1 Department of Cell and Developmental Biology, Vanderbilt University School of
Medicine, 465 21st Avenue South, Nashville, TN 37232, USA
2 Electron Microscopy Laboratory, University of California, Berkeley, 26
Giannini Hall, Berkeley, CA 94720-3330, USA
3 Department of Molecular Physiology and Biophysics, Vanderbilt University
School of Medicine, 465 21st Avenue South, Nashville, TN 37232, USA
* Author for correspondence (e-mail: david.greenstein{at}vanderbilt.edu)
Accepted 23 May 2005
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SUMMARY |
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Key words: Oogenesis, Meiotic maturation, Gamete interactions, Major sperm protein signaling, Vesicle budding, Unconventional protein secretion
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Introduction |
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In many animals, including many species of sponges, annelids, mollusks and
nematodes, sperm promote the resumption of meiosis in arrested oocytes
(Masui, 1985;
McCarter et al., 1999
). In
Caenorhabditis elegans, sperm use the major sperm protein (MSP) as a
hormone to promote oocyte meiotic maturation and gonadal sheath cell
contraction at a distance (Miller et al.,
2001
). MSP is also the key cytoskeletal element required for
amoeboid locomotion of nematode sperm
(Italiano et al., 1996
). MSP
promotes oocyte meiotic maturation, in part by binding the VAB-1 Eph receptor
protein-tyrosine kinase on oocytes, and by antagonizing an inhibitory somatic
gonadal sheath cell pathway (Miller et
al., 2003
). C. elegans hermaphrodites reproduce by either
self-fertilization or mating with males
(Hubbard and Greenstein,
2000
). Because a hermaphrodite produces only a fixed number of
sperm, oocyte meiotic maturation occurs constitutively until sperm become
limiting. In females lacking sperm, oocytes arrest in meiotic prophase until
insemination. Thus, the MSP hormone functions as the linchpin of a
sperm-sensing mechanism linking meiotic maturation and sperm availability.
Proteins with MSP domains are widespread and five human genes encode proteins
containing this domain. Recently, a mutation in the MSP domain of VAPB was
shown to cause spinal muscular atrophy and amyotrophic lateral sclerosis type
8 (Nishimura et al., 2004
).
Studies of MSP signaling, motility, or release in C. elegans may thus
provide information about the functions of this conserved domain.
MSP release probably occurs through an unconventional mechanism because
sperm lack the cellular components required in standard models of protein
secretion, such as ribosomes, endoplasmic reticulum (ER) and Golgi. Moreover,
MSP was defined as a cytoplasmic protein lacking an N-terminal leader
sequence, and there is no evidence for proteolytic processing
(Klass and Hirsh, 1981;
Miller et al., 2001
). Here, we
address the question of how sperm release MSP to signal oocytes and sheath
cells at a distance in a complex reproductive tract
(Fig. 1). We demonstrate that
spermatids and spermatozoa release MSP by a novel vesicle-budding mechanism.
Spermatids and spermatozoa differ in their signaling potencies. Spermatozoa
produce a long-range signal that is temporally labile, whereas spermatids
provide a long-acting, more local signal. We propose that differential vesicle
stability determines the physical and temporal range of signaling.
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Materials and methods |
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Oocyte meiotic maturations rates and MAPK activation were analyzed as
described (Miller et al.,
2001). Spermatozoa were labeled using 75 µM MitoTracker Red
CMXRos (Molecular Probes) by modifying the method of Hill and L'Hernault
(Hill and L'Hernault, 2001
).
spe-8(hc50) hermaphrodites were feminized using RNAi feeding of L1
larvae (Kamath et al.,
2001
).
Antibodies, western blotting and immunocytochemistry
Standard methods were used to raise, purify and characterize antibodies
(Harlow and Lane, 1988).
Peptides were purchased from Open Biosystems and purified by HPLC. Three
fixation methods were used: (1) dissected gonads with 3% paraformaldehyde
(Rose et al., 1997
); (2)
dissected gonads with methanol; or (3) wholemounts with Bouin's reagent
(Nonet et al., 1997
). Fourteen
different antibody preparations were used to examine MSP localization. The
only differences observed were the sensitivity of detection and the fixation
methods required, as indicated below. Polyclonal antibodies were
affinity-purified using peptides coupled to CNBr-activated sepharose (Amersham
Biosciences) or SulfoLink resin (Pierce). For the purification of monoclonal
antibodies, hybridomas were grown in serum-free medium and purified on protein
A/G columns (Amersham Biosciences). The N-terminal-specific antibodies were
raised to MSP (1-22) AQSVPPGDIQTQPGTKIVFNAP (2 rabbits, method 1).
