Placental Protein Expression Research Articles

Decreased placental protein expression of PHOSPHO-AKT correlates with a decrease in expression of eNOS protein at mid-gestion in an ovine model of intrauterine growth restricition (IUGR)

WITH A DECREASE IN EXPRESSION OF ENOS PROTEIN AT MID-GESTION IN AN OVINE MODEL OF INTRAUTERINE GROWTH RESTRICITION (IUGR) JUAN A. ARROYO, RUSSELL V. ANTHONY, THOMAS A. PARKER, GATES ROE, HENRY L. GALAN, University of Colorado Health Sciences Center, Obstetrics and Gynecology, Denver, Colorado, University of Colorado Health Sciences Center, Dept. of Pediatrics, Aurora, Colorado, University of Colorado Health Sciences Center, Dept. of Pediatrics, Denver, Colorado OBJECTIVE: Endothelial nitric oxidase synthase (eNOS) and NO are important regulators of blood flow in the fetal-placental circulation and are modulated by shear stress. eNOS activity is regulated by the phosphorylation (p) of ERK and AKT proteins in various tissues. In an established hyperthermic (HT) ovine model of placental and fetal growth restriction (FGR), we hypothesize that eNOS, pERK and pAKT protein levels would be decreased in the placental tissues in 95 day gestational age (dGA; term=147dGA) animals. STUDY DESIGN: 4 ewes were exposed to HT conditions for 55 days to induce FGR and 4 were used as controls. Placentomes were separated into caruncle and cotyledon components, and frozen for Western blot analysis with an antibody against eNOS, pERK, ERK, pAKT and AKT . Actin westerns on the same blots were used to account for inter-lane loading variations. RESULTS: HT animals showed, 1) smaller placentae (440G50 v. 186G18; p!0.004) while fetus weights were similar, 2) decreased O2 saturation (5.5G.06% v. 3.8G0.5% p!0.04) and pO2 levels (48.6G15.8 mmHg v. 23.2G2.34 mmHg; p!0.02), 3) a 2.5-fold decrease in caruncle eNOS protein and a 1.9-fold decrease in cotyledon eNOS protein (p!0.03), 4) a 1.5-fold increase in caruncle pERK with a 1.4-fold decrease in pAKT, 5) no differences in cotyledon pERK with a 2-fold decrease in pAKT protein (p!0.02).

Decreased placental amino acid transport and intrauterine growth restriction: which is the chicken and which is the egg?

Decreased placental amino acid transport and intrauterine growth restriction: which is the chicken and which is the egg?

Down‐regulation of placental transport of amino acids precedes the development of intrauterine growth restriction in rats fed a low protein diet

Intrauterine growth restriction (IUGR) represents an important risk factor for perinatal complications and for adult disease. IUGR is associated with a down‐regulation of placental amino acid transporters; however, whether these changes are primary events directly contributing to IUGR or a secondary consequence is unknown. We investigated the time course of changes in placental and fetal growth, placental nutrient transport in vivo and the expression of placental nutrient transporters in pregnant rats subjected to protein malnutrition, a model for IUGR. Pregnant rats were given either a low protein (LP) diet (n= 64) or an isocaloric control diet (n= 66) throughout pregnancy. Maternal insulin, leptin and IGF‐I levels decreased, whereas maternal amino acid concentrations increased moderately in response to the LP diet. Fetal and placental weights in the LP group were unaltered compared to control diet at gestational day (GD) 15, 18 and 19 but significantly reduced at GD 21. Placental system A transport activity was reduced at GD 19 and 21 in response to a low protein diet. Placental protein expression of SNAT2 was decreased at GD 21. In conclusion, placental amino acid transport is down‐regulated prior to the development of IUGR, suggesting that these placental transport changes are a cause, rather than a consequence, of IUGR. Reduced maternal levels of insulin, leptin and IGF‐1 may link maternal protein malnutrition to reduced fetal growth by down‐regulation of key placental amino acid transporters.

