17 beta-hydroxysteroid dehydrogenase activity in cultured myometrial cells: effect of serial subcultures

17 beta-Hydroxysteroid dehydrogenase (17 beta-SDH) activity was studied in culture ovine myometrial cells. After primary culture, cells were routinely subcultured (every 7th day), seeded at 5 x 10(5) cells per dish and grown in a medium with 2% of serum. 17 beta-SDH activity was measured by incubating intact cell monolayers with [3H]-estradiol (5 x 10(-9) M) in serum-free medium. Metabolites were extracted from both cells and medium, and separated by thin-layer chromatography. 17 beta-SDH was expressed as total E1 formed (cells + medium) in fmol/mg of protein as a function of time. 17 beta-SDH has an approximate Km of 5 x 10(-6) M. After 3 min of incubation, all measurable E1 is within the cells; it is progressively released but after 1 h only 40% of E1 is found in the medium. 17 beta-SDH decreases from day 2 to day 8 of each subculture, whereas total proteins increase. Subculture partially restores 17 beta-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.