Endopolyploid and proliferating trophoblast cells express different patterns of intracellular cytokeratin and glycogen localization in the rat placenta

The presence of keratin intermediate filaments is a characteristic of trophoblast differentiation. Meantime, their intracellular localization in the functionally different subtypes of placental trophoblast is poorly investigated in rodent, whereas their placentae are being broadly investigated in recent years as a model of the feto-maternal interaction. The purpose was to study the intracellular distribution of cytokeratin filaments in correlation with glycogen deposits, both being important constituents of the trophoblast cells in rat placenta. Different rat trophoblast cell populations exhibited different patterns of cytokeratin immunolocalization. The most intensive immunostaining was observed in the highly endopolyploid SGTCs (secondary giant trophoblast cells) at the border with decidua basalis. The most prominent cytokeratin-positive threads were found at the periphery of cytoplasm and in the extensive system of cytoplasmic sprouts by which the SGTC connect each other. Similar cytokeratin intensity and distribution was detected in the TSC (trabecular spongiotrophoblast cells) of the junctional zone of placenta that line the lacunae with the maternal blood. Clusters of highly proliferative pre-glycogen as well as glycogen cells showed some weaker cytokeratin signals mostly in the perinuclear and peripheral zones of cytoplasm. At the 11.5th to the 13.5th day of gestation, the interstitial and endovascular invasive endopolyploid TGTCs (tertiary giant trophoblast cells) prove the intensive cytokeratin staining throughout the cytoplasm and its sprouts. Meantime, the TGTCs were glycogen negative. By contrast, glycogen was heavily accumulated in the glycogen cells that belong both to the junctional zone of placenta and the cuff of the central arterial channel underlying the monolayer of endovascularly invading TGTCs. Thus, the TGTCs that are first to penetrate into the depth of the uterine wall do not contain glycogen but are accompanied by the glycogen-rich cells. The SGTC also contained the prominent deposits of glycogen at the periphery of cytoplasm and in the cytoplasmic sprouts. At the 16th day of gestation, an extensive interstitial invasion of the cytokeratin-positive glycogen trophoblast cells from the junctional zone was observed. The patterns of cytokeratin and glycogen intracellular localization are specific for each subtype of the rat trophoblast; that is, most probably, accounted for by the functional diversity of different trophoblast populations, i.e. patterns of invasion/phagocytosis and their involvement in a barrier at the feto-maternal interface.     

Maternal diabetes affects cell proliferation in developing rat placenta

Placentation starts with the formation of a spheroidal trophoblastic shell surrounding the embryo, thus facilitating both implantation into the uterine stroma and contact with maternal blood. Although it is known that diabetes increases the placental size and weight, the mechanisms responsible for this alteration are still poorly understood. In mammals, cellular proliferation occurs in parallel to placental development and it is possible that diabetes induces abnormal uncontrolled cell proliferation in the placenta similar to that seen in other organs (e.g. retina). To test this hypothesis, the objective of this work was to determine cell proliferation in different regions of the placenta during its development in a diabetic rat model. Accordingly, diabetes was induced on day 2 of pregnancy in Wistar rats by a single injection of alloxan (40 mg/kg i.v.). Placentas were collected on days 14, 17, and 20 postcoitum. Immunoperoxidase was used to identify Ki67 nuclear antigen in placental sections. The number of proliferating cells was determined in the total placental area as well as in the labyrinth, spongiotrophoblast and giant trophoblast cell regions. During the course of pregnancy, the number of Ki67 positive cells decreased in both control and diabetic rat placentas. However, starting from day 17 of pregnancy, the number of Ki67 positive cells in the labyrinth and spongiotrophoblast regions was higher in diabetic rat placentas as compared to control. The present results demonstrate that placentas from the diabetic rat model have a significantly higher number of proliferating cells in specific regions of the placenta and at defined developmental stages. It is possible that this increased cell proliferation promotes thickness of the placental barrier consequently affecting the normal maternal-fetal exchanges.     

Developmental changes in the intraplacental distribution of placental lactogen and alkaline phosphatase in the rat

The junctional and labyrinth regions of the rat chorioallantoic placenta during the second half of gestation showed different patterns of development with regard to DNA, protein, placental lactogen and alkaline phosphatase content. DNA and protein measurements indicated that growth of the labyrinth region was more rapid and persisted for longer during gestation than did growth in the junctional zone. At midpregnancy the junctional zone was the main source of placental lactogen whereas by late pregnancy both regions contributed considerable amounts. On Day 20 of gestation the labyrinth region contained significantly more placental lactogen than did the junctional zone. Alkaline phosphatase activity was predominant in the labyrinth zone throughout the second half of gestation. The results indicate that the chorioallantoic placenta is composed of two functionally distinct regions.     

