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First published online September 5, 2008
doi: 10.1242/10.1242/dev.025494
1 Department of Cell Biology, University of Connecticut Health Center,
Farmington, CT 06032, USA.
2 Center for Cell Analysis and Modeling, University of Connecticut Health
Center, Farmington, CT 06032, USA.
3 Department of Physiology, University of Arizona School of Medicine, Tucson, AZ
85724, USA.
4 Department of Neurobiology, Harvard Medical School, Boston, MA 02115,
USA.
5 Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Authors for correspondence (e-mails:
plampe{at}fhcrc.org;
ljaffe{at}neuron.uchc.edu)
Accepted 8 August 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Mehlmann,
2005a; Jones,
2008). LH acts on receptors on the mural granulosa cells in the
outer region of the follicle that surrounds the oocyte, and the signal is
conveyed inwards through the cumulus cells to the oocyte. By a pathway that is
incompletely understood, LH signaling results in a fall in cAMP in the oocyte
(Schultz et al., 1983;
Sela-Abramovich et al., 2006),
relieving the inhibition of cyclin dependent kinase 1 (Cdk1, also known as
Cdc2; Cdc2a - Mouse Genome Informatics) in the oocyte, and allowing the
prophase-to-metaphase transition to occur (see
Jones, 2008).
The cAMP that is required to maintain prophase arrest is produced in the
oocyte itself, by the constitutive activity of the orphan Gs-linked
receptor Gpr3 that activates adenylyl cyclase
(Mehlmann et al., 2002;
Horner et al., 2003;
Kalinowski et al., 2004;
Mehlmann et al., 2004;
Mehlmann, 2005b;
Freudzon et al., 2005;
Ledent et al., 2005;
Hinckley et al., 2005). If
Gpr3, Gs or adenylyl cyclase is absent or inhibited, cAMP decreases
and meiosis resumes. Related Gs and cAMP-dependent regulatory
systems operate in oocytes of humans
(DiLuigi et al., 2008), rats
(Hinckley et al., 2005) and
amphibians (see Gallo et al.,
1995; Ríos-Cardona et
al., 2008).
In mammals, contact of the mural granulosa cells with the cumulus-oocyte
complex is also required to maintain arrest; removal of the cumulus-oocyte
complex from the follicle (Pincus and
Enzmann, 1935; Edwards,
1965), or physical separation of these layers within the follicle
(Racowsky and Baldwin, 1989),
causes meiosis to resume. Gap junctions are required as well, as the
application of gap junction inhibitors causes meiotic resumption
(Piontkewitz and Dekel, 1993;
Sela-Abramovich et al.,
2006).
The somatic cells contribute to the maintenance of elevated cAMP in the
oocyte, because cAMP decreases when the oocyte is isolated from the follicle
(Törnell et al., 1990),
and this may occur by way of gap junctions, as the application of gap junction
inhibitors to the follicle decreases cAMP in the oocyte
(Sela-Abramovich et al.,
2006). Possibly the essential molecule entering the oocyte from
the somatic cells is cAMP itself, adding to that generated by the
Gpr3/Gs system in the oocyte. Alternatively, an inhibitor of cAMP
phosphodiesterase might diffuse into the oocyte from the mural cells
(Törnell et al., 1991).
It has been proposed that LH might cause the gap junctions in the path between
the mural granulosa cells and the oocyte to close, thus preventing the passage
of the meiosis-inhibitory molecule (Gilula
et al., 1978; Larsen et al.,
1987).
Gap junctions connect all cells of the follicle, but the connexins
comprising the gap junctions differ in the somatic cells versus the oocyte.
