Effects of Inflammatory Conditions from Coculture with Activated Macrophages on Mouse Preimplantation Embryonic Development In Vitro.

Abstract

Inflammatory conditions are involved in important reproductive events of mammals such as, ovulation, menstruation, implantation, labor and delivery. The inflammatory milieu in reproductive organs may have various effects on preimplantation embryonic development. The aim of this study was to evaluate the effects of inflammatory conditioning on mouse preimplantation embryonic development in vitro. To provide an inflammatory condition for preimplantation embryos, 2-cell stage embryos were cocultured with the RAW 264.7 cells, a mouse monocyte/macrophage cell line, which were activated by treatments of lipopolysaccharide (LPS) before coculture. The inflammatory response-like condition by LPS was evaluated and confirmed by RT-PCR for inflammation related genes (IL-1α, IL-6, iNOS, Cox-2, MCP-1 and TNF-α) and ELISA for secreted cytokines (IL-1α, IL-6 and MCP-1). Mouse 2-cell stage embryos were collected from oviduct and cultured in the Quinn's Advantage Blastocyst Medium (QABM) supplemented with 10% serum protein substitute (SPS) as control (CON), or directly cocultured with either intact (INT) or LPS activated (ACT) RAW 264.7 cells in the QABM without SPS. At post-hCG 120 h (72 h after culture), the blastulation rate of ACT (98.1%) was higher than that of CON (89.1%) and INT (94.5%) while there was no statistical significance between them. In addition, the hatching rate of ACT (54.5%) was higher than that of CON (47.3%) and INT (36.4%) without statistical significance. However, the mean cell number of blastocysts in ACT (112.0±6.9) was significantly higher (p<0.01) than those of CON (84.9±6.9) and INT (82.5±4.4), whereas there was no significant difference in apoptotic index among them. To examine whether embryotrophic effects of the inflammatory response-like condition on preimplantation embryos in vitro are induced by direct cell-to-cell contact, 2-cell embryos were indirectly cocultured with RAW 264.7 cells using cell culture inserts, which prevent direct contact between embryos and cocultured cells. Blastulation and hatching rates of ACT (94.1% and 47.1%) were slightly higher than that of CON (84.0% and 32.0%) and INT (90.9% and 32.7%) without statistical significance in indirect coculture system. Furthermore, the mean cell number of blastocysts in ACT (106.2±5.7) was also significantly higher (p<0.01) than those of CON (73.8±5.4) and INT (89.3±6.3) with no difference in apoptotic index. Collectively, this study demonstrates that the inflammatory response-like condition induced by activated RAW 264.7 cells could increase cell proliferation of mouse preimplantation embryos in vitro. Our results suggest that a certain inflammatory conditions could improve developmental competence of mouse preimplantation embryos in vitro by providing embryotrophic cytokines and factors. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0004871). (poster)