Molecular cloning and characterization of prolactin-like protein C complementary deoxyribonucleic acid.

In this report, we describe the isolation and characterization of a full length cDNA clone for rat prolactin-like protein C (PLP-C) and describe the expression of PLP-C mRNA in the developing rat placenta. Nucleotide sequence analysis of the PLP-C cDNA clone predicted a mature protein of 238 amino acids, including a 30-amino acid signal sequence. The predicted PLP-C amino acid sequence contains seven cysteine residues, three tryptophan residues, and two putative N-linked glycosylation sites. Six of the cysteine residues in PLP-C are located in positions homologous to the cysteines of pituitary prolactin (PRL). Additional sequence similarities with pituitary PRL and other members of the rat placental PRL family are evident. The PLP-C gene was localized to rat chromosome 17. Northern blot analysis showed that the PLP-C cDNA clone specifically hybridized to a 1.0-kilobase mRNA. PLP-C mRNA was first detectable between days 13 and 14 of gestation, peaked by day 18 of gestation, and remained elevated until term. In situ hybridization analysis indicated that PLP-C mRNA was specifically expressed by spongiotrophoblast cells and some trophoblast giant cells in the junctional zone region of rat chorioallantoic placenta.     

A novel secretory protein produced by rat spongiotrophoblast

The placenta secretes various factors in stage- and cell-specific manners. We have identified a cDNA encoding a novel protein with 124 amino acids, which was named spongiotrophoblast specific protein (SSP). SSP is highly homologous to mouse 4311, showing 81% and 59% similarity at the nucleotide and amino acid levels, respectively. Northern blot analysis showed that SSP mRNA was first detected on Day 14 of pregnancy, peaked on Day 16, and remained elevated until term. In situ hybridization analysis showed that SSP mRNA was specifically expressed in spongiotrophoblast cells of Day 20 placenta but not in Day 12 placenta. No expression was detected from the differentiated or undifferentiated rat choriocarcinoma Rcho-1 cell line. Native SSP was detected as a 19-kDa molecule by Western blotting in cell extracts prepared from the junctional zone. SSP was predicted to be a secretory protein, because 1) a hydropathy test revealed that SSP contained an N-terminal hydrophobic region and 2) native SSP was also detected in the cultured media of junctional zone explants. To further investigate a potential signal peptide of this protein, sets of recombinant SSP were generated using a COS7 transfection system. The N-terminal amino acid sequence of secreted recombinant SSP confirmed that the N-terminal 17 amino acids had been cleaved to produce a secretory protein. Thus, we have identified and cloned a novel secretory protein, SSP, which is specifically expressed by rat spongiotrophoblast cells during the latter half of pregnancy.     

Expression and localization of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in murine and human placentas.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is one of the scavenger receptors that recognizes oxidized low-density lipoprotein as a major ligand. The placenta is a major source of prooxidant during pregnancy, and the level of placental oxidative stress increases rapidly at the end of the first trimester and tapers off later in gestation. In our study, we evaluated placental expression of LOX-1 during different gestational stages in mice and humans. We used immunohistochemistry and ISH to identify LOX-1-expressing cells in murine and human placentas. In both species, higher expression of LOX-1 mRNA during early to midgestational stages compared with late gestation-corresponding to the increased oxidative stress in early pregnancy-was shown by real-time RT-PCR. In murine placenta, we showed that LOX-1-expressing cells were fibroblast-like stromal cells in metrial glands and decidua basalis and that they were glycogen trophoblast cells in the junctional and labyrinth zones. In the human, LOX-1 expression was detected in villous cytotrophoblasts in both first trimester and term placentas. These localization patterns of LOX-1 in murine and human placentas suggest the possible involvement of LOX-1 in high oxidative stress conditions of pregnancy.     

