317. DIFFERENTIAL EFFECTS OF cAMP AND CRH IN CELL VIABILITY AND CELL FUSION OF CYTOTROPHOBLASTIC CELLS
Y. Chen A , M. Allars A , C. Abou-Seif A , R. Smith A and R. C. Nicholson AMothers and Babies Research Centre, John Hunter Hospital, The University of Newcastle, Newcastle, NSW, Australia.
Reproduction, Fertility and Development 22(9) 117-117 https://doi.org/10.1071/SRB10Abs317
Published: 6 September 2010
Abstract
Cell fusion of cytotrophoblasts is critical for a successful pregnancy, and is marked biochemically by proteins such as human chorionic gonadotrophin (hCG) and syncytin, and morphologically as large multi-nucleated cells. Placental corticotrophin releasing hormone (CRH) plays diverse roles during pregnancy and has been linked to the length of gestation. It is well accepted that CRH expression could be upregulated by some hormones, such as hCG via cellular cAMP, and that trophoblastic cell fusion can be induced by Forskolin (increases cAMP). We have shown, by reporter gene assay in human primary placental cells, that 8-Br-cAMP increased CRH gene expression. Thus, it is of interest to determine the roles of 8-Br-cAMP and CRH on trophoblast cell viability and cell fusion. Treatment with 8-Br-cAMP for 72 h significantly inhibited BeWo cell viability (MTT assay) by 29.7% (50 μM) and 54.8% (100 μM), and increased both apoptotic and necrotic cells (FACS analysis). 8-Br-cAMP (100 μM, 48 h) resulted in the appearance of nuclear fragments (DAPI stain) and Fas ligand gene expression by 6.8-fold (real-time RT-PCR). Syncytin 1 mRNA increased by 3.2-fold (real-time RT-PCR) and hCG secretion increased by 4-fold (ELISA assay). The formation of syncytium (CellMask and Hoechst co-stain) could clearly be seen by 72 h. CRH mRNA increased with 8-Br-cAMP treatment of BeWo cells (15-fold at 48 h). Treatment with CRH (100 nM) had a mild inhibitory effect on cell growth (~18%) but no effect on cell viability, up-regulated the expression of syncytin 1 mRNA (2.3-fold at 48 h) and induced cell fusion (72 h), but had no effect on hCG secretion. In summary, we show that 8-Br-cAMP and CRH are both potential inducers of cytotrophoblast cell fusion, and there is a dissociation between morphological and biochemical differentiation. Our data also indicates that the process of cell fusion can be associated with both apoptotic and non-apoptotic events.
