Canine preprorelaxin: nucleic acid sequence and localization within the canine placenta.
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Employing uteroplacental tissue at Day 35 of gestation, we determined the nucleic acid sequence of canine preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Canine preprorelaxin cDNA consisted of 534 base pairs encoding a protein of 177 amino acids with a signal peptide of 25 amino acids (aa), a B domain of 35 aa, a C domain of 93 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the canine relaxin B domain GRDYVR contained two substitutions from the classical motif (E-->D and L-->Y). Canine preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a 1-kilobase transcript present in total RNA of canine uteroplacental tissue but not of kidney tissue. Uteroplacental tissue from two bitches each at Days 30 and 35 of gestation were studied by in situ hybridization to localize relaxin mRNA. Immunohistochemistry for relaxin, cytokeratin, vimentin, and von Willebrand factor was performed on uteroplacental tissue at Day 30 of gestation. The basal cell layer at the core of the chorionic villi was devoid of relaxin mRNA and immunoreactive relaxin or vimentin but was immunopositive for cytokeratin and identified as cytotrophoblast cells. The cell layer surrounding the chorionic villi displayed specific hybridization signals for relaxin mRNA and immunoreactivity for relaxin and cytokeratin but not for vimentin, and was identified as syncytiotrophoblast. Those areas of the chorioallantoic tissue with most intense relaxin immunoreactivity were highly vascularized as demonstrated by immunoreactive von Willebrand factor expressed on vascular endothelium. The uterine glands and nonplacental uterine areas of the canine zonary girdle placenta were devoid of relaxin mRNA and relaxin. We conclude that the syncytiotrophoblast is the source of relaxin in the canine placenta. (+info)
Expression of trophinin, tastin, and bystin by trophoblast and endometrial cells in human placenta.
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Trophinin, tastin, and bystin comprise a complex mediating a unique homophilic cell adhesion between trophoblast and endometrial epithelial cells at their respective apical cell surfaces. In this study, we prepared mouse monoclonal antibodies specific to each of these molecules. The expression of these molecules in the human placenta was examined immunohistochemically using the antibodies. In placenta from the 6th week of pregnancy, trophinin and bystin were found in the cytoplasm of the syncytiotrophoblast in the chorionic villi, and in endometrial decidual cells at the utero placental interface. Tastin was exclusively present on the apical side of the syncytiotrophoblast. Tissue sections were also examined by in situ hybridization using RNA probes specific to each of these molecules. This analysis showed that trophoblast and endometrial epithelial cells at the utero placental interface express trophinin, tastin, and bystin. In wk 10 placenta, trophinin and bystin were found in the intravillous cytotrophoblast, while tastin was not found in the villi. After wk 10, levels of all three proteins decreased and then disappeared from placental villi. (+info)
CD9 is expressed in extravillous trophoblasts in association with integrin alpha3 and integrin alpha5.
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The CD9 molecule is a 24-27 kDa cell surface glycoprotein, which may be related to Schwann cell migration and adhesion. In this study, we examined the expression of CD9 in human extravillous trophoblasts, which invade into the endometrium during implantation and placentation. CD9 was detected immunohistochemically on the extravillous trophoblasts in the cell columns of first trimester placentae, but not on villous trophoblasts. In the second and third trimester, CD9 was highly expressed on the extravillous trophoblasts in the basal plate of placentae, and in the chorion laeve in the fetal membrane of term placentae. The molecular mass of CD9 in the chorion laeve was shown to be 27 kDa by Western blotting. The mRNA of CD9 was also detected in the chorion laeve by reverse transcription-polymerase chain reaction (RT-PCR). Proteins were purified from chorion laeve by affinity chromatography with anti-integrin alpha3 and alpha5 monoclonal antibodies and Western blotting, revealed that CD9 was associated with both integrins. These findings indicate that CD9 is a differentiation-related molecule present in the extravillous trophoblasts. Since it is associated with integrin alpha5 which has been proposed to regulate trophoblast invasion, CD9 may be implicated in trophoblast invasion at the feto-maternal interface. (+info)
Characterization of human placental explants: morphological, biochemical and physiological studies using first and third trimester placenta.
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The primary objective of this study was to characterize an in-vitro model of the human placenta using morphological, biochemical and physiological parameters. Placental villi were obtained from normal first trimester and term pregnancies. The villi were incubated with Dulbecco's modified Eagle's medium: Ham's F12 nutrient mixture in a shaking water bath at 37 degrees C for up to 310 min. The viability was determined by the production of beta human chorionic gonadotrophin (HCG) and lactic dehydrogenase (LDH) and the incorporation of [3H]thymidine, [3H]L-leucine and L-[U14C]arginine, while ultrastructure was assessed by transmission electron microscopy. In the first and third trimester group, the release into the medium of the intracellular enzyme LDH remained unaltered throughout the experiment. By contrast, beta-HCG concentrations increased linearly and concentrations were higher in the first trimester than term villi (354.5 +/- 37.8 versus 107 +/- 8.1 IU/g villi protein; P < 0.001). Electron microscopy confirmed preservation of tissue viability for up to 4 h of incubation. The incorporation of thymidine (12.2 +/- 2.9 versus 5.2 +/- 0.5 nmol/g villi protein; P < 0.05), leucine (9.4 +/- 2.1 versus 1.9 +/- 0.4 nmol/g villi protein; P < 0.02) and arginine (17 +/- 4.4 versus 4.2 +/- 0.5 nmol/g villi protein; P < 0.05) were markedly higher in early than in term placenta. Furthermore, placental uptake of L-leucine by the first (9.4 +/- 2.1 versus 17 + 4.4 mol/g villi protein; P < 0.001) and third trimester placental villi (1.9 +/- 0.4 versus 4.2 + 0.5 mol/g villi protein; P < 0.001) was less than that of L-arginine. This study describes a simple technique using placental explants to determine relative rates of uptake of substrate amino acids throughout gestation. (+info)
Immunity to placental malaria. I. Elevated production of interferon-gamma by placental blood mononuclear cells is associated with protection in an area with high transmission of malaria.
