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First published online 6 June 2007
doi: 10.1242/dev.006882
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1 The Jackson Laboratory, Bar Harbor, ME 04609, USA.
2 Department of Pathology, Baylor College of Medicine, Houston, TX 77030,
USA.
3 Department of Reproductive Medicine, University of California San Diego School
of Medicine, La Jolla, CA 92093-0633, USA.
4 Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA.
5 Departments Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX 77030, USA.
* Author for correspondence (e-mail: john.eppig{at}jax.org)
Accepted 16 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: BMP15, FGF8, Cumulus cells, Glycolysis, Mouse, Oocytes
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
While fully grown oocytes regulate the proliferation, gene expression and
function of cumulus cells, nutritional support from cumulus cells is essential
for growth and development of the oocyte
(Eppig, 2001). Notably,
oocytes are deficient in carrying out glycolysis, and utilize glucose poorly
as an energy substrate. The cumulus cells surrounding the oocyte provide
products of glycolysis, such as pyruvate, to the oocyte by either secreting
them or passing them through the gap junctions that couple the two cell types
(Biggers et al., 1967;
Donahue and Stern, 1968;
Leese and Barton, 1985).
Glycolysis in cumulus cells is regulated by paracrine factor(s) secreted by
oocytes (Sugiura et al.,
2005). When compared with other follicular cell types, such as
mural granulosa cells that line the follicular wall, cumulus cells express
higher levels of mRNA encoding glycolytic enzymes, such as platelet
phosphofructokinase (Pfkp) and lactate dehydrogenase A
(Ldha). Removing oocytes from cumulus cell-oocyte complexes (COCs)
reduces the steady-state levels of these transcripts and decreases glycolytic
activity in cumulus cells, and these decreases are completely reversed by
co-culturing the cumulus cells with fully grown oocytes
(Sugiura and Eppig, 2005;
Sugiura et al., 2005).
Therefore, mouse oocytes regulate glycolysis in their companion cumulus cells,
and this is achieved, at least in part, by secreted products that promote
expression of transcripts encoding glycolytic enzymes.
Mouse oocytes secrete proteins of two growth factor families: members of
the transforming growth factor ß (TGFß) superfamily, including
growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15)
and TGFB2, and members of the FGF family. Before the luteinizing hormone (LH)
surge, recombinant GDF9 and BMP15 suppress follicle stimulating hormone
(FSH)-stimulated expression of Lhcgr mRNA, encoding
LH/choriogonadotropin receptors in granulosa cells
(Elvin et al., 1999;
Otsuka et al., 2001), and
stimulate cumulus expansion after the LH surge
(Elvin et al., 1999;
Yoshino et al., 2006). BMP15
and GDF9/TGFB2 signals in cumulus cells are mediated by phosphorylation of
SMAD1/5/8 and SMAD2/3, respectively
(Shimasaki et al., 2004).
Bmp15-null mice (Bmp15-/-) are subfertile because
of defective cumulus cell development, and reduced ovulation and fertilization
rates, an effect augmented by deficiency of GDF9
(Su et al., 2004;
Yan et al., 2001). Although
Gdf9 heterozygous mutant (Gdf9+/-) mice do not
exhibit any obvious defects in follicular and cumulus cell development,
Gdf9+/- Bmp15-/- (double mutant, DM)
mice exhibit more severe ovarian defects than those observed in
Bmp15-/- mice (Su et
al., 2004; Yan et al.,
2001).
The other family of growth factors secreted by mouse oocytes is the FGF
family, including FGF8. During embryonic development, Fgf8 is
expressed widely in a temporally and spatially regulated manner
(Crossley and Martin, 1995;
Heikinheimo et al., 1994;
Ohuchi et al., 1994); however,
detection of Fgf8 mRNA expression in adult tissues by northern blot
analysis is restricted to ovary and testis
(MacArthur et al., 1995b). In
adult mouse ovaries, Fgf8 is expressed specifically in oocytes
(Valve et al., 1997).
Expression of FGF8 mRNA was also reported in bovine oocytes
(Buratini, Jr et al., 2005).
Furthermore, expression of FGF receptors was reported in granulosa cells of
human, mouse, rat and bovine (Asakai et
al., 1994; Ben-Haroush et al.,
2005; Berisha et al.,
2004; Puscheck et al.,
1997). Although these reports suggest oocyte-derived FGF8 may be
important for granulosa cell development or function, no evidence for this has
been presented previously.
