Developmental changes in endothelial nitric oxide synthase expression and activity in ovine fetal lung

Endothelial nitric oxide (NO) synthase (eNOS) produces NO, which contributes to vascular reactivity in the fetal lung. Pulmonary vasoreactivity develops during late gestation in the ovine fetal lung, during the period of rapid capillary and alveolar growth. Although eNOS expression peaks near birth in the fetal rat, lung capillary and distal air space development occur much later than in the fetal lamb. To determine whether lung eNOS expression in the lamb differs from the timing and pattern reported in the rat, we measured eNOS mRNA and protein by Northern and Western blot analyses and NOS activity by the arginine-to-citrulline conversion assay in lung tissue from fetal, newborn, and maternal sheep. Cellular localization of eNOS expression was determined by immunohistochemistry. eNOS mRNA, protein, and activity were detected in samples from all ages, and eNOS was expressed predominantly in the vascular endothelium. Lung eNOS mRNA expression increases from low levels at 70 days gestation to peak at 113 days and remains high for the rest of fetal life. Newborn eNOS mRNA expression does not change from fetal levels but is lower in the adult ewe. Lung eNOS protein expression in the fetus rises and peaks at 118 days gestation but decreases before birth. eNOS protein expression rises in the newborn period but is lower in the adult. Lung NOS activity also peaks at 118 days gestation in the fetus before falling in late gestation and remaining low in the newborn and adult. We conclude that the pattern of lung eNOS expression in the sheep differs from that in the rat and may reflect species-related differences in lung development. We speculate that the rise in fetal lung eNOS may contribute to the marked lung growth and angiogenesis that occurs during the same period of time.     

The establishment of hepatocyte cell surface polarity during fetal liver development

Antibodies to six glycoproteins present in different domains of the hepatocyte plasma membrane were used to study the establishment of cell surface polarity during rat fetal liver development. The proteins were immunoprecipitated from fetal liver homogenates between 14 and 21 days of gestation and quantified by immunoblotting. Aminopeptidase N, CE 9, and HA 321, which reside in the apical, basolateral, and lateral plasma membrane in the adult hepatocyte, respectively, were present in high concentrations at 14 days of gestation and remained high until birth. In contrast, two apical proteins (HA 4 and dipeptidyl peptidase IV) and two basolateral proteins (ASGP receptor and EGF receptor) were first detected between 16 and 18 days of gestation and increased linearly until birth. HA 4 was the only molecule for which the fetal and adult forms differed, with the former having a faster mobility on SDS-PAGE, due to differences in N-linked oligosaccharides. With two exceptions, the localization of the molecules from earliest detection was restricted to the same domain as that in the adult. At 15 days of gestation, HA 321 and a small portion of aminopeptidase were detected on the basolateral membrane. By 21 days both molecules had assumed their adult localization pattern. Our results indicate that the biogenesis of cell surface polarity is an early event, implying that the mechanisms for sorting plasma membrane molecules are functional very early in development. Furthermore, the different patterns of appearance of the six molecules, irrespective of domain, indicate that the biochemical composition of the cell surface changes dramatically during fetal liver development.     

Regulation of the glucocorticoid receptor in fetal rat lung during development

The effect of the synthetic glucocorticoid betamethasone on the regulation of the glucocorticoid receptor mRNA and on receptor protein was studied in fetal rat lung during development. Using a glucocorticoid receptor cRNA probe, glucocorticoid receptor mRNA was examined by Northern blot hybridization and by solution hybridization. A monoclonal antibody against the glucocorticoid receptor was used to study regulation of the receptor protein by the Western immunoblotting technique. In fetal rat lungs, of 16-21 days of gestation, as well as in adult lungs, betamethasone treatment resulted in a significant decrease of glucocorticoid receptor mRNA to 50-65% of the control level. In contrast, betamethasone treatment did not down-regulate the receptor protein in rat lungs of 16-19 days of gestation, whereas a decrease of glucocorticoid receptor protein to 40-60% of control was seen in lungs of 21 days of gestation, in postnatal and adult lung. These results provide data for a change in regulation in vivo of the glucocorticoid receptor by its homologous ligand in fetal rat lung during development.     

