Localization of nitric oxide synthase in human trophoblast cells: Role of nitric oxide in trophoblast proliferation and differentiation

PROBLEM: There are conflicting reports about the isoform of nitric oxide sy nthase (NOS) present in trophoblast cells. In this study, we have examined the presence of different NOS isoforms in trophoblast cells. In addition, t he role of nitric oxide (NO) in trophoblast function has also been studied by investigating the possible role of nitric oxide in trophoblast prolifera tion and differentiation. METHOD OF STUDY: NOS isoforms in primary-term trophoblast and JEG-3 cells w ere identified by immunocytochemistry. The intracellular localization of th is enzyme was determined by confocal laser scanning microscopy. Trophoblast proliferation was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl ret rasolium bromide (MTT) conversion assay and cellular differentiation was mo nitored by human chorionic gonodotropin (hCG) and progesterone secretion, m easured by radioimmunoassay. RESULTS: The immunoreactive NOS was present in human trophoblast cells of n ormal term placenta and JEG-3 cells (a choriocarcinoma cell line) maintaine d in culture. Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase activity overlapped with the immunostaining of NOS. Specific an tibodies against the different isoforms of NOS detected the presence of neu ronal-type NOS (nNOS) only. The other two isoforms, i.e., eNOS (endothelial ) and iNOS (macrophage specific) were completely absent. The nNOS was local ized in cell cytoplasm. In culture, JEG-3 cells normally undergo proliferat ion and cytotrophoblast cells in primary culture differentiate to form horm one-secreting syncytial cells. Sodium nitroprusside (SNP), a nitric oxide d onor, when added to the culture, significantly increased proliferation of J EG-3 cells and inhibited the differentiation of cytotrophoblast cells. The arrest by SNP in the formation of syncytial cells was further evidenced by the low secretion profile of hCG and progesterone. CONCLUSIONS: Our findings suggest for the first time the presence of nNOS i n the human trophoblast cells and a previously unrecognized role of NO in t rophoblast proliferation and differentiation.