APOPTOSIS DURING MOUSE BLASTOCYST FORMATION - EVIDENCE FOR A ROLE FORSURVIVAL FACTORS INCLUDING TRANSFORMING GROWTH-FACTOR-ALPHA

Mouse blastocysts undergo cell death in the inner cell mass (ICM) as a normal feature of development, but little is known as to how this eve nt is regulated or as to the possible role of survival factors in prei mplantation development. The observation that growth factors, which ca n influence preimplantation development, can act as survival factors i n other cell types led us to investigate the effects of culture in vit ro, embryo density during culture, and transforming growth factor alph a (TGF alpha) on cell death in the blastocyst. Mouse blastocysts cultu red singly from the 2-cell stage in 25 mu I of medium KSOM + amino aci ds showed a similar to 3-fold increase in the incidence of cell death, predominantly in the ICM, relative to blastocysts formed in vivo. Inc reasing the density of embryo culture to 30 embryos per 25 mu I of cul ture medium accelerated development, increased final blastocyst cell n umber, and partially (similar to 50%) reduced the increase in cell dea th induced by culture in vitro. Addition of 0.1 pM TGF alpha to the me dium of singly cultured embryos also partially (33%) reduced this incr ease in cell death without accelerating development or increasing fina l cell number. Culturing isolated ICMs for 24 h in the presence of 0.1 pM TGF alpha also partially (33%) reduced the increase in cell death, Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of whole blastocysts confirmed that cell death as detected by fragmen ted nuclei was apoptotic, as defined by endonuclease activation. Resul ts of these experiments suggest that endogenously produced growth fact ors may function as cell survival factors during preimplantation devel opment.