Pregnancy-related changes of galactose recognition system on sinusoidal rat liver cells

We have studied the galactose specific receptor activities of sinusoidal liver cells of pregnant rat from the 13th day of gestation to the 10th day after delivery. Both liver macrophages and endothelial cells modulate the expression of hepatic binding protein during rat pregnancy and lactation. Except for endothelial cells of rats at the 22nd day of gestation, we have measured an overall decrease of binding sites. In addition, in liver macrophages the clustered distribution of galactose specific receptors, which is typical for adult animals, is reduced or completely absent. These results indicate that the galactose specific receptors on non-parenchymal liver cells are modulated in relation with the physiological state of the organ like the asialoglycoprotein receptor of hepatocytes.     

Insulin receptor gene expression during development: developmental regulation of insulin receptor mRNA abundance in embryonic rat liver and yolk sac, developmental regulation of insulin receptor gene splicing, and comparison to abundance of insulin-like growth factor 1 receptor mRNA.

Insulin gene expression has been demonstrated in nonpancreatic tissues early in development, suggesting that this hormone might have actions significant for the differentiating embryo. Because such actions imply ligand-receptor binding, we quantified mRNAs encoding the two known forms of insulin receptor in rat liver and yolk sac, two endodermally derived tissues shown to express insulin genes, between gestation days (E) 13 and E21 (mid-organogenesis to parturition). Because of its presumed importance for fetal growth, we estimated the abundance of mRNA encoding insulin-like growth factor 1 (IGF 1) receptor in the same samples for comparison. The abundance of insulin receptor mRNA exceeded that for IGF 1 receptor mRNA in liver and yolk sac at all times studied. This difference was greater in liver, where insulin receptor mRNAs were three to more than 50 times more abundant than IGF 1 receptor mRNA on gestation days E13-E16, times which antedate the development of significant hepatic metabolic actions of insulin. The marked abundance of mRNAs encoding insulin receptors is consistent with the hypothesis that insulin has significant actions in specific tissues during the organogenic period.     

Analysis of the sinusoidal endothelial cell organization during the developmental stage of the fetal rat liver using a sinusoidal endothelial cell specific antibody, SE-1

The sinusoid organization during the development of fetal rat livers was studied using a SE-1 antibody, which we have previously established as a specific monoclonal antibody against rat sinusoidal endothelial cell (SEC). Expression and localization of the SE-1 antigen in the liver tissues of 13- to 21-day-old fetuses were immunofluorescently and immunoelectron microscopically examined. The first positive fluorescence was observed in the immature liver of 15-day-old fetuses. The initial positive staining was randomly distributed in the liver parenchyma and showed no direct relation to the large vessels which may be derived from the fetal vitelline veins. The positive linear staining increased in number and connected with each other during the course of development. The SE-1 staining pattern and the sinusoidal arrangement became similar to those of the adult liver after 20th day of gestation. Immunoelectron microscopically, the immature SEC showed a weak positive reaction for the SE-1 antigen at their membrane and was observed together with immature hepatocytes and hematopoietic cells in the 15-day-old fetal liver. Along with the liver development, SEC formed a sinusoid structure closely associated with hepatocytes and came to strongly express the SE-1 antigen. These results indicate that the organization of the hepatic sinusoid may start at around 15th day of the gestation and occurs randomly in the fetal liver parenchyma. It is also suggested that the expression of SE-1 antigen is possibly regulated by the intimate association with hepatocytes.     

