151 A SIMPLE AND FAST METHOD FOR CONCURRENT DIFFERENTIAL STAINING AND TUNEL LABELLING OF BOVINE BLASTOCYSTS

Currently techniques of TUNEL labelling for detection of apoptosis and differential staining for counting the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells are used separately for assessment of embryo quality in different species. The majority of these techniques are antibody-based, and time-consuming, frequently giving inconsistent results. Here we report on the development of a simple and fast method for simultaneous differential staining and TUNEL labelling of bovine embryos. Cleaved embryos produced from in vitro-matured and fertilized oocytes were cultured to the blastocyst stage in synthetic oviductal fluid culture medium (SOF) supplemented with 4 mg mL-1 BSA and 5% FCS. Embryos were partially permeabilized in 0.5% Triton X-100 solution containing 2 µM mL-1 TOTO-3 dye (Molecular Probes, Eugene, OR, USA) for 30 s. TOTO-3 is a cell-impermeant nucleic acid dye; thus only permeabilized cells are stained red. The embryos were then quickly washed in PBS containing 3 mg mL-1 PVA, fixed for 15 min at RT in 4% paraformaldehyde containing 10 µg mL-1 Hoescht, and TUNEL-labelled using a Cell Death Kit (Roche Applied Science, East Sussex, UI) for 30 min at 37°C in a humid chamber. The embryos were then treated with RNase A (50 U mL-1) for 30 min at 37°C, washed and mounted in a small drop of glycerol on a glass slide. RQ1-DNase (3 U mL-1)-treated embryos were used as a positive control. After three-dimensional reconstruction using a Leica TCS SP2 confocal microscope, we determined the number of ICM (blue), TE (red) and apoptotic nuclei (green). Only peripheral cells of the blastocysts were labelled red, indicating that TE cells were permeabilized by the short exposure to the detergent Triton. ICM cells were consistently stained blue by the cell permeant dye Hoechst. Apoptotic nuclei were found in both types of cells. More consistent differential staining was observed in hatched blastocysts (n = 30) than in zona-enclosed blastocysts (n = 35); also, more apoptotic nuclei were observed. No differences were found in the consistency of the technique for embryos grown with or without FCS. When compared to dual staining without Tunel, no differences in cell number (74 ± 22) , and ICM/TE ratio (0.28 ± 0.06) were detected, indicating that the Tunel procedure does not affect the labeling of the DNA. Preliminary observations also indicate that this method can be successfully applied to porcine and ovine embryos. This technique has the advantage of being fast and can be applied for assessment of embryo quality. It can also be used to determine the time and origin of ICM and TE differentiation while monitoring the degree of apoptosis in different culture systems and in different species. This work was in part supported by Department of Environment Food and Rural Affairs (Defra) UK.