Morphological alterations in mouse placenta induced by diazepam / Alteraciones morfológicas inducidas en la placenta de ratón por diazepam

Diazepam (DZ) is a benzodiazepine that belongs to the group of minor tranquilizers with myo-relaxing and anticonvulsant properties. DZ and its metabolites cross the placental barrier in human, monkey, hamster, and mouse, and accumulate in the placenta. Our aim was to investigate, through histological techniques, and semifine sections if DZ induces morphological changes in the placenta. Twenty female mice of the ICR strain were distributed randomly in two groups. One group (DZ) was treated from days 6 to 17 of gestation with a single daily subcutaneous (sc) dose of DZ of 2.7 mg/kg/ (bw); the second, control group (C) was treated with saline solution. All females (10 DZ and 10 C) were killed by decapitation. Placentas were extracted and fixed in phosphates-buffered 10% formaldehyde, pH 7.3, dehydrated, and embedded in paraffin to obtain 3 µm thick sections or fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO4, embedded in epoxy resin. Histological sections were stained with hematoxylin-eosin or Weigert´s iron hematoxylin. Semifine sections were stained with toluidine blue. All sections were observed under comparative light microscopy. The DZ-group showed thinned placental barrier with multiple vacuoles. Nuclei of trophoblast cells (TCs) and trophoblast giant cells (TGCs) presented heterochromatin in coarse granules, atypically distributed in the karyolymph and conspicuous nucleoli. The cytoplasm of the TGCs was vacuolated and chromatin had a similar appearance to that observed in TCs. The total area of the placental barrier was measured in µm2/µm2; the area in the DZ group was reduced as compared with the C group (P<0.001). Alterations of TGCs could be due to an interaction of DZ with peripheral type benzodiazepine receptors involved in progesterone biosynthesis. Administration of DZ in mice alters the placental barrier and TGCs which could affect their physiology and causes teratogenic effects on the ovary and testis involved in steroid hormones biosynthesis.

El diazepam (DZ) es una benzodiazepina que forma parte de los tranquilizantes menores con propiedades miorrelajantes y anticonvulsivantes. El DZ y sus metabolitos atraviesan la barrera placentaria en el humano, mono, hámster y ratón, y se acumula en ésta. Nuestro propósito fue investigar a través de técnicas histológicas y en cortes semifinos si el DZ induce cambios morfológicos en la placenta de ratón. Hembras de ratón de la cepa ICR se distribuyeron al azar en dos grupos. Un grupo (DZ) fue tratado del día 6 al 17 de la gestación con dosis únicas diarias subcutáneas (sc) de DZ de 2.7 mg/kg (pc); el segundo grupo control (C) se trató con solución salina. Todas las hembras (10 DZ y 10 C), se sacrificaron por decapitación. Se extrajeron las placentas y se fijaron en formaldehido al 10% amortiguado con fosfatos pH 7.3, se deshidrataron y se incluyeron en parafina para obtener cortes de 3 µm, o se fijaron en glutaraldehido al 2.5%, se posfijaron en OsO4 al 1% y se embebieron en resina epóxica. Los cortes histológicos se tiñeron con hematoxilina-eosina o con hematoxilina férrica de Weigert. Los cortes semifinos se tiñeron con azul de toluidina. Todos los cortes se observaron en un microscopio óptico de comparación. El grupo DZ presentó en la barrera placentaria múltiples vacuolas. Los núcleos de las células del trofoblasto y las células trofoblásticas gigantes (TGCs) presentaron heterocromatina en grumos gruesos, distribuidos atípicamente en la cariolinfa y nucléolos conspicuos. El citoplasma de las TGCs estaba vacuolizado y la cromatina tenía una apariencia similar a la observada en las células trofoblásticas. El área total de la barrera placentaria se midió en µm2/mm2; el área en el grupo DZ era reducida en comparación del grupo C (P<0.001). Las alteraciones de las células trofoblásticas y de las TGCs podrían deberse a la interacción del DZ con los receptores benzodiazepínicos de tipo periférico involucrados en la biosíntesis de progesterona. La administración de DZ en el ratón altera la barrera placentaria y las TGCs que podrían afectar su fisiología y causar efectos teratogénicos en el ovario y el testículo involucrados en la biosíntesis de las hormonas esteroides.