Glucose as a fetal nutrient: dynamic regulation of several glucose transporter genes by DNA methylation in the human placenta across gestation

The human placenta ensures proper fetal development through the regulation of nutrient and gas transfer from the mother to the fetus and the removal of waste products from the fetal circulation. Glucose is one of the major nutrients for the growing fetus. Its transport across the placenta to the fetus is mediated by a family of facilitative transporter proteins, known as the glucose transporters (GLUTs), encoded by the SLC2A family of genes. There are 14 members of this gene family, and the expression of several of these has been shown in human placenta; however, aside from GLUT1 and GLUT3, little is known about the role of these proteins in placental function, fetal development and disease. In this study, we analysed previously generated genome-scale DNA methylation and gene expression data to examine the role of methylation in GLUT expression throughout gestation. We found evidence that DNA methylation regulates expression of GLUT3 and GLUT10, while the constitutively expressed GLUT1 showed no promoter methylation. We further analysed the level of DNA methylation across the promoter region of GLUT3, previously shown to be involved in glucose back-flux from the fetal circulation into the placenta. Using the Sequenom EpiTYPER platform, we found increasing DNA methylation of this gene in association with decreasing expression as gestation progresses, thereby highlighting the role of epigenetic modifications in regulating the GLUT family of genes in the placenta during pregnancy. These findings warrant a reexamination of the role of additional GLUT family members in the placenta in pregnancy and disease.     

Localization of erythrocyte/HepG2-type glucose transporter (GLUT1) in human placental villi

Summary The syncytiotrophoblast covering the surface of the placental villi contains the machinery for the transfer of specific substances between maternal and fetal blood, and also serves as a barrier. Existence of a facilitated-diffusion transporter for glucose in the syncytiotrophoblast has been suggested. Using antibodies to erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated-diffusion glucose transporters, we detected a 50 kD protein in human placenta at term. By use of immunohistochemistry, GLUT1 was found to be abundant in both the syncytiotrophoblast and cytotrophoblast. Endothelial cells of the fetal capillaries also showed positive staining for GLUT1. Electron-microscopic examination revealed that GLUT1 was concentrated at both the microvillous apical plasma membrane and the infolded basal plasma membrane of the syncytiotrophoblast. Plasma membrane of the cytotrophoblast was also positive for GLUT1. GLUT1 at the apical plasma membrane of the syncytiotrophoblast may function for the entry of glucose into its cytoplasm, while GLUT1 at the basal plasma membrane may be essential for the exit of glucose from the cytoplasm into the stroma of the placental villi. Thus, GLUT1 at the plasma membranes of syncytiotrophoblast and endothelial cells may play an important role in the transport of glucose across the placental barrier.     

Gestational protein restriction induces alterations in placental morphology and mitochondrial function in rats during late pregnancy

The placenta acts a regulator of nutrient composition and supply from mother to fetus and is the source of hormonal signals that affect maternal and fetal metabolism. Thus, appropriate development of the placenta is crucial for normal fetal development. We investigated the effect of gestational protein restriction (GPR) on placental morphology and mitochondrial function on day 19 of gestation. Pregnant dams were divided into two groups: normal (NP 17 % casein) or low-protein diet (LP 6 % casein). The placentas were processed for biochemical, histomorphometric and ultrastructural analysis. The integrity of rat placental mitochondria (RPM) isolated by conventional differential centrifugation was measured by oxygen uptake (Clark-type electrode). LP animals presented an increase in adipose tissue and triacylglycerol and a decrease in serum insulin levels. No alterations were observed in body, liver, fetus, or placenta weight. There was also no change in serum glucose, total protein, or lipid content. Gestational protein restriction had tissue-specific respiratory effects, with the observation of a small change in liver respiration (~13 %) and considerable respiratory inhibition in placenta samples (~37 %). The higher oxygen uptake by RPM in the LP groups suggests uncoupling between respiration and oxidative phosphorylation. In addition, ultrastructural analysis of junctional zone giant cells from LP placenta showed a disorganized cytoplasm, with loss of integrity of most organelles and intense vacuolization. The present results led us to hypothesize that GPR alters placental structure and morphology, induces sensitivity to insulin, mitochondrial abnormalities and suggests premature aging of the placenta. Further studies are needed to test this hypothesis.     

