Paracrine Actions Of Growth Differentiation Factor-9 in the Mammalian Ovary
Julia A. Elvin,
Amander T. Clark,
Pei Wang,
Neil M. Wolfman and
Martin M. Matzuk
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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Although the transforming growth factor-ß
(TGF-ß) superfamily is the largest family of secreted growth factors,
surprisingly few downstream target genes in their signaling pathways
have been identified. Likewise, the identities of oocyte-derived
secreted factors, which regulate important oocyte-somatic cell
interactions, remain largely unknown. For example, oocytes are known to
secrete paracrine growth factor(s) which are necessary for cumulus
expansion, induction of hyaluronic acid synthesis, and suppression of
LH receptor (LHR) mRNA synthesis. Our previous studies demonstrated
that absence of the TGF-ß family member, growth differentiation
factor-9 (GDF-9), blocks ovarian folliculogenesis at the primary
follicle stage leading to infertility. In the present study, we
demonstrate that mouse GDF-9 protein is expressed in all oocytes
beginning at the type 3a follicle stage including antral follicles. To
explore the biological functions of GDF-9 in the later stages of
folliculogenesis and cumulus expansion, we produced mature,
glycosylated, recombinant mouse GDF-9 using a Chinese hamster ovary
cell expression system. A granulosa cell culture system was established
to determine the role of GDF-9 in the regulation of several key ovarian
gene products using semiquantitative RT-PCR. We find that recombinant
GDF-9 induces hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2),
and steroidogenic acute regulator protein (StAR) mRNA synthesis but
suppresses urokinase plasminogen activator (uPA) and LHR mRNA
synthesis. Consistent with the induction of StAR mRNA by GDF-9,
recombinant GDF-9 increases granulosa cell progesterone synthesis in
the absence of FSH. Since induction of HAS2 and suppression of the
protease uPA in cumulus cells are key events in the production of the
hyaluronic acid-rich extracellular matrix which is produced during
cumulus expansion, we determined whether GDF-9 could mimic this
process. Using oocytectomized cumulus cell-oocyte complexes, we show
that recombinant GDF-9 induces cumulus expansion in vitro.
These studies demonstrate that GDF-9 can bind to receptors on granulosa
cells to regulate the expression of a number of gene products. Thus, in
addition to playing a critical function as a growth and differentiation
factor during early folliculogenesis, GDF-9 functions as an
oocyte-secreted paracrine factor to regulate several key granulosa cell
enzymes involved in cumulus expansion and maintenance of an optimal
oocyte microenvironment, processes which are essential for normal
ovulation, fertilization, and female reproduction.
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INTRODUCTION
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The oocyte plays an integral role in regulating folliculogenesis
within the mammalian ovary. In particular, the oocyte has been shown to
act on granulosa cells to regulate follicle formation perinatally,
stimulate granulosa cell proliferation, modulate granulosa cell gene
expression, and influence steroidogenesis (reviewed in Ref. 1).
Granulosa cells in the preovulatory follicle can be separated into two
populations with respect to their proximity to the oocyte; cumulus
granulosa cells closely surround the oocyte while the mural granulosa
cells are located around the periphery of the follicle separated from
the oocyte by an antrum. Cumulus cells secrete a hyaluronic acid-rich
matrix during cumulus expansion and are extruded with the oocyte during
ovulation. This expanded matrix is a critical factor for reproductive
integrity since it binds the oocyte and cumulus cells together,
facilitates follicular extrusion and oviductal fimbria capture, and
allows sperm penetration and fertilization (2). On the other hand,
mural granulosa cells synthesize proteases important for follicle
rupture at ovulation, remain within the ovary after the cumulus
cell-oocyte complex is released, and eventually undergo terminal
differentiation to form the corpus luteum. These positional and
functional differences in the granulosa cell populations suggests that
gradients of oocyte-secreted factors modulate gene expression and
eventual cell differentiation. In vitro studies demonstrate
that oocyte-secreted growth factors regulate granulosa cell synthesis
of hyaluronic acid, urokinase plasminogen activator (uPA) and LH
receptor (LHR) as well as steroidogenesis and luteinization (2, 3, 4, 5, 6, 7).
However, the identities of the oocyte-derived factors that regulate
these somatic cell functions remain largely unknown.
