Bone Morphogenetic Protein-15 Inhibits Follicle-stimulating
Hormone (FSH) Action by Suppressing FSH Receptor Expression*
Fumio
Otsuka,
Shin
Yamamoto,
Gregory F.
Erickson, and
Shunichi
Shimasaki
From the Department of Reproductive Medicine, University of
California, San Diego, La Jolla, California 92093-0633
Received for publication, November 3, 2000, and in revised form, January 9, 2001
 |
ABSTRACT |
We have recently reported that oocyte-derived
bone morphogenetic protein-15 (BMP-15) can directly modulate
follicle-stimulating hormone (FSH) action in rat granulosa cells. Here,
we investigate underlying mechanisms of this BMP-15 effect. Treatment
with BMP-15 alone exerted no significant effect on the basal expression
of mRNAs encoding steroidogenic acute regulatory protein, P450 side chain cleavage enzyme, P450 aromatase, 3
-hydroxysteroid
dehydrogenase, luteinization hormone receptor, and inhibin/activin
subunits. However, BMP-15 markedly inhibited the FSH-induced increases
in these messages. In striking contrast, BMP-15 did not change the forskolin-induced levels of these transcripts. Thus, the inhibitory effect of BMP-15 on FSH action must be upstream of cAMP signaling. We
next examined changes in FSH receptor mRNA expression.
Interestingly, BMP-15 severely reduced the levels of FSH receptor
mRNA in both basal and FSH-stimulated cells. To determine whether
this effect was at the level of FSH function, we investigated the
effect of BMP-15 on FSH bioactivity. Consistent with the mRNA data,
BMP-15 inhibited the biological response of FSH, but not that of
forskolin. Based on these results, we propose that BMP-15 is an
important determinant of FSH action through its ability to inhibit FSH
receptor expression. Because FSH plays an essential role in follicle
growth and development, our findings could have new implications for understanding how oocyte growth factors contribute to folliculogenesis.
 |
INTRODUCTION |
FSH1 is an essential for
normal folliculogenesis and female fertility (1, 2). In the ovary, FSH
interacts with its receptor on the granulosa cells (GCs) to initiate
cytodifferentiation and proliferation that ultimately result in the
development of preovulatory follicles (3, 4). The mechanism of FSH
action involves the activation of specific genes in the GCs through the
cAMP-dependent protein kinase-A signaling pathway
(5-7). Some of the physiologically important genes that are induced by
FSH signaling include the P450 aromatase (P450arom), luteinizing
hormone receptor (LH-R), steroidogenic acute regulatory protein (StAR),
P450 side chain cleavage enzyme (P450scc), 3
-hydroxysteroid
dehydrogenase (3
-HSD), inhibin, and activin (5, 8). The
physiological importance of FSH action is demonstrated by the fact when
restricted, the developing follicles die by apoptosis, and there are no
ovulations (9, 10). Therefore, it is important to define the mechanisms involved in FSH action.
There is a large body of evidence indicating that oocytes secrete
factors that modulate FSH action. (11-13). In vitro
experiments have shown that oocyte-derived factors can act to inhibit
FSH-induced expression of P450scc (14), progesterone (P4)
production (14), urokinase plasminogen activator (15), and LH-R
mRNA (16), while acting to stimulate mitosis (17), hyaluronic acid
(18-20), and estradiol (E2) production (21). There is
compelling evidence that growth and differentiation factor-9 (GDF-9) is
one oocyte factor involved in regulating these proliferative and
differentiation responses (22-28).
Recently, attention has been focused on another oocyte growth factor
involved in regulating FSH action: the new member of the transforming
growth factor-
superfamily designated as bone morphogenetic
protein-15 (BMP-15) or growth and differentiation factor-9B (GDF-9B)
(29-31). The cloning of the cDNAs in the mouse (29), human (29),
and rat (32) has revealed that the primary structure of BMP-15 is most
closely related to that of GDF-9 (29, 32). To date the only cell type
known to express BMP-15 is the mammalian oocyte (29-31). Regarding
function, we have recently reported that BMP-15 stimulates rat GC
proliferation and selectively inhibits FSH-induced P4, but
not E2, production (33). This is the only evidence
available to date concerning the function of the BMP-15 molecule.