Docosahexaenoic acid supply in pregnancy affects placental expression of fatty acid transport proteins

Better understanding of the mechanisms involved in docosahexaenoic acid (DHA) transfer to the neonate may contribute to improve dietary support for infants born prematurely to mothers with placental lipid transport disorders. We studied whether DHA supplements modify the messenger RNA (mRNA) expression of placental lipid transport proteins to allow a selective transfer of DHA to the fetus. Healthy pregnant women (n = 136) received, in a double-blind randomized trial, 500 mg DHA + 150 mg eicosapentaenoic acid, 400 microg 5-methyl-tetrahydrofolic acid, 500 mg DHA + 400 microg 5-methyl-tetrahydrofolic acid, or placebo during the second half of gestation. We analyzed the fatty acid composition of maternal and cord blood phospholipids and of placenta; we quantified placental mRNA expression of fatty acid-transport protein 1 (FATP-1), FATP-4, FATP-6, fatty acid translocase, fatty acid-binding protein (FABP) plasma membrane, heart-FABP, adipocyte-FABP, and brain-FABP. The mRNA expression of the lipid carriers assayed did not differ significantly between the 4 groups. However, the mRNA expression of FATP-1 and FATP-4 in placenta was correlated with DHA in both maternal plasma and placental phospholipids, although only FATP-4 expression was significantly correlated with DHA in cord blood phospholipids. Additionally, the mRNA expression of several membrane lipid carriers was correlated with EPA and DHA in placental triacylglycerols and with EPA in placental free fatty acids. Correlation of the mRNA expression of the membrane placental proteins FATP-1 and especially of FATP-4 with maternal and cord DHA leads us to conclude that these lipid carriers are involved in placental transfer of long-chain polyunsaturated fatty acids.

Altered Placental and Fetal Expression of IGFs and IGF-Binding Proteins Associated With Intrauterine Growth Restriction in Fetal Sheep During Early and Mid-Pregnancy

The insulin-like growth factors (IGFs) are postulated to be altered in association with the development of intrauterine growth restriction (IUGR). The present studies examined placental and fetal hepatic mRNA concentration of components of the IGF system at two time points (55 and 90 d gestational age, dGA; Term 147 dGA) in a hyperthermia (HT)-induced sheep model of placental insufficiency-IUGR. Maternal plasma insulin and IGF-I were constant at 55 and 90 dGA and were unaffected by treatment. Umbilical vein insulin concentrations tended to be reduced at 90 dGA following HT exposure. Caruncle IGF-I mRNA was increased at 90 dGA in HT placentae (p < 0.05), while cotyledon concentrations were constant over gestation and unaltered by treatment. In control cotyledons, IGF-II mRNA concentration increased (p < 0.01) and IGFBP-3 decreased between 55 and 90 dGA (p < 0.01). Cotyledon IGF-II and caruncle IGFBP-4 mRNA were elevated at 55 dGA in HT placentae compared with control (p < 0.01 and p < 0.05 respectively). Fetal hepatic IGF-I, IGFBP-2, -3 and -4 concentrations rose over gestation (p < 0.05), but there were no treatment effects. These data suggest that changes in placental IGF expression in early and mid gestation may predispose the pregnancy to placental insufficiency, resulting in inadequate substrate supply to the developing fetus later in gestation.

Biodiversity of multiple Pregnancy-Associated Glycoprotein (PAG) family: gene cloning and chorionic protein purification in domestic and wild eutherians (Placentalia) – a review

This review presents a broad overview of chorionic glycoproteins encoded by the Pregnancy-Associated Glycoprotein (PAG) gene family and also serves to illustrate how the recent discovery of the PAG family has contributed to our general knowledge of genome evolution, placental transcription and placental protein expression. The complex and large PAG family is restricted to the Artiodactyla order, although single PAG-like genes have also been identified in species outside the Artiodactyla. The PAGs are members of the aspartic proteinase (AP) superfamily. Unexpectedly, however, some members of the PAG family possess amino acid substitutions within and around the active site that likely render them unable to act as proteinases. This paper summarises the available information regarding biodiversity of PAG gene expression based on cDNA cloning, mRNA localisation studies and the structural organisation of the PAG genes with a particular emphasis on PAG promoters. It also compares available data regarding PAG protein purifications, sequencing and their N-glycodiversity. Finally, it discusses the scientific relevance, possible functional roles of the PAGs and describes possible profitable applications related to the detection of PAG proteins in the blood of pregnant domestic and wild species.

