Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
* Author for correspondence (e-mail: mamiller{at}uab.edu)
Accepted 9 September 2005
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SUMMARY |
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Key words: Major sperm protein, Oocyte maturation, Eph receptor, NMDA receptor, Ca2+/calmodulin-dependent protein kinase II, Inositol triphosphate receptor, Fertilization
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Introduction |
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We are using the nematode Caenorhabditis elegans as a model with
which to investigate the molecular mechanisms by which intercellular signals
regulate oocyte meiotic maturation. Oocyte development and fertilization occur
in an assembly line fashion within two U-shaped gonad arms that are connected
to a common uterus (Hubbard and
Greenstein, 2000). Oocytes in diakinesis of meiotic prophase are
located in the proximal gonad arm adjacent to the sperm storage compartment,
or spermatheca (Fig. 1A)
(McCarter et al., 1999
).
Somatic myoepithelial sheath cells surround the developing oocytes and form
gap junctions with them (Hall et al.,
1999
). Sperm promote oocyte maturation and sheath contraction,
which together facilitate ovulation
(McCarter et al., 1999
).
Fertilization occurs as ovulating oocytes enter the spermatheca
(Ward and Carrel, 1979
). In
the absence of sperm, oocytes can arrest in meiotic prophase for days. Because
the C. elegans hermaphrodite gonad produces roughly 300 sperm prior
to oogenesis, oocyte maturation and ovulation occur constitutively in adults
until sperm are depleted. In mutant hermaphrodites that do not generate sperm
(i.e. females), and in closely related nematode species with separate male and
female sexes, oocytes arrest in meiotic prophase until insemination occurs and
sperm crawl to the spermatheca (Hill and
l'Hernault, 2001
).
C. elegans sperm release an extracellular signal, the major sperm
protein (MSP), to promote oocyte maturation, MPK-1 mitogen-activated protein
kinase (MAPK) activation and sheath contraction
(Fig. 1A) (Miller et al., 2001). MSP is
the major cytoskeletal element in the sperm pseudopod, where it functions as
an actin analog during amoeboid locomotion
(Bottino et al., 2002
). MSP
leads a dual life as it is also exported from the sperm cytoplasm into the
proximal gonad by a membrane-budding mechanism
(Kosinski et al., 2005
).
Extracellular MSP binds to the VAB-1 Eph receptor protein-tyrosine kinase
(RPTK) on oocyte and sheath cell surfaces
(Miller et al., 2003
). Eph
RPTKs comprise an evolutionarily conserved receptor class that binds to
ligands called ephrins, and the C. elegans genome encodes three
ephrins that bind to VAB-1 (Chin-Sang et
al., 1999
; Wang et al.,
1999
). Ephrin/VAB-1 signaling in oocytes negatively regulates
oocyte maturation and MPK-1 MAPK activation
(Miller et al., 2003
). This
pathway acts in parallel to a sheath cell-dependent inhibitory pathway
requiring the CEH-18 POU-class homeoprotein
(Miller et al., 2003
). MSP
binding to VAB-1 and an unidentified receptor(s) antagonizes ephrin/VAB-1 and
CEH-18-dependent inhibition, resulting in oocyte maturation and MAPK
activation (Miller et al.,
2003
). MSP binds to VAB-1 on sheath cells to stimulate contraction
(Miller et al., 2003
).
Previously, we have reported that genetic regulators of oocyte meiotic
maturation and sheath contraction, including vab-1 and 28
msp loci, are physically clustered in the C. elegans genome
(Miller et al., 2004).
Building on this discovery, we now show that three clustered genes encoding
proteins involved in Ca2+-mediated signaling function in the MSP
signal transduction mechanism. Our results support a model in which VAB-1
undergoes a switch from negative to positive regulation of oocyte maturation
upon binding to MSP.
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Materials and methods |
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Phenotypic analysis and rate determination
Oocyte meiotic maturation (consisting of nuclear envelope breakdown and
cortical cytoskeletal rearrangement), ovulation and ovarian sheath contraction
were analyzed in anesthetized animals (0.1% tricaine and 0.01% tetramisole in
M9 buffer) on 2% agarose pads as previously described
(McCarter et al., 1999).
Observation and recording under DIC optics were performed using a Zeiss
Axioskop 2 plus, MRM Axiocam Hi-Res digital camera and PC for image
acquisition. Oocyte meiotic progression was evaluated by DAPI staining of
dissected gonads. For each strain, oocyte maturation and sheath contraction
were monitored by direct observation (or time-lapse recording) using DIC
optics. Basal sheath contraction rates are measured in adult hermaphrodites
and females by monitoring distal migration of the oocyte nuclear envelope,
which occurs prior to oocyte maturation and ovulatory contraction.
vab-1(dx31) is required for basal contraction in adult hermaphrodites
and females, but not in young adult hermaphrodites (<30 hours post L4 at
20°C). Oocyte maturation rates were determined by monitoring oocyte
ovulations in isolated animals on seeded plates for 4-24 hours. This method
was consistent with direct observation by DIC microscopy. Because food is
required for high maturation rates, monitoring oocyte ovulations on seeded
plates is optimal for accurate quantitation over long time periods (more than
90 minutes). itr-1(sa73) has spermathecal valve defects that impair,
but do not prevent ovulation and fertilization. In itr-1(sy290)
gain-of-function females, oocyte maturation rates are slow in young adults
(<36 hours post L4), but ovulation rates increase with age. Direct
observation by DIC microscopy indicated that spermathecal valve dilation
preceded nuclear envelope breakdown in four out of five cases. This defect is
probably due to premature spermathecal valve dilation, but it may also be due
in part to increased sheath contraction and unc-43 activation in
oocytes. unc-43(n498) mutants have vulva defects that inhibit egg
laying, so maturation rates were calculated in adults for shorter time periods
(2-4 hours). Tables show average oocyte maturation or sheath contraction
rates±standard deviation. A two-sample t-test was used to test
for significance.
RNA-mediated interference
RNAi was performed on L3 or L4 larva by the feeding method
(Timmons and Fire, 1998).
HT115 bacterial feeding strains were obtained from the genome-wide library
(Kamath et al., 2003
). To
confirm that the feeding strains contained the correct genes, we performed PCR
on DNA preps using gene specific primers internal to the cloned region. RNAi
phenotypes were compared with those of null mutants to determine
effectiveness.
Immunocytochemistry and GFP fluorescence
Monoclonal MAPK-YT (Sigma) and anti-phospho-CaM Kinase II (pThr286, Sigma)
antibodies were used to stain dissected gonads as previously described
(Miller et al., 2001).
