RESUMEN
Prostaglandin E2 (PGE2) may regulate uterine activation and cervical ripening for labor through specific contractile and relaxatory receptors (EP1-4). The aim of this study was to determine the expression of PGE2 receptor isoforms in pregnant rat cervix during RU486-induced labor and progesterone supplementation to delay labor. Localization and expression of cervical PGE2 receptors were evaluated, and quantitative real-time polymerase chain reaction (PCR) for EP1-4 was performed. EP1-4 were found in both cervical epithelium and smooth muscle. RU486 treatment increased EP2 and EP4 messenger RNA (mRNA) and protein expression. Progesterone treatment had no effect on EP2 and EP4 mRNA expression but decreased EP4 protein. Hormonal manipulation resulted in differences in cellular localization of EP1 and EP3 in cervical epithelial cells, suggesting a specific role in that cell. Progesterone differentially regulates the expression of PGE2 receptor isoforms in the cervix. Elucidating the regulation of PGE2 receptors may facilitate improved approaches to the prevention and treatment of preterm labor.
Asunto(s)
Maduración Cervical/fisiología , Cuello del Útero/fisiología , Dinoprostona/metabolismo , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E/metabolismo , Animales , Maduración Cervical/efectos de los fármacos , Epitelio/fisiología , Femenino , Expresión Génica/fisiología , Antagonistas de Hormonas/farmacología , Inmunohistoquímica , Mifepristona/farmacología , Músculo Liso/fisiología , Embarazo , Progesterona/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Subtipo EP1 de Receptores de Prostaglandina E , Subtipo EP2 de Receptores de Prostaglandina E , Subtipo EP3 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Contracción Uterina/fisiologíaRESUMEN
The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1-4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor. All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes. Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.
Asunto(s)
Amnios/fisiología , Corion/fisiología , Decidua/fisiología , Miometrio/fisiología , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Humanos , Trabajo de Parto , Trabajo de Parto Prematuro , Reacción en Cadena de la Polimerasa/métodos , Embarazo , Receptores de Prostaglandina/genética , Receptores de Prostaglandina E/genética , Subtipo EP1 de Receptores de Prostaglandina E , Subtipo EP2 de Receptores de Prostaglandina E , Subtipo EP3 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Contracción Uterina/fisiologíaRESUMEN
OBJECTIVE: The effects of prostaglandin E2 (PGE2) are mediated through G-protein coupled receptors, acting via different second messengers. The aim of this study was to characterize the temporal and tissue specific localization and expression of the PGE2 receptor subtypes (EP1-4) in uteroplacental tissues during human pregnancy. STUDY DESIGN: Placenta and fetal membranes were collected after delivery at preterm or term, each with or without labor. The localization and expression of the PGE2 receptor subtypes were determined by immunohistochemistry and Western blot. RESULTS: All 4 receptors were expressed in the placenta and fetal membranes; only EP3 was present in the syncytiotrophoblast layer. EP1 to EP4 were consistently expressed across gestation in the fetal membranes; however, a different cellular localization with labor was observed in the amnion for EP1, EP2, and EP4. CONCLUSION: The presence of these receptors in the placenta and fetal membranes may indicate autocrine roles for PGE2 in the signaling pathways associated with placental function and parturition.
Asunto(s)
Membranas Extraembrionarias/metabolismo , Trabajo de Parto/metabolismo , Placenta/metabolismo , Receptores de Prostaglandina E/metabolismo , Adulto , Amnios/metabolismo , Western Blotting , Corion/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Inmunohistoquímica , Embarazo , Subtipo EP1 de Receptores de Prostaglandina E , Subtipo EP2 de Receptores de Prostaglandina E , Subtipo EP3 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Trofoblastos/metabolismoRESUMEN
OBJECTIVE: The purpose of this study was to compare immunohistochemical expression of heat shock protein-70 (hsp70), a marker for oxidative stress, and 4-hydroxy-2-nonenal adducts (HNE), a marker for lipid peroxidation, in placental villous tissue of normotensive, preeclampsia, and intrauterine growth restricted (IUGR) pregnancies. STUDY DESIGN: Placentas were collected and flash frozen in liquid nitrogen after delivery from normotensive pregnancies (n=5), and pregnancies complicated by preeclampsia (n=5), IUGR (n=5), and preeclampsia plus IUGR (n=4). Cryosections were cut and immunostained with polyclonal anti-hsp70 and monoclonal anti-HNE antibodies using Vectastain Elite ABC kit. Normal rabbit serum or mouse IgG were used as negative controls. Three independent observers, blinded to identity of tissue, examined each slide to identify cellular localization and intensity of the immunostaining. Western blot analysis and scanning densitometry were used to quantify and compare the amount of hsp70 and HNE adducts present in tissue homogenates. RESULTS: Positive immunostaining for both antibodies was observed in cytoplasm of syncytiotrophoblasts, extravillous trophoblasts, vascular smooth muscle, and endothelial cells for all groups. Expression of hsp70 and HNE adducts was reported as observers' mean stained intensity. Overall, kappa showed good agreement between observers. Immunostaining intensity was similar in all tissue types for each group with the exception that immunostaining was significantly more intense in the vascular endothelium of the preeclamptic group for HNE adducts (P=.02) and significantly less intense in the IUGR group for hsp70 (P=.013). Scanning densitometric analysis of the Western blots showed no significant difference in total hsp70 and HNE adducts expression in all 4 tissue groups. CONCLUSION: Immunohistochemistry showed local changes for oxidative stress and lipid peroxidation in the vascular endothelium from placentas of preeclamptic and IUGR pregnancies. However, these changes were masked when studying tissue homogenates.
