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.

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.

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.

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.

[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.

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.

Specialised cell types in the chorioallantoic membrane express carbonic anhydrase during chick embryogenesis.

The expression of carbonic anhydrase in the chorioallantoic membrane (CAM) of the chick embryo was investigated by means of the histochemical localisation of the enzyme catalytic sites and the immunohistochemical identification of its isoenzymatic forms. The results show that carbonic anhydrase is developmentally expressed in a subset of cells both in the ectodermal and the endodermal epithelium. The distribution patterns from both methodological approaches indicated that carbonic anhydrase is a marker of the villus cavity cells and the mitochondria-rich cells in the ectodermal and the endodermal epithelium, respectively. Such a cell-specific pattern of the enzyme expression provides a further contribution to characterising the heterogeneous cell population of the chick CAM and supports specific functional involvement for the distinct cell types in CAM-mediated processes, such as calcium transport, maintenance of acid-base balance and water and electrolyte reabsorption, during chick embryogenesis.

CYP1A-dependent activation of xenobiotics in endothelial linings of the chorioallantoic membrane (CAM) in birds.

Metabolic activation of the heterocyclic amine 3-amino -1,4-dimethyl-5 H-pyrido[4,3-b]indole (Trp-P-1) and 7-ethoxyresorufin O-deethylase (EROD) activity were examined in the chorioallantoic membrane (CAM) of 15-day-old chicken and 18-day-old eider duck embryos. The embryos were pretreated with an Ah receptor agonist, i.e. beta-naphthoflavone (BNF) or 3,3',4,4',5-pentachlorobiphenyl (PCB 126), or vehicle in ovo. BNF and PCB 126 induced EROD activity and covalent binding of [3H]Trp-P-1 seven- to tenfold in the CAM of chicken embryos. In the CAM of eider duck embryos, which are known to be nonresponsive to coplanar PCBs, PCB 126 treatment had no effect on EROD activity or covalent binding of [3H]Trp-P-1 whereas BNF treatment increased these activities five- and threefold, respectively. Light microscopic autoradiography was used to identify the cellular localization of covalent binding of [3H]Trp-P-1 in the CAM. Preferential binding was observed in endothelial cells in intraepithelial capillaries in the chorionic epithelium and in blood vessels in the mesenchymal layer. The addition of the CYP1A inhibitor ellipticine abolished the covalent binding of [3H]Trp-P-1 in the CAM of BNF- and PCB 126-treated chicken and eider duck embryos. The results suggest that CYP1A-dependent metabolic activity can be induced in blood vessel endothelia in the CAM of bird embryos following exposure to Ah receptor agonists and that the CAM may be a target tissue for CYP1A-activated environmental pollutants. Furthermore, the highly vascularized CAM could be used as a model for studies of Ah receptor-mediated alterations in the vasculature.

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

Effects of thrombin and of the phospholipase C inhibitor, D609, on the vascularity of the chick chorioallantoic membrane.

Microvascular corrosion casting was used to assess the effects of thrombin and D609, a phospholipase C inhibitor, on the vascularity of the chick embryo chorioallantoic membrane (CAM). Discs containing vehicle, thrombin or D609 were placed on the CAM of fertilized white Leghorn eggs on Day 9 of gestation and vascularity was assessed on Day 11. Thrombin caused significant increases in the numbers (43%), diameters (5%) and lengths (17%), of both pre- and postcapillaries (first-order vessels by centripetal ordering). Conversely, D609 caused a decrease in the numbers (27%), lengths (12%) and diameters (8%) of first-order vessels. D609 decreased the total vascular volume of first- to third-order vessels by 32%, whereas thrombin increased vascular volume by 27%. Additionally, thrombin increased capillary plexus density by 6%, whereas D609 decreased capillary plexus density by 3%. These findings provide a quantitative assessment of changing vascularity in the chick CAM--a model assay system in the development of pro- and antiangiogenic agents.

Crystallization and preliminary X-ray diffraction studies of mammalian purple acid phosphatase.

The oxidized form of purple acid phosphatase from pig allantoic fluid has been crystallized in the presence of phosphate using the hanging-drop technique. The crystals belong to the space group P2(1)2(1)2(1) and have unit-cell parameters a = 66.8, b = 70.3, c = 78.7 A. Diffraction data collected from a cryocooled crystal using a conventional X-ray source extend to 1.55 A resolution. A knowledge of the three-dimensional structure of mammalian purple acid phosphatase will aid in understanding the substrate specificity of the enzyme and will be important in the rational design of inhibitors, with potential in the treatment of bone diseases.

