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Department of Pathology (J.A.E., A.T.C., P.W., M.M.M.)
Department of Molecular and Human Genetics (J.A.E., M.M.M.), and
Department of Cell Biology (M.M.M.) Baylor College of Medicine
Houston, Texas 77030
Genetics Institute
(N.M.W.) Cambridge, Massachusetts 02140
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The transforming growth factor ß (TGF-ß) superfamily is comprised
of secreted peptide growth factors which are critical for regulating a
variety of developmental events, including cell proliferation,
differentiation, matrix secretion, and apoptosis during embryogenesis
and in the adult (8, 9). These family members are synthesized as
prepropeptides which are processed to form mature, disulfide-linked
dimers. Several TGF-ß family members have been shown to be expressed
in the ovary, including Müllerian inhibiting substance, inhibin
, activin ßA, activin ßB, growth differentiation factor 9
(GDF-9), bone morphogenetic protein (BMP)-6, and BMP-15, and several of
these factors have been shown in vivo and/or in
vitro to play important roles in regulating reproductive function
(9, 10, 11, 12, 13, 14, 15). Within the ovary, GDF-9, BMP-6, and BMP-15 are expressed
specifically in the oocyte (12, 13, 14, 15). In particular, GDF-9 and BMP-15
mRNA are expressed specifically in the oocyte of the type 3a preantral
follicle (small primary follicle with one-layer of granulosa cells),
and expression persists in oocytes throughout all stages of
folliculogenesis and in cumulus cell-oocyte complexes after ovulation
(Refs. 14, 15 and J. A. Elvin and M. M. Matzuk,
unpublished data). We have shown that a knockout of the GDF-9 gene
leads to infertility due to a block at the type 3b (primary) follicle
stage, absence of thecal layer formation, and defects in oocyte meiotic
competence (16, 17, 18). However, the potential role of GDF-9 at later
stages of folliculogenesis is unknown.
In this report, we demonstrate that GDF-9 protein is synthesized in oocytes at all stages of folliculogenesis beginning at the one-layer (type 3a) follicle stage coinciding with the expression of the GDF-9 mRNA. Using an in vitro granulosa cell culture system, we demonstrate that recombinant mouse GDF-9 can mimic several of the previously reported paracrine effects of the oocyte. GDF-9 can induce cumulus expansion in the absence of the oocyte, stimulate hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2), and steroidogenic acute regulator protein (StAR) mRNA expression, suppress uPA and LHR mRNA expression, and increase progesterone synthesis in the absence of FSH or when supplemented with low levels of FSH. These effects cannot be duplicated by at least two other known oocyte-expressed TGF-ß family members (i.e. BMP-6 and BMP-15). These studies demonstrate that receptors for GDF-9 are present on granulosa cells and that GDF-9 plays multifunctional roles in oocyte-granulosa cell communication and regulation of follicular differentiation and function.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). At low-power magnification of an
immunohistochemically-stained ovary (Fig. 1A), GDF-9 immunoreactivity
is detected only in oocytes, whereas oocytes in GDF-9-deficient ovaries
do not stain (Fig. 1B). Primordial (type 2) oocytes are negative (Fig. 1C) consistent with the absence of GDF-9 mRNA expression (Ref. 14 and
J. A. Elvin and M. M. Matzuk, unpublished data). GDF-9
immunoreactivity is first seen at low (and variable) levels in oocytes
of type 3a follicles (follicles with <20 cuboidal granulosa
cells/cross-section arranged in one concentric layer around the oocyte)
and is higher in the oocytes of type 3b follicles and beyond (Fig. 1, D
and E). Full-grown oocytes of multilayer preantral follicles (Fig. 1, A, C–E) stain more intensely for GDF-9, and GDF-9 immunoreactivity is
clearly detected in oocytes of cumulus cell-oocyte complexes of large
antral and preovulatory follicles (Fig. 1, A and F). As expected for a
secreted peptide, GDF-9 immunoreactivity is excluded from the germinal
vesicle (i.e. nucleus). Interestingly, an asymmetric
staining pattern is frequently observed within the oocytes likely due
to the detection of the precursor forms of GDF-9 within the oocyte
endoplasmic reticulum and Golgi complex (19). Thus, GDF-9 mRNA and
protein are synthesized by oocytes of all growing follicles, suggesting
that it could function at all stages of folliculogenesis.
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), which were not present in
mock-conditioned medium (data not shown). Proteolytic processing of
GDF-9 at its tetrabasic R-R-R-R site would be predicted to yield a
carboxyl-terminal mature peptide of 135 amino acids with a predicted
molecular mass of approximately 15.6 kDa (20). The mature GDF-9
sequence contains a single N-linked glycosylation site
(Asn325-Leu326-Ser327). The
predominant 21-kDa form would correspond to the cleaved, mature
monomeric form of mouse GDF-9 with one N-linked oligosaccharide. This
band runs at an identical position as the recombinant human GDF-9
synthesized in CHO cells. The band at 60 kDa would correspond to the
glycosylated, unprocessed (prohormone) form (441 amino acids), which is
also secreted into the media. To confirm that the increased molecular
mass of these two forms is due to N-linked glycosylation,
GDF-9-containing medium was treated with N-glycanase to remove the
N-linked oligosaccharides. This treatment reduces the size of the
21-kDa band to 16 kDa, the same molecular mass as the GDF-9 mature
peptide produced in bacteria, and also reduces the 60-kDa band to 50
kDa. Since the 21-kDa glycosylated GDF-9 form is always the most
abundant, this strategy to produce recombinant, glycosylated GDF-9 in
mammalian cells in the presence of PACE and under reduced-serum culture
conditions appears fairly efficient.
