Essential role of the ERK/MAPK pathway in blood-placental barrier formation.

The mammalian genome contains two ERK/MAP kinase kinase genes, Map2k1 and Map2k2, which encode dual-specificity kinases responsible for ERK activation. Loss of Map2k1 function in mouse causes embryonic lethality due to placental defects, whereas Map2k2 mutants have a normal lifespan. The majority of Map2k1(+/-) Map2k2(+/-) embryos die during gestation from the underdevelopment of the placenta labyrinth, demonstrating that both kinases are involved in placenta formation. Map2k1(+/-) Map2k2(+/-) mutants show reduced vascularization of the labyrinth and defective formation of syncytiotrophoblast layer II (SynT-II) leading to the accumulation of multinucleated trophoblast giant cells (MTGs). To define the cell type-specific contribution of the ERK/MAPK pathway to placenta development, we performed deletions of Map2k1 function in different Map2k1 Map2k2 allelic backgrounds. Loss of MAP kinase kinase activity in pericytes or in allantois-derived tissues worsens the MTG phenotype. These results define the contribution of the ERK/MAPK pathway in specific embryonic and extraembryonic cell populations for normal placentation. Our data also indicate that MTGs could result from the aberrant fusion of SynT-I and -II. Using mouse genetics, we demonstrate that the normal development of SynT-I into a thin layer of multinucleated cells depends on the presence of SynT-II. Lastly, the combined mutations of Map2k1 and Map2k2 alter the expression of several genes involved in cell fate specification, cell fusion and cell polarity. Thus, appropriate ERK/MAPK signaling in defined cell types is required for the proper growth, differentiation and morphogenesis of the placenta.

Enzymatically active zinc, copper and mercury derivatives of the one-iron form of pig allantoic fluid acid phosphatase.

Derivatives of the violet, iron-containing acid phosphatase of pig allantoic fluid have been prepared in which one of the two iron atoms present in the native enzyme has been replaced by zinc, copper or mercury. The derivatives so formed are enzymatically active: the Zn-Fe, Cu-Fe and Hg-Fe enzymes have specific activities of about 80%, 25% and 17% respectively, of the maximum specific activity of the Fe-Fe enzyme in the standard assay at pH 4.9 with p-nitrophenyl phosphate as substrate. In contrast to the Fe-Fe enzyme, the mixed metal derivatives are not rapidly inactivated by H2O2. Visible absorption spectra of the derivatives confirm that all of the visible absorption of the Fe-Fe enzyme is due to one of the iron atoms. Attempts to prepare an active Cu-Cu enzyme were unsuccessful.

Mössbauer and EPR study of the binuclear iron centre in purple acid phosphatase.

Mössbauer spectra have been determined on 57Fe-enriched samples of both pink (reduced) and purple (oxidized) forms of pig allantoic acid phosphatase (EC 3.1.3.2), and EPR spectra on corresponding unenriched samples. The spectra show unambiguously that both forms of the enzyme contain two distinct, antiferromagnetically coupled, high-spin iron atoms: a ferrous-ferric ion pair in the pink, reduced form, and a pair of ferric ions in the purple, oxidized form.

Purification and characterization of particles containing RNA-dependent DNA polymerase, in the allantoic fluid of uninfected leukosis virus-free chicken eggs.

The allantoic fluid of embryonated chicken eggs regularly contains particle-associated RNA-dependent DNA polymerase, as shown by its reaction with homopolymeric and heteropolymeric RNA and by the characterization of the products. The purification of the particles is described. The purified particles are different from the known avian RNA tumor viruses in their protein composition and their sedimentation constant. They do not exhibit biological properties typical for RNA tumour viruses, such as helper activity, interfering properties or infectivity and do not show endogenous DNA synthesis. The particles are discussed as non-viral elements.

Expression of CYP4A isoforms in developing rat placental tissue and rat trophoblastic cell models.