C-terminal-specific antibodies were raised to: MSP (107-126)
EWFQGDMVRRKNLPIEYNP (2 rabbits, methods 1 and 2); and CGG-MSP (106-126)
CGGREWFQGDMVRRKNLPIEYNP [2 rabbits, method 1; 5 mice, methods 1 and 3; and 2
monoclonal hybridomas, method 1 and electron microscopy using post-embedding
immunohistochemistry (immunoEM)]. We also used mAbTR-20 raised to MSP
(Ward et al., 1986
) (methods 1
and 3, and immuno EM). Antibodies to MSD proteins were raised to CGG-MSD
(53-73) CGGDPSGSKDITITRTAGAPKEDK (2 rabbits, methods 1 and 3). Other
antibodies used were: RME-2 (Grant and
Hirsh, 1999
), and Cy2-, Cy3- or Cy5-conjugated secondary
antibodies (Jackson ImmunoResearch Laboratories).
For western blotting, protein lysates were prepared from 10 staged adults, and analyzed by electrophoresis on 4-12% NuPage gels (Invitrogen). The signal was detected with SuperSignal West Femto reagent (Pierce). Blots were quantitated using a VersaDoc imager with QuantityOne software (Bio-Rad).
Fluorescence microscopy
Wide-field fluorescence microscopy employed Zeiss Axioskop or Axioplan
microscopes using 63 x and 100 x (NA1.4) objective lenses. Images
were acquired with an ORCA ER (Hamamatsu) charge-coupled device camera using
OpenLab (Improvision) or MetaMorph (Universal Imaging) acquisition software.
Pixel intensities were measured in arbitrary fluorescent units. All exposures
were within the dynamic range of the detector. Measurements at 10 different
points within areas of interest were averaged, and background levels
subtracted as described (Miller et al.,
2003). DNA was detected with DAPI.
Confocal images were acquired on a Zeiss LSM510 microscope using a pinhole of 1.42 Airy units and 63 x and 100 x (NA1.4) objective lenses. Gain and offset were set so that all data was within the dynamic range of the PMT. Band pass filters were used to optically isolate the Cy2, Cy3 and Cy5 fluorophores, and no cross-talk was observed. For the reconstructions shown in Fig. 3B,E, serial images were smoothed with a Gaussian filter, and an isosurface and voltex were constructed using Amira (Amiravis). The images were transferred to QuickTime format using VR Worx (VR Toolbox). DNA in some samples was detected with propidium iodide (Molecular Probes).
Electron microscopy
Samples were prepared for TEM by high-pressure freezing and freeze
substitution (Howe et al.,
2001; Müller-Reichert et
al., 2003
). Wild-type (n=2), fog-2(q71)
(n=3), and spe-8(hc50) (n=3) animals were viewed in
serial longitudinal sections. For immunoEM, wild-type (n=2) and
spe-8(hc50) (n=2) samples were prepared according to
Lonsdale et al. (Lonsdale et al.,
1999
), using 0.25% glutaraldehyde as a fixative. Thin-layer
embedding in LR White (Ted Pella) was used so that tissue preservation could
be assessed by light microscopy and the sample could be oriented for
sectioning (Lonsdale et al.,
2001
). Longitudinal thin sections (70 nm) were placed on
formvar-coated grids and stained with 5-10 µg/ml mAb4D5 anti-MSP. Secondary
antibodies conjugated with 10 nm gold particles (Amersham Biosciences) were
used for detection. Grids were examined using a Philips CM-12, 120 keV
electron microscope at 80 kV. Mated (n=1) and unmated (n=1)
fog-2(q71) female samples were also prepared for TEM and immunoEM by
conventional methods (Hall et al.,
1999
). Immunolabeling of spermatozoa within mated females was
comparable to that obtained by the HPF method, but extracellular spaces in the
spermatheca were not well preserved, and MSP vesicles were not seen. No
labeling was observed in unmated females.
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Results |
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To analyze MSP release from spermatozoa in vivo, we examined gonads of mated fog-2(q71) female animals using immunofluorescence (Fig. 2B,C). In mated females, spermatozoa are only observed in the spermatheca and uterus; however, we observed MSP extending past the distal constriction into the proximal gonad arm. This staining represents MSP that is extracellular to spermatozoa. By contrast, all unmated females showed no staining (Fig. 2B, right panel; n=51). Using anti-MSP mAbTR-20 and visual inspection, 91% of mated female gonad arms exhibited extracellular MSP localization (n=36), with 39% showing extracellular MSP as far as the most proximal (1) oocyte, and the rest exhibiting extracellular MSP only within the spermatheca. Within the spermatheca, MSP staining was judged to be outside of spermatozoa if staining extended at least 5 µm beyond the pseudopod or cell body. MSP staining extended on average 33.6±19 µm (maximal range=90 µm; n=12) from spermatozoa, which are approximately 5 µm in size. When the spermatheca contained more than 50 spermatozoa, MSP extended to an average maximal distance of approximately 60 µm from spermatozoa (Fig. 2D). By contrast, when the spermatheca contained less than 50 spermatozoa, MSP extended to an average maximal distance of approximately 22 µm from spermatozoa (Fig. 2D). In adult hermaphrodites, we detected MSP outside of spermatozoa during days 1-3 of adulthood (see Fig. S1A-C in the supplementary material), but not at day 5, when no spermatozoa remain (see Fig. S1E). Thus, the distribution of extracellular MSP in the gonad correlates with sperm availability. Extracellular MSP was seen in mated C. remanei females, as well as in C. briggsae and Poikilolaimus regenfussi hermaphrodites (data not shown).