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32P]-dCTP in the PCR
reaction, identical samples were amplified for 16–22 cycles and
quantitated using photodensitometric analysis of the autoradiographic
film (data not shown). Linear increases in amplified product were
observed for HPRT over 16–20 cycles, for HAS2 over 16–20 cycles, and
for uPA over 18–22 cycles from both control and GDF-9-treated samples.
Eighteen to 20 cycles was determined to be optimal and was used to
study all three gene products in all further analyses. Similar PCR
conditions were performed for the COX-2, LHR, cytochrome P-450 side
chain cleavage (P-450 scc), and StAR studies (see below).
Next, we examined the dose-response relationship between recombinant
GDF-9 and HAS2 mRNA synthesis. The rate of hyaluronic acid synthesis by
cumulus cells or mural granulosa cells exposed to oocyte-conditioned
media peaks at 6–12 h in culture (23, 24). Thus, for the dose-response
experiment, granulosa cells were cultured for 5 h in the absence
or presence of varying concentrations of recombinant GDF-9. Using
semiquantitative RT-PCR, we can detect a small increase in HAS2
expression with 10 ng/ml of recombinant GDF-9 (Fig. 3A). Levels of recombinant GDF-9 between
30–300 ng/ml give robust HAS2 induction with a relatively linear
dose/response occurring between 30–120 ng/ml. Granulosa cells
collected from unprimed immature mouse ovaries also responded similarly
to the recombinant GDF-9 (data not shown). In contrast, recombinant
mouse BMP-15 (150 ng/ml) or recombinant human BMP-6 (50 ng/ml) was
unable to stimulate HAS2 expression or suppress uPA expression when
tested in any of the granulosa cell assays (data not shown). These
findings demonstrate that these activities are specific to GDF-9 and
that other oocyte-secreted TGF-ß family members cannot replicate
these activities.
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). Granulosa cells cultured in the absence of GDF-9 for 0–24 h
express more than 10-fold lower levels of HAS2 compared with the
GDF-9-induced peak level, indicating a very low level of basal activity
in these cells. The time course for uPA expression in control granulosa
cells cultured in the absence of GDF-9 indicates that uPA levels
increase over the first 9 h in culture (Fig. 3B). However,
granulosa cells treated with 100 ng/ml of recombinant GDF-9 maintain a
detectable but much lower level of uPA expression. At 5 h, the
band intensity from the GDF-9-treated sample was approximately 15% of
the control, and at 9 h, it was about 9% of the control-treated
sample band intensity (normalized to HPRT for each sample).
To confirm the effects of GDF-9 on HAS2 and uPA expression as seen by
RT-PCR, we examined the expression of HAS2 in primary granulosa cells
by Northern blot analysis in the presence or absence of 50 ng/ml of
GDF-9. Total RNA from each sample (at 0 or 5 h incubation in the
presence or absence of 50 ng/ml GDF-9) was subjected to Northern blot
analysis and hybridized with either an HAS2 or uPA probe and
subsequently with a glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) probe. The signals were quantitated on a phosphorimager and
HAS2 and uPA levels and normalized to GAPDH. HAS2 is barely detectable
in mural granulosa cells at 0 h or after 5 h of culture in
the control sample (Fig. 3C). However, after 5 h incubation with
GDF-9, both the 4.8-kb and 3.2-kb HAS2 mRNA forms (22) are increased
9.7-fold compared with control. In contrast, Northern blot analysis of
uPA showed that 50 ng/ml GDF-9 suppresses uPA synthesis to 40% of
control cultures (Fig. 3D). Thus, the Northern blot data confirm our
RT-PCR analyses.
Recombinant GDF-9 Causes Cumulus Expansion of Oocytectomized
Cumulus Cell-Oocyte Complexes
Intact cumulus cell-oocyte complexes were isolated from
PMSG-treated immature female mice. Using a transgenic micromanipulation
set-up, the oocytes from these complexes were punctured, and the oocyte
contents were suctioned. Before culture, these oocytectomized cumulus
complexes are spherical objects approximately 100 µm in diameter
consisting of several layers of granulosa cells that surround an empty
zona pellucida. After 18 h in culture, cumulus cells from 25 of 25
oocytectomized complexes cultured in control media (i.e.
deficient in GDF-9 but containing 10% FCS and 5 ng/ml or 150 ng/ml of
FSH) adhere to the tissue culture plate and assume a fibroblastic
appearance (Fig. 4A).
