79 THE CELL-CYCLE RELATED NUCLEAR LOCALIZATION OF PLATELET-ACTIVATING FACTOR IN BOVINE AND MURINE EMBRYO DEVELOPMENT

Abstract

Platelet-activating factor (PAF) is a lipid mediator produced by a wide variety of cells and well known for its involvement in host defense and platelet aggregation. In embryo development, PAF functions as an embryotropin and a marker of embryo quality and viability in human and mice. However, only limited information is available concerning its function during in vitro culture of bovine embryos. Bovine and murine embryos were collected at different time points according to their development (4–8-16 cells-morula-blastocyst; 8–10 embryos per replicate; 4 replicates). Phospholipid expression and intracellular localization was evaluated by immunofluorescent staining and confocal scanning microscopy. In short, the zona pellucida of the embryos was removed using 0.5% protease (from Streptomyces griseus). After a recovery period of 30 min, embryos were fixed in 4% paraformaldehyde and permeabilized (0.5% Triton X-100 and 0.05% Tween20) overnight at 4°C. Subsequently, embryos were transferred to blocking solution (30% goat serum and 0.05% Tween20) overnight at 4°C to block nonspecific binding sites. Next, embryos were incubated overnight with primary antibody against PAF (1:100; Emelca Bioscience, Breda, the Netherlands) at 4°C, followed by 1 h of incubation with secondary goat anti-rabbit (Alexa Fluor 488; 1:500; Abcam, Cambridge, MA, USA). Nuclei were visualised by 20 min incubation with Hoechst 33342 (1:500, Molecular Probes) in the dark. As a negative control, the primary antibody was replaced with rabbit IgG whole molecule (Rockland Immunochemicals, Limerick, PA, USA). Using specific lipid antibodies, we were able to show that PAF accumulates in the nuclei of pre-implantation bovine and murine embryos. Suprisingly, the localization correlated with the cell cycle: a clear nuclear signal was observed during interphase and a redistribution to the cytoplasm happened upon nuclear envelope breakdown at mitosis. To confirm this finding, bovine blastocysts (10 embryos per replicate, 3 replicates) were incubated for 12 h with the microtubule destabilising agent nocodazole (2 μM; Sigma, Germany). Embryos were analysed using immunofluorescent staining as described above. Nocodazole treatment resulted in arrest of at least 20% of the embryonic cells at mitosis. Upon treatment, we were able to reproduce the cell-cycle dependent shuttling between M-phase and interphase nuclei of PAF. We are the first to attribute this unique feature of nucleo-cytoplasmic redistribution to an important embryonic growth factor such as PAF. Further research will be needed to unravel the exact pathway and function of PAF in the nucleus and its implications on embryonic development.