Down-regulation of placental transport of amino acids precedes the development of intrauterine growth restriction in rats fed a low protein diet

Intrauterine growth restriction (IUGR) represents an important risk factor for perinatal complications and for adult disease. IUGR is associated with a down-regulation of placental amino acid transporters; however, whether these changes are primary events directly contributing to IUGR or a secondary consequence is unknown. We investigated the time course of changes in placental and fetal growth, placental nutrient transport in vivo and the expression of placental nutrient transporters in pregnant rats subjected to protein malnutrition, a model for IUGR. Pregnant rats were given either a low protein (LP) diet (n = 64) or an isocaloric control diet (n = 66) throughout pregnancy. Maternal insulin, leptin and IGF-I levels decreased, whereas maternal amino acid concentrations increased moderately in response to the LP diet. Fetal and placental weights in the LP group were unaltered compared to control diet at gestational day (GD) 15, 18 and 19 but significantly reduced at GD 21. Placental system A transport activity was reduced at GD 19 and 21 in response to a low protein diet. Placental protein expression of SNAT2 was decreased at GD 21. In conclusion, placental amino acid transport is down-regulated prior to the development of IUGR, suggesting that these placental transport changes are a cause, rather than a consequence, of IUGR. Reduced maternal levels of insulin, leptin and IGF-1 may link maternal protein malnutrition to reduced fetal growth by down-regulation of key placental amino acid transporters.

Correlations of placental perfusion and PlGF protein expression in early human pregnancy

The purpose of this study was to investigate temporal correlations between maternal serum placenta growth factor levels and placental perfusion in early human pregnancies. Systolic umbilical artery Doppler blood flow velocity indices at fetal and placental insertion sites were measured between 7 and 22 weeks of gestation from normal singleton pregnancies. Maternal serum placenta growth factor levels were determined by enzyme-linked immunosorbent assay. Maternal serum placenta growth factor levels showed an exponential increase at approximately 14 weeks of gestation. Placenta perfusion, as estimated by systolic Doppler blood flow indices, significantly increased with gestational age (P < .0001). There was a close association between placenta growth factor expression levels and evidence of increased placenta perfusion (P < .033). The significant increase in serum placenta growth factor coincides with the increased perfusion of the maternal/fetal interface at approximately 12 to 14 weeks of gestation. Correlation of placenta growth factor expression and placental perfusion suggests that placenta growth factor may contribute to assuring adequate vascular development/function of the placenta early in gestation.

The endogenous anti-angiogenic family of splice variants of VEGF, VEGFxxxb, are down-regulated in pre-eclamptic placentae at term

PET (pre-eclamptic toxaemia) has recently been linked with alterations in production of a VEGFR1 [VEGF (vascular endothelial growth factor) receptor 1] splice variant that acts as a circulating inhibitor. We have recently described a family of naturally occurring splice variants of VEGF, termed VEGFxxxb, that also appear to act as inhibitors of conventional VEGFxxx-mediated angiogenesis. To determine whether alteration in splicing of VEGF-VEGFR family members extended beyond VEGFR1, we investigated the effect of pre-eclampsia on placental VEGFxxxb mRNA and protein expression. VEGFxxx and VEGFxxxb mRNA and protein were both found in normal human term placentae. VEGFxxx protein formed the majority of the total VEGF protein (980+/-195 pg/mg), whereas VEGFxxxb (11.5 pg/mg) was found to form a small part of the total VEGF protein expression (1.5+/-0.24%). Evidence for VEGF165b, VEGF121b and VEGF145b expression was found. In pre-eclamptic placentae, there was a significant down-regulation of VEGFxxxb isoforms, but a small up-regulation of VEGFxxx isoforms. In normal placenta VEGFxxxb and VEGFxxx concentrations were positively correlated (r=0.69, P<0.02), whereas in pre-eclamptic placentae, there was a significant negative correlation between VEGFxxxb and VEGFxxx protein expression (r=-0.8, P<0.02), indicating that there was a significant uncoupling of the splicing regulation of the VEGF isoforms. Combined with previous studies showing increased soluble VEGFR1 isoforms in human pre-eclampsia, these data suggest that there may be a common mechanism in pre-eclampsia that involves dysregulation of mRNA splicing of members of the VEGF-VEGFR axis.

