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Departments of Cellular and Molecular Physiology (V.L.N., J.M.M.) and Obstetrics and Gynecology (R.S.L.). Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; Center for Research on Reproduction and Women’s Health, University of Pennsylvania (J.R.W., J.F.S.), Philadelphia, Pennsylvania 19104; Department of Pharmacology, Biochemistry, and Biophysics, University of Pennsylvania (T.M.P.), Philadelphia, Pennsylvania 19104; and Departments of Pediatrics and Medicine, University of Chicago (K.Q., R.L.R.), Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Jan M. McAllister, Ph.D., Department of Cellular and Molecular Physiology, Pennsylvania State Hershey Medical Center, 500 University Drive, Hershey, Pennsylvania 17033. E-mail: jmcallister{at}psu.edu
Abstract
Ovarian theca cells propagated from patients with polycystic ovary
syndrome (PCOS) convert steroid precursors into T more efficiently than
normal theca cells. To identify the basis for increased T production by
PCOS theca cells, we examined type I–V 17ß-hydroxysteroid
dehydrogenase (17ßHSD) isoform expression in long-term cultures of
theca and granulosa cells isolated from normal and PCOS ovaries. RT-PCR
analysis demonstrated that theca cells express type V 17ßHSD a
member of the aldo-keto reductase (AKR) superfamily (17ßHSDV,
AKR1C3), whereas expression of type I, II, and IV 17ßHSD, which are
members of the short-chain dehydrogenase/reductase superfamily, was
limited to granulosa cells. Type III 17ßHSD, the testicular isoform,
was not detected in either granulosa or theca cells. Northern and
real-time PCR analyses demonstrated that 17ßHSDV transcripts were not
significantly increased in PCOS theca cells compared with normal theca
cells. RT-PCR analysis revealed that theca cells also express another
AKR, 20
HSD (AKR1C1). Both basal and forskolin-stimulated 20HSD
mRNA levels were increased in PCOS theca cells compared with normal
theca cells. However, 17ßHSD enzyme activity per theca cell was not
significantly increased in PCOS, suggesting that neither AKR1C3 nor
AKR1C1 contributes to the formation of T in this condition. In
contrast, 17-hydroxylase/C17,20 lyase and 3ßHSD enzyme activities
were elevated in PCOS theca cells, driving increased production of T
precursors. These findings indicate that 1) increased T production in
PCOS theca cells does not result from dysregulation of "androgenic"
17ßHSD activity or altered expression of AKRs that may express
17ßHSD activity; and 2) increased synthesis of T precursors is the
primary factor driving enhanced T secretion in PCOS.
POLYCYSTIC OVARY SYNDROME (PCOS) is one of
the most common causes of infertility in women, affecting approximately
5% of women of reproductive age (1). Reproductive
endocrine abnormalities in PCOS include amenorrhea or oligomenorrhea,
infertility, hirsutism, or acne resulting from increased ovarian
androgen production (2, 3, 4, 5, 6, 7). A number of investigators have
proposed that increased levels of circulating T in patients with PCOS
are a consequence of elevated ovarian 17ß-hydroxysteroid
dehydrogenase (17ßHSD) activity. 17ßHSDs preferentially catalyze
the oxidation or reduction of specific steroid substrates. Specific
isoforms participate in the final and key steps in the formation of
androgens and estrogen, whereas others play key roles in the
inactivation of T and 17ß-estradiol. Nine human 17ßHSD isoforms
have been cloned and characterized to date (8, 9). Most
17ßHSD isoforms are members of the short-chain alcohol
dehydrogenase/reductase superfamily, with the exception of type V
17ßHSD (17ßHSDV), which belongs to the aldo-keto reductase (AKR)
family (8). Of these 17ßHSD isoforms, type III and V are
androgenic, that is, they have been shown to catalyze the reduction of
the 17-ketosteroids, androstenedione (
4A) and
dehydroepiandrosterone (DHEA), to their respective
products, T and 5-androstene-3ß,17ß-diol
(8). Type I and II 17ßHSD are expressed in human ovarian
granulosa cells (8, 10). 17ßHSDIII, the isoform that is
required for testicular androgen biosynthesis (11, 12), is
not expressed in the human ovary (13). 17ßHSDIV mRNA has
been shown to be expressed in whole human ovary, but the exact cellular
location of this enzyme is currently unknown (14).
