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1
,2
* Department of Physiology and Experimental Medicine, The George Washington University School of Medical and Health Sciences, Washington, DC 20037, USA;
Department of Clinical Pharmacology, University of Bonn, 53105 Bonn, Germany; and
Departments of Chemistry and Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892, USA.
1Correspondence: Female Health Care Research, Schering AG, Muellerstrasse 170-178, 13342 Berlin, Germany. E-mail: bernhard.lindenthal{at}schering.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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24-reduction and 4
-demethylation. Progesterone,
pregnenolone, and 17-OH-pregnenolone were potent inhibitors of
24-reduction in an in vitro cell assay and led to the
accumulation of desmosterol, a 5,24 sterol precursor of cholesterol.
A markedly different effect was observed for 17-OH-progesterone,
which caused the accumulation of sterols associated with inhibition of
4-demethylation. The flux of 13C-acetate into
lathosterol and cholesterol was decreased by progestins as measured by
isotopomer spectral analysis, whereas newly synthesized MAS
accumulated. The combined evidence that MAS concentrations can be
regulated by physiological levels of progestins and their specific
combination provides a plausible explanation for the elevated
concentration of MAS in gonads and suggests a new role for progestins
in fertility.—Lindenthal, B., Holleran, A. L., Aldaghlas, T. A., Ruan, B., Schroepfer, G. J., Jr., Wilson, W. K., and
Kelleher, J. K. Progestins block cholesterol synthesis to produce
meiosis-activating sterols.
Key Words: progesterone • 17-hydroxyprogesterone • cholesterol precursors • GC-MS • isotopomer spectral analysis • HepG2 cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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suggests that C29 precursors of
cholesterol are the long-sought (2
, 3)
meiosis-inducing
substances. Two notable examples of C29
meiosis-activating sterols (MAS) are FF-MAS
(4,4-dimethyl-5-cholesta-8,14,24-trien-3ß-ol), isolated from human
follicular fluid, and T-MAS
(4,4-dimethyl-5-cholesta-8,24-dien-3ß-ol), isolated from bull
testis (1)
. Synthetic FF-MAS (1
, 4
, 5)
and
other C29 sterols (1
, 5)
overcome
meiotic arrest produced by hypoxanthine, dbcAMP, or
3-isobutyl-1-methylxanthine in naked oocytes or cumulus-enclosed
oocytes. In contrast, lanosterol, cholesterol, 5,7- and
5-C29 sterols, and several oxysterols fail to
activate meiosis (4
5
6
7)
. The proposed regulation of
meiosis by C29 sterols is supported by the
finding that MAS are synthesized in rat and human gonads (8
, 9)
, but the mechanism by which sterols activate meiosis is
unclear (10
, 11)
. Mediation by an LXR receptor, suggested
(6)
on the basis of results that some meiosis-activating
sterols are also ligands for the LXR receptor (5
6
7
, 12)
, has been discounted (12)
.
Another unsolved problem, addressed herein, is the mechanism by which
levels of MAS are regulated. Under normal metabolic conditions, the
rapid conversion of sterol intermediates to cholesterol results in low
concentrations of precursors in liver, blood, and most other tissues.
Exceptions include the occurrence of elevated concentrations of T-MAS
in testicular tissue (1
, 11)
, of FF-MAS and T-MAS in
preovulatory follicular fluid (11)
, and of desmosterol in
spermatozoa, testis (13
, 14)
, human milk
(15)
, and the developing brain (16)
. The
significant levels of MAS in gonadal tissue could result from hormonal
regulation, and progestins have long been associated with meiotic
events (17
, 18)
and with inhibition of cholesterol
synthesis in somatic cells (19
, 20)
. Extensive results in
a variety of cell lines indicate that progesterone inhibits sterol
synthesis and that the progesterone receptor is not involved
(21
22
23)
. Lanosterol and several other sterol precursors
accumulated but were not identified or characterized beyond their TLC
or HPLC mobility.