51 PROTEOMICS ANALYSIS OF PLACENTOMEGALY IN CLONED MICE

A variety of mammalian species have been cloned during the past few years. However, the success rate of somatic cell nuclear transfer in animals has been extremely low with many problems. Particularly, placentomegaly is a frequent finding in cloned mice and cattle (Wakayma et al. 1999 PNAS USA 96, 14 984–14 989; Niemann et al. 2000 Theriogenology 53, 21–34). To assess protein expression profile in the placentomagaly of cloned mice produced by nuclear transfer of embryonic stem cells, we have used global proteomics approach by 2-D gel electrophoresis and mass-spectrometry with the differential protein patterns using the 3 placentae of cloned mice and 4 normal mouse placentae. Proteins within isoelectric point range pH 4.0~9.0 and molecular weight range of 20–100 kDa separately were analyzed by 2-D gel electrophoresis with 3 replications of each sample. A total of approximately 3500 spots were detected 2-D gel. In the comparison of normal and cloned placenta samples, a total of 49 protein spots were expressed differentially, of which 28 spots were up-regulated proteins including alpha-fetoprotein, aspartyl aminopeptidase, placental lactogen 2, tissue inhibitor of metalloproteinase 2 (TIMP-2), etc., and 21 spots were down-regulated proteins including peroxiredoxin 6, creatine kinase, pre-B-cell colony-enhancing factor 1 (PBEF), etc. Eight spots could not be identified. One of differentially up-regulated proteins in cloned mouse placenta was identified as TIMP-2 protein that is related to extracellular matrix degradation and tissue remodeling processes. Western blot was performed with placental sample used in the 2-D gel electrophoresis analysis and normal mouse placenta samples on Days 11.5 to 18.5. Indeed, Western blot analysis confirmed a significant increase of TIMP-2 protein level in cloned mouse placenta compared with normal. The expression levels of TIMP-2 in normal mouse placenta were highest at the normal mid gestation (Day 13.5) and exhibited prominent decrease at late gestation period during normal pregnancy. However, the expression levels of TIMP-2 in placenta of cloned mice appeared to be similar to levels of mid gestation normal mouse placenta. And one of down-regulated protein in NT placenta was identified as PBEF protein that is known to be related to induction of spontaneous labor. PBEF protein level in cloned mice placenta was revealed to decrease remarkably compared with normal. The expression levels of PBEF in normal mice placenta exhibited a gradual decrease between Day 11.5 and Day 18.5 without complete depletion. However, the expression levels of PBEF in placenta of cloned mice appeared to be markedly lower than those of the same day normal placenta. In conclusion, abnormal expression of placental proteins associated with tissue remodeling and labor induction may be the cases of placentomegaly in cloned mice.

Placental Deficiency of Interleukin-10 (IL-10) in Preeclampsia and its Relationship to an IL10 Promoter Polymorphism

The placenta is pivotal in the acceptance of the feto-placental unit by the maternal immune system. Imbalance at the maternal-fetal interface of tissue pro- and anti-inflammatory cytokines may be partly involved in disease causation. Previous work has shown conflicting levels of IL-10. IL-10 levels have been shown to increase, decrease, or remain unchanged in women with preeclampsia. This study examines the difference in serum and placental IL-10 expression in women with preeclampsia and investigates if the IL10 (-1082) A promoter polymorphism contributes to lower concentrations. In a prospective case-control study of 12 women with preeclampsia and 31 controls we assessed serum IL-10 by ELISA, placental mRNA by quantitative PCR and protein by immunohistochemistry as well as placental IL10 promoter genotype. Comparisons were made with non-parametric tests where necessary and chi-square. We found a significant reduction in placental IL-10 mRNA and protein expression in women with preeclampsia compared to controls. Women with the AA IL-10 promoter genotype expressed less placental IL-10 mRNA compared to women with AG or GG genotype. There was no difference in serum IL-10 concentrations between different genotypes. Preeclampsia is associated with a deficiency of placental IL-10. Placental AA genotype in the promoter region results in significantly less placental IL-10.