Discoidin domain receptors and their ligand, collagen, are temporally regulated in fetal rat fibroblasts in vitro

The biochemical regulation of collagen deposition during adult cutaneous wound repair is poorly understood. Likewise, how collagen is perceived and modulated in fetal scarless healing remains unknown. Recently, discoidin domain receptors-1 and 2 (DDR1 and DDR2) with tyrosine kinase activity have been identified as novel receptors for collagen. In light of these findings, it was speculated that the production of collagen receptors DDR1 and DDR2 by fetal fibroblasts may be temporally regulated to correlate with the ontogeny of embryonic scar formation. More specifically, because DDRs directly bind collagen and transmit the signals intracellularly, it was hypothesized that they may play an important role in fetal scarless healing by ultimately regulating and modulating collagen production and organization. As part of a fundamental assessment to elucidate the role of DDRs in scarless fetal wound repair, the endogenous expression of DDR1, DDR2, collagen I, and total collagen, as a function of fetal Sprague-Dawley rat skin fibroblasts of different gestational ages, representing scar-free (E16.5) periods was determined. Using explanted dermal fibroblasts of gestational days E13.5, E16.5, E18.5, and E21.5 (term gestation = 21.5 days) fetuses (n = 92), [3H]proline incorporation assay and Northern and Western blotting analysis were performed to compare the expressions of these molecules with scar-free and scar-forming stages of embryonic development. These results revealed a pattern of increasing collagen production with increasing gestational ages, whereas DDR1 expression decreased with increasing gestational age. This observation suggests that elevated levels of DDR1 may play an important role in scarless tissue regeneration by early gestation fetal fibroblasts. In contrast, DDR2 was expressed by fetal rat fibroblasts at a similar level throughout gestation. These data demonstrate for the first time the temporal expression of collagen and DDR tyrosine kinases in fetal rat fibroblasts as a function of gestational ages. Overall, these data suggest that differential temporal expression of the above-mentioned molecules during fetal skin development may play an important role in the ontogeny of scar formation. Future studies will involve the characterization of the biomolecular functions of these receptor kinases during fetal wound repair.     

Expression of fetal and neonatal hepatic functions by mouse hepatoma-rat hepatoma hybrids

In order to analyze the mechanisms implicated in the expression of differentiated functions during development, we have studied ten hybrid clones arising from fusion of cells of a mouse hepatoma characterized by the expression of only fetal hepatic functions with those of a rat hepatoma which express, like adult hepatocytes, a set of neonatal as well as fetal hepatic functions. The cells of most hybrid clones contain one set of chromosomes of each parent and coexpress the hepatic functions common to both parents. Among the hepatic proteins characteristic of only one parental line, some continue to be expressed while others are extinguished. The three functions out of the eight examined which are subject to extinction are expressed uniquely by the rat parental cells and appear only near or at birth during normal liver development. These results suggest that regulatory mechanisms (whose final effect is negative) operate in fetal cells to inhibit the expression of differentiated functions limited to a later stage of development.     

The development of rat alpha 2-macroglobulin. Studies in vivo and in cultured fetal rat hepatocytes

During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat alpha 2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of alpha 2-macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, visceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of alpha 2-macroglobulin expression are localized by in situ hybridization of cDNA for alpha 2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for alpha 2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. alpha 2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. alpha 2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the alpha 2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting alpha 2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.     

Developmental expression of the tissue insulin-like growth factor II/mannose 6-phosphate receptor in the rat. Measurement by quantitative immunoblotting