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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Immunohistochemical Detection of GDF-9 in Mouse Ovaries
Using a monoclonal antibody to human GDF-9, GDF-9 protein is
specifically detected in mouse oocytes (Fig. 1
). 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, CE) 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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Figure 1. Immunohistochemical Detection of GDF-9 in Mouse
Ovaries
Ovaries from 3- to 4-week-old CD1 female mice were stained with an
anti-GDF-9 monoclonal antibody. A, Low-power view of a wild-type ovary
stained with the anti-GDF-9 monoclonal antibody shows multiple oocytes
within preantral and antral follicles which stain positive
(pink). Nuclei are blue due to
hematoxylin counterstaining. B, High-power view of a GDF-9-deficient
(GDF-9 -/-) ovary stained with the anti-GDF-9 monoclonal antibody
does not show oocyte staining. CE, High-power views of wild-type
ovaries. Oocytes within primordial follicles (type 2) fail to stain
positive (C and D). Oocytes from type 3a primary follicles begin to
demonstrate weak staining in the cytoplasm (D and E), and higher level
staining is detected in the larger preantral follicles such as the
three-layer type 5a follicles (D and E). Staining is excluded from the
germinal vesicle, which stains blue. F, A preovulatory
follicle with abundant mural granulosa cells (MGC) and an oocyte
surrounded by cumulus granulosa cells (CC) demonstrate persistence of
GDF-9 protein synthesis by oocytes of late-stage follicles.
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Production of Recombinant Mouse GDF-9
To study the function of GDF-9 at later stages of
folliculogenesis, it was necessary to produce recombinant GDF-9.
Chinese hamster ovary cells (CHO) cells were stably transfected with an
expression vector containing both a full-length mouse GDF-9 cDNA and a
cDNA for PACE, a prepropeptide sequence-cleaving enzyme. Western blot
analysis of medium from CHO cells containing the mouse GDF-9 expression
vector, analyzed under denaturing conditions, demonstrated two unique
bands of approximate molecular masses 21 kDa and 60 kDa (Fig. 2
), 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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Figure 2. Western Blot Analysis of GDF-9-Conditioned Media
An antihuman GDF-9 monoclonal antibody was used for detection of GDF-9.
The lanes are as follows: lane 1, recombinant human GDF-9 synthesized
in CHO cells ( 30 ng mature form); lane 2, recombinant mouse GDF-9
synthesized in CHO cells ( 20 ng mature form); lane 3, 5 ng mouse
GDF-9 mature peptide produced in E. coli; lane 4,
recombinant mouse GDF-9 media treated with N-glycanase (+) to remove
the N-linked oligosaccharides; lane 5, mouse GDF-9 media treated as in
lane 4 in the absence of N-glycanase (-). The N-linked glycosylated
(+N) or deglycosylated (-N) propeptide (pro) and mature forms of GDF-9
are indicated.
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Regulation of HAS2 and uPA mRNA Synthesis by Recombinant GDF-9
Oocytes secrete a growth factor that is known to stimulate
hyaluronic acid synthesis, inhibit uPA, and cause cumulus expansion
in vitro (2). HAS2, which is expressed in mouse cumulus
cell-oocyte complexes after the gonadotropin surge and immediately
preceding efficient cumulus expansion (21), has been implicated as the
major hyaluronic acid synthase involved in cumulus expansion (22). To
determine whether GDF-9 is the oocyte-secreted factor responsible for
inducing cumulus expansion through increased hyaluronic acid matrix
synthesis and decreased hyaluronic acid matrix degradation, we examined
the expression levels of HAS2 and uPA in freshly isolated granulosa
cells treated with recombinant mouse GDF-9. RT-PCR incorporation of
radiolabeled nucleotides into specific products was used to monitor the
expression levels of HAS2 and uPA in control and GDF-9-treated
granulosa cell cultures. First, a linear range of product amplification
for each oligonucleotide pair of the three genes [i.e.