ERV3 and Related Sequences in Humans: Structure and RNA Expression

The ERV3 locus at chromosome 7q11 is a much studied human endogenous retroviral (HERV) sequence, owing to an env open reading frame (ORF) and placental RNA and protein expression. An analysis of the human genome demonstrated that ERV3 is one of a group of 41 highly related elements (ERV3-like HERVs) which use proline, isoleucine, or arginine tRNA in their primer binding sites. In addition to elements closely related to ERV3, the group included the previously known retinoic acid-inducible element, RRHERVI, also referred to as HERV15, but was separate from the related HERV-E elements. The ERV3-like elements are defective. The only element with an ORF among gag, pro, pol, and env genes was the env ORF of the original ERV3 locus. A search in dbEST revealed ERV3 RNA expression in placenta, skin, carcinoid tumor, and adrenal glands. Expression was also studied with newly developed real-time quantitative PCRs (QPCR) of ERV3 and HERV-E(4-1) env sequences. Results from a novel histone 3.3 RNA QPCR result served as the expression control. QPCR results for ERV3 were compatible with previously published results, with a stronger expression in adrenal gland and placenta than in 15 other human tissues. The expression of the envelope (env) of ERV3 at chromosome 7q11 was also studied by using stringent in situ hybridization. Expression was found in corpus luteum, testis, adrenal gland, Hassal's bodies in thymus, brown fat, pituitary gland, and epithelium of the lung. We conclude that ERV3 env is most strongly expressed in adrenal and sebaceous glands as well as in placenta.

DOCOSAHEXAENOIC ACID SUPPLEMENTATION DURING PREGNANCY MODULATES THE GENE EXPRESSION OF SOME FATTY ACID TRANSPORT PROTEINS IN HUMAN PLACENTA

Introduction: The placental transfer of fatty acids is a complex process mediated by specific carrier proteins from membranes and cytosol. Nevertheless, the underlying mechanisms for the preferential materno-fetal transfer of long-chain polyunsaturated fatty acids (LC-PUFA) are still poorly understood. The aim of this study is to evaluate the effects of nutritional supplements with docosahexaenoic acid (DHA) and folic acid on the placental mRNA expression of proteins that may regulate fatty acid transfer. Methods: We recruited 137 pregnant women from Granada (Spain) who received from the week 20 until delivery different nutritional supplements: placebo; MTHF supplemented with 400 ug folic acid; DHA supplemented with 500 mg DHA + 150 mg EPA; and DHA + MTHF (400ug folic acid, 500 mg DHA, 150 mg EPA ). Placental tissue was sampled at birth, fatty acid profile analyzed by gas-liquid chromatography, and mRNA expression of placental fatty acid transport proteins-1 (FATP-1) and-4 (FATP-4), heart- fatty acid binding protein (H-FABP) by real time-PCR. Results: No significant difference was found in the gene expression of fatty acid transport proteins FATP-1, FATP-4 or H-FABP among the 4 groups. There was a positive correlation of DHA % in placental phospholipids with mRNA expression of membrane proteins FATP-1 (R = +0.364, P = 0.001) and FATP-4 (R = +0.387, P < 0.001). Arachidonic acid/DHA ratio (AA/DHA) was inversely correlated with FATP-1 and FATP-4, respectively, in all placental lipid fractions except for cholesterol esters (phospholipids: R = −0.287, P = 0.007; R = −0.335, P = 0.002; NEFA: R = −0.213, P = 0.005; R = −0.235, P = 0.032; triglycerides: R= −0.310, P = 0.004; R = −0.407, P < 0.001). In contrast, the cytosolic protein H-FABP was not correlated with LC-PUFA. Conclusion: In uncomplicated pregnancies the mRNA expressions of membrane placental proteins FATP-1 and FATP-4 are related to placental accumulation of DHA and AA, respectively, whereas there is no correlation with expression of the cytosolic protein H-FABP.

The Effects of Chorioamnionitis and Betamethasone on 11β, Hydroxysteroid Dehydrogenase Types 1 and 2 and the Glucocorticoid Receptor in Preterm Human Placenta

