1 Waksman Institute and Department of Genetics, Rutgers University, Piscataway,
NJ 08854, USA
2 Department of Biology, College of William and Mary, Williamsburg, VA 23187,
USA
Author for correspondence (e-mail:
singson{at}waksman.rutgers.edu)
Accepted 18 April 2005
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SUMMARY |
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Key words: Fertilization, Sperm, Oocyte, C. elegans, Tetraspan, spe-38
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Introduction |
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Studies in marine invertebrates and mammals have strongly influenced our
current understanding of fertilization. In many species, sperm must first
interact with the extracellular matrix of the egg
(Hoodbhoy and Dean, 2004;
Kamei and Glabe, 2003
;
Yanagimachi, 1998
). This
extracellular egg coat (e.g. the zona pellucida in mammals and the vitelline
membrane in sea urchins) not only physically protects the oocyte, but also
provides a substrate for species-specific sperm binding and induces essential
sperm-specific responses (e.g. the acrosome reaction). After sperm have
penetrated the egg coat, direct gamete cell-cell interactions can occur
(Foltz and Lennarz, 1993
;
Kaji and Kudo, 2004
). In
mammals, sperm proteins that mediate egg binding and fusion are thought to
include the surface-associated protein DE
(Ellerman et al., 2002
), the
immunoglobulin-like protein Izumo (Inoue
et al., 2005
), and the sperm ADAM proteins (fertilin
,
fertilin ß and cyritestin) (Evans,
2001
). On the egg side of the equation, integrins
(Evans, 2001
), GPI-anchored
proteins (Alfieri et al., 2003
)
and the tetraspanin CD9 (Kaji et al.,
2000
) are thought to mediate sperm binding and/or fusion. The
relationship between these various molecules and their precise biochemical
function during fertilization are poorly understood and remain controversial
(Cho et al., 2000
).
Our current understanding of the molecular machinery required for the steps
of fertilization remains fragmentary and would be significantly bolstered by
identifying additional core components. The nematode C. elegans is an
excellent model system for such studies
(Singson, 2001). C.
elegans exists primarily as a self-fertile hermaphrodite that produces
both sperm and oocytes, and less frequently as a male that produces only
sperm. In fertile hermaphrodites, self and outcross sperm are stored within a
spermatheca. C. elegans oocytes are produced in an `assembly
line'-like fashion by the hermaphrodite gonad. As oocytes undergo meiotic
maturation and ovulation, they enter the spermatheca and come into contact
with the crawling sperm, which employ an amoeboid mode of cellular motility.
The coordination of cellular events and gamete presentation leads to extremely
efficient utilization of sperm; essentially every functional sperm fertilizes
an oocyte (Kadandale and Singson,
2004
; Ward and Carrel,
1979
). The zygote then completes its meiotic divisions, secretes a
protective egg shell, passes through the hermaphrodite uterus and is laid
prior to hatching.
Powerful forward and reverse genetic approaches have identified several new
genes required for fertilization in C. elegans
(Geldziler et al., 2004;
Singson et al., 1998
;
Xu and Sternberg, 2003
).
Spermatogenesis-defective (spe) hermaphrodites lay unfertilized
oocytes and are self-sterile (L'Hernault
and Singson, 2000
), but they produce viable progeny when crossed
with wild-type males, thus permitting the propagation of these mutations to
subsequent generations. A few spe genes are required for sperm
function specifically during fertilization rather than for the meiotic and
morphogenetic events of spermatogenesis
(Singson, 2001
). The first of
these genes to be cloned and phenotypically analyzed was spe-9
(Putiri et al., 2004
;
Singson et al., 1998
;
Zannoni et al., 2003
). The
spe-9 gene encodes a sperm transmembrane protein with multiple
epidermal growth factor (EGF) repeats. Because its amino acid sequence and
structural organization is similar to ligands of the Notch/LIN-12/GLP-1 family
of receptor molecules, SPE-9 is a plausible candidate for the sperm ligand for
an as yet unidentified oocyte receptor
(Singson, 2001
). Mutants of a
second gene, trp-3 (also known as spe-41), phenocopy
spe-9 mutants (Xu and Sternberg,
2003
). trp-3 encodes a TRPC-type (transient receptor
potential canonical) calcium-conducting ion channel, and is proposed to
regulate calcium flux during sperm-oocyte interactions at fertilization.
Clearly, many key components in the molecular mechanics of C. elegans fertilization have yet to be identified. Here, we report the phenotypic and molecular analysis of spe-38. Homozygous spe-38 mutants exhibit sperm-specific fertility defects that are similar to animals that lack functional copies of spe-9 or trp-3.
