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
alpha-pregnan-3 alpha or 3 beta-ol-20one. Similar studies, using
radiolabeled T, demonstrated conversion to dihydrotestosterone and two
forms of 5 alpha-androstane-diols. These data indicate the presence of
active 5 alpha-reductase and 3 alpha- and/or 3 beta-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.