The objective of the present study was to identify the oocyte factor(s) that regulate glycolysis and expression of transcripts encoding glycolytic enzymes, specifically Pfkp and Ldha, in mouse cumulus cells. Because ovaries of Bmp15-/- and DM mice exhibit abnormal follicular development, the role of BMP15 and GDF9 on glycolysis in cumulus cells was examined using knockout mouse models. We then examined the effects of recombinant proteins on the expression of genes encoding glycolytic enzymes and glycolysis in cumulus cells.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In situ hybridization
Preparation of Ldha RNA probe was reported previously
(Sugiura et al., 2005). For
the preparation of Pfkp and Fgf8 RNA probes, cDNA prepared
from mRNA isolated from either cumulus cells or fully grown oocytes of B6SJLF1
mice was amplified using polymerase chain reaction (PCR). The PCR primers used
were 5'-AACCGAGCCCGAAAAAAG-3' and
5'-GTAAAGCACAAGACAGTATCC-3' for Pfkp (GenBank accession
number NM_019703), and 5'-TGCTGTTGCACTTGCTGGTT-3' and
5'-AGTTGTTCTCCAGCACGATCTC-3' for Fgf8 (GenBank accession
number AK131980). Although the PCR primers for Fgf8 amplified
multiple alternative spliced isoforms of Fgf8 mRNA
(MacArthur et al., 1995a), the
PCR products cloned and used for template of probe production were
Fgf8b isoform [381 base pair (bp), GenBank accession number
AK131980]. The sequence of Fgf8 probe contained the common sequence
of all alternative spliced Fgf8 isoforms, therefore, the
Fgf8 probe used in the present study recognizes all known alternative
spliced isoforms of Fgf8 mRNA. In situ hybridization was performed as
reported previously (Eppig et al.,
2002), using ovaries of 22-day-old primed female mice,
approximately 44-48 hours after equine chorionic gonadotropin (eCG)
injection.
Isolation and culture of COCs, oocytes, mural granulosa cells and oocytectomized (OOX) cumulus cells
COCs, fully grown oocytes, mural granulosa cells and cumulus cells were
isolated and cultured as reported previously
(Sugiura et al., 2005). Fully
grown oocytes were maintained at the germinal vesicle (GV)-stage throughout
experiments by addition of the phosphodiesterase inhibitor, milrinone (10
µM) (Sigma). Unless otherwise indicated in the text, cells were cultured 20
hours before collection for isolation of mRNA or measurement of glycolytic
activity. OOX cumulus cells were produced by microsurgically removing oocytes,
but not the zona pellucida, from the COCs collected from 22-day-old eCG-primed
B6SJLF1 mice as described previously
(Buccione et al., 1990).
In some experiments, OOX cumulus cells were treated with recombinant human
TGFB2 (Leinco Technologies, St Louis, MO), human GDF9
(Liao et al., 2004), human
BMP15 (Otsuka et al., 2000),
mouse FGF8B (Sigma), dimethyl sulfoxide (DMSO) or SU5402 (Calbiochem, La
Jolla, CA) at several concentrations. In some experiments, a fine-glass tube
(diameter, 75-100 µm) was placed as a barrier bisecting the drop of culture
medium and preventing contact between denuded oocytes from OOX cumulus cells
while still allowing the passage of potential paracrine factors. For culturing
mural granulosa cells, the culture medium was supplemented with 100 ng/ml FSH
(Organon, Roseland, NJ) to induce expression of Lhcgr
(Eppig et al., 1997).
Assessing cumulus expansion
To induce cumulus expansion in OOX cumulus cells, the culture medium was
supplemented with 5% fetal bovine serum and 10 ng/ml of epidermal growth
factor (EGF; BD Biosciences, San Jose, CA) with various concentrations of the
recombinant proteins or with oocytes (2 oocytes/µl). Cumulus expansion
indexes were assessed as reported previously
(Eppig et al., 1993).
|
). The results
were presented as the relative expression levels to the transcript amount of a
standard sample, as indicated in the text, after normalization to the
expression levels of Rpl19 by the 2-
Ct
method (Livak and Schmittgen,
2001). To avoid false-positive signals, dissociation-curve
analyses were performed at the end of analyses. Moreover, the PCR products
were applied to agarose gel electrophoresis to confirm the sizes, and were
sequenced to verify sequence identity as preliminary experiments. The
reactions were performed at least in duplicate.
