Pregnant mice submitted to Surgical Embryo Euthanasia (SEE) induce loss of expression of inducible nitric oxide synthase isoform and NO concentration in the maternal-fetal interface

Lidia Jacinta Nunes Fernandes,Eliana Mara Oliveira Lippe

doi:10.21472/bjbs(2020)071507

Abstract

The well-succeeded pregnancy in humans and rodents is the consequence of close interaction between maternal and fetal cells with intervening of cytokines and chemical mediators. In this process a pregnant uterus subset NK cells - uterine Natural Killer cells (uNK cells) play a pivotal modulatory role under the influence of local physiological hypoxia and other alterations. The aim of the present work was to evaluate the expression and commitment of induced form of nitric oxide synthase (iNOS) and NO concentration in the homeostasis of pregnant uterus. It was used normal pregnant mice on gd 10th and those submitted to surgical intervention to induce mechanical lesion in the embryos (SEE). Uterine samples were collected at 0.5, 1, 2 and 6 h after embryo lesion and processed for paraffin embedding and tissue homogenate. The samples destinate for paraffin embedding was performed the Dolichos biflorus (DBA) lectin cytochemistry and anti-iNOS immunocytochemistry. The samples destinate to tissue homogenates were processed for SDS-PAGE and Western-blot using anti-iNOS and evaluate of NO concentration. The embryo-injured uterine segments showed hyperemia and hemorrhage at mesometrial region in which the DBA lectin reaction showed altered uNK cells suggesting the degranulation. Positive reaction with anti-iNOS was seen on uNK cells, trophoblast giant cells, endometrial stromal and decidual cells and smooth muscle cells in the normal pregnant uterus, but 1 and 2 h after embryo lesion, the iNOS labeling decreased or was absent only in uNK cells. The same results was obtained with NO concentration. These results confirm the unique constitutive expression of iNOS in the pregnant mice uterus, being the uNK cells the only one responsive against stress of embryo failure, besides showing that excessive NO produced by quick activation of uNK-iNOS should affect the local vascular permeability.

Highlights

The successful embryo implantation and development in human and rodents uterus is highly dependent of interaction and dialogue between maternal and fetal cells, with intervention of the cytokines and chemical compounds in fine balance (Dietl et al, 2006; Sojka et al, 2019)
Intriguing was the participation of uterine natural killer cells - a lymphocyte population that accumulates in the uterine environment during pregnancy (Saito et al, 2000; Lima et al, 2014)
The CD56bright/CD16dim uterine natural killer cells (uNK) cells can be distinguished from the CD56dim/CD16bright blood circulating NK cells; the comparative microarray analyses showed several qualitative and quantitative differences on gene expression profile between these two NK cell subsets (Peng et al, 2010)

Summary

Introduction

The successful embryo implantation and development in human and rodents uterus is highly dependent of interaction and dialogue between maternal and fetal cells, with intervention of the cytokines and chemical compounds in fine balance (Dietl et al, 2006; Sojka et al, 2019). This dialogue involves the blood vessels, stromal cells and leukocytes in the endometrium from maternal side, and trophoblast cells from the embryo in a complex signaling network (Burton et al, 2009). The cNK subset is more cytotoxic, has many cytolytic granules and express members of killer immunoglobulin-like receptor (KIR) family (Maynard et al, 2008; Leonard et al, 2013) while, the uNK cells in spite of high granule contents has low cytotoxicity, does not express KIRs, and experimentally it is mainly cytokine producing subset (Koopman et al, 2003)

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