In mated females, extracellular MSP exhibits a graded distribution, with a
sharp boundary between the 1 and 2 oocytes
(Fig. 2B,C). Fluorescence
intensity measurements indicate that MSP is localized in a graded manner from
the spermatheca to the 1 oocyte
(Fig. 2C, n=10).
Fluorescence intensity measurements also indicate that there is significant
MSP staining over the 2 and 3 oocytes
(Fig. 2E). MSP binds the VAB-1
MSP/Eph receptor and unidentified receptors, which are expressed in the
proximal gonad (Miller et al.,
2003). One explanation for the sharp boundary in staining
intensity between the 1 and 2 oocytes is that MSP receptors may
act as a sink for MSP vectorially presented from the spermatheca. To test this
hypothesis, we examined extracellular MSP localization in mated
emo-1/sec-61
(oz1) females, which are defective for
secretion in the germ line (Iwasaki et
al., 1996
) and MSP binding to oocytes
(Miller et al., 2003
). Mated
emo-1(oz1) females animals did not exhibit a sharp boundary between
the most proximal two oocytes, and quantitative analysis showed no significant
difference in staining intensity of the 1 to 3 oocytes
(Fig. 2E). Instead, MSP
extended further distally in mated emo-1(oz1) females, when compared
with unmated controls, frequently reaching the loop region more than 100 µm
away (data not shown). These results suggest that receptors may influence
boundary formation by restricting diffusion.
Extracellular MSP is punctate and diffuse and localizes to the oocyte cell surface
With confocal microscopy, extracellular MSP appeared both punctate and
diffuse in the spermatheca, the gonad arm and the uterus
(Fig. 2F, see Movie 1 in the
supplementary material). Analysis of 3D data stacks indicated that punctate
extracellular MSP was enriched near spermatozoa on the spermathecal walls
(Fig. 2F). The largest puncta
were at the diffraction limit of our microscope (0.5 µm) and were found
nearby spermatozoa. In the proximal gonad arm, MSP was more diffuse and
localized in focal plane slices near the oocyte surface
(Fig. 2F, Movie 1 in the
supplementary material; see below for further confirmation). In the uterus, we
observed large MSP puncta close to spermatozoa
(Fig. 2F, right panel; see
Movie 2 in the supplementary material). We also observed diffuse MSP in
extracellular spaces surrounding embryos in the uterus
(Fig. 2F, see Movie 2). We were
able to visualize MSP puncta near spermatozoa in the uterus and spermatheca
using wide-field microscopy, when these regions were less crowded with
spermatozoa (Fig. 2G). These
results are consistent with the possibility that the large MSP puncta arise
from spermatozoa and generate a diffuse MSP signal in the proximal gonad.
To pinpoint the localization of MSP at the oocyte cell surface, we
conducted a 3D confocal analysis of MSP localization in mated females using
the RME-2 yolk receptor to mark the oocyte plasma membrane and the early
endosomal compartments (Grant and Hirsh,
1999). Three-dimensional image reconstructions of the data
indicate that MSP localizes in three regions: (1) in superficial focal planes
at the oocyte cell surface with RME-2 just beneath; (2) in the same plane as
the RME-2 signal; and (3) within the oocyte beneath the plasma membrane
(Fig. 3A,B, see also Movies 3,
4 in the supplementary material). These results are consistent with data
showing that MSP is an extracellular signal that binds receptors on the oocyte
surface, and suggest the MSP signal is endocytosed.
Specificity of MSP release and apparent budding from spermatozoa
Retrospective sperm counting experiments indicate that every spermatozoa
fertilizes an oocyte (Ward and Carrel,
1979). Nonetheless, we used vital dye labeling with MitoTracker
Red to address whether MSP release results from lysis, the expectation being
that lysis would disrupt the structure and integrity of spermatozoa dispersing
the label. To label spermatozoa, males were soaked in MitoTracker Red
(Hill and L'Hernault, 2001
)
and mated to unlabeled females. Labeled spermatozoa were able to crawl to the
spermatheca of unlabeled females and produce viable progeny. The labeled
mitochondria were located in a tight cluster in the cell body surrounding the
spermatozoa chromatin (Fig.
3C). By contrast, we observed MSP release from the labeled
spermatozoa in all cases (Fig.
3C, n=12). By these criteria, the labeled spermatozoa
were intact and functional, excluding lysis.