Briefly, gonads were rapidly dissected in a watch glass in egg-salts solution,
and fixed in 2% neutral-buffered paraformaldehyde overnight at 4°C.
Following several washes, gonads were incubated with 1 mg/ml BSA in PBS for 2
hours, then with MAPK-YT (1:2000) or CaMKII-pT (1:250) antibodies for 4 hours
at room temperature. Anti-mouse FITC-conjugated secondary antibodies were used
for detection. Alexa-Fluor 660 phalloidin (Molecular Probes) and DAPI were
used to visualize F-actin and DNA, respectively. Expression was examined using
a motorized Zeiss Axioskop 2 plus equipped with epi-fluorescence, an MRM
Axiocam Hi-Res digital camera and PC computer for image acquisition. To
analyze subcellular localization, axial scans were performed, and out-of-focus
light was removed with deconvolution software (Axiovision). GFP expression of
transgenic VAB-1::GFP and NMR-1::GFP reporter strains was examined in
dissected gonads. Animals were kept in the dark for 24 hours prior to
dissection. Dissected gonads were fixed in 1% neutral-buffered
paraformaldehyde for 2 hours at 4°C in the dark. Under these conditions,
gonadal GFP expression is detectable by camera in
20% of the gonads. The
VAB-1::GFP and GFP::NMR-1 transgenes rescue the germline and/or somatic
defects of the corresponding null mutant hermaphrodites. The GFP::NMR-1
transgene does not show complete rescue, probably because expression is not at
the wild-type level.
Microinjection
MSP was microinjected into the gonad using a Zeiss Axiovert 200 microscope,
hydraulic fine type micromanipulator and Narishige IM-30 microinjector. The
least invasive method to introduce agents into the gonad is by injecting
through the vulva into the uterus. Injected animals were anesthetized and
mounted for direct observation of the gonad by DIC microscopy.
MSP purification and binding
MSP-6His was purified from E. coli under native conditions using
Ni-NTA agarose (Qiagen) (Miller et al.,
2001). MSP concentrations were determined spectrophotometrically
using
(275 nM)=3.29x104 M-1 cm-1
or by BCA protein assay (Pierce) (Miller
et al., 2001
). MSP-6His was conjugated to NHS-Fluorescein and
purified as previously described (Miller
et al., 2003
). Binding assays were performed in watch glasses
following gonad dissection. Briefly, dissected gonads were incubated with
MSP-FITC in egg-salts for 20 minutes at room temperature, washed twice with 1
ml egg-salts and fixed with 1% neutral-buffered paraformaldehyde for 10
minutes. MSP-FITC is biologically active, binding to gonads is specific and
binding sites are saturated at concentrations greater than 50 nM. MSP-FITC
binding can be out-competed with an excess of unlabelled MSP, and BSA-FITC
does not bind under the same conditions
(Miller et al., 2003
). Axial
scans of gonads incubated with 25 nM and 100nM MSP-FITC were processed with
deconvolution and 3D reconstruction software (Axiovision).
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Results |
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Ca2+/calmodulin-dependent protein kinase II [CaMKII;
(Reiner et al., 1999)] were
identified as candidate effectors (Miller
et al., 2004
).
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|
To test for site of action in the gonad, we use rrf-1(pk1417)
mutants, which are resistant to RNAi in the soma, but sensitive to RNAi in the
germ line (Sijen et al.,
2001). Using this method, vab-1 acts in the germ line to
inhibit oocyte maturation (Miller et al.,
2003
). The maturation rates of unmated itr-1 RNAi
fog-3(q443) and unmated itr-1 RNAi rrf-1(pk1417)
fog-3(q443) females are significantly faster than unmated
fog-3(q443) and rrf-1(pk1417) fog-3(q443) controls
(Table 1, compare lines 4, 11
and 3,10; P<0.001). Similarly, the MAPK activation frequencies of
itr-1 RNAi wild-type and itr-1 RNAi rrf-1(pk1417)
hermaphrodites are significantly higher than wild-type and
rrf-1(pk1417) hermaphrodites (Table S1, compare lines 10 and 11, and
lines 3 and 9; P<0.01). We conclude that itr-1 acts in
the germ line to inhibit maturation and MPK-1 MAPK activation.
nmr-1 negatively regulates oocyte maturation in the absence of MSP
The NMR-1 NMDA receptor subunit is most similar to the NR1 family of
mammalian subunits (Brockie et al.,
2001). To test whether nmr-1 negatively regulates oocyte
maturation, we constructed nmr-1(ak4); fog-2(q71) mutants. The
ak4 allele deletes the predicted pore-forming and ligand-binding
regions, and is probably a null mutation
(Brockie et al., 2001
). The
oocyte maturation rate of unmated nmr-1(ak4); fog-2(q71) females is
significantly faster than unmated fog-2(q71) females
(Table 1, compare lines 12 and
1; P<0.001). Similar results are observed in unmated
nmr-1 RNAi fog-3(q443) females
(Table 1, compare lines 13 and
3). The MAPK activation frequency of unmated nmr-1(ak4); fog-2(q71)
females is significantly higher than unmated fog-2(q71) controls
(Table S1, compare lines 12 and 1; P=0.001). These results indicate
that nmr-1 is a negative regulator of maturation and of MAPK
activation in the absence of sperm. nmr-1 is haploinsufficient for
this function, as the maturation rate of unmated nmr-1(ak4)/+;
fog-2(q71) females is significantly faster than unmated
fog-2(q71) females (Table
1, compare lines 14 and 1; P<0.001). To test whether
nmr-1 is required in germ line or somatic cells of the gonad, we used
rrf-1(pk1417) fog-3(q443) females. The maturation rates of unmated
nmr-1 RNAi fog-3(q443) and unmated nmr-1 RNAi
rrf-1(pk1417) fog-3(q443) females are significantly faster than
unmated fog-3(q443) and unmated rrf-1(pk1417) fog-3(q443)
females (Table 1, compare lines
13 and 15, and lines 3 and 10; P<0.001). Therefore, nmr-1
acts in the germ line to inhibit oocyte maturation.
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unc-43 is a positive regulator of oocyte maturation
To test whether unc-43 CaMKII negatively regulates oocyte
maturation, we examined females containing the e408 loss-of-function
and n1186-null mutations of unc-43
(Reiner et al., 1999). The
oocyte maturation rates of unmated unc-43(e408); fog-3(q443) and
unc-43(n1186); fog-3(q443) females are not significantly different
from the rate of unmated fog-3(q443) controls
(Table 1, compare lines 17, 18
and 3; P>0.10), nor are the MAPK activation frequencies different
from controls (Table S1, compare line 14 and 7; P>0.10). We
conclude that unc-43 is not a negative regulator of oocyte maturation
or of MAPK activation.