17ßHSDV has been reported to be the major 17ßHSD isoform expressed
in a human ovary library (15), and immunohistochemical
studies have shown that both human ovarian theca and corpus luteum
cells express 17ßHSDV (8, 16, 17).
Although numerous studies have demonstrated that ovarian T production
is elevated in patients with PCOS, few studies have focused on the
possible role of ovarian 17ßHSD expression in increased androgen
production (18, 19, 20, 21, 22). The studies of Bardin et
al. (22) and Barbieri et al.
(21) demonstrated that women with PCOS produce 4 times
more T than normal women, but only twice the amount of
4A. In addition, data from GnRH agonist
testing of patients with PCOS support the idea that ovarian 17ßHSD
expression is increased in PCOS (19). However, previous
studies of androgenic 17ßHSD activity in ovarian tissue have yielded
conflicting results (20). Barbieri et al.
(21) found that the ratio of T to
4A was significantly higher in the medium of
incubations of ovarian stroma from hyperandrogenic women than in that
from normally cycling women. In contrast, Pittaway et al.
(20) found no difference in 17ßHSD activity in whole
homogenates from PCOS and normal ovaries.
We recently described conditions to examine the regulation of androgen
production at the biochemical and molecular levels using normal and
PCOS theca interna cells propagated for multiple population doublings
(23, 24). Using this system, we showed that PCOS theca
cells are capable of substantial conversion of steroid precursors into
T (23). These initial observations raised the possibility
that theca cells isolated from patients with PCOS may have elevated
17ßHSD expression. To our knowledge, there are no prior reports
documenting alterations in ovarian 17ßHSD isoform expression in
patients with PCOS. Here we examined 17ßHSD isoform expression in
cultured theca and granulosa cells isolated from normal and PCOS
patients. Since 17ßHSDV is a member of the AKR family and is
identical to 3HSDII (AKR1C3) (25, 26), our studies were
expanded to include other related human HSD isoforms: 3HSDI
(AKR1C4), 3HSDIII (AKR1C2), and 20HSD (AKR1C1). Previous studies
have shown that each of these enzymes is plastic, in that they display
3-, 17ß-, and 20HSD activities to varying extents
(27). Androgenic 17ßHSD activity as well as the
activities of enzymes regulating androgen precursor synthesis were also
compared.
Materials and Methods
Patient population
PCOS and normal ovarian tissue came from age-matched women, 38–40 yr old. The diagnosis of PCOS was made according to established guidelines (23, 28, 29), including hyperandrogenemia, oligoovulation, and the exclusion of 21-hydroxylase deficiency, Cushing’s syndrome, and hyperprolactinemia. All of the PCOS theca cell preparations studied came from ovaries of women with fewer than six menses per yr and elevated serum total T or bioavailable T levels, as we previously described (23, 24). Each of the PCOS ovaries contained multiple subcortical follicles of less than 10 mm in diameter. The control (normal) theca cell preparations came from ovaries of fertile women with normal menstrual histories, menstrual cycles of 21–35 d, and no clinical signs of hyperandrogenism. Neither PCOS nor normal subjects were receiving hormonal medications at the time of surgery. Indications for surgery were dysfunctional uterine bleeding, endometrial cancer, and pelvic pain.
