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J. Biol. Chem., Vol. 276, Issue 35, 32889-32895, August 31, 2001
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From the Department of Reproductive Medicine, University of
California San Diego, School of Medicine,
La Jolla, California 92093-0633
Received for publication, April 11, 2001, and in revised form, July 10, 2001
The process of ovarian
folliculogenesis is composed of proliferation and differentiation of
the constitutive cells in developing follicles. Growth factors emitted
by oocytes integrate and promote this process. Growth differentiation
factor-9 (GDF-9), bone morphogenetic protein (BMP)-15, and BMP-6 are
oocyte-derived members of the transforming growth factor- One major direction of current research in the investigation of
mechanisms controlling folliculogenesis is the identification of the
biological functions of autocrine/paracrine factors that are produced
in the ovary. Some of these molecules are synthesized and secreted by
the oocyte (1) and act as morphogens to control follicle growth as well
as differentiation (2). There is a large body of evidence indicating
that oocyte-derived factors modulate ovarian function (1-3). In
vitro experiments have shown that these factors can act to inhibit
progesterone
(P4)1 production
(4), follicle-stimulating hormone (FSH)-induced expression of P450
side-chain cleavage enzyme (P450scc) (4), and luteinizing-hormone
receptor (LH-R) mRNA (5), while acting to stimulate estradiol
(E2) production (6) and granulosa cell (GC) mitosis (7).
Three oocyte-derived members of the transforming growth factor- Recently, there have been a number of studies published on the role of
GDF-9 and BMP-15 in the ovary. With regard to GDF-9, female mice
lacking GDF-9 have resulted in an early block in folliculogenesis leading to infertility, suggesting that GDF-9 is obligatory for normal
folliculogenesis and female fertility (8-10). In vitro studies using rat and mice GCs have demonstrated that GDF-9 regulates GC mitogenesis and steroidogenesis as well as cumulus expansion (11-14). As for BMP-15, which is most closely related in structure to
GDF-9, an intriguing finding has been reported recently by Galloway
et al. (15), in which they identified a causative point mutation in the bmp-15 gene in Inverdale sheep that
has been known to exhibit aberrant follicle development and ovulation
rate. Of interest, the homozygous Inverdale female sheep are infertile because follicle growth is arrested at the primary preantral stage, whereas the heterozygous females exhibit increased ovulation rate. Thus
this mutation causes increased ovulation and infertility in a
dosage-sensitive manner, suggesting an important role for BMP-15 in
folliculogenesis and ovulation. By in vitro studies with rat
GCs, we have shown that BMP-15 stimulates mitosis independent of FSH
and regulates steroidogenesis by inhibiting FSH receptor (FSH-R)
expression (16, 17).
In contrast, although BMP-6 mRNA is highly expressed in both
immature and mature oocytes of mice (10, 18), there have been few
studies designed to investigate the biological functions and target
cells of BMP-6 in the ovary. Mice with homozygous loss-of-function mutations in the bmp-6 gene by targeted deletion are
fertile with normal sized litters (19), which may suggest that
bmp-6 is dispensable for fertility. The lack of
perturbative reproductive phenotype seen in these mutant mice could be
the reflection of compensation by related BMP(s) expressed in the ovary
by virtue of a redundant safety system to maintain homeostasis.
Alternatively, it is possible that BMP-6 evokes no biological effects
in the ovary.
Because no information is available at present from any species about
the biological function of BMP-6 in the ovary, its role in the ovary
remains beyond speculation. In the present study, we report the
identification of GCs as a target cell type for BMP-6 in the rat ovary.
Furthermore, we report that BMP-6 evokes biological activities that are
distinct from other TGF- Reagents and Supplies--
Ovine FSH (NIDDK-oFSH-S20) was
provided by Dr. Parlow of the National Hormone and Pituitary Program
(Rockville, MD). Diethylstilbestrol (DES), forskolin, 8-bromo-cAMP
(8-Br-cAMP), 3-isobutyl-1-methylxanthine (IBMX), and
4-androstene-3,17-dione (androstenedione) were purchased from Sigma and
female Harlan Sprague-Dawley (SD) rats from Charles River Laboratories
(Wilmington, MA). Recombinant human BMP-6 was generously provided by
Dr. Kuber Sampath, Creative BioMolecules Inc., Hopkinton, MA.