Consistent with previous reports that cumulus cells have low or
undetectable levels of LHR mRNA (4), incubation of these oocytectomized
complexes (9 of 9) with LH (1 µg/ml) fails to alter their
fibroblastic appearance (Fig. 4B). In contrast, 40 of 44 oocytectomized
complexes, isolated under identical conditions and cultured in the
presence of 100 ng/ml GDF-9, maintain a spherical appearance and expand
into a three-dimensional, gelatinous sphere (Fig. 4C). These results
are similar to cumulus cell-oocyte complexes with intact oocytes
cultured in FSH-containing media (data not shown). These cells have not
detached from the plate because of cell death since the majority of
cells continue to exclude the vital dye, trypan blue (data not shown).
In contrast, incubation of the complexes with recombinant human BMP-6
or BMP-15 did not result in cumulus expansion (data not shown). These
observations indicate that GDF-9 specifically stimulates cumulus
expansion and is the oocyte-derived factor that normally mediates this
process.
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). After collection of the granulosa
cells (T=0), a low level of LHR is detected (Fig. 5A). In the absence
of GDF-9, the levels of LHR mRNA decrease at 1–3 h but increase from
5 h to 24 h (Fig. 5, A and B). In contrast, incubation of the
granulosa cells with GDF-9 (100 ng/ml) suppresses LHR mRNA synthesis at
all time points (Fig. 5A). Even in the presence of 10 ng/ml FSH, LHR
mRNA is suppressed in the presence of 100 ng/ml GDF-9 (Fig. 5B). In the
experiment shown using 10 ng/ml FSH, the control-treated granulosa
cells express about 30-fold more LHR than the GDF-9-treated granulosa
cells (P < 0.05, n = 3). In contrast, COX-2
expression is low in the absence of GDF-9, but demonstrates
dramatically elevated levels after incubation for 24 h in the presence
of 100 ng/ml GDF-9 (Fig. 5, C and E). While FSH had no significant
effect in either the control or GDF-9-treated granulosa cells, GDF-9
caused a more than 50-fold increase in COX-2 expression
(P < 0.05, n = 6). Analysis of HAS2 expression in
these samples (Fig. 5, C and F) is consistent with previous results
(Fig. 3).
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) and a RIA was used to analyze
progesterone secreted into the media. In Fig. 6A, media were collected
from granulosa cells cultured in triplicate wells for 4 h
containing varying amounts of FSH in the presence or absence of 50
ng/ml GDF-9 and assayed for progesterone by RIA. Under these
conditions, granulosa cells cultured for 4 h in the absence or at
low levels of FSH produced very little progesterone, whereas granulosa
cells cultured with 50 ng/ml GDF-9 produced 5- to 7-fold higher amounts
of progesterone (Fig. 6A). In Fig. 6B, granulosa cells were incubated
for 24 h in media containing 10% FCS with 0 or 10 ng/ml FSH in
the presence or absence of 100 ng/ml GDF-9. After incubation for
24 h, GDF-9 alone can induce significantly higher amounts of
progesterone but in the presence of high concentrations of FSH (10
ng/ml), the effect of the GDF-9 is negligible (Fig. 6B). These data
suggest that FSH and GDF-9 stimulation of progesterone are not
additive.
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, C–E). Whereas 10 ng/ml FSH causes a small increase in P-450
scc, the presence of GDF-9 does not have any effect. In contrast, GDF-9
results in 2–5 fold induction of StAR mRNA (P <
0.05). These results suggest that FSH and GDF-9 function to regulate
progesterone synthesis via different mechanisms. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Previous studies demonstrated an essential paracrine function of the
oocyte to stimulate cumulus expansion and to induce hyaluronic acid
synthesis (25, 26) and to suppress uPA expression (27) in both cumulus
and mural granulosa cells. When cumulus cells are separated from the
oocyte, they do not expand and assume an adherent, fibroblastic
appearance, producing negligible amounts of hyaluronic acid. When
oocytes are added back to the culture, or if the cumulus cells are
grown in oocyte-conditioned media (
2 oocytes/µl media), they
produce 5- to 10-fold higher levels of hyaluronic acid. Alternatively,
mural granulosa cells can be induced to synthesize hyaluronic acid
in vitro by treatment with oocyte-conditioned media (28).
Synthesis of hyaluronic acid in cumulus-oocyte-complexes can be blocked
by the addition of actinomycin D, demonstrating a dependence on gene
transcription (24). Some of the effects of the oocyte-conditioned media
could be mimicked by recombinant TGF-ß, but anti-TGF-ß antibodies
could not block the effect of the oocyte-conditioned media (24, 28).
While theca cells synthesize TGF-ßs (29), no reports of
oocyte-produced TGF-ßs exist in the literature. These data
suggest that the oocyte-produced growth factor is not one of the
TGF-ßs but is a related family member that functions in a similar
pathway.