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Materials and methods |
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Phenotypic analysis of spe-38 mutants was conducted essentially as
described for other fertility mutants
(Kadandale and Singson, 2004;
Putiri et al., 2004
;
Singson et al., 1999
;
Singson et al., 1998
;
Xu and Sternberg, 2003
). The
number of self progeny for wild type (N2), spe-38 mutants and the
transgenic rescue stocks was determined by placing single worms on separate
culture plates and counting entire individual broods. Ovulation rates were
assessed by scoring the total number of embryos and unfertilized oocytes laid
either during a specific time period or over the entire lifetime of a worm. In
male fertility tests, four spe-38 or wild-type males were crossed
with single dpy-5 hermaphrodites for 24 hours at 20°C. After two
days, the total number of Dpy (self) and Non-Dpy (outcross) progeny were
scored. Sperm competition (Singson et al.,
1999
) and mosaic analysis experiments
(Herman, 1995
;
Singson et al., 1998
) employed
the transgenic rescuing stock spe-38(eb44), asEx67 [A.1+A.2, myo-3::GFP].
spe-38 animals lacking the rescuing transgene were identified as failing
to express the myo-3::GFP reporter in either their somatic tissue or
progeny. Sperm isolation and in vitro activation protocols were performed as
described by L'Hernault and Roberts
(L'Hernault and Roberts,
1995
). In sperm motility and localization experiments, feminized
fem-1(hc17ts) or fog-2(q71) females were crossed
with spe-38(eb44); him-5(e1490) males for one day at
25°C, and male sperm within the hermaphrodite spermatheca were visualized
either by light microscopy, and/or by DAPI staining. For light microscopic
observation of ovulation and sperm-oocyte contact, animals were anesthetized
in a solution of M9 salts containing 0.1% tricaine and 0.01% tetramisole, as
described by McCarter et al. (McCarter et
al., 1999
).
Molecular biology, identification and analysis of the spe-38 gene
Molecular techniques not described in detail here can be found in Sambrook
et al. (Sambrook et al.,
1989). The position of the spe-38 gene was determined
using standard linkage mapping (Sulston
and Hodgkin, 1988
) and single nucleotide polymorphism
(SNP)-mapping (Swan et al.,
2002
) (see Table S1 in the supplementary material). Transgenic
worms (Mello et al., 1991
)
were generated carrying PCR products corresponding to different combinations
of the Y52B11A.1 and Y52B11A.2 genes. Through genetic crosses, the transgenes
were then transferred into a spe-38 mutant background where their
competence to rescue the spe-38 defect was assessed by brood size
analysis. The structure of the Y52B11A.1 and Y52B11A.2 genes was confirmed by
analysis of PCR products from a male-derived cDNA library
(Achanzar and Ward, 1997
). The
nature of the spe-38(eb44) mutation was identified by amplifying the
Y52B11A.1 gene from a lysate of spe-38(eb44) worms and comparing its
sequence with that of the N2 strain.
The eb44 mutation was mapped to linkage group I between the markers dpy-5 and unc-75. The same two markers were used for two- and three-factor mapping, and this localized eb44 within a one map unit interval on the right arm of LG I (see Table S1 in the supplementary material, Fig. 6A). Three genes associated with fertility defects (stu-10, sqv-5 and spe-9) had been previously mapped to this region, but complementation analysis indicated that the eb44 was not an allele of any of these genes (see Table S1). eb44 was thus considered to define the spe-38 gene. eb44 also complemented two deficiencies in the region, qDf7 and hDf1. This qDf7 result was surprising because qDf7 was thought to span the spe-38 region, and it suggests that qDf7, like many other C. elegans deficiencies, is molecularly complex (P. Kadandale and A.S., unpublished). Single nucleotide polymorphisms that generated restriction fragment length polymorphisms (SNIP-SNPs) between N2 and Hawaiian (H) strains of worms were used to further position spe-38 on the physical map. N2/H hybrids were generated by crossing spe-38(eb44); dpy-5(e61) and spe-38(eb44); unc-75(e950) homozygous hermaphrodites to wild-type Hawaiian males. Recombinant offspring from the hybrid worms (i.e. Dpy Non-Spe or Unc Non-Spe) were isolated and lines were established. Worm lysates were prepared for 41 such individual lines, and SNP analysis was carried out by PCR amplification using specific primers in the region of the SNP followed by restriction digestion using specific enzymes. Data from five SNIP-SNPs (see Table S1 in the supplementary material) effectively positioned spe-38 between the two cosmids F49D11 and W02B9 (Fig. 6A). This region of approximately 125 kb contains at least 14 predicted genes. After sequencing PCR products from this sub-region, we identified three new SNPs (Y52B11 SNP1-3) that can only be detected by sequencing. Using these new SNPs, analysis of several Dpy Non-Spe and Unc Non-Spe recombinants localized spe-38 to a small region on the yeast artificial chromosome (YAC) Y52B11A that contains only three predicted genes, Y52B11A.1, Y52B11A.2 and Y52B11A.3.
For the transgenic studies, PCR products were co-injected with the myo-3::gfp selectable marker (pPD118.20 Fire Lab Vector Kit). PCR products corresponding to the Y52B11A.1 and Y52B11A.2 genes were generated using the following primers:
To sequence the Y52B11A.1 gene from spe-38(eb44) the following primers were used to generate appropriate PCR products:
Sequencing confirmed that the eb44 mutation was a deletion of 270 base pairs from nucleotide position 1:10975258 to nucleotide position 1:10975528. In place of the missing sequence were 17 bases with the sequence GCCCTTTCAACCCATTT.