The maturation rates of unc-43(e408) and unc-43(n1186)
null hermaphrodites are slightly slower than the wild-type rate, raising the
possibility that unc-43 is a positive regulator
(Table 1, compare lines 19, 20
and 5; P<0.001). Mating unc-43 mutants with wild-type
males does not cause an increase in the maturation rate, demonstrating that
this defect is not due to unc-43 mutant sperm (data not shown).
Nuclear envelope breakdown, cortical cytoskeletal rearrangement, MAPK
activation and progression to metaphase occur normally in
unc-43(e408) and unc-43(n1186) hermaphrodites (Table S1,
line 15). To test whether unc-43 is a redundant positive regulator of
maturation, we examined the gain-of-function allele n498gf, which
results from an E108K mutation in the kinase domain
(Reiner et al., 1999). The
maturation rate of unmated unc-43(n498gf); fog-3(q443) females is
significantly faster than unmated fog-3(q443) females
(Fig. 2), but it is not
significantly different from unc-43(n498gf) hermaphrodites
[Table 1, compare lines 21 and
3 (P<0.001), and lines 21 and 22 (P>0.10)]. Therefore,
oocyte maturation occurs constitutively independent of sperm in
unc-43(n498gf) gonads. The maturation rate of n498gf mutants
is slower than the wild-type rate, owing at least in part to neuronal defects
that cause paralysis and prevent egg laying. To test whether unc-43
activation is sufficient to promote MPK-1 MAPK activation, we stained unmated
unc-43(n498gf) females with the MAPK-YT antibody. The MAPK activation
frequency of unmated unc-43(n498gf); fog-3(q443) females is
significantly higher than unmated fog-3(q443) females
[Fig. 1E,F; Table S1, compare
lines 16 and 7 (P<0.01)]. These results show that unc-43
is a redundant positive regulator of oocyte maturation and MPK-1 MAPK
activation.
To determine the site of unc-43 action, we constructed unc-43(n498gf); rrf-1(pk1417) fog-3(q443) mutants. The maturation rate of unmated unc-43 RNAi unc-43(n498gf); rrf-1(pk1417) fog-3(q443) females is significantly slower than the rates of unmated unc-43(n498gf); fog-3(q443) and unc-43(n498gf); rrf-1(pk1417) fog-3(q443) females (Table 1, compare lines 22, 23 and 24; P<0.001), but it is not significantly different from the rate of unmated unc-43 RNAi unc-43(n498gf); fog-3(q443) females (Table 1, cf lines 23 and 25; P>0.10). We conclude that unc-43 is required in the germ line to promote maturation. In summary, genetic and RNAi studies are consistent with itr-1, nmr-1 and unc-43 functioning in oocytes as downstream effectors of MSP signaling.
MSP signaling components are expressed at the oocyte cortex
Proximate effectors of MSP and VAB-1 signaling are predicted to localize at
or near the oocyte cell surface. A rescuing VAB-1::GFP translational reporter
shows GFP expression on oocyte and sheath cell membranes
(Miller et al., 2003). In
deconvolved mid-focal plane images, fluorescence appears particularly enriched
at membrane sites between oocytes. To test whether the distribution of
MSP-binding sites parallels the distribution of VAB-1, we incubated 25 nM
MSP-FITC with dissected gonads as described previously
(Miller et al., 2003
).
MSP-FITC is biologically active and binding to gonads is specific
(Miller et al., 2003
). Control
binding experiments performed by pre-incubating gonads with a 25-fold excess
of unlabeled MSP do not show MSP-FITC staining. In deconvolved mid focal plane
images, the fluorescent intensity at the interface between oocytes is
approximately three times greater than the sheath/oocyte interface
(Fig. 3A). Three-dimensional
reconstructions show similar results (Fig.
3B), as do incubations with saturating MSP-FITC concentrations
(data not shown). This enrichment pattern is still observed when contacting
oocytes are dissected away from the sheath
(Fig. 3C). By contrast,
MSP-FITC-binding sites are uniformly distributed around the plasma membrane in
isolated oocytes (Fig. 3D).
Mammalian Eph receptors are recruited to membrane sites between contacting
cells in culture (Marston et al.,
2003
). Our results suggest that VAB-1 and other MSP receptors are
enriched at and possibly recruited to plasma membrane sites between
oocytes.
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To investigate unc-43 expression, we incubated gonads with an
antibody that recognizes phosphorylated (pT286) rat CaMKII (CaMKII-pT). T286
is the primary autophosphorylation site and its phosphorylation correlates
with kinase activation (Colbran,
2004). The rat sequence used to generate CaMKII-pT is highly
conserved in UNC-43, including the phosphorylated threonine
(Fig. 4A, T284). The CaMKII-pT
antibody stains oocytes of wild-type gonads
(Fig. 4B), but not of
unc-43(n1186) null and unc-43(e408) loss-of-function gonads
(Fig. 4E; data not shown).
Therefore, CaMKII-pT specifically recognizes the unc-43 gene product.
To evaluate the subcellular distribution, we generated a z-series
through the gonad and processed the images with deconvolution software.
Phosphorylated T284 (pT284) UNC-43 co-localizes with F-actin at the oocyte
cortex and it is enriched at the oocyte/oocyte interface
(Fig. 4B-D), similar to the
distribution of GFP::NMR-1 and VAB-1::GFP
(Fig. 3E-J). CaMKII-pT staining
is also detected at the sheath/oocyte interface of wild-type gonads
(Fig. 4H-J). The CaMKII-pT
staining pattern contrasts with that of MAPK-YT, which is uniformly
distributed throughout the oocyte cytoplasm
(Fig. 4K). In wild-type gonads,
it is difficult to determine whether UNC-43 is expressed in sheath cells,
owing to their close proximity to oocytes and the thinness of the sheath. To
test whether pT284 UNC-43 is in sheath cells, we stained fem-3(q20gf)
mutant gonads with anti-CaMKII-pT. When grown at the restrictive temperature,
fem-3(q20gf) mutant gonads contain sperm and sheath cells, but not
oocytes (Barton et al., 1987
).
CaMKII-pT stains the somatic cells surrounding sperm in fem-3(q20gf)
gonads (Fig. 4L). These results
indicate that UNC-43 is phosphorylated in sheath cells and in oocytes when
sperm are present. In oocytes, pT284 UNC-43 co-localizes with F-actin at the
cortex.
