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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
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Abstract |
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-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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydroprogesterone are produced and few other metabolites have
been identified during short-term incubations, changes in the levels of
progesterone, as determined by RIA, have been used as a standard
measure for the overall steroidogenic activities of MA-10 cells. This
procedure has been used by nearly all investigators over a period of 15
yr. However, it has been reported that accumulated progesterone in
medium of stimulated MA-10 cells is decreased to almost basal levels
after 24 h of incubation (3). This clearly indicates
active metabolism of progesterone. Although the linear increase in
progesterone levels in the incubation medium, by stimulated MA-10
cells, during the first hours suggests that progesterone is not
metabolized at this time, progesterone levels do not further increase
after 6–8 h (1, 4, 5, 6). This has been widely interpreted
to mean that with long-term stimulation, there is an exhaustion of
steroid-synthesizing capabilities or a desensitization to trophic
stimulus in MA-10 cells, resulting in a decline in steroidogenesis.
However, levels of 20-dihydroprogesterone increase linearly over at
least 12 h (1). Moreover, normal Leydig cells
isolated from mouse testes do not show such a tendency for decreased
steroid production, not even after 24 h of stimulation (7, 8). The diminished accumulation of progesterone after 6–8 h
could also result from an increased influence of progesterone
metabolism. Although only a limited variety of progesterone byproducts
have been identified, major metabolites may have escaped detection
because of limited separation power of the previously used
chromatography systems (1, 5). If the level of
progesterone metabolism is indeed significantly underestimated,
progesterone assays will not give an accurate measure of total
steroidogenesis in MA-10 cells. In fact, they could provide misleading
data. We (see Refs. 10 and 11) have previously established conditions to measure total steroidogenesis in immature rat Leydig cells, which are very active in the metabolism of T (9). This was possible by completely inhibiting the metabolism of pregnenolone without affecting its biosynthesis (10, 11). In the presence of these inhibitors, the only steroidogenic product made by these rat Leydig cells is pregnenolone. By measuring this immediate end product of cholesterol side-chain cleavage activity, we were able to measure the true rate of steroidogenesis in rat Leydig cells without complications of steroid metabolism. In a study on LH receptor activation by different hCG preparations and using MA-10 cells, we found that the measured biological activity of the various hCG preparations depended on the type of RIA (for progesterone or for pregnenolone) used for measuring steroid production (12). We suggested that the discrepancies between the results of the two assays could result from progesterone metabolism. For this reason, we have reinvestigated the steroid metabolic enzyme activities in MA-10 cells. We have also evaluated whether the pregnenolone method, previously used for rat Leydig cells, can be applied to MA-10 cells and also how this method compares with the progesterone assay.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,5-epoxy-17ß-hydroxy-4ß,17-dimethyl-3-oxo-androstane-2-
carbonitrile) was a gift from Sterling-Winthrop (New York,
NY). Finasteride
[17ß-(N-tert-butylcarbamoyl)-4-aza-5-androstan-1-en-3-one]
was a gift from Merck & Co., Inc. (Rahway, NJ).
[7-3H]-Pregnenolone,
[1,2,6,7-3H]-progesterone (3.37 TBq/mmol),
[1,2,6,7-3H]-T (3.63 TBq/mmol) were purchased
from Amersham Pharmacia Biotech (Buckinghamshire, UK). MA-10 cells (1) were generously donated by Dr. Mario Ascoli (University of Iowa, Iowa City, IA). Cells were grown under standard conditions in RPMI-1640 medium supplemented with 10% horse serum, 100 µg/ml streptomycin sulfate, 100 IU/ml penicillin, and 2 mM L-glutamate, at 37 C, under humidified atmosphere containing 95% air-5% CO2. The cells were subcultured after trypsin treatment (0.05% wt/vol trypsin), and experimental cultures were plated and cultured for 2 d. After washing twice with RPMI-1640 containing 0.1% BSA, approximately 106 cells were used per experiment.
Steroids were measured in the incubation medium and/or in the cells
after incubation at 37 C for various periods in RPMI-1640 medium or in
Krebs Ringer buffer solutions, containing either 0.1% BSA or 1% FCS.
Depending on the type of experiments, epostane (5 µM) was
added from stock solution to inhibit 3ß-hydroxysteroid dehydrogenase
activity. In some experiments, 20 µM SU-10603 was also
added to inhibit residual activities of 17-hydroxylase/desmolase.