The sequence of the SPE-38 protein was compared with other proteins in the
database using BLASTP analysis and HHpred
(Altschul et al., 1990;
Soding, 2005
). A
Kyte-Doolittle (Kyte and Doolittle,
1982
) plot and topology algorithm
(Pasquier et al., 1999
) was
used to predict transmembrane domains within SPE-38 and PRM1. A search for
protein motifs and structural similarities with other tetraspan molecules was
carried out using the SMART web-based tool
(Schultz et al., 2000
).
Immunofluorescence, western analysis and microscopy
To generate SPE-38 antibodies, rabbits were initially pre-screened to
identify those whose sera lacked cross-reactivity with C. elegans
spermatids. Negatives were injected with keyhole-limpet hemocyanin-coupled
peptides corresponding to SPE-38 amino acids 101-114 (antibodies generated by
Zymed Laboratory).
For the isolation of sperm, animals were dissected in sperm media
containing dextrose (SM) (Machaca et al.,
1996; Nelson and Ward,
1980
). Spermatids were isolated from celibate wild-type or
spe-38 males. In vivo-activated spermatozoa were dissected directly
from the spermatheca and uteri of unmated hermaphrodites, wild-type
hermaphrodites mated to wild-type males, or females (fog-2 or
fem-1) mated to mutant (spe-38 or fer-1) males. For
in vitro-activation experiments, celibate males were dissected directly into
sperm media containing either Pronase E (200 mg/ml) or 60 mM triethanolamine
(Shakes and Ward, 1989
).
Western blots were done as described by Sambrook et al.
(Sambrook et al., 1989).
Preparations from exactly 400 him-5 or spe-38, him-5 males
were loaded into each lane. Equivalent loading was also assayed by Coomassie
blue staining. The spe-38, him-5 homozygous males were selected
individually from our transgene-balanced stock by the lack of transgene GFP
marker expression. SPE-38 localization in spermatids or mature spermatozoa was
carried out using previously described fixation and staining protocols
(Strome and Wood, 1982
;
Zannoni et al., 2003
), or with
live cell staining. For live cell staining experiments, worms were dissected
directly on Color Frost Plus slides (Fisher Scientific) in 20 µl SM
containing polyclonal anti-SPE-38 antisera (1:100 dilution), and subsequently
incubated in a humid chamber at room temperature for 30 minutes. Samples were
then fixed in 20°C methanol for 30 minutes. After three washes in
phosphate-buffered saline (PBS), the slides were blocked for 30 minutes in PBS
containing 0.5% BSA and 0.1% Tween-20, before a 1.5-hour room temperature
incubation with affinity-purified TRITC-conjugated (Jackson ImmunoResearch
Laboratories) or Alexa Fluor 488-conjugated (Molecular Probes) goat
anti-rabbit antisera (Ig Fc fragment specific). Slides were dip washed in PBS
and mounted with GelMount (Biomedia) containing DAPI. Monolayers of
cryomethanol-fixed sperm were prepared as previously described
(Golden et al., 2000
), with
the addition of a 0.2% Triton X-100 permeabilization step following the
initial post-fixation wash. In some experiments, dissected sperm were fixed,
in the absence of a freeze-crack step, in 4% paraformaldehyde in SM for 20
minutes at room temperature. Similar conditions were used for immunostaining
with either anti-SPE-9 (Zannoni et al.,
2003
) or the mouse monoclonal antibody 1CB4
(Okamoto and Thomson, 1985
).
1CB4 staining was detected using Cy3-conjugated (Jackson ImmunoResearch
Laboratories) or Alexa Fluor 488-conjugated (Molecular Probes)
affinity-purified goat anti-mouse secondary antibody. For the peptide
competition experiments, excess lyophilized SPE-38 peptide was incubated on
ice with diluted (1:100) anti-SPE-38 antibody for 30 minutes. The peptide-Ab
solution was clarified by centrifugation before use, as above.
Samples were prepared for transmission electron microscopy according to
Hall (Hall, 1999). DAPI
analysis of whole-mount and dissected hermaphrodite gonads was performed
according to Miller and Shakes (Miller and
Shakes, 1995
). Unstained images were obtained using Nomarski
differential interference contrast (DIC) microscopy. Epifluorescence images
were captured on either an Olympus BX-60 or Zeiss Axioplan compound microscope
equipped with either a Cooke or Optronics cooled CCD camera. Images were
edited in IP software, Adobe Photoshop, or Deneba Systems Canvas 9. For the
co-localization experiments, images were captured using a Nikon Eclipse TE300
microscope equipped with a Biorad Radiance 2100-AGR3Q confocal/multiphoton
system and processed using LaserSharp 2000 (v. 6.0) imaging software.
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Results |
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To assess male fertility, we compared the ability of spe-38 and
wild-type males to sire outcross progeny from morphologically marked, but
otherwise wild-type hermaphrodites. In sharp contrast to wild-type controls,
spe-38 males failed to sire any outcross progeny
(Fig. 1B,
Fig. 5B). This infertility was
not due to an inability to mate, as spe-38 mutant males were found to
transfer wild-type levels of sperm (see below) and exhibited wild-type mating
behavior (Liu and Sternberg,
1995). Therefore, the spe-38 mutant defect affects both
male and hermaphrodite sperm, and spe-38 mutant sperm are incapable
of fertilizing either spe-38 or wild-type oocytes.