The potential physiological relevance of BMP-15 in vivo is
beginning to emerge. A recent genetic study of a naturally occurring mutant in sheep termed Inverdale (FecXI) has provided
evidence for an essential role of BMP-15 in folliculogenesis and
fertility (34). In the female homozygous FecXI mutants,
follicular development arrests at the primary stage, resulting in
infertility. This phenotype has been linked to a defect in the BMP-15
gene. By contrast, the heterozygotes exhibit increased ovulation rate
and multiple pregnancies. Therefore, BMP-15 is associated with the
mechanisms of infertility and super fertility in a dosage-sensitive
manner (34). An important unanswered question is how BMP-15 regulates
these physiological functions.
The present study provides the evidence that BMP-15 inhibits major FSH
actions that are obligatory for follicle development and ovulation by
virtue of its ability to suppress FSH receptor (FSH-R) expression. The
possible mechanism by which BMP-15 controls the ovulation rate is discussed.
 |
EXPERIMENTAL PROCEDURES |
Reagents and Supplies--
Ovine FSH (NIDDK-oFSH-S20) was
provided by Dr. Parlow of the National Hormone and Pituitary Program
(Rockville, MD). Diethylstilbestrol, forskolin, and
4-androstene-3,17-dione (androstenedione, substrate of P450arom) were
purchased from Sigma and female Harlan Sprague-Dawley rats (23 day old)
from Charles-River Lab. (Wilmington, MA). Recombinant human BMP-15
tagged with a FLAG-epitope (BMP-15) was produced by 293 cells and
purified by anti-FLAG monoclonal antibody as reported previously (33).
Primary Cell Culture--
Female Harlan Sprague-Dawley rats (23 days old) were implanted with silastic capsules containing 10 mg of
diethylstilbestrol to increase GC number (3). After 4 days of
diethylstilbestrol exposure, GCs were collected from the ovaries and
cultured in serum-free McCoy's 5a medium supplemented with 2 mM L-glutamine and antibiotics as described
previously (35). The animal protocols were approved by the University
of California at San Diego Institutional Animal Care and Use Committee.
RNA Extraction and Analysis by Quantitative Competitive
RT-PCR--
GCs (2 × 106 viable cells) were cultured
in a six-well plate with 2 ml of McCoy's 5a medium containing one or a
combination of the following: 10 ng/ml FSH, 100 ng/ml BMP-15, 10 µM forskolin. One-hundred nM
androstenedione was added to the culture medium as indicated.
After 48 h culture, total RNA was extracted by guanidium acid-isothiocyanate-phenol-chlorform methods using TriZOL® (Life Technologies Inc.), quantified by measuring absorbance at 260 nm, and
stored at
80 °C until assay. Oligonucleotides used for RT-PCR were
custom-ordered from Life Technologies, Inc. PCR primer pairs were
selected from different exons of the corresponding genes to
discriminate PCR products that might arise from possible chromosome DNA
contaminants. Specifically, they are derived from the cDNA clones
at the following nucleotide numbers: 1651-1670 and 1751-1770 for StAR
(36); 148-167 and 637-656 for P450scc (37); 860-879 and 1062-1079
for P450arom (38); 317-336 and 437-456 for 3
-HSD (39); 524-543
and 684-703 for 20
-HSD (40); 30-49 and 267-286 for FSH-R (41),
962-981 and 1401-1420 for LH-R (42); 166-185 and 345-366 for
inhibin
-subunit; 428-447 and 588-607 for inhibin
A-subunit;
32-51 and 239-258 for inhibin
B-subunit (43); 401-421 and
575-595 for ribosomal protein-L19 (L19) (44). The steady state levels
of mRNA encoding StAR, P450scc, P450arom, and FSH-R (referred to
"target" mRNA) were analyzed by quantitative competitive
RT-PCR. Prior to PCR the internal control DNAs (~340 base pairs)
having target-specific primer pairs were generated by PCR as reported
previously (45). The extracted RNA (500 ng) was subjected to a RT
reaction using a First-Strand cDNA Synthesis System (Life
Technologies, Inc.) with random hexamer (2 ng/µl), reverse
transcriptase (200 units), and deoxynucleotide triphosphate (dNTP; 0.5 mM) at 42 °C for 50 min, 70 °C for 10 min. The
resultant simple strand cDNA was resuspended in 50 µl of water
for competitive PCR. The linear portion of the relationship between
target cDNAs and internal control DNAs was determined individually
for all targets. For this, a fixed amount (20 ng) of cDNA derived
from GCs treated with FSH was mixed with a series of 10-fold dilutions
of the internal control DNA, and the target and the internal control
DNAs were coamplified by PCR using a specific primer set for the
individual target (Fig. 1). Competitive PCR was performed using MgCl2 (1.5 mM), dNTP
(0.2 mM), and 2.5 units of Platinum Taq
DNA polymerase (Life Technologies, Inc.) under the following
conditions: 35 cycles of denaturation at 94 °C for 30 s,
annealing at 55 °C for 30 s, and extension at 72 °C for
30 s. Aliquots of PCR products were electrophoresed on 2% agarose
gels, visualized after ethidium bromide staining, photographed, and
scanned. The relative integrated density of each band was digitized by
multiplying the absorbance of the surface area (NIH image 1.62).