Measurement of glycolytic activity
Glycolytic activity was estimated as the ability of cells to metabolize
[5-3H]-glucose to 3H2O as reported previously
(Sugiura et al., 2005). Either
three COCs or three OOX cumulus cell complexes were used to measure glycolytic
activities as indicated in the text.
Immunoblot
Immunoblotting was performed as reported previously
(Diaz et al., 2006). Briefly,
samples were prepared from 40 OOX complexes cultured alone or with WT or
Bmp15-/- oocytes (2 oocytes/µl) for 15 hours. Samples
were simultaneously denatured by boiling in 1x loading buffer for 5
minutes followed by quenching on ice for 5 minutes. Proteins were separated on
a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and
transferred to polyvinylidene fluoride membrane. Membranes were blocked in
1x blocking buffer (Odyssey blocking buffer; Licor Bioscience, Lincoln,
NE) for 1 hour with shaking at room temperature, followed by incubation with
specific anti-phospho-SMAD2 antibody (1:1000; Invitrogen, Carlsbad, CA),
anti-phospho-SMAD1/5/8 antibody (1:1000; Cell Signaling Technology, Danvers,
MA) or ß-actin (ACTB) antibody (1:6000; Sigma) diluted in blocking buffer
with 0.1% Tween-20 for 12 hours at room temperature. Following incubation,
blots were washed three times for 10 minutes each with wash buffer
[phosphate-buffered saline (PBS), 0.1% Tween-20]. Fluorescently labeled
secondary antibodies (IRDye 800 anti-mouse or anti-rabbit; Rockland
Immunochemicals, Gilbertsville, PA) were diluted at 1:5000 and incubated with
the blots for 1 hour at room temperature. Blots were washed as above with an
additional final wash in PBS without Tween-20. Detection was accomplished with
an infrared scanner (Licor Bioscience).
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Statistical analysis
All experiments were repeated at least three times. Statistical analyses
were performed using computer software JMP (SAS Institute, Cary, NC).
Tukey-Kramer honestly significant difference tests were used to compare
multiple groups. For paired comparison, a standard t-test was used. A
P value <0.05 was considered statistically significant.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
However, in DM ovaries, a high level of Pfkp mRNA was found in theca
cells; only weak expression of either Pfkp or Ldha mRNA was
detected in cumulus cells or mural granulosa cells
(Fig. 1A). To further assess
expression levels of these transcripts in cumulus cells, real-time PCR
analyses were performed using cumulus cells of WT,
Bmp15-/-, DM and Gdf9+/- mice
(Fig. 1B).
Gdf9-/- mice were not used because follicular development
in ovaries of the Gdf9-/- mouse is blocked at the primary
follicle stage when cumulus cells have not yet developed
(Dong et al., 1996). As shown
in Fig. 1B, expression levels
of both Pfkp and Ldha mRNA in freshly isolated cumulus cells
of either Bmp15-/- or DM mice were significantly less than
that in cumulus cells of WT mice. Expression levels of Pfkp and
Ldha mRNA in cumulus cells of Gdf9+/- mice were
comparable to levels in cumulus cells of the WT mice. Furthermore, glycolytic
activities in freshly isolated COCs of Bmp15-/- and DM
mice were significantly lower than that in COCs of WT mice
(Fig. 1C).
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). However, co-culturing WT OOX cumulus cells with DM or
Bmp15-/- oocytes failed to maintain elevated Pfkp
and Ldha mRNA expression and glycolytic activity, and levels of these
transcripts were comparable to those of OOX cumulus cells cultured without WT
oocytes. Therefore, oocytes of Bmp15-/- and DM mice were
not able to sustain normal levels of Pfkp and Ldha mRNA and
glycolysis in WT cumulus cells. The culture conditions used in these experiments could have promoted elevated Pfkp and Ldha mRNA levels in cumulus cells. To test this possibility, levels of Pfkp and Ldha mRNA in cumulus cells of cultured intact COCs, OOX cumulus cells and OOX cumulus cells co-cultured with WT oocytes were compared with levels in freshly isolated cumulus cells. Culture did not increase the levels of expression of Pfkp or Ldha mRNA in intact COCs above those of freshly isolated cumulus cells. Sustaining levels of expression of these transcripts in cultured cumulus cells that were equivalent to freshly isolated cumulus cells was dependent upon the presence of WT oocytes (Fig. 2C).