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Spermatids and spermatozoa differ in temporal and spatial signaling properties
The development of both male and female gametes in the hermaphrodite gonad
provides two contexts for MSP signaling. Spermatids signal nearby oocytes
within the proximal gonad, whereas spermatozoa signal remotely from the
spermatheca (Fig. 1). To
compare the temporal and spatial signaling activities of spermatids and
spermatozoa, we analyzed spe-8(hc50) and spe-27(it110)
mutants, which are defective in hermaphrodite spermiogenesis. spe-8
and spe-27 mutants produce morphologically normal spermatids that can
be activated for spermiogenesis and fertilization by male seminal fluid
(L'Hernault, 1997). In the
wild type, meiotic maturation rates progressively decline toward unmated
female levels as spermatozoa run out (see Table S1 in the supplementary
material). By contrast, spe-8 and spe-27 mutants exhibit
maturation rates that are more constant over time (Table S1). This observation
is surprising because the mutant spermatids are rapidly cleared from the
reproductive tract (Fig. 6B,
bottom) because they cannot crawl. To compare further the signaling potencies
of spermatids and spermatozoa, we conducted a time-course analysis of MAPK
activation (Fig. 6A). In the
wild type, the percentage of gonad arms that exhibit MAPK activation in
oocytes declines as sperm are used for fertilization, paralleling the decline
in total MSP levels and sperm numbers (Fig.
6B). By contrast, in spe-8 mutants, MAPK activation
remains high at times (days 3 and 4) when sperm are depleted or no longer
present. Consistent with this observation, residual MSP was faintly detected
in spe-8 mutants at these late times
(Fig. 6B). As a control, we
feminized spe-8 (n=20) and spe-27 (n=19)
mutants using fem-1(RNAi), which resulted in low meiotic maturation
rates and a stacked oocyte phenotype comparable to fog-2(q71)
females.
We next examined MSP release from spermatids. Wild-type, spe-8, and spe-27 spermatids release MSP primarily in punctate form within the gonad arm (Fig. 6C,D; see also Movie 7 in the supplementary material; data not shown). MSP puncta appear to be widely distributed in wild-type and spe-8 proximal gonad arms (Fig. 6C,D). By contrast, extracellular MSP produced by spe-8 spermatids in the spermatheca appears more diffuse (Fig. 6C, top panels). At late times, we observed diffuse extracellular MSP in the spermatheca in spe-8 mutants, despite the absence of spermatids (Fig. 6E). However, when spermatozoa are depleted in the wild type, no extracellular MSP is observed (see Fig. S1D,E in the supplementary material). This perdurance of extracellular MSP provides an explanation for the signaling observed at late times in spe-8 and spe-27 mutants. Taken together, these results suggest that spermatids provide a temporally long-acting form of the MSP-signal, whereas spermatozoa provide a long-range, temporally labile signal.
To investigate the potential basis for the increased stability of MSP signaling in spe-8 mutants, we conducted HPF and TEM experiments. In spe-8(hc50) adult hermaphrodites, we observed MSP vesicles in the gonad arm, spermatheca and uterus (Fig. 7, and data not shown). The MSP vesicles were particularly abundant in the spermathecal-uterine junction region (Fig. 7A). In one respect, the MSP vesicles in spe-8 mutants differed from those of the wild type (Fig. 4): the outer electron-dense layer of the spe-8 MSP vesicles did not exhibit clearly distinguishable inner and outer leaflets (Fig. 7B,C, and data not shown). It is not clear whether this difference is a consequence of their increased stability, or whether it represents some fundamental difference in their assembly. One observation in spe-8 mutants, however, may shed some additional light on how the MSP vesicles may form. In fertilization-defective mutants, such as spe-8(hc50), the unfertilized oocytes sometimes lyse in the spermatheca or uterus because they do not form egg shells. In these cases, oocyte cytoplasm and organelles filled the spermathecal lumen (Fig. 7C), and we observed that the interior of the MSP vesicles contained material markedly similar to that found in the extracellular space (Fig. 7C). This observation suggests that the internal ring of the MSP vesicle may derive from the extracellular space, a possibility that will require further experiments to address it fully.
Because spe-8 mutants do not form pseudopods, we examined the morphology of the cell body in detail to uncover additional information related to the formation of the MSP vesicles. As seen for wild-type spermatids (Fig. 5D), we observed protrusions of the plasma membrane (Fig. 7D-I). These varied in length from 100 nm to 2 µm in size (Fig. 7D-I, and data not shown), and often had a bent appearance (Fig. 7E). These protrusions contained MSP labeling (Fig. 7F,I). Often, pairs of closely spaced protrusions formed in close proximity to one another (Fig. 7G-I). These observations provide additional evidence for a vesicle-budding process at the cell body of spermatids and spermatozoa, and raise the issue of whether closely spaced buds may contribute to the formation of MSP vesicles.
