental mesoderm, cytotrophoblasts and syncytiotrophoblast, and IGFBP-5 mRNA was detected in the syncytiotrophoblast. In the mid-gestation placenta, IGFBP-5 mRNA was expressed in the marginal and interlobular syncytium and IGF-II mRNA in the labyrinth. Near term, when expansion of the labyrinth was complete, IGFBP-5 mRNA was coexpressed with IGF-II mRNA in the marginal and interlobular syncytium. These observations suggest that interaction between IGF-II and IGFBP-5 plays a role in the vascularization of the placenta by fetal vessels. IGF-II mRNA was not expressed in the maternal tissues at any gestational age. IGFBP-2, -3 and -5 mRNAs were expressed in the endometrial stroma at 7-12 days of gestation but, following establishment of the placenta, IGFBP mRNAs were more abundant in the myometrium than in the decidua. IGF-II mRNA was detected in trophoblasts invading the walls of maternal vessels, and the endothelium of the preplacental vessels expressed IGFBP-4 mRNA, while IGFBP-2 and IGFBP-5 mRNAs were present in the tunica media of mesometrial arteries that had not been invaded by trophoblast. These findings suggest that IGF-II produced by the trophoblast acts in an autocrine and/or paracrine fashion to promote trophoblast invasion and that this process is modulated by interaction with IGFBPs present in maternal tissues.
To examine the relationship between the expression of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) and cell growth in a cell type with a defined IGF/IGFBP system, an ovine IGFBP-2 complementary DNA was overexpressed in C6 glioma cells. C6 cells produce IGFBP-3, IGFBP-4, a negligible amount of IGFBP-2, and IGF-I. An ovine IGFBP-2 complementary DNA was transfected into C6 cells, and nine colonies that stably expressed variable levels of IGFBP-2 messenger RNA were selected. Synthesis of corresponding levels of IGFBP-2 was confirmed by ligand blot and immunoblot analyses of conditioned media. Three clones exhibited significantly reduced growth rates, and the remainder showed growth rates similar to those of the wild-type C6 cells. The clones, which overexpressed high levels of IGFBP-2 and IGF-I, had growth rates similar to the wild-type cells, whereas the three clones that overexpressed IGFBP-2 without a concomitant increase in IGF-I had reduced growth rates. In addition, a cell-associated IGFBP was identified in the slow growing clones, but not in the wild-type or the fast growing clones. This cell-associated IGFBP was deduced to be IGFBP-5 based on its molecular size, detection of IGFBP-5 messenger RNA only in slow growing clones, and competition of its binding by heparin. Growth of the slow growing clone, C6BP2-1, could not be overcome by the addition of exogenous IGF-I, suggesting that the cell-associated IGFBP-5 was the dominant regulator of IGF action. These observations suggested that 1) in C6 glioma cells cellular growth is altered by a disturbance in the equilibrium between IGF-I and IGFBPs and/or the functional properties of the IGFBPs; and 2) C6 cells may have a limited capacity to modulate IGF/IGFBP expression in response to changes in endogenous expression of IGFBPs. Endogenous regulation of the balance between IGFs and IGFBPs may be a model of regulation of cellular growth in tumor cells.
To determine the role of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in the development of the pancreas, and specifically of the islets of Langerhans, we have examined the cellular distribution and developmental changes in the expression of IGFs and IGFBPs in the pancreas of the fetal and neonatal rat between 19.5 days of gestation and postnatal day 28. This represents a period of substantial growth and restructuring of the beta cell component in islets of this species. IGF-I, IGF-II, and IGFBPs-1 to -6 mRNAs were localized by in situ hybridization, and peptides by immunohistochemistry, in histological sections. IGF-II mRNA was highly expressed in islet cells and some ductal epithelial cells in late fetal and early neonatal life, but was barely detectable by postnatal day 28. IGF-II peptide showed a similar distribution. IGF-I mRNA was barely detected in the fetus or neonate and was localized predominantly in the ductal and acinar tissues after postnatal day 7. IGF-I immunoreactivity was associated with some islet cells in the fetus and neonate, suggesting an endocrine rather than a paracrine source. We performed co-localization studies to assess whether the distribution of IGFs within the pancreas might be due to a sequestration by locally produced IGFBPs. The presence of mRNAs for both IGFBPs-1 and -2 was minimal in the pancreas prior to postnatal day 7, although subsequently IGFBP-1 mRNA was seen in islet cells, while IGFBP-2 mRNA was localized in both islets and acinar tissues. In contrast, both IGFBPs-1 and -2 immunoreactivities were identified in islets from late fetal life, suggesting a circulatory source for these IGFBPs during early pancreatic development. IGFBPs-3 to -5 mRNAs and immunoreactivities were identified within islet cells throughout fetal and neonatal life, with IGFBPs-3 and -5 being mainly associated with the alpha cell-rich islet mantle. The results show a compartmentalization of IGFs within pancreatic tissue, reflecting both paracrine and endocrine sources. The localization and action of IGFs in pancreas likely involves sequestration and distribution by endogenous as well as circulating IGFBPs.