Asunto(s)
Aldehídos/metabolismo , Vellosidades Coriónicas/metabolismo , Retardo del Crecimiento Fetal/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Preeclampsia/metabolismo , Western Blotting , Densitometría , Femenino , Humanos , Inmunohistoquímica , Peroxidación de Lípido/fisiología , Estrés Oxidativo/fisiología , EmbarazoRESUMEN
Lipid storage droplets (LSDs) are subcellular storage depots for triglycerides (TGs) and cholesterol esters surrounded by specific populations of proteins that are necessary for their formation. We have previously described the appearance of LSDs in human fetal membranes with advancing gestation and labor. Perilipin and adipophilin are functional/structural proteins located on the surfaces of intracellular LSDs. Adipophilin and perilipin were both immunolocalized to the amnion epithelium and amnion fibroblasts in human fetal membranes. Adipophilin was also localized to the choriodecidual layer, whereas perilipin was localized to the chorion trophoblasts. Although immunohistochemical data show an apparent increase in adipophilin, but not perilipin, expression in fetal membranes with advancing gestation and labor, Western analysis of tissue homogenate supernatant revealed no significant changes in adipophilin and perilipin expression. However, Western analysis of the floating lipid-rich layer from the tissue homogenate revealed an abundance of adipophilin and perilipin as well as other enzymes (cytosolic phospholipase A2, prostaglandin endoperoxide, and microsomal-associated prostaglandin E synthase-1) involved in prostaglandin synthesis. The association of these enzymatically active proteins with LSDs suggests that LSDs may be foci for signaling via the arachidonic acid cascade in fetal membranes. The structural and functional roles of adipophilin and perilipin in gestation and labor remain to be determined.
Asunto(s)
Membranas Extraembrionarias/química , Lípidos/análisis , Proteínas de la Membrana/análisis , Fosfoproteínas/análisis , Prostaglandinas/biosíntesis , Animales , Western Blotting , Proteínas Portadoras , Ciclooxigenasa 2 , Femenino , Humanos , Inmunohistoquímica , Oxidorreductasas Intramoleculares/análisis , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Peso Molecular , Péptidos , Perilipina-1 , Perilipina-2 , Fosfolipasas A/análisis , Fosfolipasas A2 , Fosfoproteínas/genética , Fosfoproteínas/fisiología , Embarazo , Prostaglandina-E Sintasas , Prostaglandina-Endoperóxido Sintasas/análisis , ConejosRESUMEN
Increased prostaglandin (PG) synthesis by fetal membranes occurs at parturition. PGE(2) synthesis from arachidonic acid involves multiple enzymes and two isoforms of the terminal enzyme of this biosynthetic pathway, PGE synthase (PGES), were recently identified. Cytosolic PGES (cPGES) is identical to the heat shock protein 90 chaperone, p23, and is reportedly functionally coupled to constitutive PG endoperoxide H synthase-1. Microsomal PGES (mPGES) is inducible by proinflammatory cytokines such as IL-1 beta. We have studied expression and localization of both enzyme isoforms in human fetal membranes either at term or preterm, with or without labor. The cPGES was immunolocalized in the amnion epithelium, and associated with fibroblasts and macrophages in the choriodecidual layer, whereas mPGES was localized in the amnion epithelium as well as the chorion trophoblast. Both enzymes were found to be associated with lipid particles present in the amnion epithelium, which are more prevalent in term tissues. Western blot analysis of the amnion and choriodecidua showed no differences in amounts of either cPGES or mPGES at term or preterm, with or without labor, in either tissue with advancing gestation. It does not appear that expression of PGES is the rate-limiting step in PGE2 synthesis in fetal membranes at labor.