Recombinant human BMP-15 tagged with a FLAG epitope (BMP-15) was
produced by 293 cells and purified using anti-FLAG monoclonal antibody
as reported previously (16).
Primary Cell Culture--
Twenty three-day-old female SD rats
were implanted with silastic capsules containing 10 mg of DES to
increase GC number. After 4 days of DES exposure, GCs were collected
from the ovaries and cultured in serum-free McCoy's 5a medium
supplemented with 2 mM L-glutamine and
antibiotics at 37 °C in an atmosphere of 5% CO2 in air.
The animal protocols were approved by the University of California at
San Diego Institutional Animal Care and Use Committee.
Analysis of Steroid and cAMP Production--
GCs
(105 viable cells) were cultured in a 96-well plate with
200 µl of medium containing one or a combination of the following: 0-10 ng/ml FSH, 0-300 ng/ml BMP-6, 10 µM forskolin, and
1 mM 8-Br-cAMP. For the assessment of steroids, 100 nM androstenedione, a substrate for P450 aromatase
(P450arom), was added to the media. After 48 h culture, the
supernatant of culture media was collected and stored at RNA Extraction and Analysis by Quantitative Competitive
RT-PCR--
GCs (2 × 106 viable cells) were cultured
in a 6-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-6, 10 µM forskolin, and 0.2 or 1 mM 8-Br-cAMP.
Under the indicated conditions, androstenedione was added to the
culture media to a final concentration of 100 nM. After
48 h culture, total RNA was extracted by guanidinium acid/isothiocyanate/phenol chloroform methods using TRIzol® (Life Technologies Inc.), quantified by measuring absorbance at 260 nm, and
stored at Thymidine Incorporation Assay and Cell Proliferation
Analysis--
GCs (2 × 105 viable cells) were
cultured in polypropylene 1.5-ml tubes containing 200 µl (final
volume) of culture medium. After a 24-h pre-culture period, cells were
incubated with 0.5 µCi/tube of
[methyl-3H]thymidine either alone or together
with increasing doses of BMP-6 (0-300 ng/ml). After another 24 h
of culture, the GCs were washed with phosphate-buffered saline,
centrifuged (2000 × g, 30 min), and incubated with
10% ice-cold trichloroacetic acid for 30 min at 4 °C. The cell
pellet was solubilized in 0.2 M NaOH and its radioactivity
measured (16). Viable GC number was also counted by trypan blue
exclusion using hemocytometry from cells cultured for 24 h with or
without either 300 ng/ml BMP-6 or 100 ng/ml BMP-15.
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 or
unpaired t tests (StatView 5.0 software, Abacus Concept,
Inc., Berkeley, CA). p values <0.05 were accepted as
statistically significant.
We have first assumed that GCs are target cells for BMP-6 because
rat GCs express BMP receptor type IB (BMPR-IB, also known as ALK-6) and
type II (BMPR-II) and at lesser levels BMPR-IA (ALK-3) (28) as well as
activin type II and type IIB receptors (29), all to which BMP-6 can
bind (30). We have, therefore, tested whether GCs would respond to
BMP-6. Similar to our previous studies on BMP-4, BMP-7, and BMP-15 (16,
28), we examined the effect of BMP-6 on P4 and
E2 production induced by FSH using primary rat GCs cultured in serum-free medium. As observed earlier (16, 28), FSH alone increased
P4 and E2 production in a
dose-dependent manner (Fig.
1). By comparison, BMP-6 alone (up to 300 ng/ml) did not affect basal levels of P4 and E2
production. However, co-treatment of GCs with a saturated dose of FSH
(10 ng/ml) and increasing doses of BMP-6 (0-300 ng/ml) caused a marked
inhibition (80%) of the FSH-induced P4 production
(ED50 = 10 ng/ml). On the contrary, BMP-6 had no effect on
the FSH-induced E2 production. These findings indicate that rat GCs express a functional type I and type II receptor set for BMP-6
and that P4 synthesis induced by FSH is selectively
down-regulated by virtue of BMP-6.
