Fusion of villous trophoblast can be visualized by localizing active caspase 8

Villous cytotrophoblast differentiation and subsequent fusion with the overlying syncytiotrophoblast depend on multiple factors such as growth factors, cytokines, hormones, protein kinases, transcription factors, structural membrane proteins and proteases. Caspase 8, an aspartate-specific cysteine protease, is mainly known for its role in programmed cell death, but was also demonstrated to be crucial for villous trophoblast differentiation. This study aimed to localize active caspase 8 in the villous trophoblast layer of human first trimester placenta. To this end, immunofluorescence double staining was performed, using a monoclonal rabbit antibody against cleaved caspase 8 in combination with antibodies against cytokeratin 7, chorionic gonadotropin beta subunit (beta hCG), beta-actin, placental protein 13 (PP13), alpha-fodrin and Ki-67. Immunofluorescence revealed cleaved caspase 8 in one out of 422 villous cytotrophoblasts resting on the basement membrane, in one out of 759 perinuclear regions within the syncytiotrophoblast and in few trophoblasts located between these two layers. Double staining of cleaved caspase 8 and Ki-67 antigen revealed that caspase 8 is activated only in cytotrophoblasts which have left the cell cycle. The staining suggests that caspase 8 is activated in villous cytotrophoblasts just prior to fusion of these cells and escorts the nuclei from the mononucleated to the syncytial state.