Premature cranial suture fusion, or craniosynostosis, can result in gross aberrations of craniofacial growth. The biology underlying cranial suture fusion remains poorly understood. Previous studies of the Sprague-Dawley rat posterior frontal suture, which fuses at between 12 and 20 days, have suggested that the regional dura mater beneath the cranial suture directs the overlying suture's fusion. To address the dura-suture paracrine signaling that results in osteogenic differentiation and suture fusion, the authors investigated the possible role of insulin-like growth factors (IGF) I and II. The authors studied the temporal and spatial patterns of the expression of IGF-I and IGF-II mRNA and IGF-I peptide and osteocalcin (bone morphogenetic protein-4) protein in fusing posterior frontal rat sutures, and they compared them with patent coronal (control) sutures. Ten Sprague-Dawley rats were studied at the following time points: 16, 18, and 20 days of gestation and 2, 5, 10, 15, 20, 30, 50, and 80 days after birth (n = 110). Posterior frontal and coronal (patent, control) sutures were analyzed for IGF-I and IGF-II mRNA expression by in situ hybridization by using 35S-labeled IGF-I and IGF-II antisense riboprobes. Levels of IGF-I and IGF-II mRNA were quantified by counting the number of autoradiograph signals per cell. IGF-I and osteocalcin immunoreactivity were identified by avidin-biotin peroxidase immunohistochemistry. IGF-I and IGF-II mRNA were expressed in dural cells beneath fusing sutures, and the relative mRNA abundance increased between 2 and 10 days before initiation of fusion. Subsequently, IGF-I and IGF-II mRNA were detected in the suture connective tissue cells at 15 and 20 days during the time of active fusion. In contrast, within large osteoblasts of the osteogenic front, the expression of IGF-I and IGF-II mRNA was minimal. However, IGF-I peptide and osteocalcin protein were intensely immunoreactive within these osteoblasts at 15 days (during the period of suture fusion). These data suggest that the dura-suture interaction may be signaled in a paracrine fashion by dura-derived growth factors, such as IGF-I and IGF-II. These peptides, in turn, stimulate nearby osteoblasts to produce bone-promoting growth factors, such as osteocalcin.
Trophoblast growth and invasion of the uterus are tightly regulated by locally produced factors. Since insulin-like growth factor (IGF)-II is produced by the invasive human extravillous trophoblast (EVT) cells and IGF-binding protein (IGFBP)-1 by the decidual cells in situ that are in proximity to each other, we examined the possible influence of these molecules on proliferation, migration, and invasiveness of first-trimester EVT cells in culture. These EVT cell functions were respectively measured by 3H-TdR uptake, in vitro migration, and invasion assays. Secretion of invasion-associated enzymes was assessed by zymography, and IGF-binding moieties on the EVT cell were examined by affinity cross-linking. Proliferation of serum-starved EVT cells was stimulated by addition of serum but unaffected by a wide range of IGF-I, IGF-II, and IGFBP-1 concentrations. IGF-II and IGFBP-1 or their combination stimulated EVT cell invasiveness and migration, when assays were conducted in serum-reduced media. Affinity cross-linking studies failed to detect the type 1 IGF receptor, although several IGF-II-specific and IGF-II-preferring binding molecules including type 2 IGF receptor were identified on the EVT cell surface. IGF-II enhancement of invasion was unaffected in the presence of IGF-1 receptor-blocking antibody and IGF-1 failed to influence EVT cell invasion, indicating that type 1 IGF receptor was not responsible for the IGF-II effects. Secretion of gelatinases or plasminogen activators was unaltered by IGF-II or IGFBP-1. We conclude that trophoblast-derived IGF-II and decidua-derived IGFBP-1 provide autocrine/paracrine enhancement of trophoblast invasiveness largely by stimulating migration, an essential step in invasion.