Based on these findings, we hypothesized that BMP-6 may be specifically
involved in modulating the expression of steroidogenic genes in
response to FSH stimulation. To elucidate this hypothesis, we analyzed
mRNA levels for three key regulators of GC steroidogenesis StAR,
P450scc, and P450arom in the GCs treated with FSH and/or BMP-6 by a
quantitative competitive RT-PCR. We have carefully validated and
established this technique in our laboratory as an accurate technique
for the quantification of RNA expression as reported in our previous
studies (17). As shown in Fig. 2, treatment of GCs for 48 h with a saturating dose (100 ng/ml) of BMP-6 alone had no effect on the steady state mRNA levels for StAR,
P450scc, P450arom and a housekeeping gene L19.
Treatment with FSH, in contrast, markedly increased their mRNA
levels except for L19. Interestingly, BMP-6 reduced
FSH-stimulated StAR and P450scc mRNA levels to the basal levels but
had no effect on P450arom mRNA level. L19 mRNA level showed no
difference at all among these four different treatments. Given the fact
that StAR and P450scc are major rate-limiting factors of P4
synthesis in GCs (31), the suppression of their mRNA expressions are likely to reflect the selective inhibition of FSH-induced P4 production. On the other hand, the failure of BMP-6 to
inhibit FSH-stimulated P450arom (catalytic enzyme to convert
androstenedione to E2) mRNA expression is consistent
with that to inhibit FSH-induced E2 production.
Biological Function and Cellular Mechanism of Bone Morphogenetic
Protein-6 in the Ovary*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
superfamily. In contrast to the recent studies on GDF-9 and BMP-15,
nothing is known about the biological function of BMP-6 in the ovary.
Here we show that, unlike BMP-15 and GDF-9, BMP-6 lacks mitogenic
activity on rat granulosa cells (GCs) and produces a marked decrease in
follicle-stimulating hormone (FSH)-induced progesterone
(P4) but not estradiol (E2) production,
demonstrating not only the first identification of GCs as BMP-6 targets
in the ovary but also its selective modulation of FSH action in
steroidogenesis. This BMP-6 activity resembles BMP-15 but differs from
GDF-9 activities. BMP-6 also exhibited similar action to BMP-15 by
attenuating the steady state mRNA levels of FSH-induced
steroidogenic acute regulatory protein (StAR) and P450 side-chain
cleavage enzyme (P450scc), without affecting P450 aromatase mRNA
level, supporting its differential function on FSH-regulated
P4 and E2 production. However, unlike BMP-15,
BMP-6 inhibited forskolin- but not 8-bromo-cAMP-induced P4
production and StAR and P450scc mRNA expression. BMP-6 also
decreased FSH- and forskolin-stimulated cAMP production, suggesting
that the underlying mechanism by which BMP-6 inhibits FSH action most likely involves the down-regulation of adenylate cyclase activity. This
is clearly distinct from the mechanism of BMP-15 action, which causes
the suppression of basal FSH receptor (FSH-R) expression, without
affecting adenylate cyclase activity. As assumed, BMP-6 did not alter
basal FSH-R mRNA levels, whereas it inhibited FSH- and forskolin-
but not 8-bromo-cAMP-induced FSH-R mRNA accumulation. These studies
provide the first insight into the biological function of BMP-6 in the
ovary and demonstrate its unique mechanism of regulating FSH action.
INTRODUCTION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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(TGF-) superfamily, namely growth differentiation factor-9 (GDF-9),
bone morphogenetic protein-15 (BMP-15), and BMP-6 are, in particular,
potentially involved in mediating these important biological
consequences triggered by the putative oocyte factors.
superfamily members and that it utilizes
novel cellular mechanism in GCs.
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80 °C
until assay for steroids and cAMP. The levels of P4 and
E2 in the media were measured by a radioimmunoassay (16). The extracellular content of cAMP in the medium was determined by cAMP
enzyme immunoassay kit (Sigma) after the acetylation of each sample.
