17β-hydroxysteroid dehydrogenase activity in cultured myometrial cells: Effect of serial subcultures

Abstract 17β-Hydroxysteroid dehydrogenase (17β-SDH) activity was studied in culture ovine rayometrial cells. After primary culture, cells were routinely subcultured (every 7th day), seeded at 5–10 5 cells per dish and grown in a medium with 2% of serum. 17β-SDH activity was measured by incubating intact cell monolayers with [ 3 H]-estradiol (5·10 −6 ) in serum-free medium. Metabolites were extracted from both cells and medium, and separated by thin-layer chromatography. 17β-SDH was expressed as total E i formed (cells + medium) in fmol/mg of protein as a function of time. 17β-SDH has an approximate K m of 5–10 −6 M. After 3 min of incubation, all measurable E 1 is within the cells; it is progressively released but after l h only 40% of E 1 is found in the medium. 17β-SDH decreases from day 2 to day 8 of each subculture, whereas total proteins increase. Subculture partially restores 17β-SDH activity so that it is always higher on day 2 of any subculture than on day 8 of the previous one. however a progressive decline occurs with successive subcultures. This decline parallels the slowing of cell growth and overall protein synthesis and probably reflects cell ageing.