In a major extension of these (21
22
23
, 24)
and earlier
(19)
findings, we now report incubations of HepG2 cells
and rat testis with progestins at concentrations commonly found in
gonadal tissue. Our results in HepG2 cells confirm the reported
(22
23
24)
inhibition of cholesterol synthesis, the
accumulation of cholesterol precursors, and the lack of involvement of
the progesterone receptor. Moreover, we have demonstrated by GC/MS that
major accumulating sterols are identical with authentic standards of
T-MAS and FF-MAS. This critical structural information indicates a
previously unrecognized linkage between progestins and MAS. Based on
our full experimental results, we propose a mechanism leading to the
accumulation of MAS through the inhibition of two steps in the
cholesterol synthesis pathway (Fig. 1
) by specific individual progestins. Our combined results provide the
first credible explanation for the presence of micromolar levels of MAS
in gonadal tissue and bring together previously unconnected lines of
research (i.e., the biochemical role of MAS in fertility and the
effects of progestins on cholesterol biosynthesis and on meiosis).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and
used as reference compounds. 1-13C-acetate was
obtained from Cambridge Isotope Laboratories (Cambridge, Mass.). Rat
testes were obtained from Sprague-Dawley rats (weight <50 g).
Cell culture
Human hepatoma HepG2 cells were cultured to confluency in 60-mm
dishes in phenol-red-free Dulbecco‘s modified Eagle‘s medium (DMEM)
containing Pen-Strep (100 µg/ml), glucose (10 mM), sodium bicarbonate
(45 mM), and glutamine (4 mM) supplemented with 10% fetal calf serum
as described previously (24)
. At the beginning of the
experiment, the medium was changed to serum-free DMEM supplemented with
10% lipid-free controlled process serum replacement (CPSR-1).
Compounds were added from ethanolic stock solutions to produce final
ethanol concentrations of 0.1–0.3% v/v. Controls received the same
amount of ethanol. Aminotriazole was added in an aqueous solution.
Slices of rat testis (25 µm, 25–50 mg wet weight) were incubated
under the same conditions in a shaking water bath (37°C). For
isotopomer spectral analysis (ISA) of lathosterol,
1-13C-acetate was added in an aqueous solution to
a final concentration of 1 mM.
Sterol extraction and analysis
Cells were washed three times with ice-cold PBS (pH 7.4) (once
with 2 mg/ml BSA and twice with PBS alone). Epicoprostanol (1 µg in
50 µl of hexane) was added as internal standard to each sample.
Sterols were extracted in hexane/isopropanol (3:2 v/v, two times with 4
ml). After drying under nitrogen and alkaline hydrolysis (1 N NaOH in
80% ethanol, 1.5 h at 70°C), the sterols were extracted with
cyclohexane (two times with 3 ml). After drying under nitrogen, sterols
were converted to their trimethylsilyl (TMS) derivatives by adding 100
µl bis(trimethylsilyl)trifluoracetamide (Pierce)/n-decane (1:1, v/v)
and heating for 1 h at 70°C in a conical glass tube. This
solution (1–2 µl) was used for gas chromatography/mass spectrometry
(GC/MS). Rat testes (0.05–0.15 g) were homogenized manually, and
lipids were extracted in hexane/isopropanol (3:2 v/v, two times with 4
ml). The extracts were deproteinized with acetone (3 ml) and processed
as described above. The observed sterol levels in testis were
normalized to correspond to 0.1 g of tissue (wet weight).