Induction of matrix metalloproteinases and collagenolysis in chick embryonic membranes before hatching.

The membranes surrounding the chick embryo undergo striking morphological changes before hatching, which include structural degradation of the allantoic membrane. The fibrillar collagen content of the membranes declined by embryonic day (ED) 20 (the day of hatching). By ED 19, a 55-kDa matrix metalloproteinase (MMP) activity appeared in the extraembryonic fluid, and by ED 20 there was substantial 55-kDa MMP activity in embryonic membrane extracts. Reverse transcription-polymerase chain reaction was employed to clone a partial cDNA representing the chicken homologue of MMP-13, a 55- to 57-kDa enzyme. MMP-13 mRNA dramatically increased in abundance in embryonic membranes by ED 19, reaching a peak on ED 20. Introduction of the MMP inhibitor batimastat into the extraembryonic fluid prevented the structural changes in the embryonic membranes before hatching. We conclude that, like mammalian fetal membranes, chick embryonic membranes undergo terminal remodeling before hatching, in part as a result of increased MMP activity. The chicken egg system represents a novel in vivo model for exploring biochemical events leading to embryonic membrane remodeling prior to birth and to test inhibitors of MMPs for their ability to prevent collagenolysis and fetal membrane rupture.

Transcription factor AP-2gamma regulates murine adenosine deaminase gene expression during placental development.

Trophoblast cells are specialized extra-embryonic cells present only in eutherian mammals. They play a major role in the implantation and placentation processes. To understand better the molecular mechanisms that control the development and function of trophoblast cells, we sought to identify the transcription factors that regulate murine adenosine deaminase (ADA) gene expression in the placenta. Here we report a detailed characterization of a placenta-specific footprinting region (FP1) in the Ada placental regulatory element. The sequence of FP1 was mapped by DNase I footprinting and was found to match a consensus AP-2 transcription factor-binding site. Electrophoretic mobility shift assays demonstrated that FP1 interacted with AP-2-like proteins. Further analysis using AP-2 antibody confirmed that AP-2 protein was indeed present in the placenta and bound to FP1. Mutation at the AP-2 site in FP1 abolished the ability of the Ada placental regulatory element to bind AP-2 proteins and failed to target chloramphenicol acetyltransferase reporter gene expression to placentas in transgenic mice, indicating that AP-2 is required for Ada expression in the placenta. In addition, RNase protection assays demonstrated that AP-2gamma was the predominant AP-2 family member expressed in the placenta. In situ hybridization analysis revealed that AP-2gamma expression was enriched in the trophoblast lineage throughout development, suggesting that AP-2gamma may be critical for trophoblast development and differentiation.

Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity.

Angiogenesis depends on both cell adhesion and proteolytic mechanisms. In fact, matrix metalloproteinase 2 (MMP-2) and integrin alphavbeta3 are functionally associated on the surface of angiogenic blood vessels. A fragment of MMP-2, which comprises the C-terminal hemopexin-like domain, termed PEX, prevents this enzyme binding to alphavbeta3 and blocks cell surface collagenolytic activity. PEX blocks MMP-2 activity on the chick chorioallantoic membrane where it disrupts angiogenesis and tumor growth. Importantly, a naturally occurring form of PEX can be detected in vivo in conjunction with alphavbeta3 expression in tumors and during developmental retinal neovascularization. Levels of PEX in these vascularized tissues suggest that it interacts with endothelial cell alphavbeta3 where it serves as a natural inhibitor of MMP-2 activity, thereby regulating the invasive behavior of new blood vessels.

Ovine amniotic and allantoic epithelia across gestation.

BACKGROUND: Extensive studies on the regulation of the volume and composition of amniotic and allantoic fluid in the sheep have suggested that the amniotic and allantoic membranes must play an active role in these processes. Little is known of the functional morphology of the sheep amnion and allantois beyond the presence of an epithelium overlying connective tissue. METHODS: The ovine amnion and allantois were characterized at a range of gestational ages (27-140 days of gestation, where term is 145-150 days) by electron microscopy (SEM and TEM) and the presence of transporting ATPases examined by use of immunohistochemistry (Ca++-ATPase) and in situ hybridization (Na,K-ATPase). RESULTS: With increasing gestational age, the cell height of epithelium of the membranes increased, as did the number of apical microvilli and the length of zonulae occludentes. Epithelial cell cytoplasm increased in complexity, and cell shape changed from flattened to cuboidal. Proliferation of cells occurred until close to term. Immunoreactivity to Ca++-ATPase was present in the basolateral membranes at all stages of gestation examined, but hybridization with the alpha and beta subunits of Na,K-ATPase was present only at or after 100 days of gestation. CONCLUSIONS: The epithelia of the sheep amnion and allantois display characteristics typical of transporting epithelia. As the epithelia mature, changes related to increased capacity for solute and fluid transport regulation occur.

