Variability in apoptosis incidence in mouse blastocysts in relation to their age and the type of their derivation

Abstract

This study was undertaken to obtain information about apoptotic cell death incidence during embryo development up to the blastocyst stage under standard in vivo or in vitro conditions. Mouse embryos were conceived in maternal body and isolated at early or late blastocyst stage. Portion of these blastocysts was additionally cultured in vitro for 24 h. The presence of apoptotic cells in embryos was evaluated by cell death assay, based on the triple fluorescence staining, which enabled morphological assessment of nuclei (DNA staining), detection of specific DNA degradation in the nucleoplasm (TUNEL-labeling) and assessment of membrane integrity / cell viability (vital staining by propidium iodide). In our experiments, only 23 % of freshly isolated early blastocysts contained blastomeres with apoptotic features and the incidence of apoptotic cells in them was relatively low (1.19 % at average). On the contrary, groups of advanced blastocysts, derived both in vivo or by the additional culture in vitro, showed significantly higher frequency of embryos with at least one apoptotic cell (over 68 %, P<0.001) and significantly higher percentage of apoptotic cells (over 4.46 %, P<0.001). Moreover, the in vitro conditions apparently slowed down blastocysts growth and caused further elevation in cell death incidence. Our results show that the incidence of spontaneously appearing apoptosis in mouse blastocysts increases during their growth and this increase is more apparently by culture under in vitro conditions.