Theca and granulosa cell isolation and propagation
Human theca interna and granulosa cells were obtained from follicles of women undergoing hysterectomy under a protocol approved by the institutional review board of Pennsylvania State University College of Medicine. Individual follicles were dissected away from ovarian stroma. The dissected follicles were placed into serum-containing medium and bisected. Under a dissecting microscope, the theca interna was stripped from the follicle wall, and the granulosa cells were removed with a platinum loop. Granulosa cells were harvested from the medium with a Pasteur pipette. The cleaned theca shells were dispersed with 0.05% collagenase I, 0.05% collagenase IA, and 0.01% deoxyribonuclease in medium containing 10% FBS (23, 30, 31). After centrifugation the cells were placed in culture dishes that had been precoated with fibronectin by incubation at 37 C with culture medium containing 5 µg/ml human fibronectin. The growth medium used was a 1:1 mixture of DMEM and Ham’s F-12 medium containing 10% FBS, 10% horse serum, 2% UltroSer G, 20 nmol/liter insulin, 20 nmol/liter selenium, 1 µmol/liter vitamin E, and antibiotics (30). From each follicle, 12 35-mm dishes of primary theca or granulosa cells were grown until confluent, removed from the dish with neutral protease (pronase E; protease type XXIV, Sigma, St. Louis, MO) in DMEM/Ham’s F-12 (1:1), frozen, and stored in liquid nitrogen (one 35-mm dish/vial) in culture medium that contained 20% FBS and 10% dimethylsulfoxide (23, 31). In all experiments cells were thawed and propagated in the growth medium described above. Cells were collected for subculture after incubation with neutral protease as previously described (31). Sera and growth factors were obtained from the following sources: DMEM/Ham’s F-12 was obtained from Irvine Scientific (Irvine, CA); FBS and horse serum were obtained from Atlanta Biologicals (Atlanta, GA); UltroSer G was purchased from BioSepra (Cergy-Saint-Christophe, France); other compounds were purchased from Sigma. In all experiments the cells were grown in 5% O2, 90% N2, and 5% CO2. Reduced oxygen tension and supplemental antioxidants (vitamin E and selenium) were employed to prevent oxidative damage. The passage conditions and split ratios for all normal and PCOS cells were identical. Experiments comparing PCOS and normal theca cells were performed using fourth passage (31–38 population doublings) theca cells isolated from follicles obtained from age-matched subjects. All theca cell cultures were screened for aromatase activity and were determined to be free of contaminating granulosa cells.
RT-PCR
For PCR analysis, theca and granulosa cells were grown until
confluent and transferred into serum-free medium in the presence or
absence of 20 µM forskolin. At 48 h, total mRNA was
harvested as previously described (23, 31). 17ßHSD and
3HSD isoform expression was examined by RT-PCR using a Thermostable
rTth Reverse Transcriptase RNA PCR Kit (Perkin-Elmer Corp., Foster City, CA) and forward and reverse oligoprimer
pairs specific for each type of 17ßHSD (types I–V; presented in
Table 1
). Forward and reverse primers
specific for each AKR, 3HSD (types I–III), or 20HSD (presented
in Table 2) have been described
previously (15, 27, 32). The single-stranded cDNA was
synthesized using rTth DNA polymerase and downstream primers specific
for each 17ßHSD, 3HSD, or 20HSD isoform. Reactions were carried
out in a volume of 20 µl containing 2 µl 10 x reverse
transcriptase buffer, 10 mM MnCl2
solution, 5 U rTth DNA polymerase, 200 µmol/liter of each
2'-deoxynucleoside 5'-triphosphate, 10 pmol primers, and 200 ng total
RNA. All tubes were incubated at 70 C for 15 min, and the reaction was
stopped by placing the tubes on ice. Then 80 µl PCR mix were added,
containing 10 x chelating buffer, 25 mM
MgCl2, and 10 pmol upstream primers. Samples were
amplified for 25–30 cycles. Cycling conditions were an initial 3 min
at 95 C, 25–30 cycles at 95 C for 15 s and 60 C for 60 s,
and a final extension at 60 C for 7 min in a GeneAmp PCR system 9600
thermal cycler (Perkin-Elmer Corp.). Amplified DNA was
resolved on a 2% agarose gel containing 5 µg/ml ethidium bromide and
then visualized under UV light (32). A negative control
was carried out for each pair of primers, namely, a liver mRNA sample
by the same procedure without reverse transcriptase. To confirm the
RT-PCR analysis, the identities of all PCR products were confirmed by
sequence analysis.