80 °C until assay. Oligonucleotides used for reverse
transcription-polymerase chain reaction (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 were derived from the cDNA clones at the
following nucleotide numbers: 1651-1670 and 1751-1770 for
steroidogenic acute regulatory protein (StAR) (20); 148-167 and
637-656 for P450scc (21); 860-879 and 1062-1079 for P450arom (22);
487-505 and 942-959 for FSH-R (23); 166-185 and 345-366 for inhibin 
-subunit; 428-447 and 588-607 for inhibin/activin A-subunit; 32-51 and 239-258 for inhibin/activin B-subunit (24); 962-981 and
1401-1420 for LH-R (25); and 401-421 and 575-595 for ribosomal RNA
protein-L19 (L19) (26). The steady state levels of mRNA encoding
the key steroidogenic factors, StAR, P450scc, P450arom, and FSH-R
(referred to "target" mRNA), were analyzed by quantitative competitive RT-PCR as we reported previously (17). In brief, prior to
PCR the internal control DNAs having target-specific primer pairs were
generated by the method described by Siebert and Larrick (27). The
extracted RNA (500 ng) was subjected to an RT reaction using First
Strand cDNA Synthesis System® (Life Technologies, Inc.). The
resultant single 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 was mixed with a series of 10-fold dilutions of the internal
control DNA, and the target and the internal control DNAs were
co-amplified by PCR using a specific primer set for the individual
target. Quantitative 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 (for StAR,
P450scc, P450arom, and L19) or 40 (for FSH-R) 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 1.5% agarose gels, visualized after ethidium
bromide staining, photographed, and scanned. The relative integrated
density of each band was digitized by NIH image 1.62, and 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 as we
established previously (17). The following concentrations of the
internal control DNA were then selected for competitive PCR reactions:
10
5 pM for StAR; 105
pM for P450scc; 106 pM for
P450arom; 104 pM for L19; 106
pM for FSH-R. The resultant PCR products were quantified by
densitometric scanning as described above. Control analysis was
performed using L19 ribosomal protein primers as well. Steady state
levels of mRNA encoding inhibin/activin subunits (, A, and
B) and LH-R were analyzed by semi-quantitative PCR analysis, and the
levels of their PCR products were compared with that of L19 as
described previously (17).
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View larger version (22K):
[in a new window]
Fig. 1.
Effect of BMP-6 on steroidogenesis in
GCs. GCs (105 viable cells) were cultured in
serum-free media with 100 nM androstenedione in the
presence or absence of increasing doses of FSH (0-10 ng/ml) with the
combination of BMP-6 (0-300 ng/ml). After 48 h of culture, the
levels of progesterone and estradiol in the medium were measured by
radioimmunoassay. Data are shown as mean ± S.E. *,
p < 0.05 compared with FSH alone.
View larger version (48K):
[in a new window]
Fig. 2.
Effects of FSH and BMP-6 on the expression of
the mRNAs for StAR, P450scc, P450arom, and L19. GCs were
cultured either alone or together with FSH (10 ng/ml) and/or BMP-6 (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 the
upper panel, and the ratios of PCR products (target/internal
control) are graphed. Bars with different
letters indicate that group means are significantly different at
p < 0.05. i.c., internal control.
To elucidate further the mechanism by which BMP-6 suppresses
FSH-induced expression of StAR and P450scc, we utilized forskolin and
8-Br-cAMP that mimic FSH action in steroid synthesis by bypassing FSH-R
and G proteins. Forskolin is a direct activator of adenylate cyclase
(AC) and 8-Br-cAMP is a stable analog of cAMP. As shown in Fig.
3, forskolin (10 µM)
stimulated the expression of StAR and P450scc mRNA, whereas BMP-6
suppressed the forskolin-induced mRNA levels of StAR and P450scc,
similar to those had seen with the FSH treatment. However, in striking
contrast to the FSH and forskolin results, BMP-6 failed to change the
8-Br-cAMP-induced mRNA levels of these steroidogenic factors. The
mRNA level of the control housekeeping gene, L19, did
not change in response to treatment with any of the indicated reagents
and their combinations.
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We further compared the effects of BMP-6 on FSH-, forskolin-, and
8-Br-cAMP-induced P4 production by GCs (Fig.