CaMKII is activated by Ca2+/calmodulin binding and its activity
is further controlled by T286 autophosphorylation, resulting in a
Ca2+-independent form (Colbran,
2004; Hook and Means,
2001
). The unc-43(n498gf) gain-of-function mutation is
predicted to result in Ca2+-independent activation
(Reiner et al., 1999
);
however, minimal T284 phosphorylation is observed in unc-43(n498gf)
gonads (data not shown). CaMKII and many other protein kinases are capable of
signaling in the absence of activation loop phosphorylation
(Colbran, 2004
;
Kroiher et al., 2001
).
Phosphorylation is indicative of kinase activation, but lack of
phosphorylation is not necessarily indicative of an inactive state. We
conclude that UNC-43 activation does not require T284 phosphorylation and
speculate that T284 phosphorylation results in a Ca2+-independent
form.
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Mammalian NMDA receptors bind to and regulate CaMKII activity in cultured
neurons (Bayer et al., 2001;
Colbran, 2004
). To test
whether nmr-1 negatively regulates unc-43, we measured the
maturation rate of unmated unc-43 RNAi nmr-1(ak4);
fog-2(q71) females. Their rate is significantly slower than unmated
nmr-1(ak4); fog-2(q71) females
(Table 2, compare lines 8 and
9; P<0.001), indicating that loss of unc-43 suppresses
the inhibition defect of nmr-1-null mutants. We conclude that NMR-1
negatively regulates UNC-43 signaling in the absence of sperm. These results
appear inconsistent with the block model, but further investigation of the
relationship between MSP, VAB-1 and NMR-1 is necessary to draw definitive
conclusions.
NMR-1 can control the direction of VAB-1 signaling
The parallel and redundant structure of the MSP signaling mechanism raises
the possibility that crosstalk exists among pathways. To investigate the
genetic relationship between nmr-1 and vab-1 pathways, we
constructed vab-1(dx31) nmr-1(ak4); fog-2(q71) mutants. The
maturation rate of unmated vab-1(dx31) nmr-1(ak4); fog-2(q71) females
is significantly slower than unmated nmr-1(ak4); fog-2(q71) females
(Table 2, compare lines 10 and
8; P<0.001), indicating that vab-1(dx31) suppresses the
nmr-1(ak4) inhibitory defect. This relationship suggests that VAB-1
can act in a positive direction to promote oocyte maturation in the absence of
NMR-1 and MSP. When NMR-1 is present, VAB-1 acts as a negative regulator.
Genetic data support the hypothesis that ITR-1 acts downstream of ephrin/VAB-1
signaling to inhibit oocyte maturation. To test the genetic relationship
between nmr-1 and itr-1, we measured the maturation rate of
unmated itr-1 RNAi nmr-1(ak4); fog-2(q71) females. The rate
is significantly slower than unmated nmr-1(ak4); fog-2(q71) females
(Table 2, compare lines 11 and
8; P<0.001). Moreover, it is not different from unmated
fog-2(q71) controls (Table
1, line 1), indicating that both nmr-1 and itr-1
mutant defects are completely suppressed. Taken together, these results
suggest that crosstalk between NMR-1 and VAB-1/ITR-1 pathways controls the
direction of downstream signaling. Wild-type sperm stimulate a robust increase
in the maturation rates of itr-1 RNAi nmr-1(ak4); fog-2(q71)
and vab-1(dx31) nmr-1(ak4); fog-2(q71) females
(Table 2, line 12 and data not
shown). Therefore, this control mechanism must be redundant with another
sperm-responsive pathway(s).
Biochemical studies have shown that Eph and NMDA receptors directly
interact in cultured mammalian cells, leading to the recruitment of CaMKII
(Dalva et al., 2000). Our
protein localization data are consistent with NMR-1 and VAB-1 being components
of a heteromeric complex. A reduction in nmr-1 dose is sufficient to
trigger oocyte maturation in the absence of sperm
(Table 1, line 14).
Haploinsufficiency can be indicative of a balance in signaling among
components of a protein complex (Papp et
al., 2003
). To test whether a balance between NMR-1 and
VAB-1/ITR-1 signaling is important for regulating maturation, we examined the
maturation rates of unmated vab-1(dx31) nmr-1(ak4)/+ + and
nmr-1(ak4)/+; itr-1(sa73)/+ females. The rates of the double
heterozygotes are significantly slower than the rate of unmated
nmr-1(ak4)/+ females [Table
2, compare lines 15, 14 and 13 (P=0.004), and lines 17,
16, and 13 (P=0.002)]. We conclude that the level of signaling
between NMR-1 and VAB-1 pathways is crucial for the inhibitory response.
Collectively, the data suggest that the NMR-1 NMDA receptor subunit can
control the direction of VAB-1 signaling.
MSP binding to VAB-1 stimulates NMR-1-dependent UNC-43 activation
UNC-43 is phosphorylated in oocytes and sheath cells when sperm are present
(Fig. 4B-J,L). If MSP
stimulates UNC-43 activation, then UNC-43 phosphorylation should not occur in
the absence of sperm. To test this prediction, we stained unmated
fog-2(q71) and fog-3(q443) females with anti-CaMKII-pT. No
staining is observed in the absence of sperm
(Fig. 4M). To test whether MSP
is sufficient to trigger UNC-43 phosphorylation, we microinjected 100 nM MSP
into unmated fog-2(q71) females and stained their gonads with
CaMKII-pT. pT284 UNC-43 is detected in MSP microinjected gonads, but not in
control-injected gonads (Fig.
4N,O). These results support the hypothesis that MSP triggers
UNC-43 activation in the gonad. This mechanism must be redundant with another
MSP-responsive pathway(s), as 100 nM MSP can promote oocyte maturation when
microinjected into unmated unc-43(n1186); fog-3(q443) females
(Table 2, line 18). Control
injections with buffer alone did not stimulate oocyte maturation
(Table 2, line 19). Therefore,
MSP stimulates UNC-43-dependent and -independent pathways to promote oocyte
maturation.
To test the block versus switch models further, we investigated the genetic
requirements of UNC-43 T284 phosphorylation. Ephrin/VAB-1 and CEH-18-dependent
signaling act in parallel to inhibit oocyte maturation and MAPK activation
(Miller et al., 2003). The
block model predicts that T284 phosphorylation should occur in unmated
vab-1 RNAi ceh-18(mg57) females, which undergo constitutive
oocyte maturation independent of MSP presence
(Miller et al., 2003
).
CaMKII-pT staining is not observed in unmated vab-1 RNAi
ceh-18(mg57) females (Fig.