Finally, the ratios of the densitometric readings of the amplified
target cDNA and internal control DNA were plotted on the ordinate
against the serial 10-fold dilutions of internal control DNA on the
abscissa (Fig. 1). After establishing the working ranges in which
linear relationship existed, the following concentrations of the
internal control DNA were selected: 10
9
pM for StAR, 10
7 pM
for P450scc, 10
7 pM for P450arom,
10
4 pM for L19,
10
9 pM for FSH-R. For competitive
PCR reactions, 2 µl each of these internal control samples were added
to 20 ng of each cDNA sample. The resultant PCR products were
quantified by densitometric scanning as described above. Control
analysis was performed using L19 ribosomal protein primers as well. For
the other targets such as LH-R, inhibin subunits, 3
-HSD, and
20
-hydroxysteroid dehydrogenase (20
-HSD), semiquantitative PCR
analysis was performed without internal control under the same
condition, and the level of their PCR products were compared with that
of L19 target.

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Fig. 1.
Linear range analysis for quantitative
competitive RT-PCR. The linear ranges for the ratios of
coamplified PCR products were determined using cDNA
reverse-transcribed from RNA of FSH-treated GCs and competitive
internal control DNA for StAR, P450scc, P450arom, and L19 measured at
10-fold serial dilutions. A, PCR products were
gel-electrophoresed and stained by ethidium bromide. The concentrations
of the internal control DNA in each lane should refer to the number on
the abscissa of B. B, the ratios of
PCR products (target/internal control; ordinate) are plotted
against the internal control DNA (serial 10-fold dilutions;
abscissa).
|
|
Bioactvity Study--
GCs (105 viable cells) were
cultured in a 96-well plate with 200 µl of McCoy's 5a medium
containing 100 nM androstenedione together with
either 10 ng/ml FSH, 100 ng/ml BMP-15, and/or 10 µM
forskolin. After 48 h, the levels of P4 and
E2 in the media were measured by radioimmunoassay (33).
Statistical Analysis--
All results shown are mean ± S.E. of at least three separate experiments, with triplicate
determinations for each treatment. Differences between groups were
analyzed for statistical significance using analysis of variance
(StatView 5.0 software, Abacus Concept, Inc., Berkely, CA).
p values < 0.01 were accepted as statistically significant.
 |
RESULTS |
In previous experiments (33) we found that BMP-15 inhibits
FSH-induced P4, but not E2, production by
cultured rat GCs. To test the hypothesis that BMP-15 may be
specifically involved in modulating the expression of steroidogenic
genes in response to FSH stimulation, we analyzed mRNA levels for
StAR, P450scc, and P450arom by quantitative competitive RT-PCR.
As shown in Fig. 2, treating GCs for
48 h with a saturating dose of BMP-15 had no effect on the steady
state mRNA levels for StAR, P450scc, P450arom, and a housekeeping
gene L19. Treatment with FSH markedly increased the mRNA levels for
StAR, P450scc, and P450arom, whereas L19 mRNA levels were
unchanged. BMP-15 inhibited FSH-stimulated StAR and P450scc mRNA
levels by 60 and 70%, respectively, but had only a weak effect on
P450arom mRNA levels in the presence of androstenedione. L19
mRNA levels showed no difference among four different treatments.
Taken into consideration that StAR and P450scc are rate-limiting
factors of P4 synthesis in GCs (46), the suppression of
these gene expressions seems to be a major cause of the selective
suppression of FSH-induced P4 production.