4). Consistent with the results shown in
Fig. 1, FSH-induced P4 was significantly suppressed by
BMP-6 up to ~80%. As expected, BMP-6 also suppressed
forskolin-induced P4 by ~80% but did not significantly
inhibit 8-Br-cAMP-induced P4 production. These data
reinforce our findings that BMP-6 exerts its biological activity by
inhibiting FSH signaling at a site downstream of the FSH-R and upstream
of cAMP signaling.
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Therefore, we next examined the possible effect of BMP-6 on AC activity
with which cellular AMP is converted to cAMP, leading to the activation
of protein kinase A. For this analysis, GCs were treated with FSH (10 ng/ml) or forskolin (10 µM) in the presence or absence of
BMP-6 (100 ng/ml). In order to prevent the metabolic effect of
phosphodiesterase on cAMP, IBMX (specific inhibitor of
phosphodiesterase activity) was added to the culture medium at 0.1 mM (32, 33). After 48 h of culture, cAMP
concentrations in the culture media were measured. As shown in Fig.
5, the level of cAMP in the medium from
the cells without FSH or forskolin treatment was low, and BMP-6 did not
appear to alter basal cAMP production. However, cAMP levels stimulated
by either FSH or forskolin were significantly suppressed by the
co-treatment with BMP-6. The ability of BMP-6 to reduce the cAMP levels
in the presence of IBMX should be in response to decreased synthesis of
cAMP by AC rather than increased metabolic activity of
phosphodiesterase. These data suggest that BMP-6 inhibits AC activity
enhanced by FSH and forskolin, and the attenuation of FSH- and
forskolin-induced StAR and P450scc mRNA expression by BMP-6 seems
to be, at least in part, attributed to the decreased activity of
AC.
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Since FSH-R expression in GCs is known to be stimulated by FSH,
forskolin (34), or 8-Br-cAMP (35), the effect of BMP-6 on FSH-R
mRNA expression was also examined (Fig.
6). Control untreated cells spontaneously
expressed basal level of FSH-R mRNA, and BMP-6 did not alter the
level by itself. FSH treatment increased FSH-R mRNA level up to
2-fold, and BMP-6 abolished its effect. Stimulation of FSH-R mRNA
expression by forskolin was significantly suppressed by BMP-6 but that
by 8-Br-cAMP at two different concentrations was unchanged.
Furthermore, virtually identical activity of BMP-6 was obtained from
the experiments in which we tested the effect of BMP-6 on the
expression of several other genes (Fig.
7). Namely, BMP-6 inhibited the
stimulatory action of FSH and forskolin on the steady state mRNA
levels of inhibin/activin subunits (, A, and B) and LH-R, but
not that of 8-Br-cAMP.
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Since oocyte factors have been shown to stimulate GC mitosis, we also
examined whether BMP-6 is capable of regulating GC proliferation (Fig.
8). Treatment of primary cultured rat GCs
with BMP-6 (0-300 ng/ml) produced no significant change in thymidine
incorporation as well as numbers of GCs, in contrast to the positive
effect by BMP-15.
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In the present study, we first provided evidence for the
biological activity of BMP-6 in GCs. Experiments in which BMP-6 was added to cultured GCs indicated that BMP-6 is potent in suppressing FSH-induced P4 production without affecting FSH-induced
E2 production and that BMP-6 does not exhibit any mitogenic
activity in GCs. Our data demonstrate that the biological effects of
BMP-6 on GCs are distinct from those of other TGF- superfamily
members, including inhibin (36, 37), activin (37-39), TGF- (36,
40-43), BMP-4 (28), BMP-7 (28), and GDF-9 (14). However, it is
intriguing that the function of BMP-6 with respect to GC
steroidogenesis has a strong resemblance to that of BMP-15, another
oocyte-secreted factor (16). Both BMP-6 and BMP-15 selectively modulate
the biological effects of FSH on GCs by inhibiting FSH-induced
P4 production without affecting FSH-induced E2
synthesis. The maximal efficacy of BMP-6 inhibition of FSH-induced
P4 was ~30% more than that of BMP-15, but the
ED50 of both factors were similar, ~1010
M, which is within a physiological range. The biological
effects of GDF-9, a closely related factor also secreted by oocytes,
are clearly different from BMP-6. GDF-9 stimulates E2
production by cultured primary rat GCs in the absence of FSH yet
suppresses FSH-induced both E2 and P4
production (14).