Signals from the oocyte-secreted TGFß superfamily members in cumulus
cells are mediated by phosphorylation of SMAD families
(Shimasaki et al., 2004).
BMP15 and GDF9/TGFB2 stimulate phosphorylation of SMAD1/5/8 and SMAD2/3,
respectively (Moore et al.,
2003; Vitt et al.,
2002). Therefore, to assess the possibility that production of not
only BMP15, but also other factors, such as GDF9 and TGFB2, are reduced in
Bmp15-/- oocytes, the phosphorylation status of SMADs in
co-cultured WT OOX cumulus cells was examined
(Fig. 2D). When OOX cumulus
cells were co-cultured with WT oocytes, both SMAD1/5/8 and SMAD2 were
phosphorylated, indicating that both SMAD pathways in cumulus cells are
activated by oocyte-derived members of the TGFß superfamily in vitro.
However, Bmp15-/- oocytes did not stimulate SMAD1/5/8
phosphorylation in WT OOX cumulus cells, consistent with the absence of BMP15
in Bmp15-/- oocytes. However, Bmp15-/-
oocytes did promote SMAD2 phosphorylation in WT OOX cumulus cells, suggesting
that other relevant members of the TGFß superfamily are produced by
Bmp15-/- oocytes (Fig.
2D). In fact, expression levels of Gdf9, Fgf8, Tgfb2 and
Bmp6 mRNA in Bmp15-/- oocytes were comparable to
those in WT oocytes (Fig. 2E).
Therefore, the failure of Bmp15-/- oocytes to stimulate
Pfkp and Ldha expression in OOX cumulus cells is due
directly or indirectly to the absence of BMP15.
Recombinant BMP15 alone is not sufficient to promote expression of transcripts encoding glycolytic enzymes in cumulus cells
To further evaluate whether BMP15 is an oocyte-derived factor that induces
cumulus cell glycolysis, OOX cumulus cells were cultured with recombinant
BMP15, or with oocytes (2 oocytes/µl), and levels of Pfkp and
Ldha mRNA in the OOX cumulus cells were assessed
(Fig. 3). Surprisingly,
culturing OOX cumulus cells with recombinant BMP15 did not promote expression
of these transcripts or glycolysis in OOX cumulus cells
(Fig. 3D,
Fig. 8B). The inability of
BMP15 to stimulate Pfkp and Ldha mRNA expression in OOX
cumulus cells was not because of poor activity of BMP15, because this
preparation of recombinant BMP15 suppressed FSH-induced Lhcgr mRNA
expression in mural granulosa cells, and promoted cumulus expansion in a
dose-dependent manner (Fig.
3A-C) as described previously
(Otsuka et al., 2001;
Yoshino et al., 2006). The
effect of either recombinant TGFB2 or GDF9 was also assessed
(Fig. 4). Although both
recombinant TGFB2 and GDF9 stimulated cumulus expansion
(Fig. 4A,D) and the recombinant
GDF9 suppressed Lhcgr expression in mural granulosa cells
(Fig. 4D), the recombinant
proteins did not promote the expression of Pfkp and Ldha
mRNA in OOX cumulus cells (Fig.
4B,E). Taken together, these results strongly suggest that some
other factor(s), in addition to BMP15, is also required for promoting elevated
expression of transcripts encoding glycolytic enzymes and glycolytic activity
in cumulus cells.
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Because synergistic actions of BMP15 and GDF9 have been reported
(McNatty et al., 2005a;
McNatty et al., 2005b;
Su et al., 2004;
Yan et al., 2001), it was
possible that the factor required for stimulation of cumulus cell glycolysis
in addition to BMP15 might be GDF9. Therefore, the effects of co-treatment of
BMP15 and GDF9 on glycolysis in cumulus cells were examined
(Fig. 3E). Treatment of OOX
cumulus cells with BMP15, GDF9, or both had no effect on the expression levels
of Pfkp and Ldha mRNA in OOX cumulus cells
(Fig. 3E).