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Because spermatids release MSP, motility and a pseudopod are dispensable. This result prompted us to examine whether parthenogenetic nematodes, which reproduce without sperm, have MSP. We analyzed the highly divergent Cephalobid parthenogens Acrobeloides maximus and Zeldia punctata using monoclonal antibodies raised to the highly conserved MSP C terminus. We detected MSP by western blot of both A. maximus and Z. punctata (Fig. 6F). Immunostaining of A. maximus indicated that punctate immunoreactivity was widely distributed in the female germline (Fig. 6G, and data not shown). Although the functional roles of MSP in A. maximus and Z. punctata will require additional study, these results indicate that MSP can be conserved in evolution for functions unrelated to the motility of spermatozoa.
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Discussion |
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Several lines of evidence rule out alternative explanations for these observations, such as lysis, or leaching of proteins from spermatids and spermatozoa during fixation. First, MSP release is highly specific, as abundant and soluble sperm-specific components of the MSP cytoskeleton, the MSD proteins, are not observed in MSP puncta or in buds. Second, vital dye-labeling experiments indicate that spermatozoa remain intact and functional, despite releasing MSP. Third, in spermiogenesis-defective hermaphrodites, after spermatids clear from the reproductive tract, extracellular MSP staining is still detected, and thus must originate prior to fixation. Fourth, multiple MSP antibodies and fixation conditions yield consistent results. Finally, electron and light microscopy paint congruent pictures of MSP release.
Using TEM, we detected a new class of vesicle, the MSP vesicle, in the
spermatheca and uterus of adult hermaphrodites. ImmunoEM demonstrates that
these vesicles contain MSP and probably correspond to the MSP puncta observed
by confocal microscopy. The observation that MSP vesicles are more abundant in
spe-8 mutants, which produce a long-acting MSP signal, provides
correlative data that MSP vesicles may represent signaling intermediates.
While the precise steps and dynamics by which the MSP vesicles form remain to
be determined, our static observations are consistent with the possibility
that protrusions from the cell body may bend back upon themselves and pinch
off, thereby encapsulating lumenal material within a double-membraned vesicle
(see Fig. S3 in the supplementary material). MSP vesicles are likely to be
labile structures because they are not detectable by conventional electron
microscopy and they appear to fuse to generate lipid whorls. Thus, instability
of the MSP vesicles may liberate MSP in a free form able to bind oocytes and
sheath cells via the VAB-1 Eph receptor protein-tyrosine kinase and other
unidentified receptors (Miller et al.,
2003). Taken together then, these results suggest that MSP release
from spermatozoa and spermatids occurs in two stages: (1) budding of MSP
vesicles; and (2) vesicle disintegration. As both spermatids and spermatozoa
release MSP via vesicle budding, neither a pseudopod nor motility is
required.
Vesicle budding, a nexus for the motility and signaling functions of MSP
Does vesicle budding use activities of MSP that are also required for
amoeboid locomotion of nematode spermatazoa? Several features of MSP vesicle
budding suggest this is indeed the case. ImmunoEM shows that MSP is enriched
at, and associated with, the plasma membrane of the cell body of spermatids
and spermatozoa. Localized MSP filament assembly may generate the protrusive
force driving vesicle budding at the plasma membrane of the cell body,
analogous to the leading edge protrusion that drives pseudopodial extension
(Italiano et al., 1996;
Bottino et al., 2002
).
Consistent with this idea, we observed MSP in protrusions of the plasma
membrane of the cell body by immunoEM (Figs
5,
7). Confocal microscopy and
3D-image reconstructions identify MSP-containing protrusions, which are likely
to correspond to the sites of budding. Whereas MSP is concentrated at these
budding sites, the MSD proteins are absent. In vitro studies of MSP-based
motility in Ascaris identified MFD1, the ortholog of the MSD
proteins, as having an activity that decreases the rate of MSP fiber growth
(Buttery et al., 2003
). Thus,
the absence of the MSD proteins at the vesicle-budding sites may favor MSP
filament assembly and membrane protrusion.
In vesicle-budding processes, bending of the lipid bilayer is energetically
costly because of a strong hydrophobic effect
(Chernomordik and Kozlov,
2003). It is likely that the TEM views of the vesicle-budding
process demonstrate the involvement of bent and looped intermediates (Figs
5,
7). Several observations
provide initial indications of how membrane bending may be achieved during
vesicle budding. Localized polymerization of MSP filaments may provide the
protrusive force that drives membrane bending. MSP filaments are flexible and
have a short persistence length, and are thus conducive to bending
(Italiano et al., 1996
;
Bottino et al., 2002
). TEM
views of MSP vesicles indicate that they have a regular, highly bent,
scalloped appearance, suggesting the involvement of vesicle-coating proteins.