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.

Studies on the catalytic mechanism of pig purple acid phosphatase.

Several independent experiments failed to reveal any evidence in support of the involvement of a phosphoryl-enzyme intermediate in the catalytic mechanism of pig allantoic fluid purple acid phosphatase: (i) attempts to label enzyme with phosphate derived from [32P]p-nitrophenyl phosphate were unsuccessful; (ii) values of kcat for a series of phosphate derivative varied over a wide range, with the enzyme showing a marked preference for activated ester and anhydride substrates over those with a stable leaving group; (iii) burst titrations revealed a "burst" of p-nitrophenol from p-nitrophenyl phosphate only when the enzyme was added after the substrate, suggesting that this result was an artifact of the order of addition of reagents; (iv) transphosphorylation from p-nitrophenyl phosphate to acceptor alcohols could not be detected, even under conditions where a transphosphorylation to hydrolysis ratio as low as 0.015 could have been measured; (v) enzyme-catalyzed exchange of 180 between phosphate and water was demonstrated, although at a rate much slower than that observed for other phosphatases where the involvement of a phosphoryl-enzyme intermediate in the mechanism has been clearly established. The present results are compared with those obtained in similar studies on other phosphatases, particularly the highly homologous beef spleen purple acid phosphatase, and their implications for the catalytic mechanism of the purple acid phosphatases are discussed.

Ultrastructural and immunocytochemical evidence for the presence of polarised plasma membrane H(+)-ATPase in two specialised cell types in the chick embryo chorioallantoic membrane.

The chick embryo, confined in the eggshell, has to dispose/buffer the acid generated by its metabolism, as well as to release calcium from the shell which is used for growth. To localise H(+)-ATPase, electron microscope and immunocytochemical studies were conducted on chorioallantoic membranes of 15-17 d chick embryos. Ultrastructural studies of the villus cavity (VC) cells in the chorionic epithelium demonstrated that their apical plasma membrane, juxtaposed with the shell membranes, contains microvilli as well as microplicae which possess 9-10 nm studs at a density of 16,700 particles/micron2, a characteristic feature of the polarised H(+)-ATPase pump. Immunocytochemical staining, using a monoclonal antibody to the 31 kDa subunit of H(+)-ATPase, confirmed the presence of large amounts of the vacuolar H(+)-ATPase in the VC shells with a distribution highly polarised towards the eggshell membranes. Immunoelectron-microscopic localisation studies using a rabbit antiserum to whole bovine H(+)-ATPase and immunogold technique, confirmed the localisation of H(+)-ATPase at the apical microvilli/microplicae as well as in the subapical vesicles. In the allantoic epithelium, the presence of mitochondria-rich (MR) cells was confirmed; it was shown that these cells extend through the full thickness of this epithelium. The MR cells also contained large numbers of 9-10 nm studs, typical of proton secreting cells, in their apical plasma membrane. This was confirmed by immunocytochemical staining which showed abundant localisation of H(+)-ATPase in these cells; this localisation was, however, diffuse rather than apical.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional activation of cytochrome P450 side chain cleavage enzyme expression during trophoblast cell differentiation.

The purpose of this study was to investigate P450scc expression during trophoblast differentiation. Biochemical characteristics of P450scc protein and mRNA identified in rat trophoblast tissues were similar to those identified in the rat adrenal gland. Furthermore, P450scc was localized to trophoblast giant cells. This observation prompted an examination of progesterone biosynthesis and P450scc expression in Rcho-1 cells. Rcho-1 cells were derived from a transplantable rat choriocarcinoma, their differentiation can be regulated, and they have the capacity to express the trophoblast giant cell phenotype. Progesterone was produced by Rcho-1 cells and increased approximately 100-fold as the cells progressed from proliferation to differentiation. P450scc protein and mRNA accumulation also increased during trophoblast differentiation. P450scc expression within the Rcho-1 cell line was restricted to trophoblast giant cells. To further investigate the regulation of P450scc expression during trophoblast differentiation, we examined a plasmid construct, containing 894 base pairs of DNA 5' upstream from the P450scc transcriptional start site linked to a human growth hormone reporter gene, following stable transfection into Rcho-1 cells. The transfected P450scc regulatory DNA permitted the expression of human growth hormone which paralleled expression of the endogenous P450scc gene. In conclusion, transcriptional activation of the P450scc gene accompanies trophoblast giant cell differentiation.