Connexin 43 (Cx43, or Gja1) is the primary connexin in the somatic cell
junctions (see Beyer et al.,
1989; Okuma et al.,
1996; Tong et al.,
2006). Connexin 45 and a small amount of connexin 37 (Cx37, or
Gja4) are also present (Okuma et al.,
1996; Alcoléa et al.,
1999; Veitch et al.,
2004; Simon et al.,
2006), but their contribution to the overall coupling between the
somatic cells appears to be minor compared with that of Cx43 (see
Simon et al., 1997;
Tong et al., 2006). By
contrast, Cx37 is expressed by mouse oocytes and is found at the oocyte
surface in oocyte-somatic cell gap junctions, with little if any contribution
from Cx43 (Beyer et al., 1989;
Simon et al., 1997;
Kidder and Mhawi, 2002;
Veitch et al., 2004;
Gittens and Kidder, 2005;
Li et al., 2007). The
oocyte-somatic cell gap junctions are probably homotypic junctions composed of
Cx37 on both sides of the junction (Veitch
et al., 2004), with the somatic cells immediately adjacent to the
oocyte expressing Cx37 and apparently targeting it differentially to processes
that they extend across the zona pellucida
(Veitch et al., 2004;
Simon et al., 2006). Deletion
of the gene encoding Cx37 eliminates gap junction communication at the oocyte
surface, as well as gap junction plaques at the oocyte surface as seen by
electron microscopy (Simon et al.,
1997). Thus, Cx37 is essential for the junctions at the oocyte
surface, although the possibility that another unidentified connexin is also
required cannot be eliminated.
In studies of transport across the oocyte surface in cumulus-oocyte
complexes isolated from follicles after LH receptor stimulation, gap junction
permeability did not decrease before nuclear envelope breakdown (NEBD)
(Gilula et al., 1978;
Eppig, 1982;
Racowsky and Satterlie, 1985).
However, the possibility of a decrease in gap junction communication between
the somatic cells, which could also result in the inhibition of a signal
between the mural cells and oocyte (Larsen
et al., 1987), was not investigated. In support of this concept,
LH causes a rapid dispersion of the orderly packing pattern of the connexins
in the membranes of the somatic cells of the follicle
(Larsen et al., 1981), rapid
phosphorylation of Cx43 (Granot and Dekel,
1994; Sela-Abramovich et al.,
2005), and rapid closure of gap junctions between granulosa cells
grown in culture (Sela-Abramovich et al.,
2005; Sela-Abramovich et al.,
2006). But whether it causes junction closure in intact ovarian
follicles, and, if so, where and when relative to the time of meiotic
resumption, is unknown. Thus, the possible role of gap junctions in the
regulation of meiotic resumption in response to LH is unresolved.
To investigate these issues, we microinjected fluorescent tracers into intact follicle-enclosed mouse oocytes, and monitored their diffusion between the interconnected cells of the follicle by using two-photon microscopy and redistribution after photobleaching. We show that gap junction permeability between the somatic cells of the follicle decreases prior to NEBD, and establish that the decreased permeability results from MAP kinase-dependent phosphorylation of Cx43 on serines 255, 262 and 279/282. We then examine the functional relationship of these events to the reinitiation of meiosis, and show that although MAP kinase-dependent gap junction closure is one component of the mechanisms by which LH causes meiotic resumption, another signaling pathway also functions in parallel.
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MATERIALS AND METHODS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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12 follicles per plate; PICMORG50,
Millipore, Billerica, MA), in MEM
(12000-022, Invitrogen, Carlsbad, CA)
with 25 mM NaHCO3, 75 µg/ml penicillin G, 50 µg/ml
streptomycin, 5% FCS (16000-044, Invitrogen), 10 ng/ml ovine follicle
stimulating hormone [FSH, from A. F. Parlow (National Hormone and Peptide
Program, Torrance, CA; NHPP)], and 5 µg/ml insulin, 5 µg/ml transferrin
and 5 ng/ml selenium (Sigma, St Louis, MO), equilibrated with 5%
CO2 and 95% air. Meiotic resumption was stimulated with 10 µg/ml
ovine LH (NHPP). The nucleus of the oocyte was visible within the follicle on
the culture plate (see Norris et al.,
2007), allowing the observation of NEBD. For a few experiments, we
used preantral follicles (Fig.