Steroids in culture medium (300 µl) or sonified cells (see below)
were measured with RIAs previously described for pregnenolone
(10, 11), 20-dihydroprogesterone (13), and
progesterone (14). The steroid specificities of the three
antisera used are given in Table 1
. For
measurement of cellular steroid levels, cells were washed with
RPMI-1640 medium and were trypsin treated (0.05% wt/vol). After
sonification, 300 µl of the resulting homogenate was used for
measurement of steroid levels. For investigation of progesterone,
pregnenolone, or T metabolism, 0.03–30 nmol
3H-labeled steroids were added to the culture
medium. After incubation for 3 h, culture medium was extracted
three times with ethyl acetate, and the metabolites were separated by
HPLC with online radiodetection as described previously
(15). In short, metabolized steroids were separated on a
Hibar LiChrosorb Diol-column (length, 250 mm; diameter, 5 µm;
Merck & Co., Inc., Rahway, NJ), equipped with a
guard column (Resolve Silica, Waters Corp., Milford, MA).
The HPLC-system included a Waters 610 Fluid Unit, a Waters 600E System
Controller and a Waters U6K injector. The isocratic flow of the mobile
phase (hexane/propanol 96:4, vol/vol) was 1.5 ml/min. Radioactivity was
monitored with a FloOne ß Radiomatic A500 radiochromatography
detector (Packard-Canberra Benelux, Tilburg, The Netherlands) with
a 500-µl cell and a liquid scintillation flow of 1.5 ml/min
(Aqua-Luma, Lumac-LSC, Olen, Belgium).
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Data are expressed as means ± SD and analyzed for statistical significance using the t test. Values of P less than 0.01 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Results from the progesterone
assay confirm what has been observed previously for MA-10 cells:
control cells secrete very small amounts of progesterone, and the
production is increased approximately 200-fold after hCG stimulation.
When MA-10 cells were incubated in the presence of epostane and
SU-10603, which completely inhibit 3ß-hydroxysteroid dehydrogenase
and 17-hydroxylase/desmolase activity in rat Leydig cells,
progesterone synthesis was reduced by nearly 85%. The data further
confirm that MA-10 cells also, in addition to progesterone, produce
20-dihydroprogesterone. The generation of this steroid was similarly
stimulated by hCG and affected by the presence of these inhibitors of
pregnenolone metabolism. When MA-10 Leydig cells were incubated with
these inhibitors and the RIA for pregnenolone was applied, higher
amounts of steroids were measured than with the progesterone assay.
Surprisingly, when epostane and SU-10603 were omitted, there was only a
marginal effect on the outcome of the pregnenolone assay. This is
particularly remarkable because the inhibitors were active, as could be
concluded from the concomitant attenuation of progesterone production.
This apparent discrepancy can be understood if pregnenolone-like
metabolites are formed that interact with the antipregnenolone antibody
(Table 1
).
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). When 1000-fold more progesterone was
added, a level similar to that present when cells are maximally
stimulated, approximately 70%, was metabolized during 3 h of
incubation. These data show that added progesterone is extensively
metabolized by MA-10 cells and a major metabolite that is formed
is compound A. Moreover, the extent of
this metabolism and the pattern of the metabolites depends on the
concentration of progesterone or pregnenolone used (Fig. 1 and Table 3). When cells are incubated with pregnenolone, a major part of
this precursor is also converted to compound A, and only a minor
fraction to progesterone. In the presence of epostane and SU-10603,
there was a higher yield of pregnenolone, but the major steroid present
was still compound A, not pregnenolone. Higher concentrations of these
drugs failed to further inhibit pregnenolone metabolism (data not
shown).
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-pregnan-3ß-ol-20one or
5-pregnan-3-ol-20one, indicating that 3 and/or
3ß-hydroxysteroid dehydrogenase and 5-steroid reductase enzyme
activities are very active in MA-10 cells. These enzyme activities,
together with the known
5-4 isomerase and
20-hydroxysteroid dehydrogenase activities, can give rise to many
different products. We have not attempted to characterize the remaining
metabolites; but, to measure the 3 and/or 3ß-hydroxysteroid
dehydrogenase and 5-steroid reductase enzyme activities without
interference of 5-4
isomerase and 20-hydroxysteroid dehydrogenase activities,
steroid metabolism was also investigated using T as a substrate. The
results in Fig. 2 and Table 4 show that T was metabolized to
dihydrotestosterone, 5-androstane-3,17ß-diol, and probably
5-androstane-3ß,17ß-diol. From these results it can again be
inferred that both 3/ß-hydroxysteroid dehydrogenase and
5-steroid reductase enzyme activities are active in MA-10 cells.