Sperm from spe-38 mutants are indistinguishable from wild-type sperm
To determine whether the fertility defects exhibited by spe-38
worms were due to abnormal sperm morphology, we closely compared
spe-38 sperm to wild-type sperm. When examined using DIC optics,
spermatozoa from spe-38 mutant males and hermaphrodites were
indistinguishable from wild-type spermatozoa
(Fig. 2A-C). Notably,
spermiogenesis, the maturation of spherical, sessile spermatids into polar,
motile spermatozoa (Muhlrad and Ward,
2002; Shakes and Ward,
1989
), was unaffected; spe-38 sperm activated normally
both in vitro (Fig. 2B) and in
vivo (Fig. 2C). When examined
using transmission electron microscopy (TEM), spermatozoa within the
reproductive tract of adult spe-38 hermaphrodites also exhibited
wild-type morphology (Fig.
2D-F). As in wild type, the membranous organelles (MOs) in
spe-38 sperm fuse with the plasma membrane during spermiogenesis to
form permanent pores surrounded by an electron dense collar
(Fig. 2F).
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Sperm that are deposited into the hermaphrodite uterus by males must
migrate to the spermatheca. Our sperm competition data (see below) suggests
that male-derived spe-38 sperm can migrate to the spermatheca and
displace hermaphrodite sperm. However, in order to directly observe the
accumulation of male-derived sperm in the spermatheca, we crossed
spe-38 males to fem-1 females and tracked sperm accumulation
by DAPI staining of whole animals. This assay also gave us a way to roughly
compare the number of sperm transferred. Unmated wild-type hermaphrodite
controls have many sperm in the spermatheca
(Fig. 4A). Unmated
fem-1 mutant females lack endogenous sperm, have an empty
spermatheca, and accumulate mature oocytes within their proximal gonad arm.
Because of the low rates of ovulation, oocytes with endomitotically
replicating (emo) DNA are often present close to the spermatheca
(Fig. 4B,
Fig. 5A) (Doniach and Hodgkin, 1984;
Miller et al., 2003
). During
mating, wild-type (Fig. 4C) and
spe-38 (Fig. 4D,E)
males transfer comparable amounts of sperm to fem-1 females. In both
cases, transferred sperm can be detected in the spermatheca, where they
stimulate ovulation and relieve the back up of oocytes within the gonad arm.
However, fem-1 females mated to spe-38 males fail to produce
viable progeny and their uteri fill with what appears to be unfertilized
oocytes containing the characteristic emo DNA
(Fig. 4E,
Fig. 5B). Identical results
were obtained when spe-38 males were crossed to fog-2
females (see below).
To distinguish whether the spe-38 defect blocks sperm entry or an early post-fertilization step, these presumably unfertilized oocytes were isolated from the uteri of animals stained with DAPI. In the newly fertilized oocytes of wild-type hermaphrodites, the highly condensed sperm chromatin mass can be easily distinguished from the meiotically dividing oocyte chromatin (Fig. 4F). In the youngest in utero oocytes of unmated spe-38 hermaphrodites (Fig. 4G) or fog-2 females that were crossed to spe-38 males (Fig. 4I), no sperm DNA could be detected. As these older unfertilized oocytes aged, they became emo and accumulated high levels of DNA (fem-1 femalexspe-38 males, Fig. 4E; unmated spe-38 hermaphrodites, Fig. 4H; fog-2 femalexspe-38 males, Fig. 4J). By contrast, wild-type sperm successfully enter spe-38 oocytes, and the resulting embryos develop normally within the uterus (Fig. 4K). Taken together, these data indicate that, although spe-38 mutant sperm are fully motile and can migrate to the correct location in the reproductive tract, they fail to enter oocytes.
spe-38 sperm are competent to stimulate ovulation and can participate in sperm competition
In C. elegans, oocytes must undergo meiotic maturation and
ovulation in order to be fertilized
(McCarter et al., 1999). The
major sperm protein (MSP) functions as a signaling molecule to induce the
meiotic maturation and ovulation of oocytes above basal levels
(Miller et al., 2003
). This
signal helps the worms to avoid wasting metabolically costly oocytes when no
sperm are present in the reproductive tract. To examine whether
spe-38 mutant sperm are signaling competent, we quantified the total
lifetime number of ovulations of mutant and wild-type worms. Mutant
spe-38 hermaphrodites ovulated at levels slightly but not
statistically lower than the wild-type controls
(Fig. 5A). By contrast,
hermaphrodites that lack either sperm (Fig.
5A, see fem-1) or the ability to respond to the MSP
signal ovulate at only basal rates
(McCarter et al., 1999
;
Miller et al., 2003
;
Singson et al., 1998
). Because
the pattern and rates of ovulation may be affected by a multitude of
variables, such as sperm numbers, sperm aging, hermaphrodite aging and the
influence of successful fertilization events, we also examined the ability of
spe-38 mutant sperm to modulate ovulation rates in age matched
fem-1 females. As assessed by the number of eggs and/or oocytes laid
on the culture plate (Kadandale and
Singson, 2004
), spe-38 mutant sperm induced ovulation
rates that were again comparable to wild-type controls
(Fig. 5B).
