Investigation of ANXA1 role on blastocyst implantation and uterine receptivity: ex vivo assays

Abstract

Pregnancy is an extremely complex process and its success depends on formation and activation of the embryo and also on interaction of it with the uterine epithelium. These events are followed by fetal tolerance and development, which then finally ends with delivery. Gestation is controlled by distinct inflammatory profiles at each pregnancy phase. Initially, there is an intra-uterine non-aggressive inflammation, followed by a tolerance response from the mother relative to the fetus, and then a resolutive phase where recovery of uterine tissue takes place. We have solid evidences that annexin A1 (AnxA1), a 37 KDa anti-inflammatory protein which is secreted due to glucocorticoid actions, modulates pregnancy. Couples of mice genetically deficient of AnxA1 (AnxA1-/-) generate larger number of fetuses in a higher proportion of females, and this effect is dependent on higher blastocyst implantation and lower post-implantation loss. Therefore, the current project will investigate the mechanisms by which AnxA1 affects blastocyst implantation upon uterine epithelial cell culture ex vivo. For this purpose, male and female wild type (WT) or AnxA1-/- balb/c mice will mate. One male will be maintained with 2 or 3 females for matting and, daily, in the morning, the vaginal plug (VP) will be verified. On the 4th day after VP visualization, females will be euthanized by cervical dislocation and the uteri will be removed for collection of epithelium and blastocysts. The uterine tissue will be enzymatically digested and epithelial cells will be isolated for culture. The blastocysts obtained from WT or AnxA1-/- mice will be cultured with epithelial cells obtained from WT or AnxA1-/- mice. The blastocysts - epithelium interactions will be evaluated by inverted microscopy; expression of cytokeratin, 2macroglobulin and mucin will be detected by immunofluorescence in order to characterize the uterine epithelium culture and receptivity; and the supernatant of cell cultures will be stored for later quantification of inflammatory mediators. The obtained data might strengthen our hypothesis that AnxA1 controls gestation. This project is part of Thematic Project FAPESP 2014/07328-4, which investigates the endogenous pathways of inflammation control. (AU)