Similar to when progesterone or pregnenolone was used as a substrate,
the percent conversion of T decreased when higher concentrations of
substrate were used.
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-steroid reductase is a major activity and 5-reduced
steroids could be preferential substrates for 3- or
3ß-hydroxysteroid dehydrogenases, we also attempted to inhibit
conversion of radiolabeled pregnenolone through this metabolic pathway,
by inclusion of finasteride in the mixture of inhibitors.
At a concentration of 5 µM, 5-steroid reductase
activity in MA-10 cells was indeed inhibited (Table 3). Although, under
these conditions, more pregnenolone was recovered and less metabolites
were formed, the inhibition of pregnenolone conversion was still
incomplete. We have not used this compound as an additional inhibitor
for measuring endogenous steroid production, because 3 µM
finasteride can reduce cytochrome P450scc enzyme activity
by more than 50% in MA-10 cells (17).
Because the 5-pregnan-3/ß-ol-20one isomers, progesterone
(4-pregnen-3,20-dione), and 5-pregnan-3,20-dione have 50%, 20%,
and 20% cross-reactions, respectively, with the antipregnenolone
antibody, the levels of these steroids are also partly measured with
the pregnenolone RIA. Using this nonspecific assay, the total
endogenous production of progesterone, pregnenolone,
5-pregnan-3/ß-ol-20one and possibly other pregnenolone-like
steroids seemed to be linear up to 13 h (Table 5). A substantial fraction of this
steroid mixture was cellular-bound; and therefore, this fraction cannot
be ignored when assessing total steroid synthesis. The results also
showed that, in the presence of the cholesterol side-chain cleavage
enzyme inhibitor aminoglutethimide (AMG) (18), endogenous
steroid production is inhibited by more than 99%. Thus, the
nonspecific pregnenolone assay does not measure nonsteroidal compounds
in the culture medium. When MA-10 cells were preincubated in the
presence of AMG and dbcAMP for 13 h, upon removal of AMG, the rate
of steroidogenesis was 2-fold higher than in MA-10 cells treated for
only 2 or 4 h with dbcAMP alone (Table 5, ratio d/a or d/b). This
indicates that the rate of steroid production after long-term
stimulation of MA-10 cells does not decrease but shows a clear tendency
to increase.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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If this occurs in our experiments, the pattern of steroid metabolites
from added labeled pregnenolone or progesterone may be slightly
different from the endogenously produced steroids. Still, the observed
metabolism of added progesterone, pregnenolone, or T clearly shows that
there are active steroid metabolizing enzymes present in MA-10 cells.
Irrespective of the exact profile of the metabolites, active metabolism
of progesterone will lead to an underestimation of the true
steroidogenic activities of the MA-10 cells. A similar problem was
previously encountered when the steroidogenic activity of immature rat
Leydig cells (9) or rat H540 tumor cells (21)
was measured with an RIA for T. Unlike mature Leydig cells, immature
rat Leydig cells and rat H540 tumor cells are very active in
metabolizing T; and hence, the measurement of T as an end product of
steroid synthesis grossly underestimates the steroidogenic activity of
these cells. Accurate analysis of steroidogenesis in these rat Leydig
cells, however, is possible because metabolism of pregnenolone can be
abolished after inhibitors are added and pregnenolone accumulation is
assessed by RIA (10, 11). These inhibitors also reduced
pregnenolone conversion in MA-10 cells, but the inhibition was
incomplete. However, because the major metabolites,
5-pregnan-3/ß-ol-20one and progesterone, also partly
cross-react with the antipregnenolone antibody, this nonspecific RIA
gives a better, though not perfect, impression of the total
steroidogenic activity than can be derived from specific progesterone
measurements. Similar findings were reported by Purvis et
al. (9) in 1978, when they were able to show hCG
responsiveness of immature Leydig cells with a nonspecific 17ß-
hydroxyandrogen assay but not with a specific T assay.
Unfortunately, our attempts to improve the reliability of the
pregnenolone assay for MA-10 cells by adding finasteride
to further inhibit pregnenolone metabolism were unsuccessful. Although
the pregnenolone assay is imperfect, it gives a much better impression
about steroid production than can be derived from progesterone assays.
