title: Expression of NADPH oxidase by trophoblast cells: potential implications for the postimplanting mouse embryo.
abstract: Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.
authors.affiliation: Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, Brazil.
authors.forename: Sara Z
authors.initials: SZ
authors.lastname: Gomes
authors.forename: Aline R
authors.initials: AR
authors.lastname: Lorenzon
authors.forename: Juliana S
authors.initials: JS
authors.lastname: Vieira
authors.forename: Clarissa R R
authors.initials: CR
authors.lastname: Rocha
authors.forename: Clara
authors.initials: C
authors.lastname: Bandeira
authors.forename: Mara S
authors.initials: MS
authors.lastname: Hoshida
authors.forename: Lucia Rosseti
authors.initials: LR
authors.lastname: Lopes
authors.forename: Estela
authors.initials: E
authors.lastname: Bevilacqua
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D047108:Embryonic Development / Q000502:physiology*; D005260:Female; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D011247:Pregnancy; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014327:Trophoblasts / Q000166:cytology* / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; D013481:Superoxides; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C511584:Ncf2 protein, mouse; C060505:neutrophil cytosolic factor 1