6C), or antral follicles from mice that had been injected with 4
IU of pregnant mare serum gonadotropin (PMSG; NHPP) 42 hours prior to follicle
isolation (see Fig. S2 in the supplementary material).
Microinjection of follicle-enclosed oocytes
Antral follicles were placed in an injection chamber in which they were
flattened between two coverslips spaced 200 µm apart (250 µm apart for
antral follicles from PMSG-injected mice, and 100 µm apart for preantral
follicles), and quantitative microinjection of 10 pl (5% of the 200 pl volume
of the oocyte) was carried out at 22°C
(Jaffe and Terasaki, 2004;
Norris et al., 2007;
Jaffe et al., 2009).
Gap junction tracers
Alexa Fluor 350 (A10439, Invitrogen; Mr=326) and Alexa
Fluor 488 (A10436, Invitrogen; Mr=534) were dissolved in
100 mM NaCl, 5 mM PIPES, pH 6.8, with brief heating to 90°C, and
stored at -80°C. Alexa Fluor 350 was used at a stock concentration of 50
or 100 mM, resulting in an initial concentration in the oocyte of 2.5 or 5 mM;
Alexa Fluor 488 was used at a stock concentration of 2 or 5 mM, resulting in
an initial concentration in the oocyte of 100 or 250 µm. Because of its
smaller size, which results in greater permeability through the Cx37 channels
at the oocyte surface (Weber et al.,
2004), Alexa Fluor 350 was used, except where indicated.
Two-photon imaging of Alexa Fluor 350
Two-photon microscopy allowed optimal visualization at an 100 µm
depth within the follicle (Helmchen and
Denk, 2005). Follicles were imaged in the coverslip chamber in
which they had been injected with Alexa Fluor 350. We used a Zeiss LSM 510
system, with a Ti:Sapphire laser (Chameleon; Coherent, Santa Clara, CA) tuned
to 720 or 740 nm, and a 20x/0.8 NA objective. The non-descanned emitted
light was collected through a 435-485 nm filter. Images were collected at the
oocyte equator, using four different laser intensities to avoid saturation or
too low a signal in all regions. The microscope stage was maintained at
37°C, with humidified 5% CO2/air.
To quantify Alexa Fluor 350 fluorescence ratios in the mural granulosa/inner cumulus regions, the mural region was identified from a scanning transmission image, and the inner cumulus was defined as the region between the outer edge of the zona pellucida and a circle 10 µm beyond the edge of the zona; this included the inner quarter-to-half of the cumulus mass (Fig. 1E). Autofluorescence determined from corresponding regions of uninjected follicles was subtracted. Measurements from images taken at different percent laser transmissions were normalized before calculating a ratio, using empirically determined conversion factors. For example, the specimen intensity increased threefold in changing from a 5 to a 10% laser transmission.
To compare Alexa Fluor 350 efflux from follicle-enclosed oocytes
±LH, we recorded images at two to three time points between 9 and 22
minutes after injection, and calculated the percent decrease in oocyte
intensity between 12 and 20 minutes. For this approach to be valid, there
should be a large concentration gradient between the oocyte and the cumulus
cells to minimize the effect of Alexa Fluor 350 diffusing back from the
cumulus cells to the oocyte. At 20 minutes after injection, the concentration
of Alexa Fluor 350 in the oocyte was still three to 14 times that in the inner
cumulus cells (see Fig. 1A-D);
the oocyte/inner cumulus cell concentration gradient was 6.8±0.9
(mean±s.e.m., n=14 follicles) for follicles without LH, and
5.8±0.7 (n=15) for follicles exposed to LH for 1
hour.