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The spe-38 gene encodes a novel four-pass (tetraspan) integral membrane protein
To address the molecular role of SPE-38 in wild-type sperm-oocyte
interactions, we cloned the spe-38 gene. We mapped spe-38 to
a small region of chromosome I (Fig.
6A) corresponding to the yeast artificial chromosome (YAC) clone
Y52B11A. This region contains the predicted genes Y52B11A.1, Y52B11A.2 and
Y52B11A.3, but only Y52B11A.1 has been reported to have a male-enriched
transcript (Reinke et al.,
2004). An approximately 7 kb PCR product from genomic DNA that
contained both the Y52B11A.1 and Y52B11A.2 (A.1+A.2) genes was produced and
injected to generate transgenic worms. This transgene significantly rescued
spe-38 mutant sterility (Fig.
1A, Fig. 6A), as
many viable progeny were produced. In fact, this transgene allowed us to
genetically balance homozygous spe-38 mutants. We did note that our
transgenic stocks produced broods that were on average lower than wild-type
ones. Incomplete transgene rescue for germline-expressed genes is common
because such genes are often expressed poorly in simple transgenic arrays
(Putiri et al., 2004
;
Seydoux and Schedl, 2001
).
Individually, neither the Y52B11A.1 nor the Y52B11A.2 gene alone could rescue
fertility (Fig. 6A). However, a
3.7 kb genomic fragment that included the Y52B11A.1 gene plus additional
upstream sequences, including all of the last intron of the Y52B11A.2 gene
(A.1+), rescued fertility to levels comparable to rescue by the the A.1+A.2
fragment (Fig. 1A,
Fig. 6A). The structure of the
Y52B11A.1 gene (Fig. 6B) was
confirmed by sequencing of PCR products generated from a male-derived cDNA
library (Achanzar and Ward,
1997
). We confirmed that Y52B11A.1 and Y52B11A.2 are separate
genes, neither gene is transpliced, and neither gene is listed in the operon
database (Blumenthal and Gleason,
2003
).
To further confirm our identification of the spe-38 coding region and to determine the nature of the eb44 mutation, DNA from spe-38 mutant worms was amplified and sequenced. The PCR product amplified from eb44 mutant worms was significantly smaller than the corresponding PCR product amplified from wild-type worms (Fig. 6C). Sequencing confirmed that eb44 contained a deletion of 270 base pairs, in which all of exon 4 and parts of flanking introns 3 and 4 are missing (Fig. 6B). In place of the missing sequence were 17 bases with the sequence GCCCTTTCAACCCATTT. This deletion not only disrupts proper mRNA splicing but is also predicted to generate a truncated SPE-38 protein with three or four frame shift-encoded residues after amino acid 74 (Fig. 7A, and see below).
|
The spe-38 gene is predicted to encode a small protein of 179
amino acids (Fig. 7A). BLASTP
and HHpred analysis using C. elegans SPE-38 as a query did not
identify strong homologies, apart from C. briggsae SPE-38 (CBG24396
Fig. 7). The two amino acid
sequences are 55% identical and 64% similar. The identical and similar amino
acids are evenly distributed across the length of the molecules, except for an
extended loop between transmembrane domains 1 and 2 in Cb-SPE-38. When this
loop is not included in the comparison, the two molecules have 61% identity
and 71% similarity. Hydropathy and topology algorithms (see Materials and
methods) predicted that SPE-38 has four transmembrane domains
(Fig. 7A,B). Using the SMART
web-based tool and visual comparison, we identified other four-pass integral
membrane proteins that have similar domain arrangements (relatively small,
lacking channel features, and possessing two loops of varying sizes)
(Schultz et al., 2000). The
available database contains many such predicted small tetraspan integral
membrane proteins, and several have been implicated in important cell-cell
interactions (Fig. 7B).
|
SPE-38 protein localization in spermatids and spermatozoa
To analyze the cellular distribution of SPE-38, we obtained polyclonal
anti-peptide antisera to the large putative extracellular loop (amino acids
101-114) of SPE-38 (underlined sequence in
Fig. 7A). Relative to other
tetraspan proteins, this loop is unique to SPE-38 and could be In essential to
its function. cryomethanol-fixed and permeabilized spermatids, SPE-38 appears
to be enriched in large structures near the cell cortex
(Fig. 8A). Identical results
were obtained with paraformaldehyde-fixed and permeabilized spermatids (data
not shown). The specificity of our antisera was confirmed in three independent
ways. (1) Fixed and permeabilized wild-type spermatids failed to stain when
the antisera was pre-incubated with competing peptide
(Fig. 8B). (2) Fixed and
permeabilized spe-38(eb44) sperm failed to stain
(Fig. 8C). (3) A western blot
of wild-type and spe-38(eb44) males revealed a protein of the
expected molecular weight in the wild type but not in the mutant sample
(Fig. 8D).
Ultrastructural studies indicate that C. elegans spermatids
possess only four major organelles: a central condensed chromatin mass, its
associated centriole, multiple mitochondria, and numerous membranous
organelles (MOs). MOs are unique nematode sperm organelles. In spermatids they
are unfused, fully internal structures, which abut the cortex of spermatids
(Ward et al., 1981;
Wolf et al., 1978
). To test
whether the SPE-38 localization pattern reflected its presence within MOs,
permeabilized spermatids were costained with anti-SPE-38 antibody and the
monoclonal antibody 1CB4, which specifically binds to MOs
(Okamoto and Thomson, 1985
).
