Discordant secretion of placental protein hormones in differentiating trophoblasts in vitro

The regulation of trophoblast secretion of the placental proteins CG (hCG), placental lactogen (hPL), and pregnancy specific-beta-1-glycoprotein (SP-1) has not been fully elucidated. We therefore studied the secretion of hCG, hCG-beta subunit, hCG-alpha subunit, hPL, and SP-1, both in the basal state and after exposure to 8-bromo-cAMP, during the in vitro differentiation of cytotrophoblasts to syncytiotrophoblasts. Term placental tissue was enzymatically digested and cytotrophoblasts purified by Percoll density gradient centrifugation. At the time of seeding of 4-6 X 10(5) cells/ml in 35-mm flasks all of the cells were mononuclear and 48% contained hCG-alpha, but none contained hCG or hCG-beta by the avidin-biotin-peroxidase immuno-histochemical method. After 3 days in culture, hCG-alpha and hCG or hCG-beta were present in multinucleated syncytiotrophoblasts and in the mononuclear cytotrophoblasts. During the 5 days in culture, the secretion of hCG, hCG-alpha, hPL and SP-1 into the media increased and reached a maximum on day 4 followed by a decrease on day 5. Basal hCG-beta secretion was very low and did not change during culture. The ratio of hCG-alpha/hCG decreased from days 1-4 of culture. Incubation with 8-bromo-cAMP for 24 h stimulated the secretion of hCG and hCG-alpha, whereas hCG-beta and hPL levels did not change. The secretion of SP-1, however, was inhibited by 8-bromo-cAMP. These results indicate that the cytotrophoblasts secrete hCG, hCG-beta and hCG-alpha during in vitro differentiation into syncytiotrophoblasts. Since the basal ratio of hCG to hCG-alpha secretion changed during 5 days in culture and a cAMP analogue differentially modulated the secretion of the different placental protein hormones, the physiological regulation of secretion of each of the proteins also may differ.