Augmented Androgen Production Is a Stable Steroidogenic Phenotype of Propagated Theca Cells from Polycystic Ovaries
Velen L. Nelson,
Richard S. Legro,
Jerome F. Strauss, III and
Jan M. McAllister
Department of Cellular and Molecular Physiology (V.L.N.,
J.M.M.) and Department of Obstetrics and Gynecology (R.S.L.)
Pennsylvania State University College of Medicine Hershey,
Pennsylvania 17033
Center for Research on Reproduction and
Womens Health University of Pennsylvania (J.F.S.)
Philadelphia, Pennsylvania 19104
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ABSTRACT
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To test the hypothesis that the hyperandrogenemia
associated with polycystic ovary syndrome (PCOS) results from an
intrinsic abnormality in ovarian theca cell steroidogenesis, we
examined steroid hormone production, steroidogenic enzyme activity, and
mRNA expression in normal and PCOS theca cells propagated in long-term
culture. Progesterone (P4), 17
-hydroxyprogesterone (17OHP4),
and testosterone (T) production per cell were markedly increased
in PCOS theca cell cultures. Moreover, basal and forskolin-stimulated
pregnenolone, P4, and dehydroepiandrosterone metabolism were increased
dramatically in PCOS theca cells. PCOS theca cells were capable of
substantial metabolism of precursors into T, reflecting expression of
an androgenic 17ß-hydroxysteroid dehydrogenase. Forskolin-stimulated
cholesterol side chain cleavage enzyme (CYP11A) and
17
-hydroxylase/17,20-desmolase (CYP17) expression were augmented in
PCOS theca cells compared with normal cells, whereas no differences
were found in steroidogenic acute regulatory protein mRNA
expression. Collectively, these observations establish that
increased CYP11A and CYP17 mRNA expression, as well as increased CYP17,
3ß-hydroxysteroid dehydrogenase, and 17ß-hydroxysteroid
dehydrogenase enzyme activity per theca cell, and consequently
increased production of P4, 17OHP4, and T, are stable properties of
PCOS theca cells. These findings are consistent with the notion
that there is an intrinsic alteration in the steroidogenic activity of
PCOS thecal cells that encompasses multiple steps in the biosynthetic
pathway.
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INTRODUCTION
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Polycystic ovary syndrome (PCOS) is the most common cause of
infertility in women (1). PCOS ovaries are characterized by the
accumulation of small follicles 47 mm in diameter with hypertrophied
theca interna layers (2, 3). Reproductive abnormalities include
amenorrhea or oligomenorrhea, infertility, hirsutism, and acne
resulting from increased ovarian androgen production (4, 5, 6, 7). Other
frequently associated abnormalities include insulin resistance and
obesity. Familial clustering of PCOS suggests that genetic factors are
involved in the etiology of the disorder, and it has been proposed that
PCOS is an oligogenic syndrome involving genes governing steroid
hormone biosynthesis as well as insulin/glucose homeostasis
(8, 9, 10).
The molecular and cellular mechanisms underlying the excessive ovarian
androgen production associated with PCOS remain to be elucidated
(11, 12, 13, 14, 15, 16, 17, 18). There is in vivo evidence to support the concept
that excess androgen production in PCOS results from a primary
abnormality in steroid production by ovarian theca cells (19). In the
human follicle, the androgen-secreting theca cells express
17
-hydroxylase/17,20- desmolase (CYP17), cholesterol side chain
cleavage enzyme (CYP11A), 3ß-hydroxysteroid dehydrogenase (3ß-HSD),
and steroidogenic acute regulatory protein (StAR), each of which are
required for androgen and progestin production (20, 21, 22, 23, 24, 25, 26). Gilling-Smith
et al. (19) reported that after GnRH agonist (GnRHa)-induced
suppression of serum LH concentrations, ovarian androgen production in
PCOS patients was significantly higher than in controls. Because CYP17
is the key enzyme required for androgen production in theca cells,
Rosenfield and Barnes (5, 27, 28) and Nestler et al. (29, 30) proposed that excess androgen production in PCOS results from
dysregulation of CYP17 enzyme activity due to an intrinsic ovarian
defect. Subsequent studies of Gilling-Smith et al. (31)
suggested that androgen production per theca cell is increased in PCOS.