Fluorescence redistribution after photobleaching of Alexa Fluor 488
Follicle-enclosed oocytes were injected with Alexa Fluor 488 and incubated
on Millicell plates for 1-4 hours to allow the tracer to spread throughout the
follicle. After exposure to LH, the follicles were placed in a coverslip
chamber for photobleaching using a Zeiss LSM 510 microscope. We used Alexa
Fluor 488, despite its lower permeability through the Cx37 channel compared
with Alexa Fluor 350 (Weber et al.,
2004), because its higher quantum yield and longer excitation
wavelength reduce damage during photobleaching
(Galbraith and Terasaki,
2003), which occurred during initial attempts with Alexa Fluor
350.
Using a 40x/1.2 NA water immersion objective, and the 488 and 514 nm
lines of a 30 milliwatt Argon laser at 100% power, we photobleached a
60x20 µm rectangle in the mural granulosa cell layer, 20 µm
below the follicle surface. A 4.5-second exposure decreased the fluorescence
intensity by 50%. Post-bleach images were collected using the same
objective, but with the laser intensity reduced to 0.5% power, using a 505 nm
long pass filter and the confocal pinhole fully open; images were collected at
1.6-second intervals for 1 minute, and then at 10-second intervals for 2-5
minutes. These monitoring conditions did not significantly bleach the Alexa
Fluor 488. To compare the time course of fluorescence redistribution with and
without LH, we measured the change in Alexa Fluor 488 intensity in the
bleached region during the first minute (between 5 and 65 seconds) after the
end of the bleach.
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;
Lampe et al., 2006;
Sosinsky et al., 2007), were
prepared at the Fred Hutchinson Cancer Research Center Hybridoma Development
Facility (Seattle, WA). Phosphospecific antibodies for pERK (pMAPK; sc-7383)
and Cx43 phosphorylated at S255 (sc-12899) and S262 (sc-17219-R) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibodies
against pS279/S282-Cx43, pS368-Cx43, pY247-Cx43 and pY265-Cx43 were custom
prepared, affinity purified, and tested for specificity as previously
described (Solan et al., 2007;
Solan and Lampe, 2008). The
antibody against vinculin was obtained from Sigma (V4505). Two independent
Cx37 antibodies were made in rabbits against a GST fusion protein (amino acids
229-333 of rat Cx37), and were affinity purified
(Goliger and Paul, 1994;
Simon et al., 2006).
Immunoblotting
Samples for immunoblotting were prepared by washing the follicles in PBS
and sonicating them in Laemmli sample buffer containing 5%
β-mercaptoethanol, 10 mM NaF, 1 mM Na orthovanadate, 1 mM Pefabloc (Roche
Applied Science, Indianapolis) and Roche Complete protease inhibitor cocktail.
Each follicle contained 3.5 µg of protein; 5 µg of protein was
loaded per lane.
Blots of follicles were probed with various rabbit Cx43 phosphospecific
antibodies, applied together with a mouse monoclonal antibody recognizing
total Cx43 (NT1). Primary antibodies were used at 0.2-0.7 µg/ml. The rabbit
phosphospecific antibodies were detected with IRDye800-labeled anti-rabbit IgG
(611-731-127, Rockland Immunochemicals, Gilbertsville, PA) and the monoclonal
NT1 with Alexa Fluor 680 goat anti-mouse IgG (A21058, Invitrogen). Binding of
the two secondary antibodies was simultaneously quantified by using the LI-COR
Biosciences Odyssey infrared imaging system and associated software (Lincoln,
NE). Images were converted from 16 bits to 8 bits, after maximizing the
dynamic range of pixel intensity using the `levels' function in Adobe
Photoshop. Blots of isolated oocytes probed with the Cx37 antibody (0.3
µg/ml IgG) were visualized with an HRP-conjugated secondary antibody
(sc-2030, Santa Cruz Biotechnology) and ECL Plus reagents (GE Healthcare,
Piscataway, NJ).