4P). Furthermore, UNC-43 T284 is not phosphorylated in unmated
nmr-1(ak4); fog-2(q71) and vab-1(dx31); fog-2(q71) females
(data not shown). We conclude that MSP does not stimulate UNC-43 T284
phosphorylation by simply disrupting VAB-1 or NMR-1 function. These results
are not consistent with the block model and suggest that MSP functions as an
agonist in promoting UNC-43 activation.
If MSP induces a switch in VAB-1 signaling activity that triggers UNC-43
T284 phosphorylation, then VAB-1 should be required for CaMKII-pT staining in
hermaphrodites. Consistent with this prediction, no staining is observed in
vab-1(dx31) null hermaphrodite gonads
(Fig. 4Q). UNC-43 T284 is
phosphorylated in vab-1(e2) kinase-dead mutants
(Fig. 4R), indicating that
VAB-1 phosphotransferase activity is not necessary. Ca2+
mobilization by mammalian NMDA receptors can stimulate CaMKII T286
phosphorylation in cultured neuronal cells
(Bayer et al., 2001;
Colbran, 2004
). To test
whether the nmr-1 NMDA receptor subunit is required for UNC-43 T284
phosphorylation, we stained nmr-1(ak4) hermaphrodites with CaMKII-pT.
No staining is observed in nmr-1(ak4) gonads
(Fig. 4S). By contrast, T284 is
phosphorylated in the itr-1 IP3 receptor mutants
itr-1(sa73) (Fig. 4T)
and itr-1(sy290gf) (Fig.
4U), as well as in ceh-18(mg57) mutants (data not shown).
These results suggest that MSP promotes UNC-43 T284 phosphorylation by
regulating NMDA receptor-dependent Ca2+ mobilization. Because MSP
and VAB-1 directly interact (Miller et
al., 2003
) and MSP-FITC binding to oocytes is not reduced in
nmr-1(ak4) gonads (P>0.10), we favor a model in which MSP
binding to VAB-1 stimulates NMR-1-dependent UNC-43 T284 phosphorylation and
signaling. These results, together with genetic analysis of unmated females,
are consistent with a switch mechanism that controls UNC-43 CaMKII
activation.
MSP stimulates UNC-43 activation in sheath cells to regulate contraction
For the switch model to be correct, MSP should have signaling properties of
an agonist. MSP binding to VAB-1 on sheath cells acts in a positive direction
to stimulate contraction. This mechanism does not require ephrins and exhibits
less redundancy than the maturation mechanism
(Miller et al., 2003). NMR-1
and UNC-43 are expressed in oocytes and sheath cells (Figs
3 and
4), raising the possibility
that these cells share a common circuitry that promotes UNC-43 activation. To
investigate the role of nmr-1 and unc-43 in sheath
contraction, we measured the basal contraction rates of mutant and RNAi
animals in the presence and absence of sperm. The contraction rates of
nmr-1(ak4) and nmr-1 RNAi hermaphrodites are almost twice
the wild-type rate [Table 3,
compare lines 2 and 1 (P<0.001); see Table S2 in the supplementary
material, compare lines 2 and 1]. Similar results are observed in
unc-43(n1186) null, unc-43(e403) and unc-43 RNAi
hermaphrodites [Table 3,
compare lines 3, 4 and 1 (P<0.001); see Table S2, compare lines 3
and 1). If nmr-1 and unc-43 functions are dependent on MSP,
contraction defects should not be observed in unmated mutant females. The
contraction rates of unmated nmr-1(ak4); fog-2(q71) and
unc-43(n1186); fog-3(q443)-null females are not significantly
different from the rates of unmated fog-2(q71) and
fog-3(q443) controls [Table
3, compare lines 6 and 5 (P>0.10) and lines 8 and 7
(P>0.10)]. We conclude that nmr-1 and unc-43 are
negative regulators of sperm-dependent sheath contraction. RNAi studies using
rrf-1(pk1417) and fem-3(q20) mutants demonstrate that
nmr-1 and unc-43 function in the somatic gonad to control
sheath contraction (see Table S2 in the supplementary material).
|
UNC-43 T284 phosphorylation is not observed in nmr-1(ak4) hermaphrodite gonads (Fig. 4S), suggesting that UNC-43 acts downstream of NMR-1 in oocytes and sheath cells. The identical contraction defects of unc-43 and nmr-1 mutants are consistent with this relationship (Table 3, compare lines 2-4). To determine the genetic relationship between unc-43 and nmr-1, we examined hermaphrodites containing the unc-43(n498gf) gain-of-function allele and nmr-1(ak4) null allele. The contraction rate of unc-43(n498gf); nmr-1(ak4) mutants is significantly slower than the rate of nmr-1(ak4) hermaphrodites (Table 3, compare lines 12 and 2; P<0.001), but it is not significantly different from the rate of unc-43(n498gf) hermaphrodites (Table 3, compare lines 12 and 13). These results support the hypothesis that unc-43 acts downstream on nmr-1.
MSP binding to VAB-1 and an unidentified receptor(s) stimulates ITR-1
signaling in sheath cells to promote basal contraction (M.A.M., unpublished)
(Yin et al., 2004). If the
MSP/VAB-1 interaction stimulates a distinct pathway resulting in UNC-43
activation, then loss of VAB-1 should prevent UNC-43 T284 phosphorylation and
signaling. In support of this prediction, T284 is not phosphorylated in
vab-1(dx31)-null gonads (Fig.
4Q). To test whether nmr-1/unc-43 signaling is dependent
on vab-1, we examined vab-1(dx31); unc-43(e408) and
vab-1(dx31); nmr-1(ak4) hermaphrodites. The contraction rates of
these double mutants are similar to the rate of vab-1(dx31) mutants
(Table 3, compare lines 16, 15
and 14), indicating that unc-43 function is largely dependent on
vab-1. Genetic analysis of sheath contraction supports the hypothesis
that MSP binding to VAB-1 stimulates NMR-1-dependent UNC-43 activation. This
mode of action is indicative of an agonist.
![]() |
Discussion |
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Negative regulation in the absence of MSP
The ephrin efn-2 and vab-1 function in the germ line to
negatively regulate oocyte maturation and MPK-1 MAPK activation in the absence
of MSP (Miller et al., 2003).