However, it is still not known whether the increased androgen
production in PCOS is caused by dysregulation of ovarian theca cell
CYP17. Although it has been repeatedly proposed that CYP17 expression
is elevated in PCOS theca cells, no study has directly compared CYP17
enzyme activity or the regulation of CYP17 mRNA expression in normal
and PCOS theca cells. In addition, it is also possible that changes in
CYP11A, 3ß-HSD activity, or StAR expression contribute to increased
ovarian androgen production in PCOS. Moreover, the question of whether
the excessive androgen production by PCOS theca cells in culture (31)
reflects an intrinsic abnormality or whether it results from the
residual effects of the hormonal milieu to which the cells were exposed
in vivo has not been addressed.
Although a number of theories have been proposed to explain the
etiology of excess androgen production by PCOS ovaries, few studies
have focused on the regulation of steroidogenic enzyme activity and
expression in isolated theca interna cells that have been maintained in
culture in the absence of gonadotropins. We have begun to
comprehensively examine the regulation of androgen production at the
metabolic and molecular level using normal and PCOS theca interna cells
isolated from size-matched follicles and propagated for multiple
population doublings to determine whether increased androgen production
in PCOS results from abnormalities in the regulation of StAR, CYP11A,
CYP17, and 3ß-HSD gene expression in response to forskolin (22, 23, 24, 25, 32).
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RESULTS
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Normal and PCOS Theca Cells in Long-Term Culture: Steroid
Accumulation in Response to Forskolin
To compare cAMP-stimulated steroid synthesis in normal and PCOS
theca cells derived from follicles of comparable size and cultured
under identical conditions for 2226 population doubling (three
passages), we measured progesterone (P4), 17
-hydroxyprogesterone
(17OHP4), and testosterone (T) accumulation in the media of cells
treated with increasing concentrations of forskolin (Fig. 1
). In both normal
and PCOS theca cell cultures there was a dose-dependent increase in
forskolin-stimulated steroid hormone production, which was maximal
72 h after treatment with 20 µM forskolin. The
ED50 for forskolin-stimulated steroidogenesis was 3
µM in both normal and PCOS theca cell preparations. In
normal theca cells, forskolin-stimulated P4 and 17OHP4 production were
increased 40-fold and 22-fold, respectively, over basal values.
Negligible amounts of T were present in the media of normal theca cell
cultures. In PCOS theca cells maximally stimulated with forskolin, P4,
17OHP4, and T production increased 43-fold, 22-fold, and 12-fold,
respectively, over basal values. Under basal conditions, P4, 17OHP4,
and T production by PCOS theca cultures was elevated 20-fold
(P < 0.05), 22-fold (P < 0.05), and
4-fold (P < 0.05), respectively, above normal theca
cell values. Moreover, PCOS theca cells stimulated with 20
µM forskolin produced 10-fold, 25-fold, and 30-fold more
P4, 17OHP4, and T, respectively, than forskolin-stimulated normal theca
cells. Thus, while both normal and PCOS are responsive to forskolin,
PCOS thecal cells consistently produce greater quantities of steroid
hormone and have the capacity to produce substantial amounts of T.

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Figure 1. Accumulation of 17OHP4, P4, and T in the Medium of
Normal and PCOS Theca Cell Cultures Grown for 2226 Population
Doublings
Long-term cultures of normal and PCOS theca cells were grown until
subconfluence and then transferred into SFM containing 5 µg/ml low
density lipoprotein, with increasing concentrations of forskolin
(F) for 72 h. After treatment the media were collected and
analyzed by RIA. Results are presented as the means ±
SD of steroid levels from quadruplicate theca cell cultures
from three normal and three PCOS patients.
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Metabolism of P5, P4, and Dehydroepiandrosterone
(DHEA)
To further explore the biochemical differences between normal and
PCOS theca cells, the time courses and patterns of
[3H]-P5 (
Figs. 24

), [3H]-P4 (Fig. 5
), and
[3H]-DHEA (Fig. 6
) metabolism were examined
in normal and PCOS theca cells that had been grown for 2226 (three
passages) and for 3138 population doublings (four passages), and
treated for 72 h with a 20 µM dose of forskolin.
Although the metabolism profiles of cells from individual patients were
distinct, the results presented show the predominant products of
metabolism that are representative of theca cells isolated from four
normal and five PCOS patients that had been matched for follicle size
(
4 mm) and patient age (3840 yr). Similar data were also obtained
with theca cells isolated from multiple size-matched follicles from
individual patients (data not shown).