Immunofluorescence microscopy
For total Cx43 immunofluorescence, follicles were fixed with 4%
paraformaldehyde, and embedded and frozen
(Norris et al., 2007). For
pS279/S282 immunofluorescence, follicles were frozen without fixation, in
gelatin capsules containing tissue-freezing medium (Triangle Biomedical
Sciences, Durham, NC). Cryosections (10 µm) were fixed with 50% MeOH/50%
acetone at -20°C for 1-2 hours, and then probed with the IF1 antibody
(total Cx43, 1 µg/ml) and Alexa Fluor 488 goat anti-mouse IgG (A11029,
Invitrogen), or with the pS279/S282 antibody (0.5 µg/ml in a buffer
containing 0.25% Tween-20) and Alexa Fluor 488 goat anti-rabbit IgG (A11034,
Invitrogen). NaF (10 mM) and Na orthovanadate (500 µm) were included in the
fixation and processing solutions. Sections were imaged using a 40x/1.2
NA water immersion objective on a Zeiss LSM 510 or Pascal confocal
microscope.
U0126, carbenoxolone
U0126 and an inactive analog, U0124, were obtained from EMD Chemicals (La
Jolla, CA), dissolved in DMSO at 100 mM, and diluted to 10 or 100 µm for
use. Follicles were pre-incubated with U0126 for 1 hour before addition of LH.
Carbenoxolone was obtained from Sigma.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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2 hours after
application of LH (see Fig.
7C). The decrease was transient, as follicles that had been
exposed to LH for 5 hours before injecting Alexa Fluor 350 showed tracer
diffusion throughout the mural granulosa cells (see Fig. S2 in the
supplementary material).
A similar transient decrease in gap junction permeability in response to LH
was seen in follicles from prepubertal mice that had been injected with PMSG
to stimulate follicle growth and LH receptor development in vivo (see Fig. S2
in the supplementary material). However, owing to the optical density of these
500 µm diameter follicles, which made them difficult to inject and
image, we used the optically clearer follicles described above for all further
studies.
Following LH exposure, Alexa Fluor 350 was mostly restricted to the two to
three layers of cumulus cells closest to the oocyte
(Fig. 1C,D; see also Fig. S2 in
the supplementary material). These cells are directly connected to the oocyte
by processes that extend through the intervening cumulus cells and zona
pellucida to form gap junctions at the oocyte surface
(Anderson et al., 1978). Thus,
the restriction of Alexa Fluor 350 to the inner cumulus cells of LH-stimulated
follicles is most likely to indicate diffusion through the Cx37 channels that
comprise the gap junctions at the oocyte surface, but not through the Cx43
channels that are predominant throughout the somatic cells (see
Introduction).
To quantify the LH-induced changes in gap junction permeability, we measured the ratio of the average fluorescence intensity in the mural granulosa cells to that in the inner cumulus cells, at 20 minutes after injection of Alexa Fluor 350 (Fig. 1E). For follicles treated with LH for 0.5-2 hours, this ratio was less than for follicles without LH treatment. By 5 hours after application of LH, the ratio had returned to the pre-LH level (Fig. 1F).
The LH-induced permeability decrease also occurs in the junctions between mural granulosa cells
The barrier to small molecule transfer that is established between the
cumulus and mural granulosa cells could result from gap junction closure only
within this region, or from a general closure of gap junctions throughout the
somatic cell layers. To investigate if LH caused gap junctions to close
between the mural granulosa cells, we loaded the cells of the follicle with
the fluorescent tracer Alexa Fluor 488, photobleached a region within the
mural granulosa cell layer, and monitored the redistribution of fluorescence
in the bleached region (Fig.
2). At one minute after the bleach, the fluorescence intensity in
most control follicles had recovered to 40-50% of its pre-bleach value
(Fig. 2A,C,E). By contrast, in
almost all follicles that had been exposed to LH for 0.5-2 hours, the
fluorescence intensity returned more slowly, indicating that LH had caused a
decrease in gap junction permeability throughout the mural granulosa cell
layer (Fig. 2B,D,E). We were
unable to use photobleaching to investigate gap junction permeability within
the cumulus cell layer, or between cumulus cells and the oocyte, because these
regions were too deep within the tissue to bleach effectively (see Fig. S3 in
the supplementary material).