Maintaining fatty acid homeostasis during pregnancy is critical for normal fetal development. As an organ that controls nutrient supply from the mother to the fetus, the placenta plays a significant role in guiding fatty acid transfer to the developing fetus. The cytochrome P450 4A (CYP4A) subfamily of metabolizing enzymes is a group of structurally and functionally conserved proteins that are specialized in the omega/omega-1 hydroxylation of saturated and unsaturated fatty acids and their derivatives. To understand the function of the CYP4A system in the placenta and its significance in maintaining fetal fatty acid homeostasis, information about the placental expression of individual CYP4A isoforms is required. In the present study, we have elucidated the temporal and spatial patterns of expression of the four known rat CYP4A isoforms (CYP4A1, CYP4A2, CYP4A3, and CYP4A8) in the junctional and labyrinthine zones of the developing rat chorioallantoic placenta as well as two rat trophoblastic cell lines, HRP-1 and Rcho-1, using semi-quantitative RT-PCR and immunohistochemical analyses. The mRNA from the four rat CYP4A isoforms was detected in the developing rat placenta with CYP4A1 exhibiting the strongest expression (4A1 > 4A2 >> 4A3 approximately equal to 4A8). CYP4A1 was also detected by immunohistochemical staining in the developing rat placenta. We also observed CYP4A1 in both HRP-1 and Rcho-1 cells by RT-PCR, suggesting the utility of these cells as in vitro tools to study the effects of xenobiotics on placental fatty acid metabolism. Establishing the expression of CYP4A isoforms in these tissues and cell models provides a framework for further investigation of their functional and physiological significance in guiding proper fetal development.

Induction of prostaglandin endoperoxide synthase isoform-2 in ovine cotyledonary tissues during late gestation.

The present study was designed to characterize the developmental pattern of specific prostaglandin endoperoxide synthase (PGS) isoform immunoreactivity and activity in tissues of fetal origin during the last half of gestation in sheep. Fetal amnion, allantochorion, and cotyledons were collected under halothane general anesthesia on days 79-80, 105-108, 120-122, 128-131, and 140-145 (term) of pregnancy. Solubilized extracts were prepared and analyzed by immunoblots using anti-PGS antibodies previously shown to recognize PGS isoform-1 and -2 (PGS-1 and PGS-2). PGS activity from cotyledon microsomes was assayed by the measurement of prostaglandin E2 (PGE2) production under initial velocity conditions. All fetal tissues contained PGS-1 at each of the stages of gestation examined, with minimal regulation of this isoform from 79-144 days gestation. In contrast, PGS-2 increased gradually in the cotyledons from 120-139 days gestation, with the most marked expression observed at term. PGS-2 was not detected in amnion or allantochorion. PGS activity in cotyledons increased (P < 0.01) in parallel with immunoreactive PGS-2 levels; indicating that PGS-2, rather than PGS-1, is associated with increased PG synthesis in this tissue. Both the activity (n = 5/group) and the amount of PGS-2 increased significantly from 105-108 days gestation to term (P < 0.01). We conclude that the increase in PGS that occurs at term in sheep is predominantly active PGS-2 localized to fetal cotyledons.

Primary structure of the virus activating protease from chick embryo. Its identity with the blood clotting factor Xa.

Host cell proteases activating para- and orthomyxovirus fusion glycoprotein precursors play a crucial role in determining the viral tropism in infected organisms. We previously isolated such an endoprotease from the allantoic fluid of chick embryo and showed its close similarity to the activated form of blood clotting factor X (FXa) by partial amino acid sequencing. In this report, we have cloned and sequenced a cDNA of the protease, and show that it is encoded in a single gene as a preproform with all the functional and structural domains known to be characteristic of bovine or human FX, establishing the identity between the protease and FXa.

Ultrastructural localization of carbonic anhydrase in the chorioallantoic membrane by immunocytochemistry.

Carbonic anhydrase was localized in the chick embryo chorionic ectoderm at the ultrastructural level by immuno-cytochemistry. Preembedding staining of whole tissue was performed. The enzyme was present in the cytoplasm, on the membranes of apical vesicles, and on the membranes of microvilli in villus cavity cells, cells that may be involved in acid secretion and subsequent dissolution of the egg shell. In sinus covering cells, the enzyme is solely in the cytoplasm. The location of the enzyme in the thin cytoplasmic arms of the sinus covering cells is consistent with the role in nonrespiratory CO2 release.

Vascular endothelial growth factor increases heme oxygenase-1 protein expression in the chick embryo chorioallantoic membrane.

(1) Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. It has been recently suggested that the inducible heme oxygenase (HO-1) isoform may play a role in angiogenesis. (2) The aims of this study were to determine, in chicken embryo chorioallantoic membranes (CAM), whether VEGF increases HO-1 protein expression, and, if so, by which molecular mechanism, and whether HO-1 activity is required for VEGF-induced angiogenesis. (3) Treatment of CAMs with VEGF for 48 h caused a significant increase in HO-1 protein expression, simultaneously with angiogenesis. (4) VEGF-stimulated angiogenesis in CAMs was markedly attenuated by the HO inhibitor zinc mesoporphyrin (ZnMP). This inhibitory effect of ZnMP was not observed with copper mesoporphyrin (CuMP), a metalloporphyrin that has a similar structure to ZnMP but does not inhibit HO enzymatic activity. (5) Overexpression of HO-1 protein elicited by VEGF in CAMs was significantly attenuated by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). The effects of BAPTA-AM were, in turn, compensated by the calcium ionophore A-23187. (6) In addition, the protein kinase C inhibitor staurosporine significantly attenuated, in a dose-dependent manner, the VEGF-stimulated HO-1 induction observed in CAMs. (7) These results demonstrate, for the first time, that VEGF upregulates HO-1 protein expression in vivo in CAMs by a mechanism dependent on an increase in cytosolic calcium levels and activation of protein kinase C. Our findings also suggest that HO-1 activity is necessary for VEGF-induced angiogenesis in CAMs.