Preterm birth is one of the major problems faced in perinatal medicine and is often associated with underlying clinical infection. Treatment with maternal betamethasone has helped to improve neonatal morbidity and mortality. We hypothesized that betamethasone treatment and chorioamnionitis would alter the bioavailability of placental glucocorticoids through the regulation of the 11beta hydroxysteroid dehydrogenase (11beta HSD) isozymes and the glucocorticoid receptor (GR). Placental samples were obtained from three groups of women who delivered prematurely: (1) those who delivered in the absence of infection, (2) those who received betamethasone treatment before delivering without infection, and (3) those who had pregnancies complicated with chorioamnionitis. Western blotting was used to determine 11beta HSD-1, 11beta HSD-2, GRT, and GRalpha expression, and 11beta HSD-2 activity was determined in each group. JEG-3 cells were used to study the regulation of the 11beta HSD isozymes. In cases of chorioamnionitis where mothers had not been treated with betamethasone, placental 32-kd 11beta HSD-1 protein expression was increased. In cases of chorioamnionitis regardless of betamethasone treatment, placental 11beta HSD-2 expression and activity was decreased compared to controls. In these placental samples, the expression of GRT and GRalpha did not change significantly. In JEG-3 cells, 11beta HSD-1 32-kd expression was increased with interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha), while 11beta HSD-2 expression was unaffected. These data suggest that there could be an increased fetal exposure to maternal glucocorticoids in cases of chorioamnionitis as a result of changes in the expression and activity of the placental 11beta HSD isozymes.

Chorionic mRNA expression and N-glycodiversity of pregnancy-associated glycoprotein family (PAG) of the European bison ( Bison bonasus)

Placental PAG mRNA expression and N-glycodiversity of multiple PAG proteins secreted in vitro by trophectoderm (chorion epithelium) of wild pecoran Bovidae taxons was not examined previously. The study on European bison (Eb) aimed: (1) to determine placental PAG mRNA expression by in situ hybridisation; (2) to identify a profile of pecoran PAG protein family secreted in vitro by cotyledonary (CT) explants; (3) to examine N-glycodiversity of the PAG proteins in this wild taxon. In addition, we compared (4) a profile and N-glycodiversity of the PAG protein family secreted in vitro by CT and interCT-trophectoderm (intCT-TRD) explants of domestic ruminants. Cotyledonary sections of the Eb were used for in situ hybridisation (ISH) with 35S-labelled probes produced with porcine PAG cDNA as templates. Various CT and intCT-TRD explants were long-term cultured in vitro. Chorionic proteins were isolated from media, ultra-filtrated (>10 kDa MWCO) and analysed by PAGE–Western blotting with various polyclonal anti-PAG sera. Protein samples with or without enzymatic deglycosylation were examined after different times of explant cultures. Released chorionic proteins were deglycosylated by N-glycanase F (PNGase F+) and compared to glycosylated forms (PNGase F−). This is the first paper demonstrating the PAG-like mRNA transcript expression (by ISH) and N-glycodiversity of immuno-reactive PAG-like proteins (produced in vitro by chorionic explants) of European bison. Various PAG proteins of Eb (EbPAG) were secreted by CT explants during long-term in vitro studies. Major ∼78, ∼67 and ∼65 kDa EbPAG-like proteins were reduced by enzymatic deglycosylation (at least by 10 kDa). Considerably smaller amounts of ∼45 kDa EbPAG-like proteins were also observed. In addition, we have found that various PAG proteins (30–73 kDa) were secreted by bovine CT explants, during long-term in vitro cultures. Corresponding amounts of PAG proteins, similar in M r, were also secreted by intCT-TRD explants, whose tissues were not utilised for PAG protein extraction during other scientists’ previous studies. It seems that the M r-heterogeneity and N-glycodiversity of the PAG protein family can play very important role during feto–placental interactions in Bovidae species.

45 ANALYSIS OF DIFFERENTIALLY EXPRESSED PROTEINS IN THE PLACENTA OF SOMATIC CELL CLONED AND ARTIFICIAL INSEMINATION PIG PLACENTA USING PROTEOMICS