LH does not cause a detectable decrease in the permeability of the gap junctions between the oocyte and cumulus cells
Images like those shown in Fig.
1 did not show an obvious effect of LH on the permeability of gap
junctions at the oocyte surface, and measurements of the percentage decrease
in fluorescence intensity in the oocyte between 12 and 20 minutes after Alexa
Fluor 350 injection did not show a significant difference with or without an
1 hour exposure to LH [without LH, 33±5% (mean±s.e.m.),
n=14 follicles; with LH, 26±3%, n=15]. Thus, although
a small change might have been missed by this measurement method, gap junction
permeability at the oocyte surface did not show a major decrease like that
occurring in the somatic cells.
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LH causes phosphorylation of Cx43 on several regulatory serines
LH application to follicles causes a shift in the SDS-PAGE mobility of
Cx43, which is due to phosphorylation on unspecified sites
(Kalma et al., 2004)
(Fig. 3A). To determine whether
known regulatory sites on Cx43 were phosphorylated in response to LH
application, we labeled blots of follicle proteins with antibodies that
recognize particular phosphorylated serines or tyrosines of Cx43. Serines 255,
279, 282 and 368, and tyrosines 247 and 265, were of particular interest,
because phosphorylation on these sites is required for the closure of gap
junction channels by MAP kinase (S255/S279/S282)
(Warn-Cramer et al., 1998), by
PKC (S368) (Lampe et al.,
2000), and by Src family kinases (Y247/Y265)
(Swenson et al., 1990;
Lin et al., 2001).
Phosphorylation on S262 is also associated with a decreased permeability of
Cx43 gap junctions (Doble et al.,
2004).
Immunoblots using three antibodies specific for phosphoserines 279/282, 262 and 255 of Cx43 showed little phosphorylation on these sites in follicles that had not been exposed to LH (Fig. 3B-D,G), but by 15 minutes after the application of LH, phosphorylation on each of these sites increased. Phosphorylation was maximal at 0.5-1 hour after LH application, and then decreased between 2 and 5 hours; at 5 hours, the level of phosphorylation was only slightly greater than that before LH exposure (Fig. 3B-E). This time course of Cx43 phosphorylation, and subsequent dephosphorylation, paralleled the time course of changes in gap junction permeability (Fig. 1). Like the decrease in gap junction permeability, phosphorylation on S279/S282 occurred in both the mural granulosa and the cumulus cells (Fig. 3F).
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Solan and Lampe, 2008) showed
little or no signal, and no change in response to LH, whereas robust signals
were seen in positive control lanes with comparable amounts of Cx43 protein
from LA-25 cells expressing active v-Src, or NRK cells treated with phorbol
ester (see Fig. S4 in the supplementary material). This indicated that the
LH-induced decrease in Cx43 permeability was not due to phosphorylation at
these sites.
LH-stimulated Cx43 phosphorylation is MAP kinase dependent
Because S279/S282 and S255 of Cx43 are known MAP kinase substrates
(Warn-Cramer et al., 1996;
Warn-Cramer et al., 1998), and
because LH activates MAP kinase in the follicle
(Su et al., 2002;
Kalma et al., 2004;
Panigone et al., 2008), we
used the MEK-specific inhibitor U0126
(Favata et al., 1998) to test
whether the LH-induced phosphorylation of Cx43 was MAP kinase dependent. U0126
(10 µm), which inhibited the LH-induced increase in phosphorylation of MAP
kinase, also inhibited phosphorylation on Cx43 S279/S282, S262 and S255
(Fig. 3G). These results are
consistent with previous gel shift evidence for MAP kinase dependence of the
LH-stimulated phosphorylation of Cx43 in rat follicles
(Sela-Abramovich et al.,
2005).