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Normal and PCOS theca cells were grown until confluent and
transferred into serum-free medium in the presence or absence of 20
µM forskolin. At 48 h, the cells were harvested,
total mRNA was extracted, and Northern blot analysis was performed
using 50 µg total mRNA/lane, as previously described (23, 31). PCR-generated cDNAs specific to human 17ßHSDV and
20HSD 3'-untranslated regions were used as hybridization
probes. For normalization, all blots were stripped and hybridized with
a full-length 28S cDNA probe. Hybridizable mRNA species were identified
and quantitated using an SI PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). All Northern blot analysis data were
corrected for 28S mRNA.
Quantitative real-time PCR
For quantitative real-time PCR, total mRNA was isolated (23) from fourth passage normal and PCOS theca cells that were grown to subconfluence, then transferred into serum-free medium in the presence or absence of 20 µM forskolin for 48 h. To remove possible DNA contaminates, 5 µg total RNA from each sample were treated with 5 U RQ1 ribonuclease-free deoxyribonuclease in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl2 in a final volume of 25 µl for 30 min at 37 C. The deoxyribonuclease reaction was terminated with a 10-min incubation at 65 C. The deoxyribonuclease-treated RNA samples were then reverse transcribed using oligo(deoxythymidine) and 200 U Superscript II ribonuclease H- reverse transcriptase (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s directions.
17ßHSDV mRNA abundance was determined by quantitative real-time PCR
using SYBR green fluorescent indicator (PE Applied Biosystems, Foster City, CA). Briefly, equivalent dilutions of
each cDNA sample described above were combined with 300 nM
17ßHSDV-specific forward (5'-GTCTCTAAAGCCAGGTGAGGAACT-3') and reverse
(5'-TCCCAGGTGGTACAGAGATCG) primers and 2 x SYBR Green Master Mix
(PE Applied Biosystems) in a final volume of 25 µl.
Two-step PCR was carried out in triplicate for each cDNA sample in an
ABI 7700 PRISM Thermocycler (PE Applied Biosystems)
according to manufacturer’s instructions. The abundance of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was also
determined for each cDNA sample in triplicate by combining the diluted
cDNA with 20 x predeveloped TaqMan reagent endogenous control
human GAPDH and 2 x TaqMan Universal Master Mix in a final volume
of 25 µl. The resulting mean threshold cycle
(CT) for each cDNA sample was determined, in the
linear range, using Sequence Detector software (PE Applied Biosystems). To compare the abundance of 17ßHSDV between
normal and PCOS theca cells and between untreated and forskolin-treated
cells, the 17ßHSDV CT values were normalized by
subtraction of the GAPDH CT values, resulting in
a CT value.
Steroidogenic enzyme activity
For evaluation of enzyme activity, fourth passage normal and
PCOS theca cells were grown until subconfluent and transferred into
serum-free medium in the presence or absence of 20 µmol/liter
forskolin for 72 h to induce full steroidogenic capacity. For
assay of 17ßHSD, 20HSD, 17-hydroxylase, C17,20 lyase, and
3ßHSD enzyme activities, the cells were then transferred into medium
containing saturating concentrations of the appropriate tritiated
steroid substrate. For 17ßHSD activity, the cells were incubated with
10 µmol/liter
[1,2,6,7-3H]4A, and
[3H]T production was determined. For 20HSD
enzyme activity, cells were incubated with10 µmol/liter
[1,2,6,7-3H]progesterone in the presence of
SU10603 (1.0 µmol/liter), a competitive inhibitor of
17-hydroxylase enzyme activity, and production of
[3H]20-hydroxyprogesterone was
determined. For 17-hydroxylase enzyme activity, the cells were
incubated with 10 µmol/liter
[1,2,6,7-3H]progesterone, and
[3H]17- hydroxyprogesterone production
was measured. For C17,20 lyase enzyme activity, the cells were
incubated with 10 µmol/liter [7-3H]17-
hydroxypregnenolone, and the formation of
[3H]DHEA was assessed. For 3ßHSD
enzyme activity, the cells were incubated with 10 µmol/liter
[1,2,6,7-3H]DHEA, and the
conversion of substrate to
[3H]4A was
determined.
Aliquots of the medium were obtained at various time intervals, and
steroids were extracted from the medium with 4 vol dichloromethane
(HPLC grade) with an extraction efficiency greater than 90%. The
dichloromethane phase containing unconjugated steroids was evaporated.