The ability of BMP-6 to regulate specifically FSH-induced P4 biosynthesis without affecting FSH-induced E2 synthesis indicates that BMP-6 must preferentially inhibit the biochemical pathways that lead to P4 production. The present studies revealed that BMP-6 reduced the steady state levels of FSH-induced mRNAs involved in P4 synthesis, including StAR and P450scc. However, when BMP-6 was administered without FSH, no changes in the expression of these molecules were observed, indicating that BMP-6 must be acting only by regulating FSH activity. In this regard, our previous studies showed that BMP-15 regulated the sensitivity of GCs to FSH by inhibiting the expression of FSH-R (16). The present finding that BMP-6 alone had no effect on the steady state levels of FSH-R mRNA indicates that BMP-6 must work by a mechanism that is different from BMP-15 and is downstream of the FSH-R.
To elucidate the mechanism of BMP-6 regulation of FSH signaling, we
investigated whether BMP-6 would also suppress the effects of forskolin
and 8-Br-cAMP on StAR and P450scc mRNA levels in GCs. When added
alone, both forskolin and 8-Br-cAMP exhibited similar activities to FSH
in GCs. BMP-6 did inhibit forskolin-induced responses, which further
supports the hypothesis that BMP-6 is acting downstream of the FSH-R,
but did not inhibit 8-Br-cAMP-induced responses, which indicates that
BMP-6 must be acting upstream of cAMP signaling. This action of BMP-6
was broadly observed in a battery of other genes including
FSH-R, LH-R, and the inhibin/activin subunits.
Moreover, direct measurement of cAMP in GCs revealed that, when added
with FSH or forskolin, BMP-6 caused a significant decrease in the
levels of cAMP. Collectively, these data suggest that the mechanism of
BMP-6 action with respect to the biological activities identified in
the present study can be attributed to the reduction of FSH-induced
intracellular cAMP levels by BMP-6. This site of BMP-6 action presents
a novel mechanism for the modulation of FSH responses by TGF-
superfamily members. It is intriguing that BMP-15 and BMP-6, two
molecules from same family that are both produced in oocytes, modulate
FSH activity with similar physiological outcomes yet work by distinct
cellular mechanisms.
The possibilities as to how BMP-6 may modulate cAMP levels in GCs are either or both of (i) increasing activity of phosphodiesterase in the GCs and (ii) inhibiting the activity of AC in response to FSH. Although it is difficult to identify the relative contribution of the two mechanisms from the present data, the fact that BMP-6 caused a significant decrease in cAMP levels in the presence of IBMX demonstrates that BMP-6 might have the capacity to inhibit synthesis of cAMP. There is only limited data on BMP-6 regulating cAMP synthesis in other cell types. Experiments using a bone marrow-derived stromal cell line (ST2) and osteoblast-like cells (MC3T3-E1) showed that BMP-2 and BMP-4 had a synergistic effect on increasing parathyroid hormone-induced production of cAMP (44). In contrast, BMP-6, even when added at pharmacological doses, did not cause an increase in parathyroid hormone-induced cAMP levels. These data imply that BMP-6 may have different effects on AC than other BMPs.
What is the physiological relevance of BMP-6 in the ovarian function?
Our current in vitro studies may suggest the role for BMP-6
in steroidogenesis. After increases in circulating FSH during the
follicular phase of the ovarian cycle, dominant follicles are selected
and grow rapidly, resulting in a marked increase in E2
synthesis/secretion by GCs (48). In striking contrast, GCs in these
follicles do not respond to FSH to synthesize P4 in
vivo. However, once the GCs from these dominant follicles are
removed and cultured with FSH in vitro, they spontaneously secrete copious amounts of both E2 and P4.