Expression and regulation of integrin receptors in human trophoblast cells: role of estradiol and cytokines.

Embryo implantation and placentation are dynamic cellular events that require not only synchrony between the maternal environment and the embryo, but also complex cell-cell communication amongst the implanting blastocyst and the receptive endometrium through integrins, a large family of proteins involved in the attachment, migration, invasion and control of cellular functions. Integrins display dynamic temporal and spatial patterns of expression by the trophoblast cells during early pregnancy in humans. However, the precise mechanism of embryo implantation and the modulation of the integrin receptors during blastocyst attachment and further implantation remain elusive in the humans. The present study elucidates the expression and hormonal modulation of fibronectin, vitronectin and laminin integrin receptors by estradiol and IL-1alpha in human trophoblast cells. Human first trimester trophoblast cells showed the induction of the classical estrogen receptor (ER)-alpha by its own ligand, estradiol. Treatment with either estradiol or IL-1alpha induced the expressions of alpha4, alpha5, alpha6 and alpha(v) integrin receptor subunits at both the mRNA and protein levels, while expression of beta1 remained unaltered. Furthermore, estradiol upregulated the expression of IL-1alpha, thereby suggesting the possibility that estrogen may either directly or via the proinflammatory cytokine induces the expression of the cell surface integrin receptors. The findings delineate the role of hormones and the cytokines in modulating the adhesiveness and attachment of the trophoblast cells. This may reflect the in vivo scenario where the implanting embryo is surrounded by a hormone-cytokine rich uterine microenvironment that may precisely regulate the expression of integrins and thereby facilitate implantation.

Biologically active steroid and thyroid hormones stimulate secretion of sex hormone-bending globulin by human term placenta in culture

In this study, the influence of steroid and thyroid hormones and epidermal growth factor on the productin of SHBG by placenta tissue explants was investigated. Explants of trophoblastic tissue obtained from normal term placentas were cultured for 48 h in serum free culture medium, and then for an additional 24 h period in the presence or absence of various concentrations of either estradiol (0.25 - 5 nM), testoterone (0.5 - 500 nM), triiodothyronine (0.01 - 100 nM) or EGF (2-40 µM), respectively. Human SHBG concentration in culture media was estimated on each day by specific two-site time-resolved fluoroimmunometric assay and the results expressed as pmol/mg tissue protein. Binding characteristics and molecular structure of secreted SHBG were determined by [3H]5a-DHT binding assays and Western blot analysis, espectively. Estradiol and triidothyronine but not testoterone increased significantly (p<0.05 vs. control) the secretion of SHBG into the culture media. Addition of EGF did not significantly change the production of SHBG at the various concentrations studied. [3H]5a-DHT binding assays and Western blot analysis of placental SHBG resulted in identical binding affinities (Kd 2.0 ñ 0.16 x 10-9M= and molecular structure to those obtained in serum from normal pregnant women. These findings support and extend previous observations by our laboratory indicating that SHBG gene is expressed in the placenta and provide further evidence on the hormonal regulatory characteristics of this steroid-binding protein in cultured placenta

Effects of dipyrone on the placenta of albino rats: morphological and biochemical aspects

The effect of dipyrone (N-2,3-dimethyl-5-oxo-1-phenyl-3-pyrazolin-4-yl)-methyllamino methanesulfonate sodium monohydrate) on the placenta of 2 BAW albino rats was studied through Karyometry of trophoblastic giant cells and DNA, RNA and total protein determinations. The animals received a single daily dose of 50 mg/Kg body weight during different periods of pregnancy: from the 9th to the 12th, 11th to the 14th, 13th to the 16th, 15th to the 18th and 17th to the 20 thday. Control animals received a single daily dose of 0.5ml distilled water at the same time. Karyometric results showed a statistically significant increase in nuclear volumes of placental cells of rats receiving dipyrone during the first three periods, when compared to control groups. In the two groups that received the drug nearer to term there was no significant difference. Regarding DNA, RNA and total protein determinations, there was a statistically significant difference, for all of them, in the rats that received the drug from the 9th to the 12 th day of pregnancy when compared to the control group. There was no significant difference in the groups that received the drug after that period. The results show that dipyrone had a blocking effect on cell division and that this effect happens mainly in the initial period of placental development