The residue was dissolved in methanol and subjected to reverse phase
HPLC. HPLC was conducted on a computer-controlled Gilson automated
chromatogram using a Phenomenex 25-cm 5 µ Prodigy
C18 column (Milford, MA). For all enzyme assays,
the gradient solvent delivery system consisted of acetonitrile/methanol
(A/M; 1:1) and water (50:50) for 10 min, followed by a 10-min linear
gradient to 57% A/M, and an additional 4-min linear gradient to 73%
A/M for 9 min, then a 2-min linear gradient to 100% A/M. Radioactive
material was detected by an in-line liquid scintillation
spectrophotometer (IN/US System, Inc., Tampa, FL). The retention times
of authentic steroid standards were established for the
4-ene and reduced steroids at 240 and 200
nM, respectively. Conversion rates were calculated by
comparing peak areas for substrate and products, calculating the
percent conversion to product under saturation conditions, in a linear
time frame, where less than 10% of the substrate is converted to
product, and converting percent conversion to picomoles from the known
amount of substrate added. Cell number was estimated using a Coulter
counter (Coulter Electronics, Hialeah, FL) after dispersal of the cells
with trypsin. Steroidogenic enzyme activities are expressed as
picomoles per 106 cells/h.
Statistical analysis.All experiments were repeated at least four times with cells obtained from a variety of different PCOS and normal patients that had been thawed and grown to the appropriate passage. After combining the results from individual patients, unpaired two-tailed t tests and/or ANOVA were performed using StatView 5.0 from SAS Institute, Inc. (Cary, NC). Each experiment was performed with triplicate or quadruplicate replicate dishes.
Results
RT-PCR analysis of type I–V 17ßHSD mRNA expression in human ovarian cells
17ßHSD (types I–V) isoform expression was examined using RT-PCR
of total mRNA isolated from fourth passage theca and granulosa cells
cultured in the absence and presence of 20 µM forskolin
for 48 h. RT-PCR was performed at 25–30 cycles as described in
Materials and Methods, using the primers presented in Table 1
. Total mRNA isolated from liver, placenta, ovarian follicle, testis,
and adrenal was used as controls. In agreement with data previously
reported by Zhang et al. (13), the results of
this RT-PCR analysis confirmed that 17ßHSD types I, II, and IV are
expressed only in granulosa cells (Fig. 1). 17ßHSDV was the only 17ßHSD
isoform expressed in theca cells (Fig. 1). Type III 17ßHSD
(17ßHSDIII), the testicular isoform, was not detected in theca or
granulosa cells isolated from normal or PCOS subjects. Sequence
analysis of the amplified sequence confirmed that 17ßHSDV mRNA is
expressed in human theca cells (data not shown). We found no
differences in 17ßHSD isoform expression in theca or granulosa cells
isolated from normal and PCOS patients. These data also establish that
the expression of human ovarian 17ßHSDIV (14) is
restricted to the granulosa cell compartment. These results confirm
that the theca cell preparations used in our studies were free of
contaminating granulosa cells and verify that type I–V 17ßHSD
expression in granulosa and theca cells propagated for successive
population doublings is phenotypically similar to that reported in
granulosa and theca cells in vivo (8).
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Northern blot analysis of total mRNA (50 µg) isolated from theca
cells propagated from five normal and PCOS patients were cultured in
the absence or presence of 20 µM forskolin for 48 h
in serum-free medium (Fig. 2). In the
upper panel of Fig. 2 a PCR-amplified probe specific to
AKR1C3 subfamily members was used as a hybridization probe. In the
lower panel, a cDNA probe complementary to 28S was used as a
hybridization probe. There was also no significant difference in basal
or forskolin-stimulated 17ßHSDV mRNA levels in PCOS theca cells
compared with normal theca cells.
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, there was no significant difference
in the 17ßHSDV PCR product when cDNA from normal and PCOS theca cells
cultured under control conditions was used as a template (Fig. 3A).
