Progesterone and prostanoid production by bovine binucleate trophoblastic cells

Abstract

A procedure for preparing highly enriched suspensions of bovine binucleate trophoblastic cells was developed and data showing that these cells produce progesterone, prostacyclin (PGI2) and prostaglandin E2 (PGE2) were obtained. Approximately 200 x 106 enzymatically dissociated cells from bovine cotyledons were applied to the surface of a density gradient of 2% to 4% Ficoll-400 using the Wescor CELSEP sedimentation chamber. After 90-120 min of sedimentation at unit gravity, fractions containing binucleate trophoblastic cells were obtained and washed in HEPES-buffered Medium 199. Preparations of 90% to 100% binucleate trophoblastic cells were obtained routinely; variability was 50% to 80%. After incubation at 37°C, concentrations (ng/105 cells) of progesterone were greater in those fractions containing binucleate cells than in those containing primarily smaller, mononucleate cells. Total progesterone secreted (mean ± SEM) after 4 h by 1 x 105, 2 x 105, 4 x 105, 8 x 105, and 1.6 x 106 binucleate cells was 0.27 ± 0.03, 1.01 ± 0.09, 4.02 ± 0.37, 10.31 ± 0.92, and 20.96 ± 2.23 ng, respectively (r=0.997). Addition of 10% fetal bovine serum (FBS) or normal anestrous cow serum increased (P<0.05) production of progesterone by binucleate trophoblastic cells. Luteinizing hormone, follicle-stimulating hormone, prolactin, thyrotropin, and 8-bromo-adenosine 3'5'-cyclic monophosphate had no effect. Binucleate trophoblastic cells also produced PGI2 in relation to number of cells incubated (r=0.996). Time courses for production of PGI2, PGE2, and progesterone were similar. Aspirin inhibited production of PGI2 and PGE2 by about 50% at a dose of 100 μM; FBS stimulated production of both prostanoids.