Ephrin/VAB-1 signaling acts in parallel to a sheath cell-dependent pathway
requiring the CEH-18 POU-class homeoprotein
(Fig. 5A). CEH-18 is required
for the assembly or maintenance of sheath/oocyte gap junctions
(Rose et al., 1997
). Genetic
data support the hypothesis that the ITR-1 IP3 receptor acts
downstream of ephrin/VAB-1 signaling in oocytes
(Fig. 5A). Mammalian ephrins
are capable of transducing signals in a `reverse' direction (i.e. from
receptor to ligand) and our current data cannot distinguish between forward
and reverse modes of action. However, RPTKs are well known stimulators of
IP3 production in cultured mammalian cells
(Berridge, 1993
). In the
textbook model, ligand binding induces RPTK dimerization and phospholipase C
(PLC) activation. PLC hydrolyzes phosphatidyl 4,5-bisphosphate to
IP3, which binds to IP3 receptors and stimulates
Ca2+ mobilization. Increases in local Ca2+ trigger the
activation of Ca2+-responsive effectors that modulate the cell
cycle and cytoskeleton.
|
The first evidence for a switch mechanism comes from studies of the genetic relationship between NMR-1 and VAB-1 receptor pathways. Loss of vab-1 or itr-1 suppresses the inhibition defect of unmated nmr-1(ak4)-null mutant females, suggesting that vab-1 and itr-1 can act in a positive direction. When nmr-1 is present, vab-1 and itr-1 are negative regulators. One explanation for the data is that NMR-1 can control the direction of VAB-1 signaling. Reducing the nmr-1 dose is sufficient to trigger oocyte maturation in the absence of sperm, and this defect is partially suppressed by a reduction in vab-1 or itr-1 dose. These relationships are indicative of a balance in signaling between VAB-1 and NMR-1 pathways (Fig. 5A). A balanced receptor ratio could prevent the activation of maturation promoting enzymes such as UNC-43 when sperm are not available for fertilization. A shift in the balance might result in precocious activation of these enzymes. The complete loss of inhibition defects in unmated itr-1 RNAi nmr-1(ak4) females suggests that ITR-1 and NMR-1 have inhibitory and promoting activities. Alternatively, other mechanisms may be able to compensate for their inhibitory functions when both genes are inactivated. Further studies will be necessary to completely understand the mechanism by which VAB-1 Eph and NMR-1 NMDA receptor pathways control oocyte maturation in the absence of MSP.
The switch to positive regulation
MSP binding to VAB-1 and an unidentified receptor(s) antagonizes
ephrin/VAB-1 and CEH-18-dependent inhibition
(Fig. 5B)
(Miller et al., 2003).
Significant redundancy must exist in this mechanism, as MSP can still promote
oocyte maturation in unmated vab-1 and unc-43-null mutant
females (Fig. 5). The MSP/VAB-1
interaction could block ephrin/VAB-1 signaling at the receptor level or it
could trigger a switch from negative to positive regulation
(Fig. 1B). For the switch model
to be correct, MSP and VAB-1 must stimulate downstream events that are
distinct from those occurring in unmated vab-1-null mutant females.
These events should be consistent with the action of an agonist, although the
overall response is antagonistic. Switch mechanisms might exist in signaling
networks composed of interacting hierarchies of negative and positive
regulators, as there may be more opportunities during evolution to create
inducible transitions. Consistent with such a theoretical argument, some
characterized intracellular regulators of C. elegans oocyte
maturation function as positive regulators, whereas others function as
negative regulators (Boxem et al.,
1999
; Detwiler et al.,
2001
; Miller et al.,
2004
).
We show that MSP induces UNC-43 activation in oocytes and sheath cells. Results from two sets of experiments demonstrate that MSP does not promote UNC-43 activation by blocking ephrin/VAB-1 signaling at the receptor level. First, the T284 phosphorylated form of UNC-43 is not observed in unmated vab-1(dx31)-null and vab-1(dx31); ceh-18(mg57) female gonads; it is observed when MSP is present. Second, loss of UNC-43 does not suppress the inhibition defects of vab-1- and efn-2-null mutants. These experiments indicate that ephrin/VAB-1 signaling negatively regulates oocyte maturation by a mechanism independent of UNC-43 (Fig. 5A).
To determine whether MSP acts as an agonist in promoting UNC-43 activation,
we sought to identify gene products that regulate this event. Both VAB-1 and
NMR-1 are required for UNC-43 T284 phosphorylation in response to MSP, placing
these two receptors between MSP and UNC-43 in the signaling hierarchy
(Fig. 5B). Binding studies
support a direct interaction between MSP and VAB-1
(Miller et al., 2003), but not
MSP and NMR-1. Therefore, we favor a model in which MSP binding to VAB-1
initiates a transduction process resulting in NMR-1-dependent UNC-43
activation (Fig. 5B), although
we cannot exclude the possibility that another MSP receptor is involved.
Further support for this model comes from studies of the sheath contraction
mechanism, which does not require ephrin signaling. MSP induces
unc-43 activation in sheath cells to attenuate the basal contraction
rate. Genetic analysis indicates that this response requires vab-1
and nmr-1. unc-43 is epistatic to mutations in nmr-1, a
result consistent with the idea that UNC-43 acts downstream of NMR-1. From
maturation and contraction data, we infer that VAB-1 and NMR-1 act in a
positive direction to promote UNC-43 T284 phosphorylation and signaling. When
sperm are absent from the reproductive tract, VAB-1 and NMR-1 act in the
germline as negative regulators of meiotic maturation. Collectively, the data
support the hypothesis that VAB-1 switches from a negative regulator into a
redundant positive regulator of maturation upon binding to MSP. NMR-1 mediates
this switch by controlling UNC-43 activation at the cortex
(Fig. 5). Once activated,
UNC-43 stimulates oocyte MPK-1 MAPK activation and meiotic maturation, either
indirectly by downregulating ephrin/VAB-1- and CEH-18-dependent inhibition or
by a more direct mechanism (not shown in
Fig. 5B).
Clues regarding the biochemical mechanism by which VAB-1 and NMR-1 pathways
control oocyte maturation come from studies of the mammalian central nervous
system. NMDA receptors and CaMKII are integrally involved in synaptic
plasticity and they directly interact in cultured cells
(Bayer et al., 2001;
Lisman and McIntyre, 2001
).
EphB receptor activation induces a direct interaction with NMDA receptors,
resulting in the recruitment of CaMKII
(Dalva et al., 2000
). Our
results are consistent with the possibility that VAB-1, NMR-1 and UNC-43 are
components of a heteromeric complex located at the oocyte surface. Two genes
encoding proteins implicated in organizing and trafficking RPTK signaling
complexes, caveolin (cav-1) and tetraspanin (tsp-12), are
required to negatively regulate oocyte maturation and MAPK activation
(Miller et al., 2004
). We
speculate that an interactive network of negative and positive regulators
functions at the oocyte cortex, possibly in multiprotein complexes, to control
meiotic maturation rates in response to fluctuating concentrations of MSP.