To define the epithelial and mesenchymal cell types of the guinea-pig placenta, immunostaining patterns were determined for the intermediate filament proteins cytokeratin and vimentin. Chorionic and yolk sac placentae were studied at 15, 20, 25, 29-30, 44-45, 55 and 65 days of gestation. Immunohistochemistry was performed on 5-microm thick sections of paraffin embedded tissue using specific antibodies against cytokeratin, a marker for epithelial cells, including trophoblast, and vimentin, a marker for mesenchymal cells and stromal decidua. Immunostaining was identified by the avidin-biotin-peroxidase technique with diaminobenzidine as the chromogen. Most of the surface of the placenta is covered by the columnar epithelium of the parietal yolk sac, beneath which is found a layer of chorionic giant cells. In the guinea-pig, a sheet of mesenchymal cells interposed between these cell layers immunostained for vimentin, a protein that is expressed only intracellularly, and had nuclei orientated parallel to the surface of the placenta. This cell layer is quite different from Reichert's membrane in the rat or mouse, which is acellular. Within the main placenta, cytokeratin immunostaining demonstrated that the trophoblasts lining the large maternal blood sinuses are different in character from the surrounding syncytiotrophoblast, confirming earlier ultrastructural observations. In the subplacenta, some trophoblast did not immunostain for cytokeratin and there was non-specific staining of cellular debris, so that immunostaining for vimentin provided the clearest indication of the maternal-fetal interface. In later stages of gestation (30-55 days), trophoblasts invading the walls of maternal arteries immunostained for cytokeratin and were vimentin negative. In early gestation, however, trophoblast invasion of the maternal vessels was indicated by cells that were immunoreactive for both cytokeratin and vimentin.
Expression of insulin-like growth factor (IGF) I and its type I receptor is increased in the adult rat lung exposed to 85% O2. We hypothesized that there would be a parallel up- and downregulation of growth-stimulating and growth-inhibiting IGF binding proteins (IGFBPs), respectively. The normal adult rat lung expresses mRNAs for IGFBP-2, -3, -4, -5, and -6 but not for IGFBP-1. O2 exposure for 6 or 14 days reduced IGFBP-3 and -6 and increased IGFBP-4 mRNA abundance. IGFBP-5 mRNA was reduced at 6 days but increased at 14 days. IGFBP-4 mRNA was localized to perivascular and peribronchial interstitial cells and IGFBP-5 mRNA to airway and alveolar epithelial cells. IGFBP-2, -4, and -5 immunolocalized to airway epithelial cells in normal lung and to perivascular exudates after 6 days in 85% O2. IGFBP-2 was diffusely increased throughout the lung tissue only after a 6-day exposure. IGFBP-5 was reduced after a 6-day exposure but was increased and widely distributed after 14 days. IGFBP-4 increased over airway epithelium and subepithelial cells after 6 days and over perivascular interstitial cells after 14 days of 85% O2. These data are consistent with the predicted changes for IGFBPs on O2 exposure except that the generally growth-inhibitory IGFBP-4 was increased at sites of active cell proliferation.
Endothelial cells (EC) are hypoxia-tolerant and their capacity to proliferate in low oxygen tension is essential to maintain vascular endothelium integrity. The present study addresses whether hypoxia alters the expression of insulin-like growth factor (IGF) and IGF binding protein (IGFBP) genes in bovine aortic EC (BAEC) and bovine pulmonary artery EC (BPAEC). EC were cultured in normoxic (21%) conditions and exposed to 0% oxygen for 24, 48, or 72 h; some cells were reoxygenated by exposure to 21% oxygen for 24 or 48 h following hypoxia. IGF-I peptide and mRNA levels were very low in both cell types, and decreased further with exposure to hypoxia. Ligand blotting showed that both cell types synthesized 24 kDa (IGFBP-4), 30 kDa (IGFBP-5 and/or IGFBP-6), 43 kDa and 48 kDa IGFBPs (IGFBP-3 glycosylation variants). IGFBP-4 was the predominant IGFBP expressed by both cell types and did not change with exposure to hypoxia. Hypoxia caused a significant increase in IGFBP-3 secretion in BPAEC but not in BAEC. IGFBP-3 stable mRNA levels in BPAEC were increased correspondingly. IGFBP-5 was expressed only in BAEC and decreased with exposure to hypoxia. IGFBP-6 mRNA expression was low and increased in both cell types with exposure to hypoxia. These results demonstrate that EC IGFBP baseline expression as well as its expression in hypoxia vary in different vascular beds and suggest that the IGFBPs may be the dominant paracrine regulators of proliferation of EC as well as maintenance of endothelium integrity during hypoxia.
