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Original Studies |
Department of Reproductive Medicine, University of California-San Diego, La Jolla, California 92093-0633
Address all correspondence to: Dr. S. S. C. Yen, Department of Reproductive Medicine, University of California-San Diego, La Jolla, California 92093-0633. E-mail: dnye{at}ucsd.edu
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Abstract |
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-hydroxylase messenger ribonucleic acid (mRNA)
and proteins in thecal cells with follicular maturation suggest that
the intraovarian CRF system may play an autocrine role regulating
androgen biosynthesis, with a downstream effect on estrogen production
by granulosa cells. The functionality of the ovarian CRF system may be
conditioned by the relative presence of plasma-derived CRF-BP by virtue
of its localization of protein, but not transcript in thecal cells and
its ability to compete with CRF for the CRF receptor.
To further these findings, in the present study we have examined the
effect of CRF on LH-stimulated 17-hydroxylase (P450c17) gene
expression and androgen production by isolated thecal cells from human
ovarian follicles (11–13 mm). During the 48-h culture, addition of LH
(10 ng/mL) to the medium increased by 5- and 6-fold
dehydroepiandrosterone and androstenedione production by thecal cells.
Remarkably, the LH-stimulated, but not basal, androgen production was
inhibited by CRF in a time- and dose-dependent manner. The half-maximal
(ID50) effect dose of CRF occurred at 5 x
10-8 mol/L, and at a maximal concentration of
10-6 mol/L, CRF completely inhibited LH-stimulated
androgen production. This inhibitory effect of CRF became evident at
12 h (45%), and by 24 h the effect was more pronounced, with
a 70% reduction from baseline. As determined by Northern analyses, CRF
dose dependently decreased LH-stimulated P450c17 mRNA levels, with a
maximal inhibition of 85% P450c17 gene expression at a CRF
concentration of 10-6 mol/L. With the addition of
10-6 mol/L of the antagonist -helical CRF-(9–41), the
inhibitory effect of CRF was partially reversed for both P450c17 mRNA
(75%) and androgen production (50%), indicating the CRF-R1-mediated
event.
In conclusion, the present study demonstrated a potent inhibitory effect of CRF on LH-stimulated dehydroepiandrosterone and androstenedione production that appears to be mediated through the reduction of P450c17 gene expression. Thus, the ovarian CRF system may function as autocrine regulators for androgen biosynthesis in the thecal cell compartment to maintain optimal substrate for estrogen biosynthesis by granulosa cells. Further studies to define the role of CRF-BP in the endocrine modulation of the intraovarian CRF system are needed.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These earlier observations suggest that intragonadal CRF may function
to regulate androgen biosynthesis. To further this idea, we recently
characterized the CRF system, which includes ligand, receptor,
CRF-binding protein (CRF-BP), and the androgen steroidogenic enzyme
P450c17, in the human ovary using in situ hybridization and
immunohistochemistry methods (22). The thecal-stromal compartment, but
not granulosa cells, of the human ovary contain a CRF system complete
with CRF, CRF-Receptor 1 (CRF-R1), and the blood-derived CRF-BP. The
parallel increases in the intensity of gene expression and protein for
CRF, CRF-R1, and 17-hydroxylase with follicular maturation suggest
that the intraovarian CRF system may play an autocrine role in thecal
cell androgenesis, with a downstream effect on estrogen production by
granulosa cells (22). These findings provided the basis for the present
study, which sought to determine the effect of CRF on 17-hydroxylase
gene expression and androgen production by isolated thecal cells from
human ovarian follicles. Here we report these findings.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ovaries were collected from 7 women (33–41 yr old) undergoing benign gynecological surgery during the follicular phase. The stage of the menstrual cycle was determined by the recorded last menstrual period. Pathological examination of the ovaries revealed no apparent abnormalities. None of the patients had received any hormone therapy for up to 3 months before surgery. Human ovarian thecal cells were isolated from 15 healthy follicles, 11–13 mm in diameter, with an androstenedione/estradiol ratio of less than 0.9 (range, 0.11–0.9). Follicles with a androstenedione/estradiol ratio greater than 1 were considered atretic and were not used for the study. The study was approved by the committee on investigations involving human subjects at the University of California-San Diego. Informed consent was obtained from all subjects.