The discrepancies between the two assays seemed most pronounced when
low amounts of steroid were produced, as from unstimulated MA-10 Leydig
cells, or when high amounts of steroids were produced, when cells are
chronically stimulated. Some examples and consequences of inaccurate
assessment of steroidogenic activities in MA-10 cells in the literature
are discussed below.
Our observations with the pregnenolone assay of unstimulated MA-10 cells show that these cells produce more steroids than previously thought. The reported very low progesterone production by unstimulated MA-10 cells (1, 4, 5) thus seems to be more a reflection of a high metabolic rate than of a very low rate of steroidogenesis. If small amounts of progesterone are almost completely metabolized, this could also explain why progesterone synthesis in MA-10 cells responds relatively slowly to hCG stimulation, when compared with T production in normal mouse (22) or rat (23) Leydig cells.
Because unstimulated MA-10 cells are more active in steroidogenesis than heretofore recognized, this also affects the observed stimulation factor of steroid synthesis induced by the addition of trophic hormones. The stimulation factor in MA-10 cells, using the pregnenolone RIA, was approximately 30. This is very close to the data published for rat Leydig cells but much lower than the more-than-200-fold or higher acute stimulation factors derived from progesterone measurements in MA-10 cells (4, 6, 24). Thus, progesterone assays may give a false impression of the kinetics of steroid production and its responsiveness to hormonal stimulation.
Our observations with the pregnenolone assay showed that stimulated
MA-10 cells continue to produce steroids over a period of at least
13 h. This corroborates an earlier observation that the production
of 20-dihydroprogesterone, but not progesterone, is constant for at
least 12 h (1). It also seems, from our data, that
the rate of steroid production does not decrease over this time but may
instead increase, given that steroidogenesis after 13 h is higher
than the rate of steroid production during the first 4 h. This is
in line with the reported increase in cholesterol side-chain cleavage
enzyme activity after 24 h incubation with dbcAMP
(24).
Thus, the plateau of progesterone levels in culture medium after incubation of MA-10 cells for 6–8 h (1, 4) most likely results from a high rate of progesterone metabolism and does not originate from a decline in steroid synthesis. This influence of metabolism is often ignored, and changes in progesterone levels are often interpreted to be a direct consequence of changes in steroidogenesis. For instance, changes in progesterone have been presented, in a relative fashion, as a percentage of maximal expression, with the maximal level of progesterone taken as 100%, and these relative steroid values have been compared with StAR mRNA levels (4). Because progesterone and StAR mRNA levels develop in a similar fashion and both reach a plateau after approximately 6 h, it was concluded that the rate of StAR mRNA synthesis parallels that of steroid production. However, when progesterone metabolism is taken into account, the plateau probably more accurately reflects a balance between metabolism and production, not a slowdown in steroid synthesis. In addition, using the pregnenolone assay, the present data show that the true steroidogenic activity and StAR mRNA levels do not correlate after 6 h. Presumably, the early accumulation and later decline in StAR mRNA levels should yield changes in the levels of StAR protein and, thus, changes in the rate of steroid production. However, our data from the pregnenolone assay, as well as the data from the progesterone assays up to 6 h, revealed a linear increase in steroid levels and thus a constant rate of steroidogenesis. Therefore, the significance of changes in StAR mRNA levels for steroidogenic activity remains unclear at this time.
Discrepancies between the apparent and the real steroidogenic activity are very clear after prolonged incubation periods, because progesterone levels in media from stimulated cells not only reach a plateau but also diminish after 6 h of stimulation (3). Because of this metabolism of progesterone, the apparent level of steroidogenesis in MA-10 cells induced by LH was found to be 10-fold less when an incubation period of 24 h was used instead of 4 h (3).
Because of the observed discrepancies between real and apparent steroidogenic activities, not only during prolonged incubation periods of stimulated cells but also during short-term incubations of unstimulated cells, it remains to be determined whether reliable information can be obtained concerning the fine regulation of steroidogenesis from progesterone measurements. By using normal mouse Leydig cells and measuring T levels, important information on the regulation of steroid production was obtained from careful kinetic studies over 4- to 5-h periods (22). It was shown that very low concentrations of hCG that do not give detectable changes in cAMP and PKA activation can almost maximally stimulate T production, but the increase in this steroidogenic activity is only detectable after a delay of 3–4 h (22). The authors concluded from their kinetic experiments that submaximal stimulation of steroidogenesis is more the result of a delayed onset than of a reduced rate. The slow increase in the rate of T production, against a background of basal steroid production, could only be measured because T was not metabolized. Because progesterone is not a stable end product, this important conclusion about dose-dependent regulation of steroid production cannot be validated for MA-10 cells. It seems necessary to do this because it has been suggested that considerably higher cAMP concentrations are required for acute stimulation of steroidogenesis than for synthesis of P450scc in MA-10 cells (24). This conclusion was made after incubating MA-10 cells, for 24 h, with 10 µM dbcAMP and measuring progesterone accumulation and P450scc induction.