Gas chromatography/mass spectrometry
Analyses were performed on a Hewlett-Packard GC/MS system (5890
series II GC interfaced to a 5971 mass selective detector) equipped
with a DB-XLB column (30 m x 0.25 mm i.d. x 0.25 µm film; J&W
Scientific, Folsom, CA). Gas chromatography was done in the splitless
mode with temperature programming as follows: 150°C for 1 min,
followed by 20°C/min up to 260°C and 10°C/min up to 280°C (hold
for 15 min). Mass spectral data were collected either in the full-scan
mode (m/z 50–550) or by selective ion monitoring (SIM). For
SIM analyses, the electron multiplier voltage was raised by 300 V after
the elution of lathosterol to increase the sensitivity of detection for
lanosterol, dihydrolanosterol, methylsterols, dimethylsterols, and
plant sterols. The internal standard epicoprostanol was monitored at
m/z 370. In unlabeled experiments, cholesterol precursors
and plant sterols were monitored by the following ions: desmosterol
(m/z 456; 441 and 351), 8-cholestenol (m/z
458), lathosterol (m/z 458), monounsaturated methylsterols
(m/z 472), diunsaturated methylsterols (m/z 470),
monounsaturated dimethylsterols (m/z 486), diunsaturated
dimethylsterols (m/z 484), triunsaturated dimethylsterols
(m/z 482), lanosterol (m/z 498 and 393), and
dihydrolanosterol (m/z 500 and 395). Peak integration was
performed manually, and sterols were quantified from SIM analyses
against the internal standard. Unless stated otherwise, MAS amounts are
given as a percentage of the control. When
1-13C-acetate was used in the culture medium for
ISA, up to 10 ions (M+0–M+10) were monitored for the TMS derivatives
of lathosterol (m/z 458–468), T-MAS (m/z
484–494), and FF-MAS (m/z 482–492).
24-reductase assay
HepG2 cells were incubated for 12 h with 10 µM
simvastatin (a gift from M. Stapff [MSD, Germany]) to deplete
cholesterol precursors. Thereafter, the medium was changed to one
containing 10 µM simvastatin, 10 µM miconazole (an inhibitor of
14-demethylation), and 6 µg per dish lanosterol. After 12 h
incubation, neutral sterols were extracted as described above, and the
samples were monitored for lanosterol (m/z 498
[M+] and m/z 393
[M-TMSOH-CH3]) and dihydrolanosterol
(m/z 500 [M+] and m/z 395
[M-TMSOH-CH3]) by GC/MS. Dihydrolanosterol was
quantified by comparing the ratio of SIM abundances at m/z
395 and 370 (epicoprostanol) against a standard curve constructed from
SIM results on mixtures of authentic dihydrolanosterol and
epicoprostanol. Desmosterol was quantified analogously by SIM.
Isotopomer spectral analysis
Isotopomer spectral analysis (ISA) was carried out as described
previously (25)
by incubating cells with
1-13C-acetate. The isotopomer distribution in
sterols synthesized from 1-13C-acetate provided
the fractional synthesis rate g(t) of the product and the
enrichment D of the labeled precursor pool. These parameters were
calculated by nonlinear regression analysis as described previously
(25
, 26)
. Because here we used the molecular ions
containing the TMS group for the ISA calculations, the ISA program was
modified to correct for the additional C, H, and Si atoms from the
derivatization reagent. Statistical differences were assessed by
Student’s t test and considered significant for
P<0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-OH-pregnenolone led to the accumulation of desmosterol, a
cholesterol precursor in which the 24 bond has not been reduced
(Fig. 2B
and Fig. 3B
). In contrast, 17-OH-progesterone produced an
accumulation of methyl and dimethyl sterols (Fig. 2C
),
including T-MAS and 4,4-dimethyl-5-cholest-8-en-3ß-ol, a
meiosis-activating sterol (1)
that was identified by
comparison with MS data reported by Hashimoto et al. (27)
.
These effects resemble the action of aminotriazole (Fig. 2D
), which inhibits 4-demethylation (27)
and
other heme-dependent processes that may affect sterol metabolism in
peroxisomes and endoplasmic reticulum. These accumulation patterns are
consistent with the inhibition of 24-reduction (by progesterone at
10 µM) and 4-demethylation (by 17-OH-progesterone at 10 µM
and aminotriazole at 40 mM). Compared with progestins, other hormones
(testosterone, ß-estradiol, estrone, dehydroepiandrosterone, and
androstenedione at 10 µM) produced only minor changes in the
cholesterol precursor pattern (data not shown). Despite an accumulation
of desmosterol after a 12-h incubation with 10 µM progesterone,
desmosterol amounted to only 2.1% of the cholesterol content of the
cells (5.7±0.07 µg desmosterol vs. 269±3 µg cholesterol,
n=3 incubations).
