108 EFFECT OF INTERLEUKIN-1β, -3 AND -4 ON BOVINE EMBRYO DEVELOPMENT IN VITRO AND MHC-I GENE EXPRESSION

Abstract

Studies in humans and other species have shown that the MHC class I region (MHC-I) is involved at a number of levels in the establishment and maintenance of pregnancy. Expression of MHC-I and MHC-I-like molecules is regulated during embryo development and differentiation. In general, classical MHC-I gene expression appears to be down-regulated in mammalian trophoblast cells, whereas the expression of certain non-classical (NC) MHC-I genes is up-regulated. Cytokines govern uterine immunology and receptivity and are increasingly recognized for their embryotrophic roles. The aim of the current study was to investigate the regulatory effect of a number of cytokines on bovine embryo development and NC MHC-I mRNA expression at the blastocyst stage. Cumulus oocyte complexes were obtained by aspirating follicles from bovine ovaries collected from the local abattoir. At approximately 20 h postinsemination (hpi), presumptive zygotes produced following IVM/IVF were denuded by gentle vortexing and transferred to synthetic oviduct fluid medium supplemented with different concentrations (0, 0.1, 1, 10, 100 ng mL^–1) of either Interleukin (IL)-1β (n = 1759), IL-3 (n = 1053) or IL-4 (n = 1549) and cultured in an atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 100% humidity. Cleavage rates were recorded at 48 hpi and the proportion of embryos developing to the blastocyst stage was recorded from Day 6 to 8. Day 7 blastocysts were snap frozen in pools of 10 and stored at –80°C for the analysis of transcript abundance of bovine NC MHC-I genes (BOLA-NC1, NC2, NC3 and NC4, see www.ebi.ac.uk/ipd/mhc/) using quantitative real-time RT-PCR. Messenger RNA was extracted from 5 pools of 10 embryos per treatment, using Dynabeads mRNA DIRECTMicro Kit (Dynal A.S, Oslo, Norway), and cDNA was synthesized. Real-time PCR was used to compare NC MHC-I transcript abundance. The data were analyzed using the standard curve method, and means were compared by the Student t-test. There was no effect of any of the cytokines tested on cleavage or blastocyst development. However, the relative abundance of the BOLA-NC1 transcript significantly increased (P ≤ 0.05) 1.6-fold and 3-fold, respectively, as the concentration of IL-3 and IL-4 in culture increased, whereas there was no significant change (P ≥ 0.05) in the abundance of IL-1β. There was no significant difference (P ≥ 0.05) in the levels of BOLA NC2 and BOLA-NC4 in the embryos cultured with IL-3 and IL-1β, irrespective of the concentration, but the levels varied significantly (P ≤ 0.05) at higher (≥10 ng mL^–1) concentrations of IL-4, 2-fold (NC2) and 3-fold (NC4). The relative abundance of the BOLA-NC3 transcript significantly increased (1.5-fold, P ≤ 0.05) as the concentration of IL-3 in culture increased, whereas there was no significant change (P ≥ 0.05) in the presence of IL-1β or IL-4. Compared with the lack of differences in embryo developmental competence, the NC MHC-I expression data possibly suggest a preferential immunomodulatory role of these cytokines during preimplantation embryo development.

Supported by SFI PICA.