Nitric oxide synthase expression, enzyme activity and NO production during angiogenesis in the chick chorioallantoic membrane.

In order to elucidate further the role of nitric oxide (NO) as an endogenous antiangiogenic mediator, mRNA expression of inducible nitric oxide synthase (iNOS), enzyme activity and production of NO were determined in the chick chorioallantoic membrane (CAM), an in vivo model of angiogenesis. In this model, maximum angiogenesis is reached between days 9 - 12 of chick embryo development. After that period, vascular density remains constant. Inducible NO synthase (iNOS) mRNA expression, determined by reverse transcriptase polymerase chain reaction (RT - PCR), increased from the 8th day reaching a maximum (70% increase) at days 10 - 11. NO synthase activity, determined as citrulline formation in the presence of calcium, also increased from day 8 reaching a maximum around day 10 (100% increase). Similar results were obtained in the absence of calcium suggesting that the NOS determined was the inducible form. Nitric oxide production, determined as nitrites, increased from day 8 reaching a maximum around day 10 (64% increase) and remaining stable at day 13. Finally, the bacterial lipopolysaccharide LPS (which activates transcriptionally iNOS), inhibited dose dependently angiogenesis in the CAM. These results in connection with previous findings from this laboratory, showing that NO inhibits angiogenesis in the CAM, suggest that increases in iNOS expression, enzyme activity and NO production closely parallel the progression of angiogenesis in the CAM, thus providing an endogenous brake to control this process. British Journal of Pharmacology (2000) 129, 207 - 213

15-Hydroxy-prostaglandin dehydrogenase activity in uterine tissues of late gestational and parturient cows.

15-Hydroxy-prostaglandin dehydrogenase activity (PGDH) was measured in endometrium, allanto-chorion, allanto-amnion and the placenta from six cows during late gestation and six parturient cows during caesarean section. Under saturated substrate conditions PGDH activity was lower than 138 pmol g wet tissue-1 min-1 in all uterine tissues in both groups. Human placental reference samples showed a PGDH activity of 16,800 +/- 1200 pmol per g wet weight min-1 (n = 4). Under subsaturational conditions enzyme activity was demonstrated in endometrium and both fetal membranes. These results indicate that PGF2 alpha catabolism in bovine uterine tissue on day 262 and during parturition, contributes little to changing plasma PGFM concentrations.

Studies into aromatase activity associated with fetal allantochorionic and maternal endometrial tissues of equine placenta. Identification of metabolites by gas chromatography mass spectrometry.