Isolation and culture of human thecal cells were performed as
previously described by McAllister et al. (23) with minor
modification. Briefly, follicular fluid from the dissected follicle was
aspirated, and the follicle wall was opened. Granulosa cells were
gently removed with a platinum loop. After extensive washing with
medium, the theca interna was stripped from the follicle wall. A
suspension of thecal cells was obtained after dispersal for 30 min at
37 C with 0.1% collagenase and 0.01% deoxyribonuclease (Sigma
Chemical Co., St. Louis, MO) in medium containing 1% (wt/vol) BSA. The
dispersed thecal cells were further purified using a discontinuous
Percoll gradient, as previously described by Magoffin and Erickson
(24). Cells were then suspended in DMEM-Ham’s F-12 (1:1) containing 25
mmol/L HEPES, 50 IU/mL penicillin, 50 µg/mL streptomycin, an
additional 2 mmol/L L-glutamine, 10% horse serum, 10%
FBS, and 0.1% BSA and seeded in 12- or 6-well cell culture plates at a
density of 106 or 2 x 106 cells/plate,
respectively. After 24-h incubation at 37 C in humidified 5%
CO2 incubator, cells were extensively washed to remove
serum-supplemented medium and reincubated for 48 h in serum-free
DMEM-Ham’s F-12 medium containing 25 mmol/L HEPES, 1 mg/mL BSA, 1
µg/mL transferrin, 20 nmol/L selenium, 100 U/mL penicillin, and 100
µg/mL streptomycin. Thecal cells were cultured in the presence and
absence of LH (10 ng/mL), CRF (10-8, 10-7,
and 10-6 mol/L), and LH plus CRF or CRF antagonist
(-helical 9–41; 10-6 mol/L). Media of cells cultured
in 12-well plates were collected and stored at -20 C until analysis of
dehydroepiandrosterone (DHEA) and androstenedione concentrations using
RIA kits (Diagnostic System Laboratories, Webster, TX). The minimum
detection limit of the DHEA and androstenedione assays were 10 and 30
pg/mL, respectively. The intraassay variations were 3%, and 4.2%, and
the interassay variations were 6.4% and 7.6%, respectively. The total
ribonucleic acid (RNA) was extracted from cells cultured in 6-well
plates and stored at -70 C until analysis of P450c17 gene expression
by Northern blot.
Northern blot analysis
RNA was isolated from thecal cells using procedures provided by
the manufacturer (Tel-Test "B," Friendswood, TX). The RNA content
was determined by UV spectrophotometry at 254 nm. About 20 µg total
RNA from each sample were denatured in 6% formaldehyde and 50%
formamide in 1 x MOPS buffer (0.2 mol/L
3-morpholinopropanesulfonic acid, pH 7.0, containing 50 mmol/L sodium
acetate and 10 mmol/L ethylenediamine tetraacetate) for 15 min at 65 C.
Samples were then loaded onto a 1% agarose gel containing 3%
formaldehyde and subjected to electrophoresis. RNA was transferred onto
a nylon membrane (Nytran) and immobilized by UV cross-linking. The
membrane was then prehybridized for 4 h at 42 C in 50% deionized
formamide, 10% dextran sulfate, 1% SDS, 100 µg/mL sheared and
denatured salmon sperm DNA, 0.5 µg/mL transfer RNA, and 1 mol/L
sodium chloride. P450c17 transcript was detected using a
--32P-labeled human P450c17 complementary DNA fragment
that was radiolabeled by nick translation. The membrane was hybridized
with the labeled probe for 24 h at 42 C. The blot was then washed
twice with 2 x SSC (30 mmol/L M sodium citrate, pH 7.0, at 22 C
containing 0.3 mol/L NaCl) for 5 min at room temperature, twice with
2 x SSC containing 1% SDS for 30 min at 60 C, and twice with
0.1 x SSC for 30 min at room temperature. Audoradiography was
performed at -70 C with intensifying screens. Relative changes in
P450c17 messenger RNA (mRNA) level with thecal cells treated with and
without LH and in the presence and absence of CRF and CRF antagonist
were quantified using densitometric scanning of the blot and normalized
for loading of total RNA using ß-actin mRNA expression in each
treatment group.
