Estrogen synthetase (aromatase) activity in primary culture of human term placental cells: effects of cell preparation, growth medium, and serum on adenosine 3',5'-monophosphate response.
The establishment of human term trophoblast cells in culture is dependent on the method of cell preparation, growth medium used, and presence of serum. Using freshly isolated term placental cells, we investigated 1) the effects of two different methods of removal of nontrophoblast cells and two culture media on trophoblast aromatase specific activity, cellular morphology, and hCG secretion over 72 h; and 2) the sensitivity of these hormonal parameters to cAMP added 24 h after the initiation of culture. Under conditions in which aromatase is responsive to cAMP, we studied the effect of removing serum after 24 h in culture with serum. After trypsin digestion of placental villi, isolated trophoblast cells were either treated with ammonium chloride (A) or purified on Percoll density gradients (P) and then grown in monolayer culture with medium 199 plus 10% fetal bovine serum (M) or Dulbecco's Modified Eagle's Medium (DMEM) plus 20% fetal bovine serum (D). Regardless of the method of treatment or growth medium used, aromatase specific activity increased 10- to 15-fold 24 h after plating, continued to increase from 24-48 h, and then decreased (AM) or remained constant (AD, PM, and PD) from 48-72 h. Addition of cAMP significantly increased activity in DMEM-grown cells (PD or AD) at both 48 and 72 h. Aromatase activity in PM cells grown with cAMP increased at 48 h, then decreased to near-control levels by 72 h; however, in AM cells, no response to cAMP was observed relative to control cells. Secretion of hCG was suppressed for the first 48 h in all cultures, but increased by 72 h (greatest increase in AM cultures). cAMP significantly increased hCG secretion 48 h after its addition under all conditions. We further evaluated the response of the Percoll-purified DMEM-grown cells (PD) to cAMP after serum removal at 24 h. Cells deprived of serum showed significantly higher aromatase specific activity over the entire culture period compared with serum-grown cells. Secretion of hCG increased 2- or 3-fold in the presence or absence of serum, respectively, after 72 h in culture. cAMP increased aromatase specific activity by 1.8- and 1.4-fold at 48 h and by 2.5- and 2.4-fold at 72 h in serum-containing and serum-free cells, respectively. The secretion of hCG increased 11- to 14-fold at 72 h under both serum-containing and serum-free conditions, respectively. The results show that cAMP can act as an intracellular messenger in the regulation of both aromatase activity and hCG secretion.(ABSTRACT TRUNCATED AT 400 WORDS)
Lobo
J O
JO
Department of Biological Sciences, State University of New York, Buffalo 14260.
Bellino
F L
FL
D001141:Aromatase / Q000378:metabolism*; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000378:metabolism; D003470:Culture Media; D000242:Cyclic AMP / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D010920:Placenta / Q000166:cytology / Q000201:enzymology*; D011247:Pregnancy; D011506:Proteins / Q000032:analysis; D014327:Trophoblasts / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism
D006063:Chorionic Gonadotropin; D003470:Culture Media; D011506:Proteins; D000242:Cyclic AMP; D001141:Aromatase