Maternal endometrial and fetal allantochorionic tissues were separated manually from the placentae of seven healthy thoroughbred and three pony mares, ranging in gestational age from 100 to 318 days. The homogeneity of subcellular fractions prepared from these tissues was assessed initially using the marker enzymes, succinate dehydrogenase, NADPH cytochrome C reductase and lactate dehydrogenase for the mitochondrial, microsomal and cytosolic fractions, respectively. Light microscopy and histochemical analysis demonstrated that the separated fetal allantochorionic membrane, which is made up of allantoic and chorionic epithelia, contained no significant contamination of maternal tissues. The maternal endometrium, however, was found to contain appreciable amounts of fetal chorion torn off during the separation process. Tissue homogenates and subcellular fractions were incubated with testosterone together with [4-(14)C] and [(2)H5 or (2)H3] labelled analogues in either an NADPH (1 mM) or a NADPH-regenerating environment; control experiments (without additional cofactor) were also performed. After extraction of the tissue homogenates, neutral and phenolic (oestrogen) unconjugated steroids were separated by column chromatography. Radiolabelled studies revealed that in allantochorionic tissue incubations 67-77% of testosterone was converted to oestrogenic material, subcellular fractionation indicating that oestrogen production was largely confined to the microsomal fraction and time-course studies showing that the rate of formation appeared to be linear up to 90 min. In contrast, only 5-25% conversion occurred using maternal endometrial tissues, which could be accounted for by the contaminating presence of fetal chorion. No oestrogen production was detected in control incubations. These radiolabelled studies demonstrate that aromatase activity is located on the fetal allantochorionic surface and, together with the histochemical data, further delineate this activity to the chorion in mature equine placenta. Gas chromatographic-mass spectrometric (GC-MS) analysis of the phenolic extracts from allantochorionic tissue homogenate incubations indicated the presence of substrate-derived oestradiol-17beta (E2), 6-oxo-oestradiol-17beta (6-oxo-E2) and 6beta-hydroxyoestradiol-17beta (6beta-OH-E2). Whereas all three oestrogens were identified as metabolites from testosterone in incubations performed using allantochorionic tissue homogenates and post-mitochondrial suspensions (PMS), only E2 was identified from incubations performed using microsomal fractions prepared from this tissue. We conclude that both the microsomal and cytosol fractions are required for the conversion of E2 to the 6-oxygenated species in vitro. Using stable isotope-labelled substrates and GC-MS analysis the mechanism of formation of these metabolites from these in vitro incubation studies may be inferred. GC-MS analysis of the neutral extracts from allantochorionic tissue homogenate incubations confirmed the presence of small quantities of substrate-derived 5(10)-oestrenediols. No substrate-derived 5(10)-oestrene-3,17-diols were detected in extracts from microsomal preparations incubated in the absence of cytosol. These data suggest that demethylation of C19 steroids to produce C18 neutral steroids may require the synergistic action of enzymic activities that appear to reside both in the microsomal and cytosolic fractions of equine allantochorionic tissues.

Antioxidants inhibit angiogenesis in vivo through down-regulation of nitric oxide synthase expression and activity.

Although reactive oxygen species (ROS) participate in many cellular mechanisms, only few data exist concerning their involvement in physiological angiogenesis. The aim of the present work was to elucidate possible mechanisms through which ROS affect angiogenesis in vivo, using the model of the chicken embryo chorioallantoic membrane (CAM). Superoxide dismutase (SOD) and its membrane permeable mimetic tempol, dose dependently decreased angiogenesis and down-regulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. The NADPH oxidase inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and apocynin, but not allopurinol, also had a dose dependent inhibitory effect on angiogenesis and NO production in vivo. Catalase and the intracellular hydrogen peroxide (H2O2) scavenger sodium pyruvate decreased, while H2O2 increased in a dose-dependent manner the number of CAM blood vessels, as well as the expression and activity of iNOS. Dexamethasone, which down-regulated NO production by iNOS and L-NAME, but not D-NAME, dose dependently decreased angiogenesis in vivo. These data suggest that antioxidants affect physiological angiogenesis in vivo, through regulation of NOS expression and activity.

Amifostine protects blood vessels from the effects of ionizing radiation.

Amifostine (WR-2721) is a well-known radioprotective drug, selective for normal cells. The purpose of the present study was to define whether amifostine protects the vascular network from the effects of X-rays. We used the in vivo system of chicken embryo chorioallantoic membrane (CAM) as a model of angiogenesis. Amifostine reversed the early X-rays- induced decrease in the number of CAM blood vessels and reversed the early radiation-induced apoptosis of CAM cells. It also inhibited the increase in tyrosine nitration of actin and a-tubulin, which was observed 6 hours after CAM irradiation, when there was a significant decrease in non-protein SH groups. Furthermore, C6 rat glioma cells were inoculated on CAM and tumor growth, as well as tumor-induced angiogenesis, was estimated on haematoxylin-eosin-stained paraffin sections. Amifostine inhibited the post irradiation increase of C6 tumor-induced angiogenesis. These data suggest that amifostine protects CAM cells and blood vessels from the effects of X-rays, through mechanisms that do not depend solely on its free radical scavenging properties.

Autoradiographic localization of carbonic anhydrase in the developing chorioallantoic membrane.

Carbonic anhydrase was localized in the chorioallantoic membrane with labeled inhibitor autoradiography of 3H-acetazolamide at 11, 14 and 19 days of incubation. At 11 days carbonic anhydrase was present in low amounts only in the undifferentiated ectoderm cells. At 14 and 19 days, the enzyme was found in increased amounts in all three germ layers of the chorioallantois. In the chorionic ectoderm the villous cavity cells contained the highest level of carbonic anhydrase. This finding lends support to the theory of H+ production to solubilize the CaCO3 of the egg shell. Sinus covering cells showed a considerable lower concentration of the enzyme than did villous cavity cells. Carbonic anhydrase in these cells may be multifunctional, assisting in calcium transport, subserving HCO3- transport from egg shell to blood, and supporting gaseous exchange. In the allantoic endoderm carbonic anhydrase was found in granule-rich cells and might be involved in the transport of Na+ and Cl- ions from allantoic fluid into the blood. The enzyme in the undifferentiated endoderm cells may have a respiratory function. In the mesoderm carbonic anhydrase was detected in the endothelium and pericytes of blood vessels where it is interpreted to support respiratory function.