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Figure 2. Comparison of the Time Courses of Pregnenolone (P5)
Metabolism by Normal (left panel) and PCOS (right
panel) Theca Cells under Control and Forskolin-Stimulated
Conditions
Passaged theca cells (2226 population doublings) isolated from normal
and PCOS patients were treated with forskolin (20 µM) and
without (control) in SFM for 72 h and transferred into SFM
containing 1.0 µM [3H] pregnenolone.
Aliquots of the medium were removed at various intervals and extracted
with methylene chloride, and steroid products were separated by HPLC.
Disappearance of radiolabeled pregnenolone ( ) and appearance of the
indicated steroid metabolites is presented as a function of
time. Results are presented as means ± SD from
triplicate cultures from representative normal and PCOS theca cells.
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Figure 3. Time Course of Pregnenolone (P5) Metabolism by
Normal (left panel) and PCOS (right
panel) Theca Cells Propagated for 2226 Population Doublings
Passaged theca cells isolated from normal and PCOS patients were
treated in SFM with 20 µM forskolin for 72 h and
transferred into SFM containing 1.0 µM
[3H]-pregnenolone. Aliquots of the medium were removed at
various intervals and extracted with methylene chloride, and steroid
products were separated by HPLC. Disappearance of radiolabeled
pregnenolone ( ) and appearance of the indicated steroid products
is presented as a function of time. Results are presented as
means ± SD from triplicate theca cell cultures from
three normal and three PCOS ovaries.
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Figure 4. Time Course of Pregnenolone (P5) Metabolism by
Normal-2 (left panel) and PCOS-2 (right
panel) Theca Cells Propagated for 3138 Population Doublings
Passaged theca cells isolated from normal and PCOS patients were
treated in SFM with 20 µM forskolin for 72 h and
transferred into SFM containing 1.0 µM
[3H]-pregnenolone. Aliquots of the medium were removed at
various intervals and extracted with methylene chloride, and steroid
products were separated by HPLC. Disappearance of radiolabeled
pregnenolone ( ) and appearance of the indicated steroid products
is presented as a function of time. Results are presented as
means ± SD from triplicate theca cell cultures from
three normal and three PCOS ovaries.
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Figure 5. Time Course of P4 Metabolism by Normal (left
panel) and PCOS (right panel) Theca Cells in
Long-Term Culture
Passaged theca cells (2226 population doublings) isolated from normal
and PCOS patients were treated with 20 µM forskolin for
72 h and transferred into SFM containing 1.0 µM
[3H]-P4. Aliquots of the medium were removed at various
intervals and extracted with methylene chloride, and steroid products
were separated by HPLC. Disappearance of radiolabeled P4 ( ), and
appearance of the indicated steroid products is presented as a function
of time. Results are presented as means ± SD from
triplicate theca cell cultures from three normal and three PCOS
ovaries.
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Figure 6. Time Course of DHEA Metabolism by
Normal (left panel) and PCOS (right
panel) Theca Cells in Long-Term Culture
Passaged theca cells (2226 population doublings) isolated from normal
and PCOS patients were treated with 20 µM forskolin for
72 h and transferred into SFM containing 1.0 µM
[3H]-pregnenolone. Aliquots of the medium were removed at
various intervals, extracted with methylene chloride, and steroid
products were separated by HPLC. Disappearance of radiolabeled
DHEA ( ) and appearance of the indicated steroid
products is presented as a function of time. Results are presented as
means ± SD from triplicate theca cell cultures from
three normal and three PCOS ovaries.
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As shown in Fig. 2
, in theca cells isolated from normal patients and
cultured under basal conditions, the products of [3H]-P5
metabolism were primarily 17
-hydroxypregnenolone (17OHP5), and
DHEA, which are both products of CYP17 enzyme activity.