In this report, we show that GDF-9 could substitute for oocytes and oocyte-conditioned media in assays analyzing HAS2 induction and uPA suppression typical of processes occurring in preovulatory follicles. Other oocyte-expressed TGF-ß family members, BMP-15 and BMP-6, are unable to substitute for GDF-9 in these granulosa cell assays. Mural granulosa cells, isolated from antral follicles treated with recombinant GDF-9, are induced to express HAS2 in a dose-dependent and time-dependent manner. GDF-9 can induce approximately 10-fold higher levels of HAS2 mRNA in mural granulosa cells, which corresponds well to the maximum effect of oocytes on hyaluronic acid synthesis. Additionally, the dose-response curve for GDF-9 is very similar to that of the oocyte-conditioned media. Very low doses (e.g. 0.5 oocytes/µl or 10 ng/ml GDF-9) induce very low but detectable increases in hyaluronic acid synthesis or HAS2 expression whereas 1 oocyte/µl or 30–50 ng/ml GDF-9 causes a much more dramatic induction, which plateaus at 2–4 oocytes/µl or 120–300 ng/ml GDF-9 (Ref. 25 and studies presented here). The time course of GDF-9 action also agrees with previous data for the oocyte-produced factor. Whereas HAS2 mRNA is induced by 2 h in culture with GDF-9, oocyte-induced hyaluronic acid becomes detectable at low levels after 2.5 h (23). GDF-9 induces peak HAS2 mRNA levels between 3–5 h, while the rate of oocyte-induced hyaluronic acid synthesis is maximal between 6–12 h (23). Likewise, oocyte-induced hyaluronic acid synthesis drops after 12 h, and no more hyaluronic acid is made after 18 h (24); GDF-9-induced HAS2 expression is reduced by 24 h and uPA synthesis increases by 24 h. The transient nature of the activation of HAS2 expression and hyaluronic acid synthesis and the increase in uPA synthesis over this time period may be due to lability of GDF-9 in the media, down-regulation of the GDF-9 receptor, GDF-9-induced differentiation of the granulosa cells, and/or stimulation of a negative-feedback mechanism within the GDF-9 signal transduction cascade. It is interesting to note that the oocyte-secreted factor that regulates several of these processes has been noted to be labile and that continued presence of oocytes in various cocultures with granulosa cells is required for continued activity (4). Lastly, our data suggest that the difference in the in vivo phenotype of mural granulosa cells vs. expanding cumulus granulosa cells is not intrinsic to the cells themselves but is due to their proximity to the oocyte and the concentration gradient of the oocyte-produced GDF-9.
The conversion of cholesterol to pregnenolone is the rate-determining step in granulosa cell steroidogenesis. The rate of pregnenolone synthesis depends on the level and activity of the reaction-catalyzing enzyme, P-450 scc, and its access to its substrate cholesterol via stimulation of StAR (30). It is well established that FSH- and LH-induced increases in intracellular cAMP, leading to subsequent stimulation of P-450 scc and StAR mRNA synthesis and StAR protein phosphorylation, stimulate progesterone synthesis in vitro (31, 32, 33). In contrast to the effect seen with GDF-9 treatment, activin A decreased basal and FSH stimulated P-450 scc, 3ß-hydroxysteroid dehydrogenase, and progesterone synthesis by cultured granulosa cells from the diethylstilbesterol-stimulated rat. Our data confirm that FSH can stimulate P-450 scc mRNA synthesis in mouse granulosa cells but demonstrate that GDF-9 does not significantly affect P-450 scc mRNA synthesis. In contrast, FSH has only a small inductive effect on StAR mRNA, but GDF-9 with or without FSH significantly induces StAR expression. Consequently, both GDF-9 and FSH can independently increase production of progesterone by the granulosa cells and appear to function in the same pathway but via different mechanisms. It would be interesting to determine whether GDF-9 also stimulates phosphorylation of StAR protein through activation of Smads (see below) to increase its activity (33). GDF-9-stimulated local production of progesterone by the cumulus cells may be critical for achieving a perfect microenvironment for the oocyte after ovulation and before fertilization. In support of this, ovulated rat cumulus cell-oocyte complexes secrete measurable levels of both progesterone and prostaglandins (mainly PGE2) (34), and use of aminoglutethimide, which inhibits conversion of cholesterol to pregnenolone, reduces the number of normal ovine oocytes recovered after in vitro follicular maturation.