Human placental GLUT1 glucose transporter expression and the fetal insulin-like growth factor axis in pregnancies complicated by diabetes

We have previously described regulation of syncytial GLUT1 glucose transporters by IGF-I. Despite this, it is not clear what signal regulates transplacental glucose transport. In this report we asked whether changes in GLUT1 expression and glucose transport activity in diabetic pregnancies were associated with alterations in the fetal IGF axis. Cord blood samples and paired syncytial microvillous and basal membranes were isolated from normal term pregnancies and pregnancies characterized by gestational diabetes type A2 (GDM A2) and pre-existing insulin-dependent diabetes mellitus (IDDM). Circulating IGF-I, basal membrane GLUT1 expression and glucose transporter activity were correlated with birth weight, but only in control, not diabetic groups. Basal membrane GLUT1 and transporter activity were correlated with IGF-I concentrations in control, but not diabetic groups. IGF binding protein (IGFBP) binding capacity showed a ≥50% reduction in the diabetic groups compared to control; both showed a higher level of free IGF-I. The absence of a correlation between birth weight and factors such as fetal IGF-I or GLUT1 expression in the diabetic groups suggests that IGF-I-stimulated effects may have reached a limiting threshold, such that further increases in IGF-I (or GLUT1) are without effect. These data support that fetal IGF-I acts as a fetal nutritional signal, modulating placental GLUT1 expression and birth weight via altered levels of fetal circulating IGFBPs. Diabetes appears to exert its effects on fetal and placental factors prior to the third trimester and, despite good glycemic control immediately prior to, and in the third trimester, these effects persist to term.     

Regulation of Human Trophoblast GLUT1 Glucose Transporter by Insulin-Like Growth Factor I (IGF-I)

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.     

Placental changes in rats with streptozotocin diabetes]

Placentas of rats with diabetes mellitus induced by streptozotocin are investigated. Histologically, in the spongiosa zone we find dilated and congested maternal sinus as well as cysts of different size and number. These cysts contain granular eosinophilic material and cytotrophoblastic cells with large amount of glycogen. In our opinion, these cysts are large glycogen islets of the spongiosa zone respectively their remnants. However, similar findings we see in smaller extension in normal rat placentas too. The glycogen content in placentas of diabetic rats is in all localisations higher than in control cases. Comparable histological changes like in diabetic human placenta such as placental disturbances of maturation we don't find in the placental labyrinth of diabetic rats.     

Triamcinolone up‐regulates GLUT 1 and GLUT 3 expression in cultured human placental endothelial cells

The placenta is a glucocorticoid target organ, and glucocorticoids (GCs) are essential for the development and maturation of fetal organs. They are widely used for treatment of a variety of diseases during pregnancy. In various tissues, GCs have regulated by glucose transport systems; however, their effects on glucose transporters in the human placental endothelial cells (HPECs) are unknown. In the present study, HPECs were cultured 24 h in the presence or absence of 0·5, 5 and 50 µmol·l^–1 of synthetic GC triamcinolone (TA). The glucose carrier proteins GLUT 1, GLUT 3 and GC receptor (GR) were detected in the HPECs. We showed increased expression of GLUT 1 and GLUT 3 proteins and messenger RNA (mRNA) levels (p < 0·05) after 24‐h cell culture in the presence of 0·5, 5 and 50 µmol·l^‐1 of TA. In contrast, GR protein and mRNA expressions were down‐regulated (p < 0·05) with 0·5, 5 and 50 µmol·l^–1 of TA 24‐h cell culture. The results demonstrate that GCs are potent regulators of placental GLUT 1 and GLUT 3 expression through GR. Excessive exposure to GCs causes maternal and fetal hypoglycemia and diminished fetal growth. We speculate that to compensate for fetal hypoglycemia and diminished fetal growth, the expression of placental endothelial glucose transporters might be increased. Copyright © 2011 John Wiley & Sons, Ltd.     

Impact of curcumin treatment on diabetic albino rats

The current study was aimed to study the effect of curcumin on the expression levels of brain glucose transporter 1 protein (GLUT1) and femoral muscle glucose transporter 4 protein (GLUT4), in addition to study its possible therapeutic role in ameliorating insulin resistance and the metabolic disturbance in the obese and type 2 diabetic male albino Wistar rat model. Diabetes was induced by a high-fat (HF) diet with low dose streptozotocin (STZ). Curcumin was administered intragastrically for 8 weeks (80 mg/kg BW/day). The HF-diet group developed obesity, hyperglycemia, hyperinsulinemia, reduced liver glycogen content with significant dyslipidemia. In the diabetic control group, hyperglycemia and insulin resistance high calculated homeostasis model assessment (HOMA-IR-index score) were pronounced, with reductions in liver and muscle glycogen contents, concomitant with dyslipidemia and significantly elevated malondialdehyde levels in liver and pancreas. GLUT1 and GLUT4 were down-regulated in the obese and the diabetic control groups, respectively. Curcumin, showed glucose-lowering effect and decreased insulin resistance, dyslipidemia and malondialdehyde levels in both tissues, it increased liver & muscle glycogen contents, compared to the diabetic control. Curcumin significantly up-regulated GLUT4 gene expression, compared to the diabetic control group. In conclusions, these results indicate a therapeutic role of curcumin in improving the diabetic status, obesity and enhancing the expression of GLUT4 gene.     