Analysis using confocal/multiphoton microscopy revealed a significant but
incomplete overlap of the two staining patterns
(Fig. 9A). This result
indicates that, within spermatids, SPE-38 predominately localizes to
sub-compartments of the MO that only partially overlap those occupied by the
1CB4 antigen.
|
These localization patterns suggest a model in which SPE-38 moves from the
unfused MOs of spermatids to the plasma membrane of the pseudopods of
spermatozoa. In a crucial test of this model, SPE-38 remained inaccessible to
antibody binding under live-cell staining conditions of either wild-type
spermatids (Fig. 8A) or
fer-1 spermatozoa (Fig.
9B), which were previously characterized as having abnormally
short pseudopods and unfused MOs (Ward et
al., 1981). When these fer-1 spermatids and spermatozoa
were fixed and permeabilized, SPE-38 antisera bound to SPE-38 in the unfused
MOs (Fig. 9B).
|
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Discussion |
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|
In addition to channel proteins, there are hundreds of predicted tetraspan
integral membrane proteins in the current databases. Many of these molecules
have a domain organization that is similar to SPE-38 (see
Fig. 7B for examples). Several
tetraspan membrane proteins have been implicated in cell-cell interactions,
vesicle trafficking and membrane morphogenesis
(Hemler, 2003;
Hubner et al., 2002
;
Tsukita and Furuse, 1999
). One
important sub-group of tetraspans include the mammalian membrane-spanning 4A
(MS4A) gene family. As a group, these MS4A-related proteins are
expressed in many tissues (Ishibashi et
al., 2001
; Liang and Tedder,
2001
) and function within cell-surface oligomeric complexes as
signal transducers. One, TETM4, is specifically expressed in human testis
(Hulett et al., 2001
). Another
subgroup of tetraspan proteins, the tetraspanins, have been implicated in both
mammalian sperm-oocyte adhesion/fusion and other cell-cell interactions (CD9
and CD81) during both the immune response and nervous system
development/function (Bronstein,
2000
; Hemler,
2001
; Hemler,
2003
; Kaji et al.,
2002
; Kaji et al.,
2000
; Naour et al.,
2000
). Twenty C. elegans genes fit the specific criteria
for tetraspanins (Todres et al.,
2000
); spe-38 is not one of those twenty. A third group
of tetraspan molecules includes occludins and claudins, which function within
polarized epithelia to form cell-cell junctions and barriers to membrane
diffusion (Tsukita and Furuse,
1999
; Turksen and Troy,
2004
). SPE-38 also shares structural similarities with the fungal
tetraspan proteins PRM1 and PRM2, which function in membrane adhesion/fusion
during yeast mating (Heiman and Walter,
2000
). In the absence of definitive structure/function studies,
the exact molecular activities of these molecules remain poorly understood.
However, in the context of this study, it is notable that various tetraspan
molecules are proposed to function as receptors, signal transducers, fusion
proteins, and `scaffolding or membrane web' components that function to group
other specific cell-surface proteins, thus altering their activity, stability
or presentation (Ellerman et al.,
2003
; Hemler,
2003
; Kaji et al.,
2000
; Maecker et al.,
1997
; Naour et al.,
2000
; Yunta and Lazo,
2003
). The mutant phenotype of spe-38 is consistent with
it having one or more of these activities during sperm-egg interactions during
C. elegans fertilization.
The localization of SPE-38, and models for its function during fertilization
We find that SPE-38 concentrates within the membranous organelles (MOs) of
spermatids, and that a significant fraction of the protein relocalizes to the
pseudopod of the mature, motile and translationally inactive spermatozoa (Figs
8,
9). The MOs appear to be the
source of the SPE-38 protein that ends up on the pseudopods of motile
spermatozoa, as this relocalization fails to occur in mutants that are
defective in MO fusion. The observed localization pattern suggests a number of
possibilities concerning how and where SPE-38 could function to promote
successful fertilization. The localization of SPE-38 in the MOs of both
spermatids and spermatozoa suggests that it could carry out a crucial function
within MOs. However, if SPE-38 does play an essential function within MOs, it
is not required for the proper ultrastructure, MO-plasma membrane fusion, or
spermatozoan motility. As an integral membrane protein, SPE-38 might also
plausibly function to regulate the localization of other proteins. However, if
so, SPE-38 is neither functioning like an occludin to maintain large-scale
membrane domains, nor to specifically localize the sperm-specific fertility
protein SPE-9. Additional studies will be required to assess whether SPE-38 is
essential either for the localization of other proteins or for the functioning
of SPE-9. What our live-cell staining experiments do show is that SPE-38 is
present on the pseudopod membrane of mature sperm, where it is positioned to
function directly in gamete interactions.
