Discordant Secretion of Placental Protein Hormones in Differentiating Trophoblastsin Vitro

Abstract

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 .times. 105 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 immunohistochemical 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.