Expression and regulation of calcitonin gene-related Peptide receptor in rat placentas

Calcitonin gene-related peptide (CGRP), one of the most potent vasodilators known, exerts its biological action by interacting with its receptors. Recent reports suggest the existence of two types of CGRP receptors, CGRP-A and CGRP-B. The current study was designed to examine whether CGRP-B receptors are present in the rat placenta, and if they are, whether they are modulated by gestational age and by sex-steroid hormones. Placentas were obtained from timed pregnant Sprague-Dawley rats that were killed on Days 17-21 and 22 before and during labor (n = 6 for each gestational age). In addition, placentas were also obtained from pregnant rats injected with progesterone (P(4); 4 mg per rat per day s.c. on Days 20-22), antiprogesterone RU-486 (10 mg/rat s.c. on Day 17), 17beta-estradiol (5 micro g/rat s.c. on Day 17), and antiestrogen ICI 182780 (0.3 micro g/rat s.c. on Day 17). Results showed that first, immunoflourescent staining of rat placentas using monoclonal anti-CGRP-B receptor antibody revealed the presence of CGRP-B receptors in the labyrinthine layer of the placenta, specifically to the trophoblast and blood vessel endothelium and underlying smooth muscle cells. The intensity of staining was lower in placentas obtained during labor. Second, a single band of 66 kDa, reactive to CGRP-B receptor antibody, was obtained in Western blotting of the rat placenta; third, densitometric analysis of protein bands showed that CGRP-B receptors were increased from Day 17 to Day 22, with maximal levels obtained on Day 22 before labor, which was 10 times higher than that of Day 17 (P < 0.01); fourth, expression of CGRP-B receptors in rat placenta decreased during labor (8% vs. 100% on Day 22 before labor, P < 0.01); fifth, P(4) given during Days 20-22 attenuated the fall in placental CGRP-B receptors at term labor; sixth, RU-486 given on Day 17 of gestation significantly decreased expression of placental CGRP-B receptors (18% vs. 100% in controls at 6 h, P < 0.01); seventh, a significant decrease in CGRP-B receptor expression was noted 48 h after estrogen administration; and eighth, ICI 182780 treatment on Day 17 increased placental CGRP-B receptors (152% vs. 100% in control at 48 h, P < 0.01). These results indicate that CGRP-B receptors are present in rat placenta and that receptor levels are higher with gestational age and lower at term labor. Progesterone stimulated and estrogen inhibited placental CGRP-B receptor expression. Thus, elevations in placental CGRP-B receptors in late pregnancy could play a role in increasing blood flow through the fetoplacental unit associated with rapid fetal growth during late gestation.     

Relationship between [125I]RTI-55-labeled cocaine binding sites and the serotonin transporter in rat placenta

We investigated the characteristics of cocainelike binding sitesin rat placenta using[¹²⁵I]RTI-55.[³H]paroxetine bindingand immunocytochemical staining for serotonin [5-hydroxytryptamine (5-HT)] and for the 5-HT transporterwere also used to obtain evidence for rat placental 5-HT uptake.[¹²⁵I]RTI-55saturation analyses with membranes from normal gestational day 20 placentas yielded curvilinear Scatchard plots that were resolved intohigh- and low-affinity components (mean dissociation constants of 0.29 and 7.9 nM, respectively). Drug competition studies with variousmonoamine uptake inhibitors gave rise to complex multiphasicdisplacement curves, although the results obtained with the selective5-HT uptake inhibitor citalopram suggest that the 5-HT transporter isan important component of placental high-affinity[¹²⁵I]RTI-55 binding.The presence of a rat placental 5-HT uptake system was additionallysupported by the[³H]paroxetine bindingexperiments and by the presence throughout the placenta ofimmunoreactivity for 5-HT and the 5-HT transporter. Immunostaining withboth antibodies was most intense in the junctional zone, whereas thedensity of[¹²⁵I]RTI-55 bindingsites was greater in the placental labyrinth. This discrepancy may bedue to the fact that[¹²⁵I]RTI-55 appearsto be labeling additional cellular components besides the 5-HTtransporter. The presence of cocaine- and antidepressant-sensitive 5-HTtransporters in the placenta has important implications for thepossible effects of these compounds on pregnancy and fetal development.