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Fig. 2.
Effects of FSH and BMP-15 on the expression
of the mRNAs for StAR, P450scc, and P450arom. GCs were
cultured either alone or together with FSH (10 ng/ml) and/or
BMP-15 (100 ng/ml) in the presence of androstenedione (100 nM) for 48 h, after which total RNA was extracted and
then subjected to quantitative competitive RT-PCR analysis as described
under "Experimental Procedures." The PCR products are shown in
insets, and the ratios of PCR products (target/internal
control) are graphed. Bars with different letters
indicate that group means are significantly different at
p < 0.01.
|
|
We next investigated the effect of BMP-15 on forskolin-stimulated GC
responses. As shown in Fig. 3, forskolin
(10 µM) stimulated the levels of StAR, P450scc, and
P450arom mRNAs similar to that seen with FSH. However, in striking
contrast to the FSH results, BMP-15 failed to change the
forskolin-induced mRNA levels of these steroidogenic factors. L19
mRNA levels had no change by the forskolin treatment regardless of
the presence of BMP-15 among four groups. These results suggest that
the inhibitory effect of BMP-15 on FSH-induced StAR and P450scc
mRNA expression is mediated by a pre-cAMP signaling event.

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Fig. 3.
Effect of forskolin (10 µM) and BMP-15 (100 ng/ml) on
the expression of the mRNAs for StAR, P450scc, and P450arom.
The experimental design is as described in the legend to Fig. 2.
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|
The failure of BMP-15 to inhibit FSH-induced P450arom mRNA
expression is paradoxical. Given that androgens can up-regulate FSH-induced P450arom gene activity (47, 48), we wondered whether the
androstenedione in the culture medium could explain this paradox. As
seen in Fig. 4, FSH stimulated P450arom
mRNA expression in the absence of androstenedione and importantly,
this increase was significantly inhibited by BMP-15. As expected (see
Fig. 3), the forskolin-induced P450arom expression was not changed by
BMP-15 in the absence of androstenedione. The steady state mRNA
levels of StAR, P450scc, and L19 were unaffected by androstenedione
(data not shown). Thus, it is clear that in the absence of
androstenedione, P450arom mRNA induced by FSH was also inhibited by
BMP-15 at a site upstream of the cAMP signaling event.

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Fig. 4.
Effect of FSH (10 ng/ml) and forskolin
(10 µM) alone and
together with BMP-15 (100 ng/ml) on the expression of P450arom mRNA
in GCs cultured in the absence of androstenedione. The experiment
was performed as described in the legend of Fig. 2.
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|
To investigate whether the BMP effect involves changes in FSH-R
expression, we measured the mRNA levels for FSH-R by quantitative competitive PCR. First, we established a linear relationship between the expression of target FSH-R and internal control (Fig.
5A) and then selected
10
9 pM concentration of
internal control to perform the competitive reactions. Control
untreated cells expressed FSH-R mRNA spontaneously. Interestingly,
the level of FSH-R mRNA in control cells was markedly reduced
(80%) following treatment with BMP-15 (Fig. 5B). FSH
treatment increased FSH-R mRNA levels ~2-fold, and this increase
was completely abolished by cotreatment with BMP-15, actually
decreasing the FSH-R mRNA to that seen with BMP-15 alone. Thus,
both basal and FSH-induced FSH-R mRNA expression is negatively
regulated by BMP-15.

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Fig. 5.
Effect of FSH (10 ng/ml) and BMP-15 (100 ng/ml) on FSH-R mRNA levels in cultured GC. A, the
linear ranges for the ratios of coamplified PCR products were
determined as described in the legend to Fig. 1. B, FSH-R
mRNA levels in GCs with the indicated treatments are presented
together with those of L19 mRNA. Bars with different
letters indicate that group means are significantly
different at p < 0.01. i.c., internal
control.
|
|
To determine whether BMP-15 modulates other FSH-dependent
functions in GCs, we examined the possible role of BMP-15 in the regulation of LH-R, the inhibin subunits (
,
A, and
B),
3
-HSD, and 20
-HSD. As seen in Fig.