5-Pregnene,3ß,20
-diol (20
-OHP5), a product of
20
-hydroxysteroid dehydrogenase (20
-HSD) activity, was also
produced to a limited extent. After forskolin stimulation, the rate of
[3H]-P5 metabolism to 17OHP5 and DHEA was
increased, as was the production of 17,20
-dihydroprogesterone
(4-pregnene-17
, 20
-diol-3-one, 17,20
-DHP4) and
5-pregnene-3ß,17
,20
-triol (17,20
-P5). 17,20
-DHP4 and
17,20
-DHP5 are produced by the action of 20
-HSD on 17OHP4 and
17OHP5, respectively. In contrast, in PCOS theca cells
[3H]-P5 was further metabolized to T, Adione, and
androstenediol (Adiol) as well as to larger amounts of 17OHP4,
17,20
-DHP4, and 17,20
-DHP5. The rate of [3H]-P5
metabolism was increased after forskolin treatment in both normal and
PCOS theca cells, but was more markedly enhanced in PCOS theca
cells.
To determine whether increased [3H]-P5 metabolism is a
property retained by PCOS theca cells over longer culture periods,
[3H]-P5 metabolism was examined in forskolin-stimulated
theca cells isolated from three normal and three PCOS patients grown
for 2226 population doublings (Fig. 3
)
and 3138 population doublings (Fig. 4
).
The metabolism profiles were similar for cells grown for 2226 and
3138 population doublings but PCOS cells differed markedly from
normal theca cells. In normal theca cells, 90% of labeled pregnenolone
was metabolized to 17
-hydroxypregnenolone and DHEA in
812 h, while at 2448 h only low levels of Adione were produced
(left panel). In contrast, in PCOS theca cells (right
panel), the rate of [3H]-P5 metabolism was much
faster; more than 90% of labeled P5 was converted to 17OHP5 and
DHEA within 36 h, and within 1224 h it had been
metabolized to T, Adione, and Adiol. These data suggest that 3ß-HSD
and/or CYP17 enzyme activities are elevated in PCOS cells as compared
with normal theca cells. Although 20
-HSD activity appeared to be
stimulated by forskolin in both normal and PCOS theca cells, 20
-HSD
activity/cell was not elevated in PCOS theca cells.
To further explore differences in CYP17 enzyme activity in PCOS
and normal theca cells, [3H]-P4 metabolism was examined
in cells grown for 2226 population doublings (Fig. 5
) and 3138 population doublings (data
not shown). In theca cells isolated from normal ovaries, we previously
reported that P4 is metabolized predominantly to 17OHP4, 17,20
-DHP4,
and 16
-hydroxyprogesterone (16OHP4) (25). 16
-Hydroxylation of P4
to 16OHP4 was a side reaction associated with CYP17 enzyme activity. P4
was also metabolized to a limited extent to
4-pregnene-20
-hydroxy-3-one (20
-OHP4), which is a product of the
20
-HSD reaction. In the present studies utilizing normal theca cells
after forskolin stimulation (left panel), 80% of
[3H]-P4 was metabolized to 17
-hydroxylated products
within 2436 h, whereas in PCOS theca cells (right panel)
80% of [3H]-P4 was metabolized to similar products
within 48 h. Thus, the rate of labeled P4 metabolism was accelerated
and CYP17 enzyme activity/theca cell was elevated in PCOS cells as
compared with normal theca cells. Moreover, the metabolism profile
presented for theca cells grown for 2226 population doublings (Fig. 5
) was similar to that observed for cells grown for 3138 population
doublings. These data again indicate that CYP17 enzyme activity per
theca cell is increased in PCOS, and that increased CYP17 activity is a
stable property of PCOS theca cells in long-term culture.
To compare 3ß-HSD activity in normal and PCOS theca cells, as well as
establish the predominant intermediates of androgen biosynthesis,
[3H]-DHEA metabolism was examined in cells
grown for 2226 population doublings (Fig. 6
) and 3138 population doublings (data
not shown). In normal theca cells (left panel), 30% of
[3H]-DHEA was converted to Adione and, to a
limited extent, Adiol within 48 h. In agreement with the data
presented in Fig. 1
examining de novo T production in normal
theca cells, T was not a product of DHEA metabolism during
the 48-h incubation period, suggesting the absence of androgenic
17ß-hydroxysteroid dehydrogenase (17ß-HSD) activity. In contrast,
in PCOS theca cells (right panel), 60% of
[3H]-DHEA was converted to Adione, Adiol,
and T within 12 h. Further metabolism of T to 5
-reduced
steroids was not observed during the 48 h incubation period, indicating
the absence of 5
-reductase. Moreover, the metabolism profile
presented for theca cells grown for 2226 population doublings (Fig. 6
) was similar to that observed for cells grown for 3138 population
doublings (data not shown). These observations confirm that increased
3ß-HSD enzyme activity per theca cell is a stable property of PCOS
theca cells in culture and demonstrate that normal thecal cells are
relatively deficient in androgenic 17ß-HSD. Labeled
DHEA (Fig. 6
), P4 (Fig. 5
), and P5 (
Figs. 24

) were not
converted into estradiol by either normal or PCOS thecal cells. The
absence of detectable estradiol formation is consistent with the
lack of granulosa cell contamination of our theca cell
preparations.