     

Glucose transporter isoform-3 mutations cause early pregnancy loss and fetal growth restriction

Glucose transporter isoform-3 (GLUT3) is the trophoblastic facilitative glucose transporter. To investigate the role of this isoform in embryonic development, we created a novel GLUT3-null mouse and observed arrested early embryonic development and loss at neurulation stage when both alleles were mutated. This loss occurred despite the presence of other related isoforms, particularly GLUT1. In contrast, when a single allele was mutated, despite increased embryonic cell apoptosis, adaptive changes in the subcellular localization of GLUT3 and GLUT1 in the preimplantation embryo led to postimplantation survival. This survival was compromised by decreased GLUT3-mediated transplacental glucose transport, causing late-gestation fetal growth restriction. This yielded young male and female adults demonstrating catch-up growth, with normal basal glucose, insulin, insulin-like growth factor-I and IGF-binding protein-3 concentrations, fat and lean mass, and glucose and insulin tolerance. We conclude that GLUT3 mutations cause a gene dose-dependent early pregnancy loss or late-gestation fetal growth restriction despite the presence of embryonic and placental GLUT1 and a compensatory increase in system A amino acid placental transport. This critical life-sustaining functional role for GLUT3 in embryonic development provides the basis for investigating the existence of human GLUT3 mutations with similar consequences during early pregnancy.     

Expression and localisation of GLUT1 and GLUT12 glucose transporters in the pregnant and lactating rat mammary gland

Glucose plays a major role in mammary gland function during lactation as it is used both as a fuel and as a precursor of milk components. In rats, previous studies have shown that the facilitative glucose transporter GLUT1 is expressed in mammary epithelial cells. We have used confocal immunofluorescence to localise GLUT1 and GLUT12, a recently identified member of the sugar transporter family, in pregnant and lactating rat mammary gland. GLUT12 staining was observed in the cytoplasm of mammary epithelial cells at day 20 of pregnancy, and at 1 and 6 days postpartum. Furthermore, GLUT12 staining was present at the apical plasma membrane of epithelial cells during lactation. In contrast, GLUT1 protein localised to the cytoplasm and basolateral surface of mammary epithelial cells. Forced weaning resulted in decreased cytoplasmic GLUT1 staining intensity, but no change in GLUT12 staining. The results suggest a possible role for GLUT12 in the metabolism of mammary epithelial cells during pregnancy and lactation.     

Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (FLT-1) pre-mRNA is important for the regulation of VEGF activity

Angiogenesis is essential for normal mammalian development and is controlled by the local balance of pro- and antiangiogenic factors. Here we describe a novel mouse cDNA sequence encoding sFLT-1 that is a potent antagonist to vascular endothelial growth factor (VEGF) and show for the first time its in vivo production. In situ hybridization and Northern blot analysis with probes specific for sFLT-1 or FLT-1 showed that the relative abundance of their mRNAs changed markedly in spongiotrophoblast cells in the placenta as gestation progressed. On day 11 of pregnancy, sFLT-1 mRNA was undetectable but FLT-1 readily apparent, and by day 17 sFLT-1 mRNA was abundant but FLT-1 barely detectable. sFLT-1 was identified in conditioned medium of cultured placenta from day 17 pregnant mice and likely to be present in the circulation, as there is a substantial increase of VEGF-binding activity in the serum from day 13 of pregnancy, which coincides with the abundant sFLT-1 expression in placenta. Expression of sFLT-1 was also observed in adult lung, kidney, liver, and uterus. These data suggest a novel mechanism of regulation of angiogenesis by alternative splicing of FLT-1 pre-mRNA. Treatment of pregnant mice with exogenous VEGF from day 9 to 17 of pregnancy, which alters the ratio of VEGF to sFLT-1, resulted in an increase in the number of resorption sites and fibrin deposition in the placenta of ongoing pregnancies. These findings have important implications for understanding placental function and may be relevant in a range of disease states.