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Effect of progestins on 24 reduction and desmosterol
accumulation
To isolate 24 reduction from other steps of the pathway, the
conversion of lanosterol to dihydrolanosterol was studied in HepG2
cells by blocking further metabolism with added miconazole (Fig. 3A
). Incubations with various progestins showed the
following order of suppression of 24 reduction: pregnenolone >
progesterone > 17-OH-pregnenolone >> 17-OH-progesterone.
In the absence of miconazole, the 24 reduction step was
strongly suppressed by the antiestrogen drug tamoxifen and by
pregnenolone, progesterone, and 17-OH-pregnenolone, but not by
17-OH-progesterone (Fig. 3B
). The observed accumulation
of desmosterol in HepG2 cells in the absence of miconazole (Fig. 3B
) indicates that all steps of cholesterol biosynthesis are
intact except the 24 reduction.
Identification of T-MAS and FF-MAS
Treatment of HepG2 cells with a higher concentration of
progesterone (40 µM) produced increased levels of methylsterols
(compounds 4 and 7, Fig. 4
A) in addition to desmosterol (compound 1, Fig. 4A
). The dominant cholesterol precursor was a diunsaturated
C29 sterol also found in adult rat testis (Fig. 4B
). These sterols were both identified as T-MAS
(4,4-dimethyl-5-cholesta-8,24-dien-3ß-ol) by comparison of their
GC retention times and mass spectra (Fig. 4C
, 4D
)
with those of a synthetic standard (5)
for T-MAS. FF-MAS
(4,4-dimethyl-5-cholesta-8,14,24-trien-3ß-ol) (1)
was also detected in the HepG2 cells (Fig. 4A
, compound 8)
and testis extracts and identified by comparison of its GC retention
time and mass spectrum with those of the reference compound
(5)
. The identities of T-MAS and FF-MAS were further
confirmed by analysis of testis extracts spiked with various amounts of
authentic standards of MAS. The observed pattern of cholesterol
synthesis intermediates was consistent with inhibition of both
4-demethylation and 24 reduction.
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Synergistic and additive effects of progestins and drugs
Incubation of HepG2 cells and rat testis slices with individual
progestins and drugs altered the levels of the three
C29-sterols (Table 1
). In many cases, combinations of 17-OH-progesterone with other
progestins produced additive and/or synergistic effects (Table 1)
.
These effects were less pronounced in testis slices, possibly as a
consequence of a lower total cholesterol synthesis rate relative to
that of the highly metabolic HepG2 cells, the higher background of
pre-existing MAS in testes, and/or the shorter incubation time. As
shown in Table 1
, the effects of progestins, both alone and in
combination, were similar to those caused by aminotriazole, a known
inhibitor of 4-demethylation (27)
; tamoxifen, an
inhibitor of 24-reduction and 7-8 isomerization
(26)
; and verapamil, a calcium blocker used in treating
cardiovascular diseases. Medroxyprogesterone, a synthetic progestin
used as a contraceptive, also led to large increases in
C29-sterols. A combination of ß-estradiol (10
µM), the second major hormone in follicular fluid, and progesterone
(10 µM) did not alter the accumulation of cholesterol precursors
observed with progesterone alone (data not shown).
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T-MAS levels in the developing rat testis
To detect changes in cholesterol precursors in developing
rat testis, neutral sterols were measured by GC-MS in young rats (body
weight <60 g) and adult rats (body weight 100–350 g). The results
(Fig. 5A
) show that T-MAS levels were approximately four times
higher in the adult rats than in young rats and that T-MAS becomes the
dominant cholesterol precursor (Fig. 4B
). No other
cholesterol precursor showed such a pronounced change during
maturation. In fact, cholesterol precursors later in the biosynthetic
pathway (e.g., lathosterol and 8-cholestenol) were reduced by
50% in adult rats. Interestingly, the relationship between
4,4-dimethyl-5-cholest-8-en-3ß-ol (no 24 bond) and T-MAS (24
double bond present) exhibited a strong inverse correlation, as shown
in Fig. 5B
. This suggests that the shift of cholesterol
precursors in the developing rat testis involves impaired 24
reduction.