These findings thus led to the proposition that there should be
inhibitor(s) of FSH-stimulated P4 production present in the
ovary in vivo. By using rabbit dominant follicles in
situ, El-Fouly et al. (49) demonstrated that removal of
the oocyte caused granulosa and theca cells to luteinize spontaneously and secrete large quantities of P4 equivalent to that
produced by normal corpora lutea, suggesting the presence of such
inhibitory molecule(s) in the oocytes. They may be present in
developing follicles and function to prevent GCs from secreting
P4. Our current findings that oocyte-derived BMP-6 inhibits
FSH-induced P4 production by GCs suggest that BMP-6 may
contribute to preventing the premature luteinization of the dominant
follicles. In this regard, we have previously reported that theca
cell-derived BMP-4 and -7 can regulate FSH-dependent
steroid synthesis in GCs (28). In contrast to activins and TGF-s
that stimulate P4 and E2 accumulation in the
presence of FSH (36-43), these BMPs appeared to inhibit and stimulate
FSH-dependent P4 and E2
accumulation, respectively. Based on these findings we hypothesized
that BMP-4 and -7 are putative (theca cell-derived) luteinization
inhibitors (28). Collectively, our findings suggest that factors that
inhibit premature luteinization of GCs in dominant follicles may come
from two directions within the follicle, namely BMP-4 and -7 from the
theca cells and BMP-6 and BMP-15 from the oocyte. Given that each of
these factors has similar, yet distinct, biological functions and
mechanisms of action in GCs, one can imply that the intrafollicular
regulation of luteinization is controlled by complex and redundant mechanisms.
Recently, increased attention has been paid to the Booroola strain of Marino ewes which, like the heterozygous Inverdale, is considered highly prolific (50). Heterozygous Booroola ewes exhibit higher litter sizes than wild-type ewes due to increased ovulation rates, similar to Inverdale heterozygotes. However, in contrast to homozygous Inverdale ewes that are infertile, homozygous Booroola ewes have even higher ovulation rates and litter sizes than the heterozygotes. Recently, three independent research groups have identified that the increased ovulation rate seen in the Booroola Merino ewe is associated with a point mutation in the gene encoding BMPR-IB (51-53). Notably, this mutation was located in the highly conserved intracellular kinase signaling domain of the BMPR-IB. To date, the functional ligand that binds to this receptor and the cellular mechanism of how this mutation causes an increase in ovulation rate have not been established; however, several BMP family members including BMP-6, BMP-7, BMP-4, and GDF-5 have been shown to bind to the BMPR-IB in various cell types (30, 54). Our present findings on the biological activities and cellular mechanism of BMP-6 suggest that the Booroola phenotype may in fact be caused by the inability of Booroola GCs to properly elicit BMP-6 signaling. This hypothesis is supported by the earlier observations that, when cultured for 48 h in the presence of FSH and LH, follicles dissected from the ovaries of Booroola ewes produce increased amounts of P4 than comparable follicles from wild-type ewes, yet there is no change in E2, androstenedione, nor testosterone production (55). Also, Booroola follicles were found to be more responsive to FSH and LH stimulation with respect to cAMP production than wild-type follicles (55). Further studies demonstrated that the changes in steroidogenesis and cAMP levels in GCs of the Booroola ewes are not caused by changes in FSH binding capacity of GCs (56). Collectively, the enhanced gonadotropin responsiveness of the follicles from Booroola ewes, compared with those from wild-type ewes, could be explained by the incapable BMPR-IB signaling triggered by endogenous BMP-6. Because of the loss of BMPR-IB signaling in the Booroola ewes, BMP-6 would not be able to inhibit cAMP synthesis, which would result in an increase in the sensitivity of GCs to FSH (and the subsequent selective increase in P4 production) without affecting the number of FSH-R on the surface of the GCs. Wilson et al. (52) have suggested that BMP-15 could be one of the candidate ligands for the defective BMPR-IB signaling in the Booroola ewes. We assume, however, that BMP-6 is a more likely candidate based on our previous finding that BMP-15 action is dependent on its ability to down-regulate FSH-R expression. This hypothesis can be supported by comparing the phenotypes of Booroola homozygotes to Inverdale homozygotes. Homozygous Booroola ewes have higher ovulation rates than heterozygous Booroola ewes, whereas Inverdale homozygotes are infertile with a block in folliculogenesis at the primary follicle stage. Our previous investigations demonstrated that BMP-15 is potent in stimulating mitosis of GCs, and we proposed that lack of the mitotic properties of BMP-15 may be the cause of arrested follicle development in Inverdale homozygotes. In Booroola homozygotes the mitotic capacity of the GCs seems to be intact, which is consistent with the lack of BMP-6 exhibiting any effect on GC mitosis.