FGF activity is required for elevated expression of Pfkp and Ldha in OOX cumulus cells
As shown above, in addition to BMP15, some other factor(s), other than
GDF9, secreted by oocytes, is also required for stimulating cumulus cell
glycolysis. One of the other growth factors secreted by oocytes is FGF8
(Valve et al., 1997). Using in
situ hybridization, we confirmed specific expression of Fgf8 mRNA in
oocytes of secondary to large antral follicles
(Fig. 6A). Because cooperation
between BMP and FGF signals has been observed during embryonic development
(Hayashi et al., 2003;
Hayashi et al., 2001;
Nakamura et al., 2005;
Reinhold et al., 2004;
Warren et al., 2003), it was
possible that FGF8 secreted by oocytes might cooperate with BMP15 to promote
glycolysis in cumulus cells.
There are eight possible alternative spliced isoforms of Fgf8
(Fig. 6B)
(MacArthur et al., 1995a). To
assess which isoforms of Fgf8 mRNA are expressed in mouse oocytes,
PCR analysis was performed to amplify oocyte cDNA using Fgf8-specific
primers indicated in Fig. 6B.
After agarose electrophoresis, eight bands, whose sizes are well-correlated
with the expected PCR product sizes of each Fgf8 isoforms, were
detected (Fig. 6C). Identities
of bands 8a, 8b and 8e were confirmed by sequencing; however, expression of
the other Fgf8 isoforms in oocytes was not confirmed (see Materials
and methods).
Because Fgf8b appeared to be the most abundant isoform expressed in mouse oocytes, effects of recombinant FGF8B on the expression levels of Pfkp and Ldha mRNA in OOX cumulus cells were tested. As shown in Fig. 7A, when OOX cumulus cells were treated with recombinant FGF8B alone, there were no significant effects on the expression levels of Pfkp and Ldha mRNA. However, when OOX cumulus cells were co-cultured with a low density of oocytes (0.1 oocytes/µl, 20-fold lower than the previous oocyte co-culture experiments, i.e. Figs 2, 3, 4 and 5), FGF8B stimulated cumulus cell expression of Pfkp and Ldha transcripts in a dose-dependent manner (Fig. 7A). This FGF8B effect is not because of the additive effect of recombinant FGF8B and endogenous oocyte-secreted FGF8B, because higher concentrations of FGF8B (up to 1 µg/ml) did not promote expression of these genes to levels comparable to those in OOX cumulus cells co-cultured with oocytes (2 oocytes/µl) (data not shown). Therefore, these results strongly suggest that FGF8B is capable of inducing Pfkp and Ldha mRNA expression in cumulus cells, but some other oocyte-secreted factor(s) is also required.
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), was
used. Concentrations of this inhibitor below 60 µM have no cross-reactivity
between platelet-derived growth factor, EGF or insulin signaling
(Mohammadi et al., 1997). When
OOX cumulus cells co-cultured with oocytes (0.5 oocytes/µl) were treated
with SU5402 for 24 hours, Pfkp and Ldha mRNA in the OOX
cumulus cells was decreased in a dose-dependent manner compared with OOX
cumulus cells treated with only the solvent, DMSO
(Fig. 7B). The effect of SU5402
is reversible, because when OOX cumulus cells treated with SU5402 (25 µM)
for 24 hours were washed and cultured for an additional 20 hours with oocytes,
elevated levels of Pfkp and Ldha mRNA were restored
(Fig. 7C). These results
provide supporting evidence that FGF signals are necessary for increased
expression of transcripts encoding glycolytic enzymes in cumulus cells.
Co-treatment with recombinant FGF8B and BMP15 promotes Pfkp and Ldha mRNA expression and glycolysis in OOX cumulus cells
Effects of co-treatment with recombinant BMP15 and FGF8B on glycolysis by
OOX cumulus cells were examined (Fig.
8). Although recombinant FGF8B alone had no effect on levels of
Pfkp and Ldha mRNA by OOX cumulus cells, co-treatment with
BMP15 and FGF8B promoted expression of these transcripts by OOX cumulus cells
to levels comparable to those promoted by oocytes (2 oocytes/µl)
(Fig. 8A). Co-treatment with
FGF8B and GDF9 seemed to have slight effects on the expression of
Ldha mRNA, however, the difference was not significant and the mRNA
level was not comparable to that stimulated with BMP15 and FGF8B together.
