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

Abstract

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.

Steroidogenic activities in MA-10 Leydig cells are differentially altered by cAMP and Müllerian inhibiting substance

“…The 5␣-reduced forms of progesterone are the major steroid hormones produced by MA-10 cells [21]. By inducing Cyp17 expression, cAMP essentially switches the MA-10 Leydig cell from being a cell producing predominantly 5␣-reduced progesterone metabolites to one that is much more efficiently converting progesterone to androstenedione and its metabolites (Fig.…”

“…MA-10 cells are a mature mouse Leydig cell line that we and others have used to recapitulate the effects of exogenous treatments on steroidogenesis [20,21]. We have previously shown that there is a several-fold induction of Cyp17 mRNA in MA- 3 Hprogesterone substrate as described in Section 2 with or without MK386, a 5␣-reductase type 1-specific inhibitor.…”

“…MA-10 cells express the MIS type II receptor (MISRII) [4], which is required for MIS signal transduction [4,15] and they respond to LH and cAMP by increasing steroid hormone production [20], including low but measurable amounts of testosterone [6]. The major metabolites of MA-10 cells with and without induction by hCG have been described [21] but their steroidogenic responses to MIS have not been thoroughly investigated.…”

“…The elution fraction of tritiated standards are shown with P, progesterone; A, androstenedione; T, testosterone; 17-OHP, 17-hydroxyprogesterone. 5␣,3␣/␤-P are known metabolites of progesterone[21].…”

Effects of Estradiol and Methoxychlor on Leydig Cell Regeneration in the Adult Rat Testis

“…Because testosterone is the major androgen produced by adult rat Leydig cells and DIOL is the major androgen produced by immature Leydig cells [10,27], serum testosterone and DIOL concentrations were measured with a tritium-based radioimmunoassay, as described [28]. Inter-assay and intra-assay variations of the testosterone and DIOL were between 7% and 8%.…”