Inhibition of gap junction permeability is sufficient to cause meiotic resumption
The experiments described above provide the first direct evidence that LH
action on the intact follicle decreases gap junction permeability between the
somatic cells, prior to NEBD, and that this is linked to the MAP
kinase-dependent phosphorylation of Cx43 on multiple serine residues. The
closure of the junctions isolates the inner cumulus-oocyte complex from
signals that pass through the junctions from the mural granulosa cells.
Because mechanical isolation of the cumulus-oocyte complex is sufficient to
cause meiotic resumption (Pincus and
Enzmann, 1935; Racowsky and
Baldwin, 1989), we examined whether the inhibition of gap junction
communication between the mural granulosa cells and the oocyte would cause
meiotic resumption.
In the absence of a method to rapidly and selectively close only the Cx43
channels, as occurs in response to LH, we examined the effect of applying the
general gap junction inhibitor carbenoxolone (CBX)
(Rozental et al., 2001). A
previous study had shown that 100 µm CBX inhibits gap junction permeability
between rat granulosa cells in culture
(Sela-Abramovich et al.,
2006), and, likewise, we found that 100 µm CBX blocked gap
junction communication between the somatic cells and the oocyte in intact
mouse follicles (Fig. 5A). At a
concentration of 10 µm, CBX only partially inhibited gap junctional
communication (Fig. 5B). In rat
follicles, 100 µm CBX has been found to cause NEBD, as assayed at 5 hours
after CBX application (Sela-Abramovich et
al., 2006). Similarly, we found that 100 µm CBX caused NEBD in
mouse follicles, and determined that, in most follicles, this occurred after
1-2 hours (Fig. 5C). A
concentration of 10 µm CBX did not cause NEBD
(Fig. 5C).
We also used an antibody against the C-terminal cytoplasmic domain of Cx37
(Fig. 6A) to decrease gap
junction communication between the cumulus cells and the oocyte, and thus
indirectly to decrease communication between the mural cells and the oocyte.
Injection of this antibody into follicle-enclosed oocytes decreased Alexa
Fluor 350 diffusion from the oocyte (Fig.
6B), and the inhibition developed over a period of several hours
(Fig. 6C). This suggested an
effect on Cx37 turnover (Laird,
2006), which could decrease the number of channels in the plasma
membrane, rather than an effect on individual channel permeability.
Corresponding to the reduction in gap junction communication, NEBD occurred at
6-12 hours after injection of the antibody
(Fig. 6D).
These two different ways of reducing gap junction communication in the follicle both resulted in meiotic resumption, supporting the conclusion that the signal between the mural granulosa cells and the oocyte that maintains meiotic arrest is conveyed by way of gap junctions. Thus, the junction closure that occurs in response to LH would have the consequence of releasing the inhibition.
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However, as reported in a previous study of mouse follicles
(Su et al., 2003), 10 µm
U0126 caused little, if any, decrease in the percentage of oocytes undergoing
NEBD in response to LH, with inhibition seen only when the U0126 concentration
was increased to 100 µm (Fig.
7C) (see Su et al.,
2003). U0126 treatment at a concentration of 10 µm also caused
no significant delay in the time course of NEBD
(Fig. 7C). Thus, LH stimulated
NEBD even under conditions where gap junction closure was inhibited. This
finding supports the conclusion that although gap junction closure is
sufficient to cause meiotic resumption
(Sela-Abramovich et al., 2006)
(Figs 5,
6), LH also activates an
additional meiosis-stimulatory pathway that does not require the MAP
kinase-dependent closure of gap junctions.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) or the regulation of steroidogenesis
(Borowczyk et al., 2007).
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It has been proposed that the mural granulosa cells produce a small
molecule that passes through gap junctions into the oocyte and inhibits cAMP
degradation in the oocyte, and that this molecule could be cGMP
(Törnell et al., 1991).