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Influence of progestins on flux of 13C-acetate to
lathosterol and cholesterol
To investigate possible mechanisms of action of the progestins, we
used ISA to determine the flux of 13C-acetate to
cholesterol precursors later in cholesterol synthesis. We measured the
precursor pool enrichment D and the fractional synthesis rate
g(t) of lathosterol (5-cholest-7-en-3ß-ol) in HepG2
cells in the presence of various progestins (Fig. 6
). Progesterone, 17-OH-progesterone and pregnenolone reduced de
novo lathosterol synthesis by >60%, and the total amount of
lathosterol was lowered by 50%. De novo fractional
cholesterol synthesis g(t) was 4.2% ± 0.2 (n=3)
for controls but not detectable under the influence of progestins (data
not shown). Reduced flux of 13C-acetate to
cholesterol was also indicated by the markedly lower fractional
abundance of the 13C isotopomers
([M+3]+ to [M+8]+) of
cholesterol in the treated cells (Fig. 7
). Despite the very small MS responses, we observed a reduced fractional
abundance for [M+5]+ in progestin-treated cells
(Fig. 7B
), with a dose-dependent lowering for progesterone
(Fig. 7A
). The combined results show that progestins block
de novo cholesterol synthesis at steps before lathosterol.
In contrast to lathosterol, 13C-T-MAS did
accumulate (Fig. 8
), indicating that de novo synthesis of intermediates before
24 reduction and 4-demethylation was not affected by the
progestins.
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All the added progestins produced an acetate precursor pool enrichment
D (proportion of 13C-acetate in the acetate pool
for sterol synthesis) of 0.2–0.3 (Fig. 6B
), with a slight
but statistically significant difference for progesterone and
17-OH-progesterone. This difference might arise from differing
concentrations of cholesterol precursors, which, through feedback
inhibition of HMG-CoA reductase, may ultimately affect the acetate
precursor pool. If so, investigation of effects of inhibitors of
biosynthetic pathways on precursor pools might offer a useful tool for
understanding metabolic regulation.
Accumulation of 13C-labeled T-MAS and FF-MAS in
progestin-treated HepG2 cells
To test whether the accumulating T-MAS and FF-MAS are derived from
newly synthesized sterols, we incubated HepG2 cells with progestins (10
µM) and 1-13C-acetate (1 mM) for 12 h. The
isotopomer distribution of T-MAS after incubation with
17-OH-progesterone showed a preponderance of
13C-containing species. The fit of the T-MAS
isotopomer distribution to the ISA model indicated that T-MAS is
derived primarily from new synthesis (Fig. 8A
). The effect
of various progestins on the accumulation of T-MAS and FF-MAS is shown
in Fig. 8B
and C
. These results show that highly labeled and
therefore newly synthesized MAS accumulated during incubation with
progesterone and 17-OH-progesterone.
The progesterone receptor is not involved in blocking cholesterol
synthesis
To investigate the possible involvement of the progesterone
receptor in the actions of progesterone, we incubated HepG2 cells with
the progesterone antagonist RU486 (2 µM) alone or in the presence of
progesterone (10 µM). As shown in Fig. 9A
, RU486 did not counteract the dramatic decline in the ratio
of dihydrolanosterol to lanosterol nor the accumulation of desmosterol
observed on incubation with progesterone (Fig. 9B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-OH-pregnenolone produced a
pattern of precursors consistent with inhibition of 24 reduction
(Fig. 2B
and Fig. 3
). In contrast, 17-OH-progesterone
produced a different pattern (notably methyl and dimethyl sterols)
corresponding to inhibition of 4-demethylation (Fig. 2C
).