Given the functional diversity of tetraspan proteins, the best clues
regarding the biochemical function of SPE-38 may ultimately come from the
analysis of its binding partners. For instance, identifying an oocyte binding
partner(s) would suggest an egg receptor/ligand function analogous to a model
proposed by Ellerman et al. (Ellerman et
al., 2003) for the immunoglobulin superfamily (IgSF)/CEA subfamily
protein PSG17 binding to the tetraspanin CD9. Alternately, identifying
sperm-protein binding partner(s) might support a role for SPE-38 as either a
single subunit within a key, but larger, protein complex, or as an essential
modulator of key sperm receptor proteins. Additional clues will come from a
detailed structure function analysis of SPE-38 in transgenic worm strains. In
any case, the current study demonstrates that SPE-38 is a new component of the
sperm fertilization machinery, whose function is absolutely required for
C. elegans sperm-egg interactions. Continued analysis of SPE-38
activity in C. elegans promises to give us new insights regarding,
not only the molecular mechanisms of fertilization, but also the molecular
functions of tetraspan proteins.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/12/2795/DC1
* Present address: Laboratory of Genetics, University of Wisconsin, Madison,
WI 53706, USA
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Achanzar, W. E. and Ward, S. (1997). A nematode
gene required for sperm vesicle fusion. J. Cell Sci.
110,1073
-1081.
Alfieri, J. A., Martin, A. D., Takeda, J., Kondoh, G., Myles, D.
G. and Primakoff, P. (2003). Infertility in female mice with
an oocyte-specific knockout of GPI-anchored proteins. J. Cell
Sci. 116,2149
-2155.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215,403 -410.[CrossRef][Medline]
Blumenthal, T. and Gleason, K. S. (2003). Caenorhabditis elegans operons: form and function. Nat. Rev. Genet. 4,112 -120.[Medline]
Boucheix, C. and Rubinstein, E. (2001). Tetraspanins. Cell Mol. Life Sci. 58,1189 -1205.[Medline]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Bronstein, J. M. (2000). Function of tetraspan proteins in the myelin sheath. Curr. Opin. Neurobiol. 10,552 -557.[CrossRef][Medline]
Cho, C., Ge, H., Branciforte, D., Primakoff, P. and Myles, D. G. (2000). Analysis of mouse fertilin in wild-type and fertilin beta(/) sperm: evidence for C-terminal modification, alpha/beta dimerization, and lack of essential role of fertilin alpha in sperm-egg fusion. Dev. Biol. 222,289 -295.[CrossRef][Medline]
Doniach, T. and Hodgkin, J. (1984). A sex-determining gene, fem-1, required for both male and hermaphrodite development in Caenorhabditis elegans. Dev. Biol. 106,223 -235.[CrossRef][Medline]
Ellerman, D. A., Da Ros, V. G., Cohen, D. J., Busso, D.,
Morgenfeld, M. M. and Cuasnicu, P. S. (2002). Expression and
structure-function analysis of de, a sperm cysteine-rich secretory protein
that mediates gamete fusion. Biol. Reprod.
67,1225
-1231.
Ellerman, D. A., Ha, C., Primakoff, P., Myles, D. G. and
Dveksler, G. S. (2003). Direct binding of the ligand PSG17 to
CD9 requires a CD9 site essential for sperm-egg fusion. Mol. Biol.
Cell 14,5098
-5103.
Evans, J. P. (2001). Fertilin beta and other ADAMs as integrin ligands: insights into cell adhesion and fertilization. BioEssays 23,628 -639.[CrossRef][Medline]
Foltz, K. R. and Lennarz, W. J. (1993). The molecular basis of sea urchin gamete interactions at the egg plasma membrane. Dev. Biol. 158,46 -61.[CrossRef][Medline]
Geldziler, B., Kadandale, P. and Singson, A.
(2004). Molecular genetic approaches to studying fertilization in
model systems. Reproduction
127,409
-416.
Golden, A., Sadler, P. L., Wallenfang, M. R., Schumacher, J. M.,
Hamill, D. R., Bates, G., Bowerman, B., Seydoux, G. and Shakes, D. C.
(2000). Metaphase to anaphase (mat) transition-defective mutants
in Caenorhabditis elegans. J. Cell Biol.
151,1469
-1482.
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]
Harris, T. W., Chen, N., Cunningham, F., Tello-Ruiz, M.,
Antoshechkin, I., Bastiani, C., Bieri, T., Blasiar, D., Bradnam, K., Chan, J.
et al. (2004). WormBase: a multi-species resource for
nematode biology and genomics. Nucl. Acids Res.
32,D411
-D417.
Heiman, M. G. and Walter, P. (2000). Prm1p, a
pheromone-regulated multispanning membrane protein, facilitates plasma
membrane fusion during yeast mating. J. Cell Biol.
151,719
-730.
Hemler, M. E. (2001). Specific tetraspanin
functions. J. Cell Biol.
155,1103
-1107.
Hemler, M. E. (2003). Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu. Rev. Cell Dev. Biol. 19,397 -422.[CrossRef][Medline]
Herman, R. K. (1995). Mosaic analysis. Methods Cell Biol. 48,123 -146.[Medline]
Hoodbhoy, T. and Dean, J. (2004). Insights into
the molecular basis of sperm-egg recognition in mammals.
Reproduction 127,417
-422.