In summary, these studies provide the first insight into the biological
activities of BMP-6 in the ovary. BMP-6 is able to suppress selectively
FSH-induced P4 production and the relevant steroidogenic
factors. This physiological effect is similar to BMP-15; however,
unlike BMP-15, BMP-6 does not inhibit FSH-R expression; instead BMP-6
has a novel mechanism of modulating FSH signaling, namely the
attenuation of FSH-stimulated cAMP production. Also, unlike BMP-15 and
GDF-9, BMP-6 does not have proliferative properties in GCs. Taken
together our current studies demonstrate that oocyte-derived BMP-6
exerts a distinct function among multiple members of the TGF-
superfamily that are expressed in the ovary and may play an important
role in FSH-dependent follicle development.
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FOOTNOTES |
|---|
* This work was supported in part by the University of California San Diego Academic Senate Grant RY 440M and NICHD Grant U54HD12303 from the National Institutes of Health 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.
Supported by a fellowship grant from the Lalor Foundation.
§ Supported by National Institutes of Health Training Grant T32 HD07203-17.
¶ To whom correspondence should be addressed: Dept. of Reproductive Medicine, University of California, School of Medicine, 9500 Gilman Dr., La Jolla, CA 92093-0633. Tel.: 858-822-1414; Fax: 858-822-1482; E-mail address: sshimasaki@ucsd.edu.
Published, JBC Papers in Press, July 10, DOI 10.1074/jbc.M103212200
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ABBREVIATIONS |
|---|
The abbreviations used are:
P4, progesterone;
AC, adenylate cyclase;
BMP-6, bone morphogenetic
protein-6;
BMP-15, bone morphogenetic protein-15;
DES, diethylstilbestrol;
E2, estradiol;
FSH, follicle-stimulating hormone;
FSH-R, follicle-stimulating hormone
receptor;
GC, granulosa cell;
GDF-9, growth differentiation factor-9;
LH-R, luteinizing hormone receptor;
P450arom, P450 aromatase;
P450scc, P450 side-chain cleavage enzyme;
StAR, steroidogenic acute regulatory
protein;
TGF-, transforming growth factor-;
8-Br-cAMP, 8-bromo-cAMP;
IBMX, 3-isobutyl-1-methylxanthine;
RT-PCR, reverse
transcription-polymerase chain reaction.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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R. A. Taft, J. M. Denegre, F. L. Pendola, and J. J. Eppig Identification of Genes Encoding Mouse Oocyte Secretory and Transmembrane Proteins by a Signal Sequence Trap Biol Reprod, September 1, 2002; 67(3): 953 - 960. [Abstract] [Full Text] [PDF]
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 F. Otsuka and S. Shimasaki A negative feedback system between oocyte bone morphogenetic protein 15 and granulosa cell kit ligand: Its role in regulating granulosa cell mitosis PNAS, June 11, 2002; 99(12): 8060 - 8065. [Abstract] [Full Text] [PDF]
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 N. Yamamoto, L. K. Christenson, J. M. MCAllister, and J. F. Strauss III Growth Differentiation Factor-9 Inhibits 3'5'-Adenosine Monophosphate-Stimulated Steroidogenesis in Human Granulosa and Theca Cells J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2849 - 2856. [Abstract] [Full Text] [PDF]
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 J. S. Richards, D. L. Russell, S. Ochsner, M. Hsieh, K. H. Doyle, A. E. Falender, Y. K. Lo, and S. C. Sharma Novel Signaling Pathways That Control Ovarian Follicular Development, Ovulation, and Luteinization Recent Prog. Horm. Res., January 1, 2002; 57(1): 195 - 220. [Abstract] [Full Text] [PDF]
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