Bovine in vitro produced embryos induce modifications of gene expression profile of bovine oviduct epithelial cells in vitro
Résumé
In vivo, the ovicluct provicles the optimal environment to allow the sucœssful clevelopment of the carly mammalian embryo. A molecular dialogue may take place between the cmbryo and its maternai milieu, moclulating the embryo microenvironment clnring its migration towm*ds the implantation site. ln vitro co-culture witb bovine ovicluct epithelial œlls (BOEC) bas been widely used to mimic this materna] environment and to study its effect on embryo development. The exact mechanisms of BOEC action on embryo development have not been l'ully elucidated yet. Therefore, the pUL*pose of this research was to evaluate the BOEC in vitro rcsponsiveness to embryos, accorcling to the regional origin of the oviduct cells (isthmus vs. ampulla). Oviducts ipsilateral to ovnlies with sign of recent ovulation were brought to the laboratory. Exp. l: Ampulla and istbmus regions were dissected, washed thoroughly in TCM199 and epithelial cells werc scrappecl out using a sterile slide. BOEC from Arnpulla (A-BOEC) or lsthmus (1-BOEC) were seeded separately in 4 weil NUNC plates and cultured to confluence (7 days) to be used for in vitro embryo development (IVD). Immature cumulus oocyte complexes were aspirated from slaughterhouse avaries. Zygotes produced by in vitro maturation and fcrtilization wcrc culturecl in SOF medium supplementecl with 10% FCS in the presence of A-BOEC, l-BOEC or without cells (control). Somc A- and I-BOEC wells were culturecl without embryos. At Day 8 p.i., RNA were extracted (Trizol). Exp. 2) Confluent BOEC in 4 well NUNC plates were stimulated by synthetic INFtau (0. l, 10 and lOO ng/mL) for 6 or 24 hours and RNA were exa*acted (Trizol). Ali RNA srm1ples were treated with DNAsc and RT was petformed (MMLV RT kit). The leve! of expression of some known ovicluct exprcssecl genes (GPX4, C3, OVGP), as well as some genes related to IFN signaling (STATl, !FIT5, ISG15, OASl, IFITMl, MXl, OASl, USP18), wcre cvaluatcd by RT-qPCR. Data were analyzed by Mann Whitney non parmnetric test using PRISM 5 software. TI!C relative abun dance of ali IFNt related genes was significantly upregttlatecl whcn epithelial cells (either A-BOEC or I BOEC) were exposecl to embryos duling 8 days in vitro. The IFNI stimulation reproduced tllis embryo clfect, whatever the concentration tmcl duration of treatment. Furthennore, a regional difference in exprcssionlcvcl was found for OVGP (higher in I-BOEC, p<0.05) and C3 (bighcr in A-BOEC, p<0.05), without effect of the presence of embryos. GPX4 mRNA abundanœ was not significantly different among the culture conditions tested. In conclusion, these data confirm the specializatiou of ovicluct regions and show the ability of oviduet œlls to respond to emlHyo signaling. at !east ù1rough lFN-Iike pathway in our in vitro mode!, by modulating gene expression profile, thus suppo1ting the existence of a rerdialog betwcen carly embryo tmd oviduct.