6, FSH induced marked increases in the
levels of mRNA for each of these end points, except for 20
-HSD. Although BMP-15 alone had no effect on the basal levels of these mRNAs, it totally abolished the stimulatory effects seen with FSH
(Fig. 6). BMP-15, either alone or together with FSH, did not change the
basal levels of 20
-HSD mRNA. As illustrated in Fig. 7, forskolin induced the expression of
high levels of the mRNAs encoding LH-R, inhibin
,
A and
B
subunits, and 3
-HSD. Consistent with our earlier results with StAR,
P450scc and P450arom, cotreatment with BMP-15 failed to alter the
forskolin-induced expression of these mRNAs (Fig. 7).

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Fig. 6.
Effect of BMP-15 on other FSH-induced
cellular responses. GCs were cultured for 48 h with FSH (10 ng/ml) and/or BMP-15 (100 ng/ml), after which total RNA was extracted
and subjected to semiquantitative RT-PCR analysis using specific
primers for the indicated mRNAs. Two distinct bands of LH-R are due
to alternatively spliced transcripts (53). The representative data are
presented from three independent experiments.
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Fig. 7.
Effect of BMP-15 on the indicated forskolin
(10 µM)-induced
biological responses in GCs. The experimental design is as
described in the legend to Fig. 6.
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|
To determine whether this BMP-15 inhibition was at the level of FSH
function, we tested the effect of BMP-15 on FSH bioactivity, specifically, P4 and E2 production in the GCs.
In agreement with our recent study (33), FSH-induced P4,
but not E2, production was suppressed by BMP-15 (Fig.
8). In contrast, the forskolin-induced
steroidogenic responses were unaffected by BMP-15 (Fig. 8). These
results fit our hypothesis that BMP-15 inhibits FSH action by
suppressing functional FSH-R in GCs.

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Fig. 8.
Effect of BMP-15 on FSH- and
forskolin-induced steroidogenesis. GCs (105 viable
cells) were cultured in a 96-well plate with 200 µl of McCoy's 5a
medium containing 100 nM androstenedione and one or a
combination of the following: 10 ng/ml FSH, 100 ng/ml BMP-15, 10 µM forskolin. After 48 h, the concentrations of
P4 and E2 were measured by radioimmunoassay.
Bars with different letters indicate that group
means are significantly different at p < 0.01.
|
|
 |
DISCUSSION |
In a recent study we showed that BMP-15 stimulates proliferation
of rat GCs and negatively regulates FSH-induced P4, but not
E2, production in vitro (33). The objective of
the present study was to begin to explore the molecular basis by which BMP-15 modulates FSH action. The major new findings of the present study are as follows. First BMP-15 reduced the steady state levels of
mRNA induced by FSH, including StAR, P450scc, 3
-HSD, LH-R, inhibin/activin subunits, and FSH-R. These observations suggest that
BMP-15 can act broadly to inhibit the expression of a large battery of
genes induced by FSH. Accordingly, BMP-15 can be considered a negative
regulator of the major actions of FSH in the rat ovary. Second, in
striking contrast to the FSH action, BMP-15 did not affect the
stimulation of this battery of mRNAs in response to forskolin.
Therefore, one can conclude that BMP-15 exerts its inhibitory effect on
FSH action upstream of cAMP signaling. Third, BMP-15 markedly decreased
both the basal and FSH-induced increases in FSH-R mRNA. And fourth,
FSH-induced, but not forskolin-induced, steroidogenesis was suppressed
by BMP-15, a finding that is consistent with the decrease in FSH-R
message. Based on these findings, we propose that the negative
regulation of FSH-R expression is the primary cause of BMP-15
inhibition of FSH action. It is important to note, however, that
because we did not measure FSH protein levels directly, we cannot
exclude the possibility that BMP-15 might exert its effects by a
mechanism other than reducing the number of FSH-R.
The finding that BMP-15 is a negative regulator of FSH-R expression
together with our earlier report that BMP-15 stimulates DNA synthesis
(33) establishes the concept that oocyte-derived BMP-15 is functionally
involved in regulating two major aspects of GC development,
namely stimulating proliferation and inhibiting FSH-dependent cytodifferentiation. This suggests a role for
BMP-15 in coordinating GC proliferation with FSH-dependent
cytodifferentiation during follicle growth and development. Our
observation that FSH stimulates the expression of FSH-R mRNA is
consistent with the previous reports demonstrating that FSH itself
increases FSH-R gene transcription and cell surface FSH receptors (49,
50). Our results clearly demonstrated that BMP-15 completely blocks this FSH effect. Therefore, we propose that BMP-15 is a determinant of
FSH action by exerting inhibitory effects on FSH-R expression.