Effects of influenza virus replication on neutral proteolytic activities in embryonated egg fluids.

The levels of neutral protease activity associated with allantoic and amniotic fluids of embryonated eggs during the replication of influenza strains A/PR/8/34 (H1N1) and A/turkey/Ontario/7732/66 (H5N9) were investigated. A sensitive fluorometric technique proved useful for characterization and monitoring changes of protease activities in egg fluids. The predominant type of protease in allantoic and amniotic fluids had trypsin-like specificities. Variation in protease levels of both fluids occurred throughout the course of virus replication irrespective of the virus strain or the route of inoculation used. Concomitant with the production of high levels of infectious virus there was a marked decrease in neutral protease activity in the fluid from the cavity initially infected. Translocation of virus also occurred especially with amniotically infected eggs, as evidenced by high infectious virus titers and decreased protease activities in allantoic fluids.

Allantoic fluid protease activity during influenza virus infection.

Neutral protease activity of allantoic fluid from embryonated chicken eggs was quantified during the course of influenza virus infection. Antigenic subtypes of influenza A viruses selected for study were H1N1 strains PR/8/34, Brazil/8/78, FM/1/47, the H3N2 strain Bangkok/1/80 and the H5N9 Turkey/ /Ontario/66 as well as the Sendai strain of parainfluenza type 1 virus. Three different types of profiles of allantoic fluid proteases could be readily distinguished after infection of eggs with various virus strains. In all profiles, periodic peaks of protease activity always preceded the partial shut down of protamine cleaving proteases which paralleled the production of near maximum titers of infectious virus. To determine the mechanism involved in this reduction of proteolytic activity, infectious allantoic fluids were analysed for the presence of protease inhibitors. Acid heat treated 48 hour virus-infected allantoic fluids of different influenza strains could inhibit the activities of subtilisin and allantoic fluid proteolytic enzymes.

[Sequential changes of extracellular matrix metalloproteins in pregnancy and labor in the rat chorio-allantois]. / Cambios secuenciales de metaloproteinasas de matriz extracelular durante la gestación y el trabajo de parto en el corioalantoides de la rata.

Participation of matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) in the rupture of fetal membranes (FM) during labor has been proposed. We describe and characterize sequentially the activity of the enzymes that degrade connective tissue in the FM of the rat during pregnancy and labor. Pregnant Wistar rats were sacrificed on different days of gestation and samples of amniotic fluid (AF) and MCA were collected to be analyzed for proteolytic activity, zymography, western blot and northern blot. Results showed that during labor, there is a significant increase in the proteolytic activity in the MCA and in the AF. MMP-2 was identified from day 15 of pregnancy and it increased near labor. MMP-9 was only identified two days before labor. MMP-2 and MMP-9 mRNA expression were coincident with enzymatic activity and western blot data. During labor, TIMP-1 decreased and TIMP-2 did not change. These results allow us to conclude that there exists a quantitative and qualitative differential expression of MMPs during gestation, especially during labor.

[Delivery of secreted placental alkaline phosphatase (SEAP) gene in vitro and in vivo as a component of recombinant avian adenovirus (CELO)]. / Dostavka in vitro i in vivo gena sekretiruemoi platsentarnoi shchelochnoi fosfatazy (SEAP) v sostave rekombinantnogo adenovirusa ptits (CELO).

Recombinant adenoviruses capable of expressing the gene of secreted placentary alkaline phosphatase (SEAP) under control of CMV-promoter was obtained on the basis of CELO avian adenovirus and human adenovirus-5 (Ad5) genomes. The efficiency of the CELO vector was determined in experiments with transduction of human (293, A549, and H1299), mouse (B16), and avian (LMH) cell cultures. It was shown in C57BL/6 mice in vivo that SEAP gene is expressed under conditions of intravenous, intranasal, and intratumoral application of recombinant adenovirus CELO-SEAP. The duration of expression of the alkaline phosphatase CELO = SEAP gene in immunocompetent mouse body was 21 days. The level of SEAP gene expression was measured in the allantois fluid of chicken embryo infected with recombinant adenovirus CELO-SEAP.