Similarly, when cDNA from forskolin-treated normal and PCOS theca cells
was used as a template, there were no differences in 17ßHSDV product
detected. Restriction digestion (Fig. 3B) and sequence analysis (data
not shown) of the 17ßHSDV PCR products verified amplification of only
17ßHSDV and not any other member of the AKR family.
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HSD and 20HSD mRNA
expression
RT-PCR was used to examine 3HSD (type I–III 3HSD and
20HSD) isoform expression in normal and PCOS theca cells. Using the
primers presented in Table 2, RT-PCR was performed for 30 cycles, as
described in Materials and Methods, using total mRNA
isolated from fourth passage theca and granulosa cells treated without
or with 20 µM forskolin in serum-free medium
for 48 h. Total mRNA isolated from liver, placenta, ovarian
follicle, testis, and adrenal was used as controls. As shown in Fig. 4, the results of this analysis confirm
that both normal and PCOS theca cells express 3HSDII (AKR1C3), which
is 99.9% identical to 17ßHSDV. Both normal and PCOS theca cells also
expressed 20(3)HSD, a protein that shares 84% sequence identity to
17ßHSDV. Type I and III 3HSD were not expressed in normal or PCOS
theca cells. Sequence analysis of the amplified sequences confirmed
20(3)HSD mRNA is expressed in human theca cells (data not
shown).
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HSD mRNA expression
Northern blot analysis of total mRNA (50 µg/lane) isolated from
five individual normal and PCOS patients after 48 h treatment in
serum-free medium in the absence or presence of 20 µM
forskolin. As shown in Fig. 5, 20HSD
mRNA levels were increased in response to forskolin treatment in normal
theca cells (P < 0.05). In nontreated PCOS theca
cells, 20HSD mRNA transcripts were comparable to those found in
forskolin-stimulated normal theca cells. 20HSD mRNA expression
was significantly increased in PCOS theca cells compared with normal
theca cells, under basal and forskolin-stimulated conditions (Fig. 5).
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HSD, 17-hydroxylase, C17,20 lyase,
and 3ßHSD enzyme activities
To quantitate 17ßHSD enzyme activity in normal and PCOS theca
cells, fourth passage cells were transferred into serum-free medium in
the presence and absence of 20 µM forskolin. After
72 h, the medium was removed, and 17ßHSD enzyme activity was
determined after exposing the cells to serum-free medium containing a
saturating concentration of
[3H]4A (10
µM) for 12–24 h and measuring
[3H]T formation. As shown in Fig. 6, in theca cells isolated from four
individual normal and PCOS patients, 17ßHSD enzyme activity/theca
cell was not significantly increased in response to forskolin
treatment. There was also no significant increase in basal or
forskolin-stimulated 17ßHSD activity in PCOS theca cells compared
with normal theca cells.
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HSD enzyme activity in normal and PCOS theca cells,
fourth passage cells were transferred into serum-free medium in the
presence and absence of 20 µM forskolin. After 72 h,
the medium was removed, and 20HSD enzyme activity was assayed after
exposing the cells to serum-free medium containing a saturating
concentration of [3H]progesterone (10
µM) as substrate in the presence of SU10603 (1
µM), a competitive inhibitor of 17-hydroxylase for
12–24 h, and measuring the formation of
[3H]20-hydroxyprogesterone. As shown in Fig. 7, 20HSD enzyme activity/cell was
significantly increased (
2-fold) in response to forskolin treatment
in theca cells isolated from five individual normal and PCOS patients
(P < 0.05). The 20HSD enzyme activity per theca
cell was also significantly increased in PCOS theca cells compared with
normal theca cells under basal and forskolin-stimulated
conditions.
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- hydroxylase
and C17,20 lyase activity (23, 33). To independently
quantitate 17-hydroxylase and C17,20 lyase activities in normal and
PCOS theca cells, fourth passage cells isolated from five individual
normal and PCOS patients were transferred into serum-free medium in the
presence and absence of 20 µM forskolin. After 72 h,
the medium was removed, and 17-hydroxylase enzyme activity was
determined by exposing theca cells to a saturating concentration of
[3H]progesterone (10 µM) for
12 h, followed by quantitation of
[3H]17-hydroxyprogesterone production.