It was found that BMP-15 does not inhibit the amplifying effect of
androstenedione on FSH-stimulated P450arom mRNA expression. But, it
did suppress the ability of FSH to induce P450arom in the absence of
androstenedione. This is consistent with the failure of BMP-15 to
inhibit FSH-induced E2 production in the presence of added
androstenedione. The potential significance of this finding is unknown.
In this regard, however, a fundamental principle in ovarian physiology
is that FSH stimulates the expression of P450arom and E2
production in the dominant follicle that contains a high concentration
of androstenedione in the follicular fluid. Therefore, the finding that
BMP-15 does not inhibit FSH-induced P450arom mRNA and
E2 production in the presence of androstenedione may be
physiologically relevant.
Genetic and physiological studies in Inverdale (FecXI)
sheep have identified a point mutation in the BMP-15 gene that has
profound effects on follicle development and ovulation quota (34).
Homozygous FecXI mutant females are infertile because
follicle growth is arrested at the primary preantral stage.
Consequently there are no ovulations in these animals. This provides
compelling evidence for a requirement of bioactive BMP-15 in follicle
cell proliferation and differentiation in sheep (51). Surprisingly, the
heterozygous FecXI mutants exhibit increased ovulation
rate. In this connection, several interesting abnormal features of the
ovaries of the heterozygotes have been identified: (i) there are more
healthy estrogenic follicles, ii) the number of GCs in these developing
follicles is significantly smaller, iii) these GCs have a higher mean
LH responsiveness at smaller follicle stages, and iv) the corpora lutea
are smaller (52). The fact that the plasma FSH and LH levels in the
heterozygotes are normal would imply that the mechanisms responsible
for these unusual features in the heterozygote reside in the ovary. Our observation that BMP-15 inhibits FSH-R expression could explain the
cause of the abnormal phenotype of the Inverdale ewe. In the heterozygotes, we propose that reduced levels of BMP-15 result in
higher levels of FSH-R in the GCs, which in turn lead to more developing healthy estrogenic follicles with more LH-R. The end result
of this sequence of events would be increased ovulation rate. At the
opposite extreme, follicle development in the homozygotes is arrested
at the primary follicle (FSH-independent) stage. In these animals we
propose that the absence of BMP-15 results in the cessation of GC
proliferation, which in turn leads to arrested follicle development.
In summary, the conclusion emerging from these findings is that BMP-15
can inhibit some of the most important events in the process of
FSH-dependent GC cytodifferentiation, including the expression of the FSH-R itself. Consequently the oocyte can play an
important role in determining GC proliferation and FSH sensitivity in
developing follicles.
 |
ACKNOWLEDGEMENTS |
We appreciate constructive discussion with
Drs. Kelly Moore and Woo-Sik Lee and the technical assistance of Mei
Wang and typing of Andrea Hartgrove.
 |
FOOTNOTES |
*
This work was supported in part by The Lalor Foundation,
University of California, San Diego Academic Senate Grant RY
440M, and NICHD/National Institutes of Health through cooperative
agreement U54HD12303 as part of Specialized Cooperative Centers Program in Reproduction Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Reproductive
Medicine, University of California at San Diego, School of Medicine,
9500 Gilman Dr., La Jolla, CA 92093-0633. Fax: 858-822-1482; E-mail
address: sshimasaki@ucsd.edu.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M010043200
 |
ABBREVIATIONS |
The abbreviations used are:
FSH, follicle-stimulating hormone;
BMP-15, bone morphogenetic protein-15;
E2, estradiol;
FSH-R, follicle-stimulating hormone
receptor;
GC, granulosa cell;
GDF-9, growth differentiation factor-9;
3
-HSD, 3
-hydroxysteroid dehydrogenase;
20
-HSD, 20
-hydroxysteroid dehydrogenase;
LH, luteinizing hormone;
LH-R, luteinizing hormone receptor;
P4, progesterone;
P450arom, P450 aromatase P450scc, P450 side chain cleavage enzyme;
StAR, steroidogenic acute regulatory protein;
RT-PCR, reverse
transcription-polymerase chain reaction.
 |
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