Coordinating responses and generating signaling specificity
RPTK activation at the cell surface can result in a variety of tissue
specific-responses. However, many of these responses are mediated by common
downstream pathways, such as the Ras/MAPK and phospholipase
C/IP3 cascades. MSP stimulates parallel responses in oocytes
and ovarian sheath cells to ensure that oocyte maturation, ovulation and
fertilization are tightly coupled. To coordinate these responses, MSP binds to
the VAB-1 Eph RPTK on oocyte and sheath surfaces. Our results, together with
those from previous studies, indicate that NMR-1 NMDA and ITR-1 IP3
receptors act downstream of MSP in both cell types
(Yin et al., 2004
) (M.A.M.,
unpublished). These pathways regulate meiotic maturation in oocytes and smooth
muscle-like contraction in sheath cells. NMDA and IP3 receptors
mobilize intracellular Ca2+, which is a ubiquitous second messenger
that can regulate the cell cycle and cytoskeleton, depending on the
Ca2+-responsive effectors that are present in a given cell. The
parallel structure of the MSP transduction mechanism allows oocytes and sheath
cells to respond differently to the same stimulus with minimal genetic
complexity.
Specificity in signaling can be achieved through the activation of distinct
downstream effectors. The cyclin-dependent kinase CDK-1 and polo-like kinase
PLK-1 are two downstream mediators of oocyte maturation in C. elegans
(Boxem et al., 1999;
Chase et al., 2000
). The
actin-linked regulators tropomyosin and troponin probably function in the
sheath cells to regulate contraction (Ono
and Ono, 2004
). Therefore, Ca2+ mobilization acts
upstream of CDK-1 and PLK-1 in oocytes and tropomyosin and troponin in sheath
cells. itr-1 acts downstream of the let-23 EGF RPTK homolog
in the spermatheca to control valve dilation during ovulation
(Clandinin et al., 1998
;
Ono and Ono, 2004
). Taken
together, RPTK activation of the phospholipase C
/IP3 pathway
regulates oocyte meiotic maturation, ovarian sheath contraction and
spermathecal valve dilation. These opposing cell types must be programmed to
respond differently to Ca2+ mobilization. The collective action of
shared RPTK signaling cascades and cell type-specific effectors is predicted
to help drive fertilization in the C. elegans gonad.
![]() |
ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/23/5225/DC1
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barton, M. K., Schedl, T. B. and Kimble, J.
(1987). Gain-of-function mutations of fem-3, a sex-determination
gene in Caenorhabditis elegans. Genetics
115,107
-119.
Bayer, K. U., De Koninck, P., Leonard, A. S., Hell, J. W. and Schulman,H. (2001). Interaction with the NMDA receptor locks CaMKII in an active conformation. Nature 411,801 -805.[CrossRef][Medline]
Baylis, H. A., Furuichi, T., Yoshikawa, F., Mikoshiba, K. and Sattelle, D.B. (1999). Inositol 1,4,5-trisphosphate receptors are strongly expressed in the nervous system, pharynx, intestine, gonad and excretory cell of Caenorhabditis elegans and are encoded by a single gene (itr-1). J. Mol. Biol. 294,467 -476.[CrossRef][Medline]
Berridge, M. J. (1993). Inositol trisphosphate and calcium signalling. Nature 361,315 -325.[CrossRef][Medline]
Bottino, D., Mogilner, A., Roberts, T., Stewart, M. and Oster,
G. (2002). How nematode sperm crawl. J. Cell
Sci. 115,367
-384.
Boxem, M., Srinivasan, D. G. and van den Heuvel, S.
(1999). The Caenorhabditis elegans gene ncc-1 encodes a
cdc2-related kinase required for M phase in meiotic and mitotic cell
divisions, but not for S phase. Development
126,2227
-2239.
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Brockie, P. J., Mellem, J. E., Hills, T., Madsen, D. M. and Maricq, A. V. (2001). The C. elegans glutamate receptor subunit NMR-1 is required for slow NMDA-activated currents that regulate reversal frequency during locomotion. Neuron 31,617 -630.[CrossRef][Medline]
Chase, D., Serafinas, C., Ashcroft, N., Kosinski, M., Longo, D., Ferris, D.K. and Golden, A. (2000). The polo-like kinase PLK-1 is required for nuclear envelope breakdown and the completion of meiosis in Caenorhabditis elegans. Genesis 26, 26-41.[CrossRef][Medline]
Chin-Sang, I. D., George, S. E., Ding, M., Moseley, S. L., Lynch, A. S. andChisholm, A. D. (1999). The ephrin VAB-2/EFN-1 functions in neuronal signaling to regulate epidermal morphogenesis in C. elegans. Cell 99,781 -790.[CrossRef][Medline]
Clandinin, T. R., DeModena, J. A. and Sternberg, P. W. (1998). Inositol trisphosphate mediates a RAS-independent response to LET-23 receptor tyrosine kinase activation in C. elegans. Cell 92,523 -533.[CrossRef][Medline]
Colbran, R. J. (2004). Targeting of calcium/calmodulin-dependent protein kinase II. Biochem. J. 378,1 -16.[CrossRef][Medline]
Dal Santo, P., Logan, M. A., Chisholm, A. D. and Jorgensen, E. M. (1999). The inositol trisphosphate receptor regulates a 50-second behavioral rhythm in C. elegans. Cell 98,757 -767.[CrossRef][Medline]
Dalva, M. B., Takasu, M. A., Lin, M. Z., Shamah, S. M., Hu, L., Gale, N.W. and Greenberg, M. E. (2000). EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell 103,945 -956.[CrossRef][Medline]
Detwiler, M. R., Reuben, M., Li, X., Rogers, E. and Lin, R. (2001). Two zinc finger proteins, OMA-1 and OMA-2, are redundantly required for oocyte maturation in C. elegans. Dev. Cell 1,187 -199.[CrossRef][Medline]
Ellis, R. E. and Kimble, J. (1995). The fog-3
gene and regulation of cell fate in the germ line of Caenorhabditis elegans.
Genetics 139,561
-577.
Eppig, J. J. (2001). Oocyte control of ovarian
follicular development and function in mammals.
Reproduction 122,829
-838.