HAS2, uPA, and hypoxanthine phosphoribosyltransferase (HPRT)] was
established. Using radiolabeled [
32P]-dCTP in the PCR
reaction, identical samples were amplified for 1622 cycles and
quantitated using photodensitometric analysis of the autoradiographic
film (data not shown). Linear increases in amplified product were
observed for HPRT over 1620 cycles, for HAS2 over 1620 cycles, and
for uPA over 1822 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 612 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
30300 ng/ml give robust HAS2 induction with a relatively linear
dose/response occurring between 30120 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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Figure 3. Differential Regulation of HAS2 and uPA by GDF-9
A, HAS2 dose-response curve. RT-PCR analysis of RNA from granulosa
cells cultured for 5 h in the presence or absence (control) of
varying concentrations of GDF-9 using primer sets for HAS2 and HPRT. B,
HAS2 and uPA time courses. RT-PCR analysis was performed with primer
sets for HAS2, uPA, and HPRT using RNA from granulosa cells cultured in
the presence or absence of 100 ng/ml of GDF-9 for 024 h. C, Northern
blot analysis of HAS2. Each lane contains 15 µg total RNA from
granulosa cells immediately after isolation (T = 0) or after
5 h of culture in the presence or absence of 50 ng/ml GDF-9. The
blot was hybridized first with an HAS2 cDNA probe, and then stripped
and rehybridized with a GAPDH cDNA probe. D, Northern blot analysis of
uPA. Each lane contains 15 µg total RNA from granulosa cells
immediately after isolation (T = 0) or after 5 h culture in
the presence or absence of 50 ng/ml GDF-9. The blot was hybridized
first with a uPA cDNA probe, and then stripped and rehybridized with a
GAPDH cDNA probe.
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A time course analysis (024 h) of the HAS2 expression pattern using
100 ng/ml of GDF-9 demonstrated a small induction of HAS2 mRNA at
2 h (data not shown), a peak induction between 35 h in culture,
and by 9 h in culture, the HAS2 expression level is decreasing
(Fig. 3B
). Granulosa cells cultured in the absence of GDF-9 for 024 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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Figure 4. GDF-9 Induces Cumulus Expansion in the Absence of
Oocytes
Morphological comparison of oocytectomized
cumulus-oocyte-complexes cultured for 18 h in the absence of GDF-9
or LH (A), the presence of LH (B), or the presence of 100 ng/ml GDF-9
(C). All cultures contained 10% FCS and 100 ng/ml FSH. Cells of the
control-treated complexes (A) or the complexes treated with 1 ng/ml LH
(B) appear flattened and polygonal and have adhered to the plate
forming a monolayer. The oocytectomized complexes incubated with GDF-9 (C) have undergone cumulus
expansion: cells appear round and glistening and are suspended in a
three-dimensional matrix. The plane of the image is above the level of
the culture surface, approximately at the level of the empty zona
pellucida.
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Regulation of LHR and COX-2 by GDF-9
Based on the above findings, we determined the effect of
recombinant mouse GDF-9 on LHR and COX-2 expression in granulosa cell
cultures using semiquantitative RT-PCR (Fig. 5
). 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 13 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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Figure 5. Regulation of LHR, COX-2, and HAS2 mRNA Synthesis
A, Time course (in hours) of LHR mRNA synthesis in granulosa cells
using semiquantitative RT-PCR. Granulosa cells were cultured with 5
ng/ml FSH in the presence or absence (control) of l00 ng/ml recombinant
mouse GDF-9. B, Effects of FSH and GDF-9 on LHR mRNA synthesis in
granulosa cells after 24 h of culture. Semiquantitative RT-PCR
analysis of LHR mRNA in triplicate wells treated ± FSH (10 ng/ml)
and ± GDF-9 (100 ng/ml) is shown. C, Effects of FSH and GDF-9 on
COX-2 or HAS2 mRNA synthesis in granulosa cells after 24 h of
culture. RT-PCR analysis of COX-2 or HAS2 mRNA in duplicate or
triplicate wells treated ± FSH and ± GDF-9 is shown. In
panels AC, semiquantitative RT-PCR analysis of HPRT mRNA under
identical conditions is used as the internal control. D and E,
Quantitation of the LHR, COX-2, and HAS2 mRNA levels from the above
experiments normalized to HPRT levels. Each value is the mean ±
SEM of duplicate or triplicate wells.
