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Implications of Progesterone Metabolism in MA-10 Cells for Accurate Measurement of the Rate of Steroidogenesis

Department of Endocrinology and Reproduction (F.F.G.R.), Erasmus University, 3000 DR Rotterdam, The Netherlands; Scott Department of Urology (S.R.K.), Huffington Center on Aging, and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030; and Department of Chemical Endocrinology (P.N.S.), University Medical Center Sint Radboud, 6500 HB Nijmegen, The Netherlands

Address all correspondence and requests for reprints to: F. F. G. Rommerts, Department of Endocrinology and Reproduction, Erasmus University, dr. Molewaterplein 50, 3000 DR Rotterdam, The Netherlands. E-mail: rommerts{at}endov.fgg.eur.nl

In virtually all studies with MA-10 cells, progesterone RIAs have been used to measure steroid synthesis. To test whether progesterone is a stable end product, we investigated the metabolism of added tritiated progesterone and pregnenolone in MA-10 cells over a period of 3 h. Steroids were then extracted, separated by HPLC, and identified by GC/MS. We found that more than 70% of radiolabeled steroids were converted to at least five different metabolites. A major metabolite (40%) was 5-pregnan-3 or 3ß-ol-20one. Similar studies, using radiolabeled T, demonstrated conversion to dihydrotestosterone and two forms of 5-androstane-diols. These data indicate the presence of active 5-reductase and 3- and/or 3ß-hydroxysteroid dehydrogenase activities in MA-10 cells.

Because these results suggest that progesterone is an unstable end product, to gauge the level of active metabolism, we incubated cells in the presence of inhibitors of pregnenolone metabolism and assessed pregnenolone levels by RIA. We discovered that basal levels of steroidogenesis in MA-10 cells were considerably higher than previously estimated. Moreover, dibutyryl cAMP-stimulated steroid production was linear over more than 13 h, in contrast to previous findings that measured progesterone levels. Other consequences of inaccurate assessment of steroidogenic activity in MA-10 cells because of the application of the progesterone assay are discussed.

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