Hall, D. H., Winfrey, V. P., Blaeuer, G., Hoffman, L. H., Furuta, T., Rose,K. L., Hobert, O. and Greenstein, D. (1999). Ultrastructural features of the adult hermaphrodite gonad of Caenorhabditis elegans: relations between the germ line and soma. Dev. Biol. 212,101 -123.[CrossRef][Medline]
Hassold, T. and Hunt, P. (2001). To err (meiotically) is human: the genesis of human aneuploidy. Nat. Rev. Genet. 2,280 -291.[CrossRef][Medline]
Hill, K. L. and l'Hernault, S. W. (2001). Analyses of reproductive interactions that occur after heterospecific matings within the genus Caenorhabditis. Dev. Biol. 232,105 -114.[CrossRef][Medline]
Hook, S. S. and Means, A. R. (2001). Ca(2+)/CaM-dependent kinases: from activation to function. Annu. Rev. Pharmacol. Toxicol. 41,471 -505.[CrossRef][Medline]
Hubbard, E. J. and Greenstein, D. (2000). The Caenorhabditis elegans gonad: a test tube for cell and developmental biology. Dev. Dyn. 218,2 -22.[CrossRef][Medline]
Hunt, P. A. and LeMaire-Adkins, R. (1998). Genetic control of mammalian female meiosis. Curr. Top. Dev. Biol. 37,359 -381.[Medline]
Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M.,Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M. et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421,231 -237.[CrossRef][Medline]
Kosinski, M., McDonald, K., Schwartz, J., Yamamoto, I. and
Greenstein,D. (2005). C. elegans sperm bud vesicles to
deliver a meiotic maturation signal to distant oocytes.
Development 132,3357
-3369.
Kroiher, M., Miller, M. A. and Steele, R. E. (2001). Deceiving appearances: signaling by `=dead' and `fractured' receptor protein-tyrosine kinases. BioEssays 23,69 -76.[CrossRef][Medline]
Lisman, J. E. and McIntyre, C. C. (2001). Synaptic plasticity: a molecular memory switch. Curr. Biol. 11,R788 -R791.[CrossRef][Medline]
Marston, D. J., Dickinson, S. and Nobes, C. D. (2003). Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion. Nat. Cell Biol. 5, 879-888.[CrossRef][Medline]
Masui, Y. (1985). Meiotic arrest in animal oocytes. In Biology of Fertilization, Vol.1 (ed. A. Monroy and C. B. Metz), pp.189 -219. Orlando, FL: Academic Press.
Masui, Y. and Clarke, H. J. (1979). Oocyte maturation. Int. Rev. Cytol. 57,185 -282.[Medline]
McCarter, J., Bartlett, B., Dang, T. and Schedl, T. (1999). On the control of oocyte meiotic maturation and ovulation in Caenorhabditis elegans. Dev. Biol. 205,111 -128.[CrossRef][Medline]
Miller, M. A., Nguyen, V. Q., Lee, M. H., Kosinski, M., Schedl,
T., Caprioli,R. M. and Greenstein, D. (2001). A sperm
cytoskeletal protein that signals oocyte meiotic maturation and ovulation.
Science 291,2144
-2147.
Miller, M. A., Ruest, P. J., Kosinski, M., Hanks, S. K. and
Greenstein, D. (2003). An Eph receptor sperm-sensing control
mechanism for oocyte meiotic maturation in Caenorhabditis elegans.
Genes Dev. 17,187
-200.
Miller, M. A., Cutter, A. D., Yamamoto, I., Ward, S. and Greenstein, D. (2004). Clustered organization of reproductive genes in the C. elegans genome. Curr. Biol. 14,1284 -1290.[CrossRef][Medline]
Ono, K. and Ono, S. (2004). Tropomyosin and
troponin are required for ovarian contraction in the Caenorhabditis elegans
reproductive system. Mol. Biol. Cell
15,2782
-2793.
Papp, B., Pal, C. and Hurst, L. D. (2003). Dosage sensitivity and the evolution of gene families in yeast. Nature 424,194 -197.[CrossRef][Medline]
Pasquale, E. B. (2005). Eph receptor signalling casts a wide net on cell behaviour. Nat. Rev. Mol. Cell. Biol. 6,462 -475.[CrossRef][Medline]
Pincus, G. and Enzmann, E. V. (1935). The comparative behaviour of mammalian eggs in vivo and in vitro.J. Exp. Med. 62,655 -675.
Poliakov, A., Cotrina, M. and Wilkinson, D. G. (2004). Diverse roles of eph receptors and ephrins in the regulation of cell migration and tissue assembly. Dev. Cell 7,465 -480.[CrossRef][Medline]
Reiner, D. J., Newton, E. M., Tian, H. and Thomas, J. H. (1999). Diverse behavioral defects caused by mutations in Caenorhabditis elegans unc-43 CaM kinase II. Nature 402,199 -203.[CrossRef][Medline]
Rose, K. L., Winfrey, V. P., Hoffman, L. H., Hall, D. H., Furuta, T. andGreenstein, D. (1997). The POU gene ceh-18 promotes gonadal sheath cell differentiation and function required for meiotic maturation and ovulation in Caenorhabditis elegans. Dev. Biol. 192,59 -77.[CrossRef][Medline]
Schedl, T. and Kimble, J. (1988). fog-2, a
germ-line-specific sex determination gene required for hermaphrodite
spermatogenesis in Caenorhabditis elegans. Genetics
119, 43-61.
Sijen, T., Fleenor, J., Simmer, F., Thijssen, K. L., Parrish, S., Timmons,L., Plasterk, R. H. and Fire, A. (2001). On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107,465 -476.[CrossRef][Medline]
Timmons, L. and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395, 854.[CrossRef][Medline]
Wang, X., Roy, P. J., Holland, S. J., Zhang, L. W., Culotti, J. G. andPawson, T. (1999). Multiple ephrins control cell organization in C. elegans using kinase-dependent and -independent functions of the VAB-1 Eph receptor. Mol. Cell 4, 903-913.[CrossRef][Medline]
Ward, S. and Carrel, J. S. (1979). Fertilization and sperm competition in the nematode Caenorhabditis elegans. Dev. Biol. 73,304 -321.[CrossRef][Medline]
Yin, X., Gower, N. J., Baylis, H. A. and Strange, K.
(2004). Inositol 1,4,5-trisphosphate signaling regulates rhythmic
contractile activity of myoepithelial sheath cells in Caenorhabditis elegans.
Mol. Biol. Cell 15,3938
-3949.
Yoshikawa, F., Morita, M., Monkawa, T., Michikawa, T., Furuichi,
T. andMikoshiba, K. (1996). Mutational analysis of the ligand
binding site of the inositol 1,4,5-trisphosphate receptor. J. Biol.
Chem. 271,18277
-18284.
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