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In areas in which malaria is holoendemic, primigravidae and secundigravidae, compared with multigravidae, are highly susceptible to placental malaria (PM). The nature of gravidity-dependent immune protection against PM was investigated by measuring in vitro production of cytokines by placental intervillous blood mononuclear cells (IVBMC). The results demonstrated that interferon (IFN)-gamma may be a critical factor in protection against PM: production of this cytokine by PM-negative multigravid IVBMC was elevated compared with PM-negative primigravid and secundigravid and PM-positive multigravid cells. Low IFN-gamma responsiveness to malarial antigen stimulation, most evident in the latter group, was balanced by increased interleukin (IL)-4 production, suggesting that counter-regulation of these two cytokines may be a crucial determinant in susceptibility to PM. A counter-regulatory relationship between IL-10 and tumor necrosis factor-alpha was also observed in response to malarial antigen stimulation. These data suggest that elevated production of IFN-gamma, as part of a carefully regulated cytokine network, is important in the control of PM. (+info)
Characterization and expression of the laminin gamma3 chain: a novel, non-basement membrane-associated, laminin chain.
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Laminins are heterotrimeric molecules composed of an alpha, a beta, and a gamma chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel laminin, composed of known alpha and beta chains but containing a novel gamma chain, gamma3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that gamma3 contains all the expected domains of a gamma chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that gamma3-containing laminins are likely to exist in a stable matrix. Studies of the tissue distribution of gamma3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, gamma3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The gamma3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, ductus deferens, and seminiferous tubules. The distribution of gamma3-containing laminins on the apical surfaces of a variety of epithelial tissues is novel and suggests that they are not found within ultrastructurally defined basement membranes. It seems likely that these apical laminins are important in the morphogenesis and structural stability of the ciliated processes of these cells. (+info)
Rapid detection of chromosome aneuploidies by quantitative fluorescence PCR: first application on 247 chorionic villus samples.
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We report the results of the first major study of applying quantitative fluorescence polymerase chain reaction (QF-PCR) assays for the detection of major chromosome numerical disorders. The QF-PCR tests were performed on a total of 247 chorionic villus samples, which were analysed blind, without any knowledge of the results obtained using conventional cytogenetic analysis. The aims of this investigation were to evaluate the detection power and accuracy of this approach by testing a large number of fetal samples and to assess the diagnostic value of each of the chromosome specific small tandem repeat (STR) markers used. In addition, we introduced three more markers specific for chromosomes 13, 18, and X to allow an accurate analysis of samples homozygous for a particular STR. Fluorescent labelled primers were used to amplify 12 STRs specific for chromosomes 21, 18, 13, X, and the amylogenin-like DNA sequence AMXY, expressed on the X and Y chromosomes. In this blind study of 247 fetal samples, 222 were correctly diagnosed by QF-PCR as normal for each of the five chromosomes investigated; 20 were diagnosed by QF-PCR as trisomic for chromosomes 21, 18, or 13, in agreement with the cytogenetic tests. Only one false negative result was observed, probably owing to the mishandling of the sample, which had been transferred through three laboratories before being analysed by QF-PCR. The 247 samples also included four cases of mosaicism or translocation; one case of mosaic trisomy 21 was detected by QF-PCR and the other cases were not identified by QF-PCR. The results of this investigation provide clear evidence that the QF-PCR assays are powerful adjuncts to conventional cytogenetic techniques and can be applied for the rapid and accurate prenatal diagnosis of the most frequent aneuploidies. (+info)
Trisomy/tetrasomy 21 mosaicism in CVS: interpretation of cytogenetic discrepancies between placental and fetal chromosome complements.
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Trisomy/tetrasomy 21 mosaicism was found in chorionic villi (semidirect preparation) obtained from a 40 year old pregnant woman. Since both cell lines were abnormal, the couple elected for pregnancy termination. Placenta and fetal tissue samples were obtained for cytogenetic study. Long term cultured villi showed a non-mosaic trisomy 21 karyotype, while other tissues showed either a normal karyotype or normal/trisomy21 mosaicism. These discrepancies could be explained by a modified "bottle neck" embryogenic model with a trisomic zygote and a non-disjunction event taking place in one of the first divisions. Our case emphasises the need for confirmatory studies in other tissues when mosaicism is encountered in chorionic villi, even if all cell lines are abnormal. (+info)