In situ hybridization analysis of LHR in preovulatory
follicles demonstrates that LHR is suppressed in the cumulus cells but
not the mural granulosa cells, whereas after LH treatment in
vivo, COX-2 expression is highest in the cumulus cells (Ref. 18
and J. A. Elvin and M. M. Matzuk, unpublished data). We show here
that recombinant GDF-9 also suppresses LHR mRNA but induces COX-2
expression, mimicking the normal expression of these genes in the
cumulus cells. In addition, Eppig and colleagues (4, 35) elegantly
demonstrated that full-grown oocytes suppress LHR mRNA expression, but
that oocytes from preantral follicles, metaphase II-arrested oocytes,
or two-cell embryos were not as effective. We have shown in our
knockout studies (16, 18) and in the current studies using recombinant
GDF-9 that GDF-9 can stimulate changes in cell morphology, gene
expression, and steroid production, indicating that granulosa cells, at
least from primary follicles and from antral follicles, possess
receptors that bind GDF-9 (see Fig. 7 for
a summary of our findings). Since GDF-9 dramatically increases the
level of COX-2 expression, it is unclear why COX-2 expression is not
expressed at earlier stages of folliculogenesis or why earlier stage
oocytes (4, 5, 35) are less effective in suppressing LHR. Although
GDF-9 protein is detected at the immunohistochemical level at all
stages of folliculogenesis, this does not necessarily prove that GDF-9
is active at all of these stages. One possibility is that the GDF-9
precursor is only processed to an active mature dimer at the type 3b
stage and at the antral follicle stage. The regulation of the
GDF-9-processing enzyme would be one way to regulate the activity of
GDF-9 posttranslationally. An alternative explanation is that both
GDF-9-regulated and GDF-9-independent transcription factors function
together to regulate the synthesis of COX-2. At least one regulator of
COX-2 is the transcription factor enhancer-binding protein ß
(C/EBPß). C/EBPß, which is induced between 4 and 7 h
after hCG treatment, binds to the COX-2 promoter to down-regulate COX-2
mRNA expression. COX-2 expression in the ovary normally peaks
at 4 h after hCG treatment whereas COX-2 protein continues to be
present in the cumulus cells after ovulation (36). However, in the
C/EBPß knockout mouse, which are infertile (36A ), levels of
COX-2 mRNA remain elevated. COX-2 knockout mice are also infertile due
to defects in ovulation and impaired oocyte maturation (36). These
studies from the C/EBPß and COX-2 knockout models suggest that
appropriate regulation of COX-2 in the cumulus cells is necessary to
maintain the optimal microenvironment of PGs around the oocyte. GDF-9
appears to be one of the factors involved in induction of COX-2, and
C/EBPß plays a role in down-regulation of COX-2. Our studies and
others (37, 38) also suggest that LH regulates the expression of both
COX-2 and C/EBPß in cumulus cells indirectly because LHRs are not
present on these cells. However, it is unclear how this is achieved or
how COX-2 is turned on rapidly and C/EBPß more slowly. Future studies
will be necessary to determine the critical interplay between LH and
GDF-9 signaling in the preovulatory follicle and their regulation of
C/EBPß and COX-2.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Production of Recombinant Mouse GDF-9
A full-length mouse GDF-9 cDNA (44) was subcloned into the
expression vector pHTop containing the processing gene PACE (a gift
from Dr. Monique Davies, Genetics Institute). The GDF-9
expression vector was lipofectin transfected into CHO cells under
standard conditions (Gibco BRL, Grand Island, NY).
Expression of mouse GDF-9 in CHO cells was subsequently driven by a
tetracycline-regulatable promoter while an SV40 promoter regulated
expression of PACE. Stable, positive clones were selected in the
presence of 0.02 µM methotrexate in -modified Eagles’
medium (-MEM) containing 10% heat- inactivated dialyzed FBS, 100
µg/ml G418-sulfate (Gibco BRL; Life Technologies) and the antibiotics gentamicin, penicillin, and
streptomycin. After clonal selection and expansion in 0.02
µM methotrexate, the GDF-9-expressing cells were
incubated for 24 h in Opti-MEM- reduced serum collection media
containing 100 mg/ml heparin (Sigma Chemical Co., St.
Louis, MO). The media were harvested, and GDF-9 protein levels were
determined by SDS-PAGE with subsequent immunoblotting (see next
section). N-linked oligosaccharides were removed by incubation with
N-glycanase (Oxford GlycoSciences, Wakefield, MA) overnight at 37 C
according to manufacturer’s protocol.
Western Blot Analysis
Samples of GDF-9-containing media were electrophoresed on a 5%
stacking/15% resolving SDS polyacrylamide gel in a Mini-Subcell
apparatus (Bio-Rad Laboratories, Inc., Hercules, CA) as
previously described (45) and subsequently transferred to
polyvinylidenedifluoride membrane. The membranes were blocked
overnight in a 5% nonfat milk in 1x Tris-buffered-saline with 0.05%
Tween 20. Mouse antihuman GDF-9 monoclonal antibody (described above)
was used at a 1:1000 dilution in blocking solution, and an antimouse
secondary antibody conjugated to horseradish peroxidase (Southern Biotechnology Associates, Birmingham, AL) was used at a 1:2500
dilution in blocking solution. Chemiluminescence using enhanced
chemiluminescence Western detection reagents (Pierce Chemical Co., Rockford, IL) and autoradiographic film detected signal.
Bands were quantitated using a densitometer (Molecular Dynamics, Inc., Sunnyvale, CA) and Imagequant software, and the
concentration of GDF-9 in the conditioned media was determined by
comparing the signal intensity of GDF-9 in the conditioned media to
known concentrations of GDF-9 standards run concurrently. Several
batches of recombinant mouse GDF-9 were produced during the course of
these studies, all of which appeared to have similar activities based
on Western blot quantitation (i.e. immunoreactivity
correlated with bioactivity).