Statistical analysis
Means were compared by one-factor ANOVA. Values are given as the mean ± SD. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the 48-h culture, the addition of LH (10 ng/mL) to culture
medium induced an approximately 6-fold increase (P <
0.001) in DHEA and androstenedione production by thecal cells compared
to control values (Fig. 1
). Remarkably,
CRF inhibited LH-stimulated DHEA and androstenedione production in a
dose-dependent manner, with half-maximal (ID50) effect at
5 x 10-8 mol/L (P < 0.05). At a
maximum concentration of 10-6 mol/L, CRF suppressed
LH-stimulated androgen production to a level comparable to the basal
value (P < 0.001; Fig. 1). Although CRF profoundly
attenuated LH-stimulated androstenedione production, it had no effect
on basal levels (data not shown). The time course of the inhibitory
effect of CRF on LH-stimulated androstenedione production by thecal
cells was evident at 12 h of culture (45% inhibition;
P < 0.01), and by 24 h, inhibition of
LH-stimulated androstenedione production by CRF became more pronounced,
with a 70% reduction (P < 0.001) from baseline (Fig. 2). The inhibitory effect of CRF on
LH-stimulated androgen production appears to be receptor mediated, as
it was partially reversed (50%; P < 0.01)) by the
addition of the CRF receptor antagonist (10-6 mol/L;
-helical 9–41; Fig. 3).
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To determine whether CRF exerts its inhibitory action on
LH-stimulated androgen production by affecting gene expression of the
key enzyme for androgen biosynthesis, P450c17, Northern blot analysis
was performed to quantify changes in the P450c17 mRNA level. Thecal
cells were cultured for 48 h with and without LH and in the
presence and absence of CRF and CRF antagonist. Relative levels of
P450c17 mRNA in each treatment group were quantified using
densitometric scanning and normalized for loading of total RNA from
thecal cells using ß-actin mRNA expression (Fig. 4). CRF decreased the LH-stimulated
P450c17 mRNA level in a dose-dependent manner, with a maximum
inhibition at a concentration of 10-6 mol/L CRF, which
decreased P450c17 gene expression by 85% (P < 0.001)
compared to the control value. This CRF-induced inhibition of P450c17
gene expression appears to be receptor mediated because it was
partially reversed (75%; P < 0.001) by addition of
the -helical CRF-(9–41) receptor antagonist to culture medium (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Northern blot analyses showed that CRF also reduced LH-stimulated
P450c17 mRNA levels in a dose-dependent manner, with maximum inhibition
occurring at a concentration of 10-6 mol/L CRF, at which
point P450c17 gene expression was decreased by 85% compared to the
control value. The inhibitory effect of CRF on LH-stimulated of both
P450c17 mRNA and androgen production appears to be mediated by CRF-R1,
as the addition of 10-6 mol/L -helical CRF-(9–41)
receptor antagonist partially reversed the inhibition of thecal cell
production of DHEA and androstenedione (50%) and P450c17 gene
expression (75%). Thus, the inhibitory effect of CRF on androgen
production by thecal cells in culture appears to be mediated through
the reduction of androgen biosynthetic enzyme P450c17 mRNA. This was
further indicated by the parallel decline in mRNA expression for
P450c17 and the attenuation of LH-stimulated androgen production by
CRF. It is known that LH stimulates androgen production in gonads by
affecting several steps in the steroidogenic pathway. The binding of LH
to its receptor induces a conformational change in the receptor,
leading to G protein binding, and activates adenlylate cyclase and
cAMP, which subsequently stimulate steroidogenic enzymes, P450scc and
P450c17, and androgen production (25). However, our data cannot exclude
the possibility that CRF may also exert its inhibitory action on
LH-stimulated androgen production by human thecal cells at P450scc
and/or at a step proximal to this enzyme. Further studies are required
to address this issue. Although we have previously shown that the
transcripts and protein for CRF, CRF-R1, and 17-hydroxylase are
colocalized in the thecal-stromal compartment (22), consideration
should be given that resident ovarian macrophages, a major cellular
component of the interstitial compartment, may subserve an additional
source of CRF (18) as well as providing IL-1, through which it can
inhibit androgen production (26).