Thus, MSP polymerization may provide the energy driving membrane protrusion
and bending, while uncharacterized coat proteins may store this energy and
stabilize the bent configuration. As MSP cytoskeletal dynamics powers
retraction in amoeboid motility (Miao et
al., 2003
), an attractive idea is that disassembly of MSP
filaments at the base of the projection may play a role in scission.
How is MSP vesicle budding regulated? Vesicle budding results in loss of MSP and plasma membrane from spermatids and spermatozoa; therefore, there must be a trade-off between MSP signaling and motility. The best evidence that MSP vesicle budding is regulated comes from two sets of related observations: first, MSP release does not occur from spermatids within or dissected from males; and second, extracts of female animals promote vesicle budding from spermatids in vitro (data not shown). The possibility that MSP release may depend on extracellular cues from the hermaphrodite reproductive tract may have precedents in MSP-based motility, because spermatozoa are likely to sense directional cues as they navigate from the uterus to the spermatheca. In this view then, MSP cytoskeletal dynamics would drive pseudopodial extension and crawling in response to one set of cues, and vesicle budding in response to another. Alternatively, a single signal from the hermaphrodite could elicit vesicle budding and directional movement by activating divergent downstream effectors. Identification of the putative cues will provide the most direct test of these hypotheses.
Vesicle budding provides a basis for long- and short-range MSP signaling
Our results suggest two modes of MSP signaling: spermatids appear to
provide a temporally long-acting form of the MSP signal; and spermatozoa
provide a long-range, labile signal. This plasticity is well adapted for the
developmental stages of MSP signaling. Spermatids signal neighboring oocytes
from within the gonad, and spermatozoa must signal from far-flung regions
including the spermatheca and the uterus. For the sperm-sensing mechanism
(Miller et al., 2003) to
generate a biologically meaningful output, extracellular MSP levels must be
valid and reliable indicators of sperm availability. A block to spermiogenesis
short-circuits the control mechanism.
Our results suggest that differential MSP vesicle stability may provide a
mechanistic basis for the distinct signaling activities of spermatids and
spermatozoa. In spe-8 mutants, MSP vesicles are more stable, and
signaling persists after the spermatids are swept from the reproductive tract
by ovulated oocytes. While it is not possible to completely exclude the
possibility that spe-8 mutant spermatids release the MSP signal in a
qualitatively or quantitatively different manner from wild-type spermatids in
the gonad, the isolation of a large class of sperm-defective mutants on the
basis that they lay unfertilized oocytes in high quantity suggests that many
mutants that disrupt spermiogenesis may have this property
(L'Hernault, 1997). If
wild-type spermatids do indeed produce a long-acting signal within the gonad
arm, then some mechanism must exist to eliminate this form of the MSP signal
after ovulations have commenced and the spermatids have entered the
spermatheca and undergone spermiogenesis. Otherwise, meiotic maturation might
continue at a brisk pace after sperm are depleted and thus oocytes would be
wasted. Possibly, the presence of spermatozoa may destabilize MSP vesicles
from spermatids in trans. Although the actual determinants of MSP vesicle
stability are unclear, both intrinsic and extrinsic factors may contribute.
During spermiogenesis, ER/Golgi-derived organelles, the membranous organelles
(MOs), fuse with the plasma membrane to transfer their contents to the cell
surface and the extracellular environment
(L'Hernault, 1997
;
Xu and Sternberg, 2003
). MO
fusion is not required for the budding process because spermatids, which have
unfused MOs bud vesicles, and fer-1 mutants, which are defective in
MO fusion (Achanzar and Ward,
1997
), are able to signal
(McCarter et al., 1999
).
Nonetheless, MO fusion generates a difference between the plasma membrane
protein composition of spermatids and spermatozoa that might affect the
stability of their respective MSP vesicles.
MSP vesicle budding and unconventional secretory mechanisms
How general is the MSP vesicle-budding mechanism? MSPs are highly conserved
in nematodes where they play both cytoskeletal and signaling roles. Proteins
with MSP domains are also found in fungi, plants and animals. Genetic studies
demonstrate that a MSP domain protein, DVAP-33A, functions as an instructive
signal during bouton formation at the neuromuscular junction in
Drosophila (Pennetta et al.,
2002). A mutation in the MSP domain of VAPB causes late-onset
spinal muscular atrophy and amyotrophic lateral sclerosis type 8 in humans
(Nishimura et al., 2004
). Our
observation that MSP localizes to membranes and apparently drives vesicle
budding may define a general activity for the MSP domain in other
proteins.