C17,20 enzyme activity was determined in replicate cultures exposed to
a saturating concentration of
[3H]17-hydroxypregnenolone (10
µM) for 12 h and quantitation of
[3H]DHEA production. 17-
Hydroxylase and C17,20 lyase activities per cell were significantly
increased in response to forskolin treatment approximately 5-fold in
both normal and PCOS theca cells (P < 0.05). Both
17-hydroxylase and C17,20 lyase activities were significantly
increased in PCOS theca cells compared with normal theca cells under
basal and forskolin-stimulated conditions. The results presented in
Fig. 8 also demonstrate that C17,20 lyase
activity is not disproportionally increased in PCOS theca cells. These
data are consistent with increased T biosynthesis in PCOS theca cells
resulting from increased precursor flux, in part due to increased P450
17-hydroxylase (P450c17) enzyme activity.
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4A.
Forskolin-stimulated 3ßHSD activity increased about 3-fold in both
normal and PCOS theca cells. As shown in Fig. 9, representative data from five normal
and PCOS patients demonstrate that 3ßHSD enzyme activity/cell was
significantly increased (20-fold) in PCOS theca cells compared with
normal theca cells under basal and forskolin-stimulated conditions.
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The final step in the conversion of androstenedione to T is
catalyzed by androgenic (reductive) 17ßHSD. Although there are
immunohistochemical studies documenting 17ßHSD isoform expression in
the intact human ovary (8) and Northern and RT-PCR
analysis studies using whole human ovarian tissue (10), a
comparison 17ßHSD isoform expression in granulosa and theca cells
isolated from normal and PCOS patients has yet to be reported. In this
study we compared 17ßHSD and 20HSD isoform expression in theca
cells isolated from normal cycling women and patients with PCOS that
have been propagated for multiple population doublings. Our results
indicate that 17ßHSDV (3HSDII; AKR1C3) or possibly 20(3)HSD
(AKR1C1) catalyzes the reduction of the 17-keto group in the
biosynthetic pathway of theca androgen synthesis. The finding that
17ßHSDIII is not expressed in either granulosa or theca cells
propagated from normal and PCOS ovaries (8, 13) rules out
the possibility that 17ßHSDIII is inappropriately expressed in the
PCOS ovary. A role for the other known 17ßHSDs in theca androgen
biosynthesis has been excluded based on our failure to detect their
transcripts in normal and PCOS theca cells by sensitive RT-PCR.
In view of our previously published data demonstrating that 1) T
biosynthesis was increased per PCOS theca cell, and 2) the rate of
conversion of pregnenolone and DHEA into T was markedly
increased in PCOS theca cells, we anticipated finding increased
17ßHSDV expression. However, after an extensive comparison of
17ßHSDV transcript levels in normal and PCOS theca cells, we
demonstrated for the first time that 17ßHSDV mRNA expression is not
significantly elevated in PCOS theca cells compared with normal theca
cells. In contrast, 20HSD expression was augmented in PCOS theca
cells.
A comparison of 17ßHSD and 20HSD activities in normal and PCOS
theca cells demonstrated that androgenic 17ßHSD activity per theca
cell was not different in PCOS theca cells, whereas 20HSD activity
was significantly augmented. Because our analysis of total androgenic
17ßHSD activity per theca cell revealed no differences between normal
and PCOS theca cells, it appears that although 20HSD mRNA and
activity are increased in PCOS theca cells, it is unlikely that this
enzyme makes a major contribution to androgenic 17ßHSD activity. In
the absence of selective inhibitors of 17ßHSDV (3HSDII) and
20HSD, we cannot quantitate the individual contribution of each of
these enzymes to T synthesis. In our previous studies of labeled
steroid precursor metabolism (23) the concentration of
4A used may not have been saturating, thus
preventing us from obtaining an accurate assessment of 17ßHSD
activity. The present study has addressed this deficiency, clearly
indicating that when saturating levels of substrate are provided
17ßHSD activity is not significantly elevated. Our new data support
those of Pittaway et al. (20) and argue that
increased T production in PCOS theca cells is not a consequence of
increased 17ßHSD mRNA accumulation or enzyme activity.