Furthermore, the glycolytic activity of OOX cumulus cells treated with both
BMP15 and FGF8B was significantly greater than that of cells treated with
either FGF8B alone or co-treated with FGF8B and GDF9, or cultured without
oocytes (Fig. 8B).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the finding that Bmp15-/- oocytes did not promote the expression of Pfkp and Ldha mRNA levels and glycolysis in cumulus cells is strong evidence for the role of BMP15, it could be argued that defective oocyte-cumulus cell interactions throughout follicular development could have impaired development of Bmp15-/- oocytes and resulted in deficiencies in production of other factors that could influence cumulus cell glycolysis. However, this does not appear to be the case. In collaboration with Drs H. Pan and R. M. Schultz (University of Pennsylvania), we compared the transcriptomes of WT and DM oocytes by microarray analysis. The overall gene expression patterns were similar between these groups of oocytes, except that Bmp15 mRNA was not detected and approximately half the amount of Gdf9 mRNA was detected in DM oocytes as expected (H. Pan, R. M. Schultz, M.M.M. and J.J.E., unpublished). Furthermore, we have confirmed that levels of transcripts encoding other known oocyte-secreted factors, Bmp6, Gdf9, Tgfb2 and Fgf8, were not significantly changed between Bmp15-/- and WT oocytes. Moreover, the finding that Bmp15-/- oocytes stimulated SMAD2 phosphorylation in co-cultured WT cumulus cells strongly suggests that Bmp15-/- oocytes develop normally to the extent that they can produce ligands that stimulate SMAD2 phosphorylation in co-cultured WT cumulus cells. Therefore, the absence of BMP15 probably accounts for the inability of DM and Bmp15-/- oocytes to stimulate glycolysis in cumulus cells.
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). Moreover, Gueripel et al. have reported that although
mature BMP15 protein is present in mouse GV oocytes before LH surge, it is not
secreted (Gueripel et al.,
2006). Consistent with these reports, Gilchrist et al. suggest
that mouse oocytes are not capable of activating the SMAD1/5/8 pathway in
monolayer cultured mural granulosa cells using the reporter gene assay system
(Gilchrist et al., 2006). By
contrast, the present results showed that WT GV-stage oocytes promoted
SMAD1/5/8 phosphorylation in co-cultured OOX cumulus cells. Moreover,
Pfkp and Ldha mRNA levels and glycolytic activity in
Bmp15-/- cumulus cells were lower when compared with those
in WT mice before LH surge. Therefore, although the levels of secreted BMP15
are low before the LH surge, the BMP15 that is present plays a biologically
significant role during follicular development. The discrepancy in SMAD1/5/8
activation might be because of the different cell types used (e.g. OOX cumulus
cells versus monolayer cultured mural granulosa cells), the methods used to
detect activation of the SMAD1/5/8 pathway (e.g. immunoblot versus reporter
gene assay), or the concentration of oocytes used (2 versus 0.24
oocytes/µl).
Some of the defects in Bmp15-/- mice could be explained
by lower glycolysis in the cumulus cells. In vivo, resumption of meiosis in
Bmp15-/- oocytes is slower when compared with that in
Bmp15 heterozygous mutant (Bmp15+/-) oocytes, and
less than half of the Bmp15-/- oocytes reach meiotic
metaphase II stage at ovulation, whereas more than 90% of
Bmp15+/- oocytes reach meiotic metaphase II stage
(Su et al., 2004). Because
oocytes require pyruvate, which is produced by glycolysis in cumulus cells,
for their resumption of meiosis (Biggers et
al., 1967; Eppig,
1976), it is possible that lower glycolysis in
Bmp15-/- cumulus cells results in reduced availability of
pyruvate in the Bmp15-/- follicles, and this accounts for
the slower resumption of meiosis in Bmp15-/- oocytes. In
fact, when Bmp15-/- oocytes were cultured in a medium
containing pyruvate, they matured at the same rate as WT oocytes
(Su et al., 2004).
Furthermore, lower developmental ability of the Bmp15-/-
oocytes (Su et al., 2004)
could also be attributed to the lower nutritional support from cumulus cells
during their development.
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;
Galloway et al., 2000;
Hanrahan et al., 2004).
Whether glycolysis in cumulus cells before the LH surge is regulated by BMP15
and FGFs also in women or ewes remains to be determined. However, LH promotes
increased glycolytic activity in bovine COCs but not in OOX cumulus cells
(Zuelke and Brackett, 1992),
suggesting that factors derived from oocytes regulate glycolysis in cumulus
cells after the LH surge in the bovine. Moreover, apoptosis of bovine cumulus
cells is prevented by oocyte-derived BMP signals, including BMP15
(Hussein et al., 2005).