We used quantitative immunoblotting to measure the total tissue insulin-like growth factor II/mannose 6-phosphate (IGF-II/Man-6-P) receptor in the rat. Whole embryos (6-15 days of gestation) and tissues from 16- and 20-day-old fetal and 5-, 10-, 20-, and 40-day-old postnatal rats were placed in liquid nitrogen, extracted with 2% Triton X-100, and boiled in 2% sodium dodecyl sulfate. The extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis together with aliquots of a highly purified rat IGF-II/Man-6-P receptor standard. The protein bands were transferred from the gel to nitrocellulose sheets by electroelution. The nitrocellulose sheets were incubated with a specific IGF-II/Man-6-P receptor antiserum (3637). The immunoblots were developed with 125I-protein A and an immunoperoxidase stain. Stained areas were cut from the immunoblots, and radioactivity was measured in a gamma-counter. IGF-II/Man-6-P receptor levels were high in fetal tissues and in most tissues declined dramatically in late gestation and/or in the early postnatal period. Concentrations in 16-day-old fetal tissues, expressed as percent of total protein in the extract, were: heart, 1.7; placenta, 1.0; lung, 0.7; intestine, 0.7; muscle, 0.5; kidney, 0.5; liver, 0.3; and brain, 0.1. In whole embryos (6-15 days of gestation), the IGF-II/Man-6-P receptor ranged between 0.1 and 0.4% of total protein in the extract. The IGF-II/Man-6-P receptor size varied within approximately 20 kDa among different tissues and also varied with developmental age in the same tissue. We conclude that the IGF-II/Man-6-P receptor is a major cellular protein in some fetal tissues and that the developmental pattern of receptor expression suggests that the receptor plays an important role in fetal growth and development.     

Development of ER-alpha and ER-beta expression in the developing ovine brain and pituitary

Fetal neuroendocrine development in late gestation is critical for maintenance of fetal homeostasis, growth, and readiness for birth. We designed the present study to identify the regional patterns of expression of the two main isoforms of the estrogen receptor, ER-alpha and ER-beta, in the developing ovine fetal brain. Fetal (80, 100, 120, 130, and 145 days gestation), neonatal (1 and 7 days), and adult sheep were euthanized and the following tissues were collected: pituitary, hypothalamus, hippocampus, cerebral cortex, and brainstem. Both ER's are expressed in the ovine brain as early as 80 days gestation, and the expression of both receptors appears to be developmentally regulated. We conclude that both forms of the estrogen receptor are expressed in fetal brain and pituitary throughout the latter half of gestation.     

Differential regulation of suppressor of cytokine signaling-3 in the liver and adipose tissue of the sheep fetus in late gestation

It is unknown whether the JAK/STAT/suppressor of cytokine signaling-3 (SOCS-3) intracellular signaling pathway plays a role in tissue growth and metabolism during fetal life. We investigated whether there is a differential profile of SOCS-3 expression in the liver and perirenal adipose tissue during the period of increased fetal growth in late gestation and the impact of fetal growth restriction on SOCS-3 expression in the fetal liver. We also determined whether basal SOCS-3 expression in the fetal liver and perirenal adipose tissue is regulated by endogenous fetal prolactin (PRL). SOCS-3 mRNA abundance was higher in the liver than in the pancreas, spleen, and kidney of the sheep fetus during late gestation. In the liver, SOCS-3 mRNA expression was increased (P < 0.05) between 125 (n = 4) and 145 days (n = 7) gestation and lower (P < 0.05) in growth-restricted compared with normally grown fetal sheep in late gestation. The relative expression of SOCS-3 mRNA in the fetal liver was directly related to the mean plasma PRL concentrations during a 48-h infusion of either a dopaminergic agonist, bromocriptine (n = 7), or saline (n = 5), such that SOCS-3 mRNA expression was lower when plasma PRL concentrations decreased below approximately 20 ng/ml [y = 0.99 - (2.47/x) + (4.96/x(2)); r(2) = 0.91, P < 0.0001, n = 12]. No relationship was shown between the abundance of phospho-STAT5 in the fetal liver and circulating PRL. SOCS-3 expression in perirenal adipose tissue decreased (P < 0001) between 90-91 (n = 6) and 140-145 days (n = 9) gestation and was not related to endogenous PRL concentrations. Thus SOCS-3 is differentially expressed and regulated in key fetal tissues and may play an important and tissue-specific role in the regulation of cellular proliferation and differentiation before birth.     