To investigate the extent to which increased T production results from
increased substrate flux, as opposed to increased 17ßHSD enzyme
activity, we compared the relative amounts of P450 c17 and 3ßHSD
enzyme activities. Although a number of investigators have proposed
that the C17,20 lyase activity of P450 c17 is dysregulated in PCOS
(34, 35, 36, 37, 38, 39), no reports have directly compared
17-hydroxylase vs. C17,20 lyase activities in normal and
PCOS theca cells. In these studies we independently examined both
17-hydroxylase and C17,20 lyase activities in replicate cultures of
normal and PCOS theca cells. Both 17-hydroxylase and C17,20 lyase
activities are coordinately increased in PCOS theca cells under basal
and forskolin-stimulated conditions. In agreement with our previous
studies demonstrating that P450c17 gene transcription is increased in
the absence of cAMP stimulation in PCOS theca cells, both basal
17-hydroxylase and C17,20 lyase activities in PCOS theca cells were
comparable to the levels observed in normal theca cells after maximal
forskolin stimulation. The ratio of C17,20 lyase to 17-
hydroxylase activity was similar in normal and PCOS theca cells.
These are the first biochemical data to demonstrate that C17,20 lyase
activity is not disproportionately increased in PCOS theca cells.
3ßHSD enzyme activity per theca cell was also markedly increased in
PCOS theca cells under basal and forskolin-stimulated
conditions. Because P450c17 and 3ßHSD enzyme activities are
markedly elevated by more than 500% and more than 1000%,
respectively, in PCOS theca cells compared with normal cells, whereas
17ßHSD enzyme activity is unaffected, and 20HSD enzyme activity is
increased only about 75% in PCOS theca cells, it is likely that the
increased production of T by PCOS theca cells is driven by increased
androgen precursor production and not by altered 17ßHSD activity.
As the expression of multiple genes appears to be affected in PCOS theca cells, we have begun to use long-term cultures of normal and PCOS theca cells to characterize the repertoire of differentially expressed genes and altered signal transduction cascade(s) that are characteristic of PCOS theca cells. The identification of the network of genes that are differentially expressed in PCOS, and ultimately the regulatory pathway(s) responsible for altered gene expression, will provide valuable information that we believe will be applicable to the clinical management of excessive ovarian androgen production and follicular growth arrest in PCOS.
Acknowledgments
We thank Drs. Walter L. Miller and Richard J. Auchus for their
valuable advice regarding the analysis and comparison of
17-hydroxylase and C17,20 lyase activities in normal and PCOS theca
cells. We also thank Dr. Diane M. Thiboutot for her helpful advice
related to our analysis of 17ßHSD isoform expression.
Footnotes
This work was supported by NIH Grants HD-34449 (to J.F.S. and J.M.M.), HD-0118 (to R.S.L.), HD-33852 (to J.M.M.), RR-00055 (to R.L.R.), HD-39267 (to R.L.R.), HD-07305 (to J.R.W.), and DK-47015 (to T.M.P.) and gifts from Lilly Research Laboratories (to K.Q.) and the Children’s Research Foundation (to K.Q.).
Abbreviations: 4A, Androstenedione; AKR,
aldo-keto reductase; A/M, acetonitrile/methanol; CT,
threshold cycle; DHEA, dehydroepiandrosterone; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; 17ßHSD;
17ß-hydroxysteroid dehydrogenase; 17ßHSDV, type V 17ßHSD; PCOS,
polycystic ovary syndrome.
Received June 28, 2001.
Accepted September 6, 2001.
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) or presence (
) of forskolin was determined by
quantitative real-time PCR. 17ßHSDV mRNA levels were normalized by
GAPDH mRNA levels for each sample and depicted graphically as the
mean ± SEM of these normalized values
(
) to the
right of the figure. Mol wt are indicated in base pairs
to the left. 17ßHSDV mRNA values, quantitated using
real-time PCR, were not significantly increased in PCOS theca cells
compared with normal theca cells. There was no significant difference
in forskolin-stimulated 17ßVHSD mRNA accumulation in either normal or
PCOS patients.