Expression of Steroidogenic Enzyme mRNAs
To determine whether the increased production of steroids and
rates of precursor metabolism characteristic of PCOS theca cells
resulted from increased steady state levels of steroidogenic enzyme
mRNAs, Northern analyses were performed. Total mRNA was harvested from
theca cells isolated from four normal and four PCOS patients that were
cultured with and without 20 µM forskolin for 48 h.
In Fig. 7
, a representative Northern
analysis of mRNA (50 µg/lane) isolated from theca cells from two
normal and two PCOS patients, hybridized with complementary probes for
human CYP17, CYP11A, StAR, and 28S rRNA, is presented. Results from
these experiments demonstrated that forskolin-stimulated CYP17 and
CYP11A mRNA accumulation is markedly augmented in PCOS cells. As shown
in Fig. 8
, 48 h of forskolin
treatment of control theca cells resulted in modest (1.5- to 2-fold)
but significant increases in steady state levels of CYP17 and CYP11A
mRNA over the basal levels. In contrast, under identical conditions
CYP17 mRNA levels were increased 6-fold and CYP11A levels were
increased 5-fold over basal levels in PCOS theca cells. However, StAR
mRNA levels were increased to similar extents in normal and PCOS theca
cells (
4-fold) in response to forskolin treatment.

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Figure 7. cAMP-Stimulated CYP17, CYP11A, StAR, and 28S mRNA
Expression in Normal and PCOS Theca Cells
Normal and PCOS theca cells (2226 population doublings) were grown
until confluent and transferred into SFM in the presence (F) or absence
(C) of 20 µM forskolin. At 48 h, total mRNA was
harvested and Northern blot analysis was performed using 50 µg of
total mRNA per lane. A riboprobe complementary to human CYP17 mRNA was
used as a hybridization probe. Multiprime labeled cDNAs homologous for
human CYP11A, StAR, and 28S rRNA were also used as probes. Human
adrenal mRNA (hA) was used as a positive control.
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Figure 8. Relative Abundance of CYP17, CYP11A, and StAR mRNA
in Normal and PCOS Theca Cells
Cumulative data from Northern blot analyses for steady state CYP17,
StAR, and CYP11A mRNA levels in theca cells propagated for 2226
population doublings from four normal and four PCOS patients normalized
to 28S rRNA levels. Values presented are means ± SEM
for fold increases above background. Forskolin treatment significantly
increased (P < 0.05) CYP17, StAR, and CYP11A mRNA levels in
normal and PCOS theca cells (*).
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DISCUSSION
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The observations of Gilling-Smith et al. (31) suggested
that androgen production per cell is elevated in PCOS theca cells in
primary culture. However, it was not known whether the excessive
androgen production resulted from increased CYP17 expression, changes
in other proteins involved in steroid synthesis, or the hormonal milieu
to which the isolated thecal cells had been exposed in vivo.
Yet, these observations raised the possibility that the excessive
androgen production in PCOS results from intrinsic abnormalities in
theca cell steroidogenesis. If the abnormalities that underlie PCOS are
indeed intrinsic, then increased androgen production should persist in
PCOS theca cells maintained in long-term culture. Our data provide
support for the hypothesis that excessive androgen production by PCOS
theca cells results from increased expression of the enzymes involved
in steroid synthesis. Using culture conditions developed for normal
theca cells, we established methods to passage normal and PCOS theca
cells for successive population doublings with the maintenance of
regulatable CYP17, CYP11A, 3ß-HSD, and StAR gene expression (23, 25, 26, 32). The ovaries from which PCOS theca cells were isolated had a
characteristic gross morphology. The diagnosis of PCOS was established
by menstrual history (<6 menses per year) and elevated total serum T
and/or bioavailable T levels. It is important to recognize that
previous studies comparing steroid production by normal and PCOS theca
cells used primary cultures of theca cells that were pooled from
multiple follicles and/or patients (30, 31). To our knowledge, all of
these studies examined steroid production within a 4872 h period
after cell isolation, under conditions in which the influence of prior
in vivo stimulation may have persisted. In marked contrast,
in our studies theca cells were propagated from individual follicles
isolated from normal and PCOS patients, matched for follicle size and
patient age. Cells from individual follicles were initially frozen and
stocked in multiple vials. From each vial of frozen theca cells,
second-passage cells could be propagated, frozen, and stocked, or
subsequently cultured through a third passage (i.e. 2226
populations doublings) or fourth passage (3138 population doublings).