Isolation and Culture of Granulosa Cells
Female CD-1 (ICR) mice 21–24 days of age (Baylor College of
Medicine) were injected with 7.5 IU Gestyl (Diosynth B.V., Oss,
Holland), and ovaries were harvested 44–48 h later, dissected free of
fat and surrounding tissue, and placed in minimal essential media with
25 mM HEPES supplemented with 0.3 mg/ml
L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin
(Gibco BRL), and 0.3% BSA (Sigma Chemical Co.). Mural granulosa cells were released by puncturing large
antral follicles. Oocytes and cumulus cell-oocyte complexes (COCs) were
carefully removed (see below). Granulosa cells from multiple ovaries
were pooled, centrifuged, and resuspended in 2x granulosa cell culture
media (GCM): -MEM (Gibco BRL) with 0.6 mg/ml
L-glutamine, 200U/ml penicillin, 0.2 mg/ml streptomycin,
and 2x insulin-transferrin-selenite (Gibco BRL) in the
presence or absence of 20% FBS (HyClone Laboratories, Inc., Logan, UT), and the presence or absence of ovine FSH
(NIDDK-o-FSH-20 kindly provided by Dr. Parlow of the National Hormone
and Pituitary Program). GDF-9-containing media or control conditioned
media were diluted to 2x the final concentration in -MEM. Equal
volumes of 2x GDF-9-containing media or control media were combined
with granulosa cells in 2x culture media and cultured at 37 C in a
humidified atmosphere with 5% CO2. After varying periods
of culture, nonadherent cells were pelleted from the media, and the
media were stored at -20 C. Granulosa cells were lysed, and total RNA
was isolated using RNA Stat-60 (Leedo Medical Laboratories, Houston,
TX) following the manufacturer’s protocol.
Semiquantitative RT-PCR Analysis
Oligo-dT-primed cDNA from each RNA sample was synthesized using
Superscript reverse transcriptase (Gibco BRL) following
the manufacturer’s protocol. One microliter of each RT reaction (1/20
of total) was used in each 25 µl PCR reaction primed with
gene-specific oligonucleotides. Mouse HAS2 mRNA expression was detected
using 5'-GCTTGACCCTGCCTCATCTGTGG-3'(sense) and
5'-CTGGTTCAGCCATCTCAGATATT- 3' (antisense) primers (21), which span a
1.4-kb intron. A PCR product of 403 bp is amplified from RNA, easily
distinguished from amplification of contaminating DNA. Mouse uPA mRNA
expression was detected using 5'- GTTCAGACTGTGAGATCACTGG-3' (sense) and
5'-CAGAGAGGACGGTCAGCATGG- 3' (antisense) primers that span two introns
of 1.4 kb total length. A PCR product of 434 bp is amplified from RNA.
Mouse HPRT was amplified using 5'-CCTGGTTAAGCAGTACAGCC 3' (sense) and
5'-TACTAGGCAGATGGCCACAG-3' (antisense) primers, which span three
introns of unknown sizes and give an expected mRNA-derived product size
of 309 bp from RNA. Mouse StAR mRNA expression was detected using
5'-TCGCTTGGAGGTGGTGGTAGAC-3'(sense) and 5'-GCAGGTCAATGTGGTGGACAGT-3'
(antisense) primers, which span multiple small introns and give an
mRNA-derived 522-bp product. Mouse cholesterol P-450 scc mRNA
expression was detected using 5'-GCCAACATTACCGAGATGC-3'(sense) and
5'-CGAACACCCCAGCCAAAGCC-3' (antisense) primers and give an mRNA-derived
426-bp product. Mouse COX-2 mRNA expression was detected using
5'-CTCCTTTTCAACCAGCAGTTCC-3'(sense) and 5'-TCTGCAGCCATTTCCTTCTCTC-3'
(antisense) primers and give a 377-bp product. Mouse LHR mRNA
expression was detected using 5'-CTTATACATAACCACCATACCAG-3'(sense) and
5'-ATCCCA-GCCACTGAGTTCATTC-3' (antisense) primers, which span
multiple introns and give a 516-bp product. PCR products amplified from
granulosa cell cDNA were initially isolated, subcloned, and sequenced
to confirm that they matched published sequences. In later studies,
[32P]-dCTP was added to each PCR reaction, and
products were separated by electrophoresis on a 4% polyacrylamide gel.
The gels were dried and exposed to autoradiography, and radioactive
bands were quantitated on a Molecular Dynamics, Inc.
phosphorimager (Storm 860).
Northern Blot Analysis
Total RNA was isolated from granulosa cells and quantitated by
fluorometry using Ribogreen RNA quantitation reagents (Molecular Probes, Inc., Eugene, OR) on a VersaFluor fluorometer
(Bio-Rad Laboratories Inc.) using a 485–495 nm excitation
filter and 515–525 nm emission filter. Fifteen micrograms of total RNA
of each sample were electrophoresed on a 1.2% agarose/7.6%
formaldehyde gel and transferred to Hybond N nylon membrane
(Amersham, Arlington Heights, IL). Probes for HAS2 and uPA
were generated from the aforementioned subcloned PCR products by random
priming with [32P] dATP using the Strip-EZ probe
synthesis kit (Ambion, Inc., Austin, TX). The membrane was
hybridized, washed, and subjected to autoradiography as described (46).
The probe was removed from the membrane using the Strip-EZ removal
reagents (Ambion, Inc.) following the manufacturer’s
protocol. The same blots were then reprobed with GAPDH as a loading
control. Signals for each probe were quantitated on a Molecular Dynamics, Inc. phosphorimager.