From the available evidence, it is now clear that, during prolonged
incubations of stimulated MA-10 cells, many (mainly pregnane-derived)
steroids accumulate, because of the metabolism of progesterone. In a
recent publication on the inhibitory effect of Müllerian
inhibiting substance on the induction of steroidogenic enzymes in MA-10
cells, it was shown, for the first time, that the cyp17 gene
can be activated strongly in MA-10 cells after 18 h of stimulation
with 50 µM dbcAMP (25). This is in contrast
to previous findings that cyp17 gene and
17-hydroxylase/desmolase activity were absent in MA-10 cells and
could not be induced after exposure to dbcAMP (2).
Teixeira et al. (25) also showed that this
induction was completely quenched in the presence of müllerian
inhibiting substance. However, the reported shifts in this gene
activity, after 18 h of incubation, were not reflected in changes
in progesterone levels. Because the levels of progesterone at 24 h
and 48 h were low and not different, there was no indication of
active steroid production. Only a small increase in the very low levels
of T was observed after 48 h. From our study with MA-10 cells and
also from incubation studies in the past with immature rat Leydig cells
(20) and H540 rat tumor Leydig cells (21), it
is evident that there are many possibilities for steroid metabolism.
Chronic exposure to dbcAMP could not only further increase the activity
of 17-hydroxylase/desmolase in MA-10 cells, but that of other
enzymes as well. Under these conditions, it seems almost impossible to
relate changes in T levels to changes in cyp17 gene
activity.
Since their introduction, MA-10 cells have proven very useful for
studies on hormonal regulation of steroidogenesis. However, as this
paper shows, accurate measurement of steroidogenesis because of
progesterone metabolism is a limiting factor. The described
pregnenolone assay gives a better impression about the real
steroidogenic activity than the progesterone assay; but, for a truly
accurate assessment of steroid synthesis, further improvements in
methodology are necessary. The observed underestimation of progesterone
metabolism may be attributable to the limited capacity of TLC used in
the past to separate steroids. Only 20-dihydroprogesterone was
identified as a major metabolite (1, 5). With HPLC, more
metabolites could be identified; but even with HPLC, the major
metabolite 5-pregnane-3/ß-ol-20-one could not be completely
separated from progesterone. It is therefore possible that this
metabolite was also formed in the MA-10 cells used in the previous
studies but could not be detected with the TLC system. Also, it remains
to be investigated whether sublines of MA-10 cells with different
steroid (progesterone) metabolizing capacities now exist. If indeed,
sublines of MA-10 cells exist, with different activities of
steroid-metabolizing enzymes, this will further complicate quantitative
assessment of steroidogenesis via progesterone measurements.
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Acknowledgments |
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Footnotes |
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Abbreviations: AMG, Aminoglutethimide; dbcAMP, dibutyryl cAMP; hCG, human CG.
Received April 2, 2001.
Accepted for publication August 15, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase/C17–20
lyase cytochrome P450 expression. Endocrinology 131:2165–2172[Abstract]
in the regulation of mouse Leydig cell steroidogenesis.
Endocrinology 132:2438–2444[Abstract]
-reductase kinetic characteristics: extreme
pH-dependency of the type II isozyme in prostate and epididymis
homogenates. J Steroid Biochem Mol Biol 54: 185–192
-dihydrotestosterones mesterolone and drostanolone; gas
chromatographic/mass spectrometric characterization of the urinary
metabolites. J Steroid Biochem Mol Biol 42:411–419[CrossRef][Medline]
-hydroxylase and C
17–20 lyase activities in cultured mouse Leydig cells is enhanced
by inhibition of steroid biosynthesis. Endocrinology 118:1222–1228[Abstract]
,3ß-hydroxysteroid
dehydrogenase/5–4 isomerase, and steroid 5-reductase in rat H540
Leydig tumor cells. Mol Cell Endocrinol 74:R11–R17
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