The ability to propagate functional theca cells for successive
population doublings provides a reproducible system to repetitively
compare theca cells isolated from individual patients and individual
follicles. Moreover, this system permits us to examine cells under
conditions that are distant from their in vivo paracrine and
endocrine milieu to investigate whether there are unique molecular or
biochemical phenotypes in PCOS ovarian cells.
Data from our experiments clearly establish that P4, 17OHP4, and T
production are markedly elevated per cell in PCOS theca cultures. In
these experiments, both androgen and P4 levels were found to be
increased in PCOS theca cells, and the androgen/P4 ratio was elevated,
indicating that androgen production predominates. Since the
ED50 for forskolin-stimulated steroid accumulation in both
normal and PCOS theca cells was identical (
3 µM), it
does not appear that the differences in steroidogenic activity can be
attributed to differences in forskolin-stimulated adenylate cyclase
activity. It is notable that the absolute amounts of P4, 17OHP4, and T
produced per PCOS theca cell (passaged for 2238 population doublings)
were similar to those reported by Gilling-Smith et al. (31),
which were obtained from primary (nonpassaged) PCOS theca cells. In
both our study and that of Gilling-Smith et al. (31),
steroid production as well as the androgen-progestin ratio were
increased in PCOS theca cells. Our findings, however, demonstrate that
the rates of basal and forskolin-stimulated P5, P4, and
DHEA metabolism are all dramatically increased in PCOS
cells. These data definitively demonstrate for the first time that
CYP17 and 3ß-HSD enzyme activities per theca cell are increased under
both basal and forskolin-stimulated conditions in PCOS theca cells. The
metabolic profiles also indicate that an androgenic 17ß-HSD activity
is increased in PCOS theca cells. Thus, the distinctive biochemical
phenotype of PCOS theca cells in long-term culture encompasses
increased activities of multiple steroidogenic enzymes. The evidence
for increased androgenic 17ß-HSD is of particular interest because
normal human ovaries are reported not to express type III 17ß-HSD,
the enzyme responsible for testicular T production (33). Thus, either
PCOS theca cells aberrently express the type III enzyme or an
alternative activity capable of reducing the 17-keto group of C19
steroids must be activated in PCOS.
The data presented in
Figs. 26



provide new and significant
information about the principal intermediates and the predominant
steroidogenic pathways involved in steroid biosynthesis in normal and
PCOS theca cells. Specifically, normal and PCOS theca cells do not have
the capacity to convert 17OHP4 to Adione. Androgen production in normal
theca cells, as well as increased androgen production by PCOS theca
cells, evidently involves the initial conversion of cholesterol to P5
(via CYP11A) and the subsequent conversion of P5 to DHEA
(via CYP17). DHEA is then further metabolized to Adione
(via 3ß-HSD) and/or Adiol (via 17ß-HSD), which are finally
converted to T. Thus, in agreement with our previous reports (22, 24),
the
5-steroid pathway is the predominant pathway used
for androgen biosynthesis by both normal and PCOS theca cells. The
4-steroids, P4 and 17OHP4, are not precursors for Adione
or T production by the normal or PCOS ovary. As we previously reported
(23), estradiol was not found to be a product of steroid metabolism in
normal or PCOS theca cells, substantiating the purity of the theca cell
preparations used in these studies. Of importance is the fact that the
steroid metabolism profiles observed in normal and PCOS theca cells
grown for 2226 population doublings were similar to those observed
when cells are grown for 3138 population doublings. These data verify
that increased androgen production is a stable phenotype of the
cultured PCOS theca cells.