Progesterone RIA
Progesterone in the culture media was measured in duplicate by a
specific, solid-phase RIA using a kit from Diagnostic Products (Los Angeles, CA) according to the manufacturers
instructions. The sensitivity of this assay is 0.02 ng/ml, and
calibration standards between 0.1 and 40 ng/ml were used.
Expansion of Oocytectomized Complexes
Cumulus cell-oocyte complexes were collected as described above.
The oocyte was removed from each complex using a microinjection
apparatus as previously described (47). Successful oocytectomy was
assessed by the removal of the germinal vesicle along with the majority
of ooplasm. Oocytectomized complexes were incubated for 18 h in
groups in 20 µl droplets of granulosa cell culture media supplemented
with 10% FBS and 5 ng/ml or 100 ng/ml of oFSH with or without 1
µg/ml oLH in the presence or absence of 100 ng/ml GDF-9. Photographs
were taken on a Nikon (Melville, NY) inverted
microscope.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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These studies were supported in part by sponsored research grants from Genetics Institute and Metamorphix and NIH Grant HD-33438 (to M.M.M). Julia A. Elvin is a student in the Medical Scientist Training Program supported in part by NIH Grants GM-07330 and GM-08307 and the Baylor Research Advocates for Student Scientists (BRASS) organization.
Received for publication February 1, 1999. Revision received March 22, 1999. Accepted for publication March 24, 1999.
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A. Salustri, C. Garlanda, E. Hirsch, M. De Acetis, A. Maccagno, B. Bottazzi, A. Doni, A. Bastone, G. Mantovani, P. B. Peccoz, et al. PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization Development, April 1, 2004; 131(7): 1577 - 1586. [Abstract] [Full Text] [PDF]
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D. Mukhopadhyay, A. Asari, M. S. Rugg, A. J. Day, and C. Fulop Specificity of the Tumor Necrosis Factor-induced Protein 6-mediated Heavy Chain Transfer from Inter-{alpha}-trypsin Inhibitor to Hyaluronan: IMPLICATIONS FOR THE ASSEMBLY OF THE CUMULUS EXTRACELLULAR MATRIX J. Biol. Chem., March 19, 2004; 279(12): 11119 - 11128. [Abstract] [Full Text] [PDF]
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S. Mazerbourg, C. Klein, J. Roh, N. Kaivo-Oja, D. G. Mottershead, O. Korchynskyi, O. Ritvos, and A. J. W. Hsueh Growth Differentiation Factor-9 Signaling Is Mediated by the Type I Receptor, Activin Receptor-Like Kinase 5 Mol. Endocrinol., March 1, 2004; 18(3): 653 - 665. [Abstract] [Full Text] [PDF]
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S. K. Bristol and T. K. Woodruff Follicle-Restricted Compartmentalization of Transforming Growth Factor {beta} Superfamily Ligands in the Feline Ovary Biol Reprod, March 1, 2004; 70(3): 846 - 859. [Abstract] [Full Text] [PDF]
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 S. Shimasaki, R. K. Moore, F. Otsuka, and G. F. Erickson The Bone Morphogenetic Protein System In Mammalian Reproduction Endocr. Rev., February 1, 2004; 25(1): 72 - 101. [Abstract] [Full Text] [PDF]
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C. Glister, C F. Kemp, and P. G Knight Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin Reproduction, February 1, 2004; 127(2): 239 - 254. [Abstract] [Full Text] [PDF]
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N. Minami, A. Aizawa, R. Ihara, M. Miyamoto, A. Ohashi, and H. Imai Oogenesin Is a Novel Mouse Protein Expressed in Oocytes and Early Cleavage-Stage Embryos Biol Reprod, November 1, 2003; 69(5): 1736 - 1742. [Abstract] [Full Text] [PDF]
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D. L. Russell, K. M. H. Doyle, S. A. Ochsner, J. D. Sandy, and J. S. Richards Processing and Localization of ADAMTS-1 and Proteolytic Cleavage of Versican during Cumulus Matrix Expansion and Ovulation J. Biol. Chem., October 24, 2003; 278(43): 42330 - 42339. [Abstract] [Full Text] [PDF]
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K. H. Burns, G. E. Owens, S. C. Ogbonna, J. H. Nilson, and M. M. Matzuk Expression Profiling Analyses of Gonadotropin Responses and Tumor Development in the Absence of Inhibins Endocrinology, October 1, 2003; 144(10): 4492 - 4507. [Abstract] [Full Text] [PDF]
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A. Balla, N. Danilovich, Y. Yang, and M. R. Sairam Dynamics of Ovarian Development in the FORKO Immature Mouse: Structural and Functional Implications for Ovarian Reserve Biol Reprod, October 1, 2003; 69(4): 1281 - 1293. [Abstract] [Full Text] [PDF]
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K. L. Loveland, M. Bakker, T. Meehan, E. Christy, V. von Schonfeldt, A. Drummond, and D. de Kretser Expression of Bambi Is Widespread in Juvenile and Adult Rat Tissues and Is Regulated in Male Germ Cells Endocrinology, September 1, 2003; 144(9): 4180 - 4186. [Abstract] [Full Text] [PDF]