Because glycolytic enzymes participate in anti-apoptotic pathways
(Kim and Dang, 2005), it is
possible that BMP15 prevents apoptosis of cumulus cells through promoting
expression of genes encoding glycolytic enzymes in the bovine.
FGFs produced by oocytes probably function redundantly in promoting
glycolysis in cumulus cells. In bovine oocytes, expression of multiple
FGF mRNA species, including FGF8 mRNA, were detected by
microarray analysis, and expression of FGF8, FGF10, FGF17, FGF18 and
FGF22 transcripts was confirmed using RTPCR
(Zhong et al., 2006).
Moreover, our microarray analysis also detected transcripts encoding multiple
FGF family members, including Fgf8, Fgf17 and Fgf18, in
mouse oocytes (Su et al.,
2007). FGF family members are classified into seven subfamilies,
and within subfamilies each member possesses similar receptor specificity
(Zhang et al., 2006). Because
FGF8, FGF17 and FGF18 are classified in the same FGF8 subfamily, FGF8 is
probably not the only oocyte-derived FGF family member that regulates cumulus
cell glycolysis. Consistent with this idea, neutralizing antibodies against
FGF8 did not efficiently inhibit cumulus cell expression of genes encoding
glycolytic enzymes induced by oocytes (data not shown), whereas the FGF
receptor-dependent protein kinase inhibitor, SU5402, did.
Recent studies have focused on the role of oocyte-secreted TGFß
superfamily members, especially BMP15 and GDF9, because these paracrine
factors are specifically expressed by oocytes and because informative knockout
mutations have been produced (Dong et al.,
1996; Su et al.,
2004; Yan et al.,
2001). Interestingly, several FGF mutant mice also exhibit
reproductive deficiency. For example, whereas Fgfr2-knockout mice die
before birth (Xu et al.,
1998), mutant mice that carry an activating mutation of
Fgfr2 survive, but are infertile
(Chen et al., 2003). By
contrast, Fgfr3- and Fgfr4-knockout mice are fertile
(Colvin et al., 1996;
Deng et al., 1996;
Weinstein et al., 1998),
however, mice that carry a point mutation in Fgfr3 or
double-homozygous mutants of Fgfr3 and Fgfr4 are infertile
(Amsterdam et al., 2001;
Wang et al., 1999;
Weinstein et al., 1998). Null
mutations of Fgf8 produce embryonic lethality with embryo resorption
by E10.5 (Meyers et al.,
1998). Despite these observations, no studies have focused on the
importance of oocyte-derived FGF signaling on the development of granulosa
cells and follicles. Therefore, the results presented here are the first
showing a requirement of oocyte-secreted FGFs in the regulation of granulosa
cell function.
How do BMP15 and FGF signals cooperate to regulate glycolysis in cumulus
cells? During development, there are several organs in which BMP and FGF
signals cooperate to regulate cell differentiation. For example, during
calvarial suture osteogenesis, FGF2 augments the BMP4 signal by suppressing
expression of Nog mRNA, encoding the BMP antagonist noggin
(Warren et al., 2003). A
similar mechanism was observed during chondrogenesis, where FGF18 facilitates
the BMP2 signal by suppressing Nog mRNA expression
(Reinhold et al., 2004). Other
examples of cooperation of BMP and FGF signals were reported in nervous system
and ectopic bone formation (Hayashi et
al., 2003; Hayashi et al.,
2001; Nakamura et al.,
2005). During FGF-induced differentiation of
pheochromocytoma-derived PC12 cells, BMP2 facilitates FGF signaling by
promoting expression of Fgfr1 mRNA, encoding FGF receptor 1
(Hayashi et al., 2003;
Hayashi et al., 2001).
Moreover, during ectopic bone formation, low-dose FGF2 augments BMP2-induced
ectopic bone formation by stimulating expression of Bmpr1b, encoding
BMP receptor, type 1B in the bone-forming progenitor cells
(Nakamura et al., 2005). These
reports suggest the possibility that either BMP15 or FGFs modulate the
expression of antagonists and receptors for FGFs or BMP15 in cumulus cells.
Involvement of expression of BMP and FGF antagonists and receptors is
currently under investigation. Further research on these mechanisms will
clarify our knowledge of oocyte-cumulus cell communication as well as oocyte
and follicular development.
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ACKNOWLEDGMENTS |
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REFERENCES |
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