The ability to propagate normal and PCOS theca cells in long-term
culture has and will continue to facilitate the examination of the
molecular basis for increased androgen production in PCOS. In this
report we present, for the first time, Northern analyses on RNA
obtained from theca cells isolated from individual follicles from
normal and PCOS patients. Data from these experiments definitively
demonstrate that the magnitude of forskolin-stimulated CYP17 and CYP11A
mRNA induction is greater in PCOS than in normal theca cells. These
data support the hypothesis that increased steady state levels of CYP17
and CYP11A mRNAs contribute to increased levels of steroidogenic
enzymes involved in androgen production by PCOS theca cells. Although
StAR mRNA levels were increased in response to forskolin, the magnitude
of StAR mRNA induction by forskolin was not different between PCOS and
control theca cells. These data suggest that increased androgen
production in PCOS does not result from overall differences in cAMP or
adenylate cyclase regulation, but rather selective alterations in
steroidogenic enzyme expression.
The persistent differences in steroidogenic activity of PCOS theca
cells could reflect an intrinsic (i.e. genetic) abnormality
in these cells or a stable biochemical imprint resulting from the
endocrine milieu experienced by the cells in vivo.
Unfortunately, at present, no definitive experiment can distinguish
between these two possibilities. However, there is increasing evidence
for a genetic basis for the hyperandrogenemia associated with PCOS (8).
If the stable biochemical phenotype in PCOS cells that we observed is
the result of a genetic variation, it evidently influences the
expression of multiple genes in the steroidogenic machinery, suggesting
that the putative abnormality most likely involves a signal
transduction pathway. PCOS theca cells may generate autocrine factors
that enhance steroidogenesis. Alternatively, PCOS cells may have
increased sensitivity to some component of the culture medium that
stimulates expression of steroidogenic enzymes. The factor is not
likely to be insulin since we have found, in unpublished experiments,
that both normal and PCOS theca cells respond equivalently to insulin
in terms of steroid (170HP4) production per cell and that increased
forskolin-stimulated steroid secretion by PCOS cells is observed in the
absence of insulin in the culture medium. The thecal cell system we
have employed is well suited for the study of the biochemical locus
that results in the stable steroidogenic phenotype of PCOS cells.
In conclusion, our analyses of normal and PCOS theca cells maintained
in long-term culture suggest that increased expression of CYP17 and
CYP11A mRNAs, as well as increased CYP17 and 3ß-HSD and 17ß-HSD
activities per theca cell, are stable properties of PCOS theca cells.
These data are consistent with the concept that increased androgen
production by PCOS theca cells is an intrinsic and, possibly
genetically determined, property of the cells. In future studies we
will attempt to define the locus of this abnormality through a
comprehensive analysis of the molecular mechanisms involved in the
transcriptional and posttranscriptional regulation of CYP17, CYP11A,
3ß-HSD, and 17ß-HSD expression.
 |
MATERIALS AND METHODS
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Theca Cell Isolation and Propagation
Human theca interna tissue was obtained from follicles of women
undergoing hysterectomy. The PCOS and normal ovarian tissue came from
age-matched women 3840 yr of age. The diagnosis of PCOS was made
according to established guidelines (34) including hyperandrogenemia,
oligo-ovulation, and the exclusion of 21-hydroxylase deficiency,
Cushings syndrome, and hyperprolactinemia. All of the theca cell
preparations studied came from ovaries of women with fewer than six
menses per year and elevated serum total T or bioavailable T levels as
we have previously described (8). Each of the PCOS ovaries contained
multiple subcortical follicles of less than 10 mm in diameter. The
control theca cell preparations came from ovaries of fertile women with
normal menstrual histories and no clinical signs of
hyperandrogenism.
Individual follicles were dissected away from ovarian stroma. The
isolated follicles were size selected for diameters ranging from 35
mm so that theca cells derived from follicles of similar size from
normal and PCOS subjects could be compared. 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. 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). Dispersed 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 media used for cell plating
was a 1:1 mixture of DMEM and Hams F-12 medium containing 10% FBS,
10% horse serum, 2% UltroSer G, 20 nM insulin, 20
nM selenium, 1 µM vitamin E, and antibiotics
(23). From each follicle twelve 35-mm dishes of primary theca interna
cells were grown until confluent, removed from the dish with neutral
protease (pronase-E; protease type XXIV, Sigma Chemical Co., St. Louis, MO) in DMEM/F12 (1:1), frozen, and stored in
liquid nitrogen (one 35-mm dish per vial) as previously described (23)
in culture medium that contained 20% FBS and 10% dimethyl sulfoxide.