The regulation and functional role of the human ovarian CRF system are complicated by the presence of high affinity CRF-BP in human plasma. We have previously shown an intense immunostaining for CRF-BP in the thecal-stromal compartment of mature follicles and in the lumen of capillary vessels, but mRNA encoding CRF-BP was not detected in the human ovary by either in situ hybridization or RT-PCR (22). Thus, the IrCRF-BP found in the thecal-stromal layer is not derived from the ovarian transcript, and in all probability, it originated from the peripheral circulation. CRF-BP was initially isolated from human plasma (27), and its complementary DNA was subsequently cloned from a liver DNA library (28). The binding affinity of CRF-BP to CRF exhibits a Kd an order of magnitude lower than that displayed by the CRF receptors (29). CRF-BP was shown to inhibit CRF-induced ACTH secretion from the pituitary (28) and placental tissue (30) in vitro. Although CRF-BP is expressed in many areas of the rat brain, CRF-BP has not been found in the peripheral plasma of several species studied (2, 29). Thus, CRF-BP in plasma appears to be unique to humans, and women have higher concentrations than men (31). It is likely, therefore, that the circulating CRF-BP may partake an endocrine role in the modulation of the ovarian CRF system by controlling the amount of free CRF available to interact with CRF receptors in the thecal compartment. This quenching effect of circulating CRF-BP on the ovarian CRF system in vivo may dictate the optimal degree of androgenesis by the thecal cell compartment, a proposition that may explain the paradox of an inhibitory effect of CRF on P450c17 mRNA in vitro and the concomitant increases in CRF and P450c17 mRNA in ovarian thecal-stromal cells with follicular maturation in vivo (22).
Recently, an inhibitory effect of CRF on FSH-stimulated estradiol production by human granulosa-luteal cells in vitro has been reported (32). This finding is inconsistent with the absence of CRF and CRF receptor transcript and protein in granulosa cells in vivo (22). The presence of CRF in follicular fluid (18) may reflect a paracrine action of CRF from thecal cells to granulosa cells. Further, CRF action on granulosa cells may be mediated by the IL-1 receptor rather than CRF-R1, as reported by Ghizzoni et al. (33).
In summary, the present in vitro findings together with our
recent in vivo data have established the presence of a CRF
system replete with ligand and receptor in the thecal-stromal cell
compartment, with enhanced expression of both proteins and transcripts
in parallel with the steroidogenic enzyme, 17-hydroxylase, in the
maturing follicle. The inhibitory effect of CRF on LH-stimulated
androgen production appears to be mediated through the reduction of
17-hydroxylase gene expression. Circulating CRF-BP may function as
an extraovarian modulator by virtue of its ability to neutralize CRF
action. As such, CRF-BP may be a determinant of the net effect of
intraovarian CRF bioactivities. Thus, the ovarian CRF system may be
viewed as an autocrine and paracrine regulator of steroidogenesis with
an endocrine component of modulation. Additional studies are required
to further characterize the functionality of the ovarian CRF system and
its aberrations in conditions with excessive androgen production and
enhanced P450c17 enzymatic expression, such as polycystic ovary
syndrome (34).
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Acknowledgments |
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Footnotes |
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2 Former Research Fellow in Reproductive Endocrinology.
3 Former Clinical Fellow in Reproductive Endocrinology.
4 Investigator with the Clayton Foundation.
Received August 4, 1997.
Revised September 23, 1997.
Accepted October 14, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase/lyase activities in proliferating human theca interna
cells in long term monolayer culture. Endocrinology. 125:1959–1966.This article has been cited by other articles:
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