Incubations of progestins in the presence of miconazole (Fig. 3A
), which blocks 14-demethylation, demonstrated that the
effects of the progestins on 24 reduction are not mediated through
other cholesterol precursors that may accumulate in cells when the
entire pathway is intact. 17-OH-progesterone led to the accumulation
of the same dimethyl and methyl cholesterol precursors as aminotriazole
(Fig. 2C
and D
), a known (albeit less potent) inhibitor of
4-demethylation (27)
. Our findings represent the first
report of the effects of 17-OH-progesterone on cholesterol
synthesis. These effects are distinct from the actions of other
progestins described herein. Incubations with progestins in the
presence of 13C-acetate led to the accumulation
of 13C-MAS (Fig. 8)
. These results demonstrate
that the MAS arose from de novo synthesis rather than
interconversion of pre-existing sterols. Progestins were also capable
of increasing the levels of MAS in testicular slices (Table 1)
.
We hypothesize that simultaneous inhibition of 24 reduction and
4-demethylation by progestins leads to the accumulation of MAS.
Additive and synergistic effects of 17-OH-progesterone with either
pregnenolone or progesterone on the levels of T-MAS and FF-MAS (Table 1)
are compatible with this hypothesis. Furthermore, we found that
progesterone, pregnenolone, and 17-OH-progesterone in HepG2 cells
(Figs. 6
and 7)
and rat testis slices (data not shown) suppressed the
incorporation of 13C-acetate into sterols
requiring both 24 reduction and 4-demethylation (e.g.,
lathosterol and cholesterol) (Figs. 6
and 7)
and led to the
accumulation of labeled MAS (Fig. 8)
. It is also notable that the
dominant cholesterol precursor in rat testis is T-MAS (Fig. 4B
) and that T-MAS accumulates in the developing rat testis
(Fig. 5A
), whereas levels of
4,4-dimethyl-5-cholest-8-en-3ß-ol decline during maturation (Fig. 5B
). These observations support our hypothesis that
24-reductase activity is suppressed in the adult rat testis.
The effects described herein of progestins on cholesterol synthesis
occurred at normal physiological concentrations for gonadal tissue.
Increases in T-MAS (ninefold) and FF-MAS (fourfold) occurred in the
presence of 1 µM progesterone or 1 µM 17-OH-progesterone (Table 1)
. These concentrations are comparable to reported levels in human
testes (505 ng/g [1.6 µM] pregnenolone; 306 ng/g [0.9 µM]
17-hydroxyprogesterone; and 81 ng/g [0.3 µM] progesterone
[wet weight]) (28)
and human follicular fluid (8–25
µM progesterone; 3–6 µM 17-OH-progesterone) (29)
.
In evaluating the ramifications of progestin levels, it should be noted
that a permanent induction of meiosis is required in men, whereas the
resumption of meiosis in women coincides with the progestin surge
induced by lutenizing hormone (30)
. We also found that the
effects of progesterone on the accumulation of desmosterol and on the
ratio of dihydrolanosterol to lanosterol were not modulated by the
progesterone receptor blocker RU486 (2 µM) (Fig. 8)
. This result and
our other observations are in agreement with previous reports
(19
, 21
22
23
, 32)
that steroid hormones affect cholesterol
synthesis in various cell types, leading to an accumulation of
cholesterol precursors, and that the progesterone receptor is not
involved.