Identification of candidate growth-regulating genes that are overexpressed in late gestation fetal liver in the rat

We have shown previously that hepatocyte proliferation in the late gestation fetal rat is mediated by growth factor-independent mechanisms that are distinct from the signaling pathways that promote proliferation of adult rat hepatocytes. In the present studies, we identified six candidate growth-regulating genes that are overexpressed in fetal rat liver (embryonic day 19, 2 days pre-term) relative to adult rat liver using suppressive subtractive hybridization. These included the following: Grb10, a growth factor receptor binding protein; eps15, a growth factor receptor substrate; nuc2+, a retinoblastoma protein binding protein; cdc25B, a cell cycle tyrosine phosphatase; the peroxisome proliferator-activated receptor PPAR alpha; and a deoxyuridine triphosphatase that functions as a PPAR alpha binding partner. In every case, the ontogeny of the expression of these genes declined postnatally in a manner consistent with the transition from a fetal to an adult hepatocyte phenotype. None were found to be cell cycle-dependent, in that they did not show expression that followed perinatal changes in hepatocyte cell cycle activity. Based on our identification of these genes and previous work characterizing their role in growth regulation, we conclude that they may contribute to the mitogenic signaling phenotype of fetal rat hepatocytes.     

LXR is crucial in lipid metabolism

Liver X receptors (LXRalpha and LXRbeta) are members of the nuclear receptor superfamily and are activated by oxysterols and intermediates in the cholesterol synthetic pathway. The pivotal role of LXRs in the metabolic conversion of cholesterol to bile acids is well established. Analysis of gene expression in LXRalpha and LXRbeta deficient mice have confirmed that LXR regulates a number of target genes involved in both cholesterol and fatty acid metabolism in liver, macrophages and intestine. The observation that LXRalpha is responsive to fatty acids and is expressed in metabolic tissues suggests that it also plays a general role in lipid metabolism. Adipose tissue is the main storage site for fat in the body and plays a crucial role in overall lipid handling. Both LXRalpha and LXRbeta are expressed and activated by endogenous and synthetic ligands, which lead to lipid accumulation into adipocytes. This indicates an important regulatory role of LXR in several metabolic signaling pathways in the adipose tissue, such as glucose uptake and de novo fatty acid synthesis. Here, we review recent studies that provide new insights into the mechanisms by which LXRs act to influence fatty acid synthesis in liver and adipose tissue.     

Expression of aldolase isozyme mRNAs in fetal rat liver

The regulation of aldolase isozyme expression during development was studied by measuring the concentrations of mRNAs coding for aldolase A and B subunits in fetal and adult rat liver. Poly(A)-containing RNAs were extracted from livers at various stages of development of fetal rats, and the aldolase A and B subunits in the in vitro translation products of these RNAs were analyzed immunologically. The content of aldolase B mRNA in 14-day fetal liver, measured quantitatively as translational activity, was somewhat smaller than that of aldolase A mRNA; immunologically precipitable aldolase B and A amounted to 0.06% and 0.25% respectively, of the total products. Similar experiments using RNAs from fetuses at later stages, however, showed that aldolase B mRNA increased during development, whereas aldolase A mRNA decreased. In newborn rat liver, aldolase B constituted 0.56% of the total translation products of mRNA, but there was little detectable aldolase A (0.03%). The changes of aldolase mRNA levels were analyzed further by northern blot and dot-blot hybridization experiments using cloned aldolase A and B cDNAs. The content of aldolase B mRNA increased in the fetal stage, and that in newborn rat liver was about 12 times that in 14-day fetal liver. In contrast, the aldolase A mRNA content decreased during gestation and that in newborn rat liver was about one-eighth of that in 14-day fetal liver. These observations suggest that the switch of aldolase isozyme expression in fetal liver is controlled by the levels of the respective mRNAs.