Hypoxemia represents a major stress for the fetus, and is associated with alterations and adaptations in cardiovascular, metabolic and endocrine responses, which in turn may affect tissue growth and differentiation. To determine the effects of hypoxemia on fetal adrenal activity and growth, we subjected sheep fetuses at days 126-130 and 134-136 (term 145 days) to reduced PaO2 by reducing the maternal fraction of oxygen for 48 h (mean reduction of 6.8 mmHg), without change in arterial pH or PaCO2. This stimulus resulted in similar increases in the plasma immunoreactiveACTH response at both ages. Among adrenal steroids, plasma cortisol (C21Delta4) rose in both groups of animals, but plasma androstenedione (C19Delta4) declined marginally, resulting in a pronounced increase in the cortisol:androstenedione ratio in the plasma that was greater and more sustained in the older fetuses. In the younger fetuses, after 48 h of hypoxemia, there were no significant changes in mRNAs encoding steroidogenic enzymes in the fetal adrenal gland. However, in the older fetuses, hypoxemia resulted in significantly increased levels of mRNAs encoding P450scc, P450C21 and 3beta-hydroxysteroid dehydrogenase, but not for P450C17, in the fetal adrenal gland. Levels of IGF-II mRNA in the fetal adrenal gland fell in both groups of fetuses, and this response was greater at the later gestational age. We conclude that sustained hypoxemia is a potent stimulus which activates adrenal steroidogenesis in the late gestation fetal sheep. The resultant increase in cortisol synthesis is associated with decreased expression of adrenal IGF-II mRNA. We speculate that this relationship might influence patterns of fetal organ growth and differentiative function in response to fetal stress such as hypoxemia.
Bovine oviductal monolayer and vesicle primary cultures express insulin-like growth factor (IGF)-I and -II mRNAs and polypeptides. Early bovine embryos also express IGF-I, IGF-II, IGF-I receptor, IGF-II receptor, and insulin receptor mRNAs. This study reports the expression of IGF binding protein (IGFBP) mRNAs and polypeptides in bovine oviduct primary cultures and IGFBP mRNAs in preattachment embryos. Release of immunoreactive IGF-I and IGF-II by oviduct cultures and bovine blastocysts was also determined. IGFBP-2, -3, -4, and -5 transcripts were observed in oviduct primary cultures throughout an 8-day interval. IGFBP-1 and -6 mRNAs were consistently not detected in the oviduct. Messenger RNAs encoding IGFBPs -2, -3, and -4 were detected throughout bovine preattachment development, while transcripts encoding IGFBP-5 were detected only in blastocysts. IGFBP-1 and -6 transcripts were not detected in early embryos. Ligand blot analysis with 125I-labeled IGF-II revealed the presence of four prominent polypeptide bands of approximate molecular masses 24, 31, and 36 kDa, and a broad band extending from 46 to 53 kDa, in conditioned media samples prepared from oviduct primary cultures. Western immunoblot analysis confirmed the identity of the 24-kDa, 31-kDa, and 36-kDa species as IGFBP-4, -5, and -2, respectively. Levels of the release of IGF-II from oviductal vesicle cultures were significantly greater than levels observed for monolayer cultures (p < 0.005). No significant difference in the levels of IGF-I release between monolayer and vesicle cultures was observed. Pools of 10 blastocysts released on average 36.2 +/- 3.9 pg of IGF-II per embryo, while the release of embryonic IGF-I was below the levels of detection for our assay. The results suggest that maternally derived IGF may be regulated by IGFBPs to support bovine preattachment development.