The predominant cAMP phosphodiesterase in the oocyte is PDE3A
(Masciarelli et al., 2004),
and PDE3A is competitively inhibited by cGMP
(Hambleton et al., 2005). A
role for cGMP in maintaining meiotic arrest is supported by the findings that
cGMP injection into isolated oocytes delays meiotic resumption
(Törnell et al., 1990)
and that treatment of rat follicles with an inhibitor of soluble guanylate
cyclase causes meiotic resumption
(Sela-Abramovich et al.,
2008). In addition, inhibitors of inosine monophosphate
dehydrogenase (IMPDH), which is required in the pathway leading to formation
of cGMP, caused meiotic resumption when injected into mice
(Downs and Eppig, 1987) or when
applied to cultured follicles (Eppig,
1991). If gap junction closure reduced the supply of cGMP to the
oocyte, this would increase the activity of PDE3A. Although such an increase
was not seen in response to carbenoxolone treatment
(Sela-Abramovich et al.,
2006), a cGMP-mediated change in PDE activity might have been
undetectable by this method, owing to the dilution of cGMP in the assay. If
PDE activity did increase, the resulting decrease in cAMP would relieve the
inhibition of Cdk1, linking closure of somatic cell gap junctions to meiotic
resumption in the oocyte.
The concept of an alternative pathway linking LH action to meiotic
resumption, which is independent of gap junction closure, and which involves a
positive stimulus rather than a reversal of the mural cell inhibition, is
supported by studies of isolated cumulus-oocyte complexes. Oocytes within
their cumulus masses resume meiosis spontaneously, but this can be prevented
by incubation with dbcAMP or the cAMP phosphodiesterase inhibitor IBMX. Under
these conditions, EGF receptor stimulation, which is an intermediate in LH
signaling (see Panigone et al.,
2008), overcomes the inhibition imposed by dbcAMP or IBMX and
causes meiotic resumption (Downs et al.,
1988; Downs and Chen,
2008). Importantly, the percentage of cumulus-enclosed oocytes
that resume meiosis in response to EGF is greater than that seen in isolated
oocytes in the same dbcAMP- or IBMX-containing medium, implying that the
stimulation of meiotic resumption by LH/EGF signaling results in part from a
positive stimulus, in addition to the release of the mural cell
inhibition.
The identity of this positive stimulus, and how it reaches the oocyte, is
unknown, but it appears that the signal might pass through the Cx37 gap
junctions between the cumulus cells and the oocyte, based on evidence that in
the presence of the gap junction inhibitor glycerrhetinic acid, oocytes within
dbcAMP-arrested cumulus complexes fail to resume meiosis in response to EGF
(Downs and Chen, 2008). This
finding suggests that both pathways linking LH to meiotic resumption could
depend on gap junctions, although in different ways.
The functional redundancy in this regulatory system (release of inhibition
by gap junction closure, as well as a positive stimulus that is independent of
gap junction closure) is reminiscent of the dual pathways by which
progesterone causes meiosis to resume in Xenopus oocytes
(Haccard and Jessus, 2006). In
Xenopus, progesterone increases the synthesis of both cyclin B and
MOS, but synthesis of either protein is sufficient to cause NEBD; thus the
identified redundancy occurs in the oocyte itself, rather than in the somatic
cells of the follicle. Redundant signaling mechanisms occur in many other
physiological and developmental processes as well, such as chemokine signaling
in the immune system (Mantovani,
1999) and the specification of dorsal structures in vertebrate
development (Khokha et al.,
2005), and such redundancy is thought to both confer robustness
and facilitate evolutionary change
(Kirschner and Gerhart, 1998).
The evolutionary modification of molecules required for meiosis, which might
occur to optimize their functions in other tissues, could be deleterious for
reproduction, so redundancy at multiple levels in meiotic signaling pathways
would appear to be advantageous.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/19/3229/DC1
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ACKNOWLEDGMENTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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