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GDF-9 Regulation of Progesterone Synthesis
To study the effects of GDF-9 and FSH on the regulation of
progesterone synthesis, we performed dose-response and time course
experiments. Semiquantitative RT-PCR was performed to analyze P-450 scc
mRNA synthesis and StAR mRNA synthesis (Fig. 6C
) 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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Figure 6. Progesterone Production and Regulation of Enzymes
Involved in Progesterone Synthesis
A, Granulosa cells were cultured for 14 h in serum-free media in
the absence or presence of 50 ng/ml GDF-9 in the presence of varying
concentrations of 0, 0.5, or 1 ng/ml FSH. Progesterone in the media was
assayed by RIA. The error bars represent the SEM. *,
P < 0.05 for control vs. 50 ng/ml
GDF-9 at FSH = 0 ng/ml, FSH = 0.5 ng/ml, or FSH = 1 ng/ml. B,
Progesterone in the media after 24 h of treatment of cells ±
GDF-9 (100 ng/ml) or ± FSH for triplicate samples in 10% of FCS
containing media. *, P < 0.05 for control
vs. GDF-9-treated cells cultured without FSH. C, Effects
of FSH and GDF-9 on P-450 scc or StAR mRNA synthesis in granulosa cells
after 24 h of culture. Semiquantitative RT-PCR analysis of P-450
scc or STAR mRNA in duplicate or triplicate wells treated ± FSH
or ± GDF-9. Semiquantitative RT-PCR analysis of HPRT mRNA under
identical conditions is used as the internal control. D and E,
Quantitation of the P-450 scc and StAR mRNA levels from the above
experiment normalized to HPRT levels. Each value is the mean ±
SEM of duplicate or triplicate wells.
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To determine how FSH and GDF-9 regulate progesterone synthesis in the
granulosa cell cultures, the levels of P-450 scc and StAR mRNA were
analyzed by semiquantitative RT-PCR after incubation for 24 h in
the presence or absence of FSH or the presence or absence of GDF-9
(Fig. 6
, CE). 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 25 fold induction of StAR mRNA (P <
0.05). These results suggest that FSH and GDF-9 function to regulate
progesterone synthesis via different mechanisms.
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DISCUSSION
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We previously demonstrated that GDF-9 knockout mice have a block
in folliculogenesis at the one-layer type 3b follicle stage,
demonstrating that GDF-9 protein is essential at this stage (16).
Consistent with these studies and the GDF-9 mRNA expression detected by
in situ hybridization (Ref. 14 and J. A. Elvin and M.
M. Matzuk, unpublished data), we show here by immunohistochemical
analysis that GDF-9 protein is first detected at low levels within
growing oocytes of primary follicles (type 3a follicles), is present at
higher levels in full-grown oocytes of type 3b follicles, and is
detected in oocytes of every subsequent developmental stage. Critical
to the other studies presented in this manuscript, GDF-9 protein is
also synthesized by oocytes of large antral and preovulatory follicles
in which the oocyte is closely associated with cumulus cells. Although
GDF-9 protein is detected in oocytes of type 3a follicles, it only
becomes essential for folliculogenesis at the type 3b-type 4 follicle
transition. This suggests that the GDF-9 signal transduction cascade is
not active before the type 3b follicle stage [e.g. GDF-9
receptors or downstream proteins may be absent in granulosa cells at
the type 3a stage or GDF-9 prohormone may not be processed correctly to
bioactive dimers (see below)].
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 3050 ng/ml GDF-9 causes a much more dramatic
induction, which plateaus at 24 oocytes/µl or 120300 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 35 h, while the rate of
oocyte-induced hyaluronic acid synthesis is maximal between 612 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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Figure 7. The Multifunctional Roles of GDF-9 in the
Mammalian Ovary (Model)
Based on GDF-9 knockout mouse studies, GDF-9 is required for granulosa
cell proliferation and differentiation leading to the formation of the
two-layer preantral follicle and subsequent recruitment of the thecal
layer. In the preovulatory follicle, GDF-9 induces HAS2 [and
subsequently hyaluronic acid (HA)], and suppresses uPA, resulting in
cumulus expansion (bottom box). GDF-9 may play an
important role in cumulus-oocyte-complex survival and fertilization by
stimulating cumulus cell progesterone production via induction of StAR
and prostaglandin synthesis via induction of COX-2. In the absence of
GDF-9 (top box), uPA and LHR are induced leading to
follicular rupture and luteinization, respectively.
