Inhibitory effect of IGF-I on induced apoptosis in mouse preimplantation embryos cultured in vitro

Abstract

Insulin-like growth factor I (IGF-I) has been shown to promote mammalian early embryo development. Increased cell division or decreased cell death have been proposed as two main possible mechanisms in its effect. Here we examine the nature of this promoting effect in a model situation. Camptothecin (0.01 μg/ml) and actimomycin D (0.005 μg/ml) were used to induce apoptosis. Four-cell mouse embryos were cultured in vitro to blastocyst stage in the temporary (15 h) presence or absence of apoptotic inductors and in the permanent presence or absence of IGF-I (100 ng/ml). Embryos were assessed by morphological triple staining (Hoechst 33342, propidium iodide, Calcein AM) and comet assay on Day 5, 120 h after administration of hCG. The number of nuclei, the blastocyst formation, the proportion of embryos containing fragmented DNA and the percentage of apoptotic and secondary necrotic nuclei were assessed. Both inductors of apoptosis significantly increased the percentage of apoptotic and secondary necrotic cells and reduced total cell counts (camptothecin, P>0.001; actinomycin D, P>0.001). When IGF-I was added to the culture medium in the presence of an apoptosis inductor, apoptosis incidence was significantly decreased ( P<0.001). The addition of IGF-I into control samples also decreased the percentage of apoptotic and secondary necrotic cells. In contrast, IGF-I addition had no significant influence on embryo development ( P>0.05). Our data suggest a primary role for IGF-I as an apoptotic survival factor in mouse preimplantation embryos in specific conditions.