Somatic cell cloning has been hampered by biological and technical problems. A major limitation of cloning procedures is the extreme inefficiency of producing healthy offspring for reasons such as sudden intrauterine unexplained death and infant death syndrome. In this study, we analyzed differentially expressed protein profiles in the placenta of somatic cell cloned (SCNT) and artificial insemination (AI; control) pigs by proteomics. Protein expression pattern of placentas was established in the pH range 4–7, IEF cell system, and 7.5–17.5% gradient 2-dimensional polyacrylamide gel electrophoresis. Image analysis following silver staining used the spot and PDQuest analysis system. Peptide mass fingerprinting with matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and SWISS-PROT database search were utilized to identify proteins. Around 1000 protein spots were recognized by image analysis on each placenta. In SCNT pig placentas, 37 spots were changed compared with those of AI pig placentas. Among them 11 proteins such as serum albumin precursor and annexin A5 showed an increased protein expression level whereas the expression level of 26 other proteins such as aldose reductase and tropomyosin decreased. Annexin A5 and aldose reductase are implicated in the apoptosis process: the former is an apoptotic marker of oxidative stress; on the other hand, the latter is a critical regulator of TNF-α-induced apoptotic signaling in endothelial cells. Also, tropomyosin is implicated in stabilizing cytoskeleton actin filaments. The expression of these proteins was confirmed by western blot and real-time PCR analysis. These results suggest expression of abnormal placental protein and apoptotic-related protein in SCNT pig placentas affects the regulation of placental growth and development. This work was supported in part by a grant program from RDA(Biogreen21) and Cho-A, Republic of Korea.

Influence of gestational age and fetal iron status on IRP activity and iron transporter protein expression in third-trimester human placenta.

Placental iron transport during the last trimester of pregnancy determines the iron endowment of the neonate. Iron transport is a function of the major iron transport proteins: transferrin receptor-1 (TfR-1) and ferroportin-1 (FPN-1). The mRNAs for TfR-1 and, potentially, FPN-1 are posttranscriptionally regulated by iron regulatory protein (IRP)-1 and IRP-2. We assessed the effect of gestational age and fetal iron status on IRP-1- and IRP-2-binding activity and on the localization and protein expression of TfR-1 and FPN-1 protein at 24-40 wk of gestation in 21 placentas obtained from iron-sufficient nonanemic mothers. Gestational age had no effect on cord serum ferritin concentration, IRP-2 RNA-binding activity, transporter protein location, and TfR-1 or FPN-1 protein expression. IRP-1 activity remained constant until full term, when it decreased (P = 0.01). Placental ferritin (r = 0.76, P < 0.001) and FPN-1 (r = 0.44, P < 0.05) expression increased with gestational age. Fetal iron status, as indexed by cord serum ferritin concentration, was inversely related to placental IRP-1 (r = -0.66, P < 0.001) and IRP-2 (r = -0.42, P = 0.05) activities. Placental ferritin protein expression correlated better with IRP-1 (r = -0.45, P = 0.04) than with IRP-2 (r = -0.35, P = 0.10) activity. Placental TfR-1 and FPN-1 protein expression was independent of fetal or placental iron status and IRP activities. Iron status had no effect on transport protein localization. We conclude that, toward the end of the third trimester of iron-sufficient human pregnancy, the placenta accumulates ferritin and potentially increases placental-fetal iron delivery through increased FPN-1 expression. IRP-1 may have a more dominant role than IRP-2 activity in regulating ferritin expression.

PC63 EXPRESSION PATTERN OF FATTY ACID TRANSPORT PROTEIN-1 (FATP-1), -4 (FATP-4), AND HEART-FATTY ACID BINDING PROTEIN (H-FABP) GENES IN HUMAN TERM PLACENTA

Introduction: Placental fatty acid transfer is critical to meet the fetal requirements for biosynthesis of biological membranes, myelin and various signaling molecules. Increasing evidence suggests that the direction and magnitude of fatty acid flux is mainly determined by the availability of transfer proteins (Dutta-Roy, 2000). However, the relative expression of these proteins in human placental tissue is not known. The aim of this study was to elucidate the human placental mRNA expression of proteins that may regulate placental fatty acid transfer. Methods: We collected placental tissue from five pregnant women undergoing caesarean section at 38+/−1.1 weeks of gestation and from five different placenta locations. We quantified the fatty acid composition at the individual locations by gas-liquid chromatography (Klingler et al., 2003) and mRNA expression of placental fatty acid transport proteins-1 (FATP-1) and −4 (FATP-4), heart-fatty acid binding protein (H-FABP) and liver-FABP (L-FABP) by real time-PCR. The mRNA expression of these proteins was expressed relative to that of the house keeping gene beta-2-microglobulin. Results: The mRNA expression of plasma membrane proteins FATP-1 and FATP-4 (39.5+/−6.4a, and 16.4+/−2.8b) was significantly higher than that of the cytosolic proteins H-FABP (9.3+/−0.7c) and L-FABP. The expression of L-FABP was too low in placenta for accurate quantification. No significant differences were detected in the expression of FATP-1, FATP-4 and H-FABP between the different placental locations. Conclusion: Our results indicate the feasibility of reproducible quantification of placental mRNA expression of some fatty acid transport proteins and provide first results on their expression in human placentas at term. Although a higher expression of membrane bound transport proteins than of cytosolic transport proteins was demonstrated, further studies are needed to establish the role of the individual placental proteins in the fetal accretion of fatty acids and their response to maternal diet and disease.