As a molecular mechanism, vesicle budding provides a general means for
releasing cytoplasmic proteins from cells. It is now becoming clear that
diverse intracellular proteins can be secreted from cells by novel means,
independent of a signal peptide or the ER/Golgi system
(Nickel, 2003). Proteins
released by non-classical secretory pathways fit into two broad groups: those
that are located within vesicular compartments within the cell; and those that
are cytoplasmic. For example, IL-1ß is associated with secretory
lysozymes and is released by an unconventional mechanism
(Stinchcombe et al., 2004
). By
contrast, galectin 1 and 3 (Cooper and
Barondes, 1990
), fibroblast growth factors 1 and 2
(Mignatti et al., 1992
), and
HIV-Tat (Chang et al., 1997
)
are probably cytoplasmic, yet are exported from cells. For some members of
both groups (e.g. IL-1ß, galectin 1, FGF-2), there is evidence for
release within vesicles (Cooper and
Barondes, 1990
; MacKenzie et
al., 2001
).
With classical ER/Golgi-dependent protein secretion mechanisms so robust,
it is reasonable to ask why cells should bother with unconventional export
pathways? In the case of MSP, nematode spermatids and spermatozoa simply do
not have any other option, having jettisoned their ribosomes, ER/Golgi and
actin during meiosis II. A similar argument explains why spermatozoa from many
vertebrate and invertebrate species rely on the acrosome reaction for zona
penetration. A variety of highly specialized cells (e.g. melanocytes,
platelets, cytotoxic T-lymphocytes, mammary gland cells and sweat gland cells)
rely on unconventional protein export pathways
(Nickel, 2003;
Stinchcombe et al., 2004
).
Possibly, non-canonical secretion mechanisms provide highly specialized cells
with a greater flexibility in dynamic environments in which the cell positions
or developmental status are changeable, as for MSP vesicle budding.
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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/132/15/3357/DC1
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REFERENCES |
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---|
Achanzar, W. E. and Ward, S. (1997). A nematode
gene required for sperm vesicle fusion. J. Cell Sci.
110,1073
-1081.
Bottino, D., Mogilner, A., Roberts, T., Stewart, M. and Oster,
G. (2002). How nematode sperm crawl. J. Cell
Sci. 115,367
-384.
Buttery, S. M., Ekman, G. C., Seavy, M., Stewart, M. and
Roberts, T. M. (2003). Dissection of the Ascaris
sperm motility machinery identifies key proteins involved in major sperm
protein-based amoeboid locomotion. Mol. Biol. Cell
14,5082
-5088.
Chang, H. C., Samaniego, F., Nair, B. C., Buonaguro, L. and Ensoli, B. (1997). HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region. AIDS 11,1421 -1431.[CrossRef][Medline]
Chernomordik, L. V. and Kozlov, M. M. (2003). Protein-lipid interplay in fusion and fission of biological membranes. Annu. Rev. Biochem. 72,175 -207.[CrossRef][Medline]
Cooper, D. N. and Barondes, S. H. (1990). Evidence for export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism. J. Cell Biol. 110,1681 -1691.[Abstract]
De Ley, P., Geraert, E. and Coomans, A. (1990). Seven cephalobids from Senegal. J. Afr. Zool. 104,287 -304.
Eisenbach, M. and Tur-Kaspa, I. (1999). Do human eggs attract spermatozoa? BioEssays 21,203 -210.[CrossRef][Medline]
Grant, B. and Hirsh, D. (1999).
Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte.
Mol. Biol. Cell 10,4311
-4326.
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]
Hardy, D. M. (ed.) (2002). Fertilization. San Diego: Academic Press.
Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.
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]
Howe, M., McDonald, K. L., Albertson, D. G. and Meyer, B. J.
(2001). HIM-10 is required for kinetochore structure and function
on Caenorhabditis elegans holocentric chromosomes. J. Cell
Biol. 153,1227
-1238.
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]
Italiano, J. E., Jr, Roberts, T. M., Stewart, M. and Fontana, C. A. (1996). Reconstitution in vitro of the motile apparatus from the amoeboid sperm of Ascaris shows that filament assembly and bundling move membranes. Cell 84,105 -114.[CrossRef][Medline]
Iwasaki, K., McCarter, J., Francis, R. and Schedl, T. (1996). emo-1, a Caenorhabditis elegans Sec61p gamma homologue, is required for oocyte development and ovulation. J. Cell Biol. 134,699 -714.[Abstract]
Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G. and Ahringer, J. (2001). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2, 1-10.
Klass, M. R. and Hirsh, D. (1981). Sperm isolation and biochemical analysis of the major sperm protein from C. elegans. Dev. Biol. 84,299 -312.[CrossRef]
L'Hernault, S. (1997). Spermatogenesis. InC. elegans II (ed. D. L. Riddle, T. Blumenthal, B. J. Meyer and J. R. Priess), pp. 271-294. New York: Cold Spring Harbor Laboratory Press.
L'Hernault, S. W., Shakes, D. C. and Ward, S.
(1988). Developmental genetics of chromosome I
spermatogenesis-defective mutants in the nematode Caenorhabditis elegans.Genetics 120,435
-452.