|
|
Members of the TGF-ß superfamily bind to a distinctive
combination of type II and type I serine/threonine kinase receptors and
transduce signals through phosphorylation of specific Smad proteins to
alter transcription. TGF-ßs and activins induce phosphorylation of
Smad2 and Smad3, whereas BMPs stimulate phosphorylation of Smad1 and
Smad5 (39). Multiple TGF-ß superfamily type II receptors
(e.g. TGF-ß type II, activin receptor type II, activin
receptor type IIB), type I receptors [e.g. Alk2, Alk4
(activin type IB), Alk5 (TGF-ß type I)], and Smads (e.g.
Smad2 and Smad4), and unique receptors (e.g. the inhibin
receptor) are detected in mouse, rat, and human granulosa cells or
granulosa cell tumors (40, 41, 42). GDF-9 may be binding to and signaling
through already characterized type II and type I receptors and Smad
proteins, or through a novel GDF-9-specific signaling pathway. The lack
of phenocopy of GDF-9-deficient mice (i.e. block at the
one-layer primary follicle) and the activin type II receptor-deficient
mice (i.e. block at the antral follicle stage) (43),
indicates that GDF-9 is not signaling exclusively through the activin
receptor type II and suggests that GDF-9 and activins signal through
distinct receptor and Smad pathways in the same granulosa cells.
Further identification and characterization of the GDF-9 receptors, the
intracellular Smad signal transduction cascade, and the downstream
target genes for GDF-9 in granulosa cells is one continued interest of
our laboratory.
 |
MATERIALS AND METHODS
|
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Immunohistochemistry
Ovaries from CD1 (ICR) mice (generated at Baylor College of
Medicine from a stock from Charles River Laboratories, Inc., Wilmington, MA) were fixed in 10% neutral buffered
formalin, processed, and embedded in paraffin. Ovarian sections (4 µm
thick) were dewaxed then rehydrated in a graded series of ethanol
solutions. Nonspecific binding was reduced by preincubation for 30 min
in 1x universal blocking buffer (BioGenex Laboratories, Inc., San Ramon, CA) diluted in 0.1 M PBS and 0.1%
BSA. The primary antibody, mouse antihuman GDF-9 monoclonal antibody
was added to each section and incubated for 2 h at a final
concentration of 3060 ng/µl. Sections were washed twice in 0.1%
BSA in PBS for 5 min followed by incubation for 20 min in biotinylated
goat antimouse IgG (BioGenex Laboratories, Inc.). After
washing as above, the sections were incubated for 20 min in alkaline
phosphatase-conjugated streptavidin (BioGenex Laboratories, Inc.), washed twice in 0.1% BSA in PBS, and incubated with New
Fuchsin alkaline phosphatase substrate as per manufacturers
instructions (BioGenex Laboratories, Inc.). After
detection of a positive reaction, sections were counterstained with
hematoxylin and mounted in glycerol.
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 manufacturers 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 2124 days of age (Baylor College of
Medicine) were injected with 7.5 IU Gestyl (Diosynth B.V., Oss,
Holland), and ovaries were harvested 4448 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 manufacturers protocol.
Semiquantitative RT-PCR Analysis
Oligo-dT-primed cDNA from each RNA sample was synthesized using
Superscript reverse transcriptase (Gibco BRL) following
the manufacturers 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 485495 nm excitation
filter and 515525 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 manufacturers
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.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. David Albertini and Mary Jo Carabatsos for teaching
us the techniques of follicle and granulosa cell culture; Drs. Kathy
Tomkinson and Monique Davies for advice on the production of
recombinant GDF-9 and use of anti-GDF-9 antibodies; Dr. T. Rajendra
Kumar for critical reading of the manuscript; Kim Paes for help with
the figures; Shirley Baker for aid in manuscript preparation; Drs.
Monty Krieger and Helena Miettinen for interesting discussions; and
Dr. Albert Parlow and the National Hormone and Pituitary Program for
the gift of the ovine FSH.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Martin M. Matzuk, M.D., Ph.D., Professor, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: mmatzuk{at}bcm.tmc.edu
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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