O0077 DIETARY POLYUNSATURATED FATTY ACIDS AFFECT THE CELL CYCLE AND INHIBIT APOPTOSIS IN THE RAT PLACENTA

Introduction: Maternal nutrition is one of the determining factors for the intrauterine environment, which plays an important role in fetal and placental development. Among these dietary fatty acids, especially the long-chain polyunsaturated fatty acids, are essential. Whereas the effects on fetal development of changes in the availability of lipid components, like those produced by changes in dietary fatty acids, are widely established, little is known about their influence on placental development and function. This study investigated the effects of dietary fatty acids on placental development in rat. Methods: Sprague-Dawley rats were fed diets containing 10% (wt/wt) of palm- (saturated), sunflower- (18:2), olive- (18:1) or fish (w-3) oil. At day 20 of gestation, placental fatty acid composition and protein expression levels of the cell cycle regulating proteins PCNA (S-phase), p21 (inhibitor of G2M & G1S transition), cyclin E (G1-phase), p53 (masterswitch for apoptosis) and PARP (split 89 & unsplit 116 kDa) were determined. The proteins were also immunolocalized and the proportion of labelled cells was counted. Results: Litter size, placental weight and gross morphology were unaltered by the diet. Placental fatty acid composition in the phospholipid and triglyceride fractions reflected the diet composition except for saturated fatty acids. PCNA levels do not differ between the groups. The higher proportion of labyrinthine-trophoblasts (fetal side) in G1 (cyclin E) in the fish oil group was associated with low p21, but unaltered PCNA levels, suggesting p21-independent control of the G1/S transition. p21 absence in spongiotrophoblasts (maternal side) demonstrates that cell cycle must be regulated by other inhibitors. The higher p53 levels in sunflower and olive oil groups were associated with a higher level of apoptosis, low levels of apoptosis in the fish oil group were associated with low p53 levels. This contrasts the palm oil group, in which a high level of apoptosis was found despite relatively lower levels of p53, suggesting other p53 independent control mechanisms to account for this effect. Conclusion: Dietary fatty acids do not have gross effects on reproductive performance and placental development. However, subtle changes of the cell cycle and apoptosis do occur, which are most pronounced when the oils contain long-chain polyunsaturatd fatty acids. These appear to protect the placenta from apoptosis.

Human placental taurine transporter in uncomplicated and IUGR pregnancies: cellular localization, protein expression, and regulation.

Transplacental transfer is the fetus' primary source of taurine, an essential amino acid during fetal life. In intrauterine growth restriction (IUGR), placental transport capacity of taurine is reduced and fetal taurine levels are decreased. We characterized the protein expression of the taurine transporter (TAUT) in human placenta using immunocytochemistry and Western blotting, tested the hypothesis that placental protein expression of TAUT is reduced in IUGR, and investigated TAUT regulation by measuring the Na(+)-dependent taurine uptake in primary villous fragments after 1 h of incubation with different effectors. TAUT was primarily localized in the syncytiotrophoblast microvillous plasma membrane (MVM). TAUT was detected as a single 70-kDa band, and MVM TAUT expression was unaltered in IUGR. The PKC activator PMA and the nitric oxide (NO) donor 3-morpholinosydnonimine decreased TAUT activity (P < 0.05, n = 7-15). However, none of the tested hormones, e.g., leptin and growth hormone, altered TAUT activity significantly. PKC activity measured in MVM from control and IUGR placentas was not different. In conclusion, syncytiotrophoblast TAUT is strongly polarized to the maternal-facing plasma membrane. MVM TAUT expression is unaltered in IUGR, suggesting that the reduced MVM taurine transport in IUGR is due to changes in transporter activity. NO release downregulates placental TAUT activity, and it has previously been shown that IUGR is associated with increased fetoplacental NO levels. NO may therefore play an important role in downregulating MVM TAUT activity in IUGR.