In all experiments, cells were thawed and propagated in the growth
medium described above.
To obtain successive passages of normal and PCOS theca cells, cells
were thawed, propagated, and frozen at consecutive passages. The
passage conditions and split ratios for all normal and PCOS cells were
identical. Experiments comparing PCOS and normal theca were performed
utilizing third passage (i.e. 2226 population doublings)
and fourth passage (3138 population doublings) theca cells isolated
from size-matched follicles obtained from age-matched subjects.
At confluence the cells were transferred into serum-free medium (SFM)
containing DMEM/F12, 1.0 mg/ml BSA, 100 µg/ml transferrin, 20
nM insulin, 20 nM selenium, 1.0
µM vitamin E, and antibiotics. Sera and growth factors
were obtained as follows: FBS was obtained from Irvine Scientific
(Irvine, CA): horse serum was obtained from Gibco BRL
(Gaithersburg, MD); UltroSer G was from Reactifs IBF
(Villeneuve-la-Garenne, France): other compounds were from Sigma Chemical Co. In all experiments the gas phase used was 5%
O2, 90% N2, and 5% CO2. Reduced
oxygen tension and supplemental antioxidants (vitamin E and selenium)
were employed to prevent oxidative damage to CYP17 and CYP11A (22, 23).
Subculture was performed by incubation with neutral protease.
Assays for P5, P4, 17OHP4, and T
For evaluation of steroid production, normal or PCOS theca cells
were grown until subconfluent and transferred into SFM with 5 µg/ml
low-density lipoprotein in the presence and absence of 20
µM forskolin for 72 h to induce full steroidogenic
capacity. At 72 h the media were collected. RIAs for 17OHP4, P,
and T were then performed without organic solvent extraction using RIA
kits from ICN Biochemicals, Inc. (Irvine, CA).
Steroid Metabolism Assays
Long-term theca cultures (normal and PCOS) were grown until
subconfluent and transferred into SFM in the presence or absence of 20
µM forskolin for 72 h to induce full steroidogenic
capacity. The cells were then transferred into medium containing
saturating concentrations of [3H]-pregnenolone (1.0
µM), [3H]-P4 (1.0 µM), or
[3H]-DHEA (1.0 µM). Aliquots
of the medium were obtained at various time intervals (i.e.
3, 6, 12, 24, 36, 48, and 72 h). 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 automated chromatogram (Gilson Medical Electronics,
Inc., Middleton, WI) using a Phenomenex 25-cm 5
µm Prodigy C18 column (Milford, MA). The gradient solvent
delivery system consisted of 1:1 acetonitrile/methanol (A/M) 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, and 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 nonreduced and reduced steroids at 240 and 200
nM, respectively.
Determination of CYP17, CYP11A, StAR, and 3ß-HSD mRNA
Levels
Total RNA was extracted from theca cells as previously described
(23). Specific mRNA levels were quantitated using standard Northern
techniques. Human CYP17, CYP11A, 3ß-HSD, and StAR cDNAs were used as
hybridization probes. Hybridizable mRNA species were identified by
autoradiography and normalized using 28S ribosomal RNA.
Statistical Analysis
Statistical analysis was performed using unpaired two-tailed
t tests after combining the results from individual
patients. Each experiment was performed using triplicate or
quadruplicate replicate dishes. Experiments were repeated several times
with cells obtained from various PCOS and normal patients, that had
been thawed and grown to the appropriate passage.
 |
ACKNOWLEDGMENTS
|
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We would like to thank Drs. Ronald E. Estabrook and John M.
Trant for their valuable advice in the establishment of our HPLC
separation technique for identifying the numerous metabolic products of
steroid metabolism in normal and PCOS theca cells. We also thank
Jessica Wickenheisser for collecting media samples at various time
points throughout the steroid metabolism experiments.
 |
FOOTNOTES
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Address requests for reprints to: Jan M. McAllister Ph.D., Department of Cellular and Molecular Physiology, Pennsylvania State University, Hershey Medical Center, 500 University Drive, Hershey, Pennsylvania 17033.
This work was supported by NIH Grants U54 HD-34449 (J.F.S. and J.M.M.)
and R01 HD-33852 (J.M.M.)
Received for publication February 16, 1999.
Revision received March 31, 1999.
Accepted for publication April 1, 1999.
 |
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