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D. M. Duffy Growth Differentiation Factor-9 Is Expressed by the Primate Follicle Throughout the Periovulatory Interval Biol Reprod, August 1, 2003; 69(2): 725 - 732. [Abstract] [Full Text] [PDF]
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S. A. Ochsner, D. L. Russell, A. J. Day, R. M. Breyer, and J. S. Richards Decreased Expression of Tumor Necrosis Factor-{alpha}-Stimulated Gene 6 in Cumulus Cells of the Cyclooxygenase-2 and EP2 Null Mice Endocrinology, March 1, 2003; 144(3): 1008 - 1019. [Abstract] [Full Text] [PDF]
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D. L. Russell, S. A. Ochsner, M. Hsieh, S. Mulders, and J. S. Richards Hormone-Regulated Expression and Localization of Versican in the Rodent Ovary Endocrinology, March 1, 2003; 144(3): 1020 - 1031. [Abstract] [Full Text] [PDF]
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C. Glister, N. P. Groome, and P. G. Knight Oocyte-Mediated Suppression of Follicle-Stimulating Hormone- and Insulin-Like Growth Factor-Induced Secretion of Steroids and Inhibin-Related Proteins by Bovine Granulosa Cells In Vitro: Possible Role of Transforming Growth Factor {alpha} Biol Reprod, March 1, 2003; 68(3): 758 - 765. [Abstract] [Full Text] [PDF]
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N. Suzumori, K. H. Burns, W. Yan, and M. M. Matzuk RFPL4 interacts with oocyte proteins of the ubiquitin-proteasome degradation pathway PNAS, January 21, 2003; 100(2): 550 - 555. [Abstract] [Full Text] [PDF]
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S. Jabara, L. K. Christenson, C. Y. Wang, J. M. McAllister, N. B. Javitt, A. Dunaif, and J. F. Strauss III Stromal Cells of the Human Postmenopausal Ovary Display a Distinctive Biochemical and Molecular Phenotype J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 484 - 492. [Abstract] [Full Text] [PDF]
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H. Chang, C. W. Brown, and M. M. Matzuk Genetic Analysis of the Mammalian Transforming Growth Factor-{beta} Superfamily Endocr. Rev., December 1, 2002; 23(6): 787 - 823. [Abstract] [Full Text] [PDF]
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L. L. Espey and J. S. Richards Temporal and Spatial Patterns of Ovarian Gene Transcription Following an Ovulatory Dose of Gonadotropin in the Rat Biol Reprod, December 1, 2002; 67(6): 1662 - 1670. [Abstract] [Full Text] [PDF]
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J. L. Juengel, N. L. Hudson, D. A. Heath, P. Smith, K. L. Reader, S. B. Lawrence, A. R. O'Connell, M. P.E. Laitinen, M. Cranfield, N. P. Groome, et al. Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 Are Essential for Ovarian Follicular Development in Sheep Biol Reprod, December 1, 2002; 67(6): 1777 - 1789. [Abstract] [Full Text] [PDF]
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A. E. Stock, N. Bouchard, K. Brown, A. P. Spicer, C. B. Underhill, M. Dore, and J. Sirois Induction of Hyaluronan Synthase 2 by Human Chorionic Gonadotropin in Mural Granulosa Cells of Equine Preovulatory Follicles Endocrinology, November 1, 2002; 143(11): 4375 - 4384. [Abstract] [Full Text] [PDF]
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N. C. Zachos, R. B. Billiar, E. D. Albrecht, and G. J. Pepe Developmental Regulation of Baboon Fetal Ovarian Maturation by Estrogen Biol Reprod, October 1, 2002; 67(4): 1148 - 1156. [Abstract] [Full Text] [PDF]
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A.L. Johnson, E.V. Solovieva, and J.T. Bridgham Relationship Between Steroidogenic Acute Regulatory Protein Expression and Progesterone Production in Hen Granulosa Cells During Follicle Development Biol Reprod, October 1, 2002; 67(4): 1313 - 1320. [Abstract] [Full Text] [PDF]
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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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U. A. Vitt, S. Mazerbourg, C. Klein, and A. J.W. Hsueh Bone Morphogenetic Protein Receptor Type II Is a Receptor for Growth Differentiation Factor-9 Biol Reprod, August 1, 2002; 67(2): 473 - 480. [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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S. Varani, J. A. Elvin, C. Yan, J. DeMayo, F. J. DeMayo, H. F. Horton, M. C. Byrne, and M. M. Matzuk Knockout of Pentraxin 3, a Downstream Target of Growth Differentiation Factor-9, Causes Female Subfertility Mol. Endocrinol., June 1, 2002; 16(6): 1154 - 1167. [Abstract] [Full Text] [PDF]
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W. Yan, A. Rajkovic, M. M. Viveiros, K. H. Burns, J. J. Eppig, and M. M. Matzuk Identification of Gasz, an Evolutionarily Conserved Gene Expressed Exclusively in Germ Cells and Encoding a Protein with Four Ankyrin Repeats, a Sterile-{alpha} Motif, and a Basic Leucine Zipper Mol. Endocrinol., June 1, 2002; 16(6): 1168 - 1184. [Abstract] [Full Text] [PDF]
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