Lonsdale, J. E., McDonald, K. L. and Jones, R. L. (1999). High pressure freezing and freeze substitution reveal new aspects of fine structure and maintain protein antigenicity in barley aleurone cells. Plant J. 17,221 -229.[CrossRef]
Lonsdale, J. E., McDonald, K. L. and Jones, R. L. (2001). Microwave polymerization in thin layers of LR white allows selection of specimens for immunogold labeling. In Microwave Techniques and Protocols (ed. R. T. Giberson and R. S. Demaree, Jr), pp. 139-153. New Jersey: Humana Press.
MacKenzie, A., Wilson, H. L., Kiss-Toth, E., Dower, S. K., North, R. A. and Surprenant, A. (2001). Rapid secretion of interleukin-1beta by microvesicle shedding. Immunity 15,825 -835.[CrossRef][Medline]
Masui, Y. (1985). Meiotic arrest in animal oocytes. In Biology of Fertilization (ed. C. B. Metz and A. Monroy), pp. 189-219. Florida: Academic Press.
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]
McDonald, K. (1999). High-pressure freezing for preservation of high resolution fine structure and antigenicity for immunolabeling. Methods Mol. Biol. 117, 77-97.[Medline]
Miao, L., Vanderlinde, O., Stewart, M. and Roberts, T. M.
(2003). Retraction in amoeboid cell motility powered by
cytoskeletal dynamics. Science
302,1405
-1407.
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.
Mignatti, P., Morimoto, T. and Rifkin, D. B. (1992). Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex. J. Cell Physiol. 151,81 -93.[CrossRef][Medline]
Müller-Reichert, T., O'Toole, E. T., Hohenberg, H. and McDonald, K. L. (2003). Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructure. J. Microsc. 212,71 -80.[CrossRef][Medline]
Neill, A. T. and Vacquier, V. D. (2004).
Ligands and receptors mediating signal transduction in sea urchin spermatozoa.
Reproduction 127,141
-149.
Nickel, W. (2003). The mystery of nonclassical
protein secretion. A current view on cargo proteins and potential export
routes. Eur. J. Biochem.
270,2109
-2119.
Nishimura, A. L., Mitne-Neto, M., Silva, H. C. A., Richieri-Costa, A., Middleton, S., Cascio, D., Kok, F., Oliveira, J. R. M., Gillingwater, T., Webb, J., Skehel, P. and Zatz, M. (2004). A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am. J. Genet. 75,822 -831.[CrossRef]
Nonet, M. L., Staunton, J. E., Kilgard, M. P., Fergestad, T.,
Hartwieg, E., Horvitz, H. R., Jorgensen, E. M. and Meyer, B. J.
(1997). Caenorhabditis elegans rab-3 mutant synapses
exhibit impaired function and are partially depleted of vesicles.
J. Neurosci. 17,8061
-8073.
Pennetta, G., Hiesinger, P., Fabian-Fine, R., Meinertzhagen, I. and Bellen, H. J. (2002). Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner. Neuron 32,291 -306.[CrossRef]
Riddle, D. L., Blumenthal, T., Meyer, B. J. and Priess, J. R. (eds) (1997). C. elegans II. New York: Cold Spring Harbor Laboratory Press.
Rose, K. L., Winfrey, V. P., Hoffman, L. H., Hall, D. H., Furuta, T. and Greenstein, 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]
Spehr, M., Gisselmann, G., Poplawski, A., Riffell, J. A.,
Wetzel, C. H., Zimmer, R. K. and Hatt, H. (2003).
Identification of a testicular odorant receptor mediating human sperm
chemotaxis. Science 299,2054
-2058.
Stinchcombe, J., Bossi, G. and Griffiths, G. M.
(2004). Linking albinism and immunity: the secrets of secretory
lysosomes. Science 305,55
-59.
Thorne, G. (1925). The genus Acrobeles von Linstow, 1877. Trans Am. Microsc. Soc. 44,171 -209.
Ward, G. E., Brokaw, C. J., Garbers, D. L. and Vacquier, V. D. (1985). Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer. J. Cell Biol. 101,2324 -2329.[Abstract]
Ward, S. and Carrel, J. S. (1979). Fertilization and sperm competition in the nematode Caenorhabditis elegans. Dev. Biol. 73,304 -321.[CrossRef][Medline]
Ward, S. and Klass, M. (1982). The location of the major protein in C. elegans sperm and spermatocytes. Dev. Biol. 92,203 -208.[CrossRef][Medline]
Ward, S., Roberts, T. M., Strome, S., Pavalko, F. M. and Hogan, E. (1986). Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple C. elegans sperm-specific proteins. J. Cell Biol. 102,1778 -1786.[Abstract]
Wassarman, P. M., Jovine, L. and Litscher, E. S. (2001). A profile of fertilization in mammals. Nat. Cell Biol. 3,E59 -E64.[CrossRef][Medline]
Xu, X. Z. and Sternberg, P. W. (2003). A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization. Cell 114,285 -297.[CrossRef][Medline]
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