In seeking an explanation for the effects of progesterone on
cholesterol synthesis, some authors have investigated mechanisms based
on the interference of progesterone with intracellular cholesterol
trafficking (23
, 31
32
33
34
35)
. Our results suggest an
additional or alternative mechanism involving direct inhibition of
specific steps of sterol synthesis. In efforts to understand the
synthetic origins of MAS, two groups have reported up-regulation of
sterol 14-demethylase (P45014DM) (8
, 9)
. Such an increase in the activity of the 14-demethylase
would likely result in simultaneous accumulation of dimethylsterols
with and without a 24 bond, whereas we observed an actual decrease
in levels of monounsaturated dimethylsterols and a concomitant increase
in levels of T-MAS in developing rat testes (Fig. 5B
). Our
results indicate impaired 24 reduction rather than enhanced activity
of the 14-demethylase. The observed pattern of precursors is
compatible with a downstream mechanism of MAS regulation consisting of
inhibition of later steps in the cholesterol biosynthetic pathway to
divert flux into MAS. Under normal circumstances this pathway operates
such that cholesterol precursors do not accumulate. Interestingly,
progesterone (10 µM) has been shown to increase the activity of
HMG-CoA reductase in human fibroblasts (33)
. This
observation is consistent with decreased feedback inhibition of
cholesterol (or C27-oxysterols) on HMG-CoA
reductase, effects that would be anticipated from our finding that
added progesterone decreases flux from
13C-acetate to lathosterol.
Although the suggested connections between progestins, MAS, and
fertility represent an attractive explanation that unifies a
considerable body of observations, our results do not exclude
alternative scenarios. For example, low micromolar MAS accumulation
produced by progestins might be physiologically irrelevant to the
regulation of meiosis (or any other process). Related to this caveat is
our inability to fully explain the accumulation of FF-MAS in follicular
fluid and ovarian tissue, a process that may involve inhibition of
14-reductase. We cannot exclude mediation by a metabolite of the
progestins. Although hepatocytes contain enzymes not expressed in
gonadal tissue, HepG2 cells are otherwise a useful experimental model
because of their high rate of cholesterol synthesis in lipid-deficient
media and their low background of pre-existing MAS. It should also be
noted that, although our GC/MS evidence for FF-MAS and T-MAS exceeds
commonly accepted standards for sterol identification, reported
limitations of GC/MS (36)
point to the value of more
definitive analyses in future work, which should also include
measurements of MAS levels relative to cell protein.
The mechanism of action suggested by our results may bear
directly on the role of progestins in human fertility. The importance
of changing from an estrogen/androgen-secreting status to a
predominately progesterone-secreting status in human ovaries has been
emphasized by several investigators (29
, 37
38
39)
and
coincides with the resumption of meiosis (30
, 40)
.
Impairment of progesterone secretion is associated with atretic
follicles, and low progesterone levels have been reported in the empty
follicle syndrome (41
, 42)
. In some oligospermic men,
abnormal steroidogenesis with lower levels of testicular
17-OH-progesterone has been reported (43)
. These
requirements of progestins in normal fertility may be explained by our
findings that progestins stimulate the production of sterols known to
activate meiosis (1
, 5
, 12)
. Increases in desmosterol
levels in primate testis parallel the onset of changes in testosterone
synthesis (14)
, and activation of meiosis is associated
with the onset of spermatogenesis (44)
. Evidence for the
synthesis of MAS in rat gonads has recently been presented by Yoshida
et al. (8)
. Our data further suggest that elevated
progestin levels, leading to inhibition of 24 reduction, provide a
coherent explanation for the relatively high concentrations of
desmosterol in semen, testes (13
, 14)
, mothers’ serum
before and after delivery (45)
, and human milk
(15)
.
In summary, we provide evidence that progestins may be natural
regulators of MAS. Our findings point to a new role for progestins in
fertility through synergistic actions of 17-OH-progesterone with
other progestins and suggest potential targets for possible
pharmacological intervention. However, the relevance of our
experimental results to the regulation of meiosis presupposes a
physiological role for MAS in fertility, a matter that remains to be
demonstrated conclusively.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication May 8, 2000.
Revision received August 23, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Nature (London) 383,728-731[Medline]
is positively and negatively regulated by distinct products of mevalonate metabolism. Proc. Natl. Acad. Sci. USA 94,10588-10593-demethylase (CYP51) and the synthesis of oocyte meiosis-activating sterols in postmeiotic germ cells of male rats. Endocrinology 139,2314-2321This article has been cited by other articles:
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