Hubner, K., Windoffer, R., Hutter, H. and Leube, R. E. (2002). Tetraspan vesicle membrane proteins: synthesis, subcellular localization, and functional properties. Int. Rev. Cytol. 214,103 -159.[Medline]
Hulett, M. D., Pagler, E., Hornby, J. R., Hogarth, P. M., Eyre, H. J., Baker, E., Crawford, J., Sutherland, G. R., Ohms, S. J. and Parish, C. R. (2001). Isolation, tissue distribution, and chromosomal localization of a novel testis-specific human four-transmembrane gene related to CD20 and FcepsilonRI-beta. Biochem. Biophys. Res. Commun. 280,374 -379.[CrossRef][Medline]
Inoue, N., Ikawa, M., Isotani, A. and Okabe, M. (2005). The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 434,234 -238.[CrossRef][Medline]
Ishibashi, K., Suzuki, M., Sasaki, S. and Imai, M. (2001). Identification of a new multigene four-transmembrane family (MS4A) related to CD20, HTm4 and beta subunit of the high-affinity IgE receptor. Gene 264,87 -93.[CrossRef][Medline]
Kadandale, P. and Singson, A. (2004). Oocyte production and sperm utilization patterns in semi-fertile strains of Caenorhabditis elegans. BMC Dev. Biol. 4, 3.[CrossRef][Medline]
Kaji, K. and Kudo, A. (2004). The mechanism of
sperm-oocyte fusion in mammals. Reproduction
127,423
-429.
Kaji, K., Oda, S., Shikano, T., Ohnuki, T., Uematsu, Y., Sakagami, J., Tada, N., Miyazaki, S. and Kudo, A. (2000). The gamete fusion process is defective in eggs of Cd9-deficient mice. Nat. Genet. 24,279 -282.[CrossRef][Medline]
Kaji, K., Oda, S., Miyazaki, S. and Kudo, A. (2002). Infertility of CD9-deficient mouse eggs is reversed by mouse CD9, human CD9, or mouse CD81; polyadenylated mRNA injection developed for molecular analysis of sperm-egg fusion. Dev. Biol. 247,327 -334.[CrossRef][Medline]
Kamei, N. and Glabe, C. G. (2003). The
species-specific egg receptor for sea urchin sperm adhesion is EBR1, a novel
ADAMTS protein. Genes Dev.
17,2502
-2507.
Kyte, J. and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157,105 -132.[CrossRef][Medline]
L'Hernault, S. W. and Roberts, T. M. (1995). Cell biology of nematode sperm. Methods Cell Biol. 48,273 -301.[Medline]
L'Hernault, S. W. and Singson, A. (2000). Developmental genetics of spermatogenesis in the nematode Caenorhabditis elegans. In The Testis: From Stem Cell to Sperm Function, Serono Symposium USA (ed. E. Goldberg), pp.109 -119. New York: Springer-Verlag.
LaMunyon, C. W. and Ward, S. (1998). Larger sperm outcompete smaller sperm in the nematode Caenorhabditis elegans.Proc. R. Soc. London Ser. B 265,1997 -2002.[CrossRef][Medline]
Liang, Y. and Tedder, T. F. (2001). Identification of a CD20-, FcepsilonRIbeta-, and HTm4-related gene family: sixteen new MS4A family members expressed in human and mouse. Genomics 72,119 -127.[CrossRef][Medline]
Liu, K. S. and Sternberg, P. W. (1995). Sensory regulation of male mating behavior in Caenorhabditis elegans.Neuron 14,79 -89.[CrossRef][Medline]
Machaca, K., DeFelice, L. J. and L'Hernault, S. W. (1996). A novel chloride channel localizes to Caenorhabditis elegans spermatids and chloride channel blockers induce spermatid differentiation. Dev. Biol. 176, 1-16.[CrossRef][Medline]
Maecker, H. T., Todd, S. C. and Levy, S.
(1997). The tetraspanin superfamily: molecular facilitators.
FASEB J. 11,428
-442.
McCarter, J., Bartlett, B., Dang, T. and Schedl, T. (1999). On the control of oocyte meiotic maturation and ovulation in C. elegans. Dev. Biol. 205,111 -128.[CrossRef][Medline]
Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1991). Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10,3959 -3970.[Abstract]
Miller, D. M. and Shakes, D. C. (1995). Immunofluorescence microscopy. Methods Cell Biol. 48,365 -394.[Medline]
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.
Muhlrad, P. J. and Ward, S. (2002).
Spermiogenesis initiation in Caenorhabditis elegans involves a casein
kinase 1 encoded by the spe-6 gene. Genetics
161,143
-155.
Naour, F. L., Rubinstein, E., Jasmin, C., Prenant, M. and
Boucheix, C. (2000). Severely reduced female fertility in
CD9-deficient mice. Science
287,319
-321.
Nelson, G. A. and Ward, S. (1980). Vesicle fusion, pseudopod extension and amoeboid motility are induced in nematode spermatids by the ionophore monensin. Cell 19,457 -464.[CrossRef][Medline]
Okamoto, H. and Thomson, J. N. (1985). Monoclonal antibodies which distinguish certain classes of neuronal and supporting cells in the nervous tissue of the nematode Caenorhabditis elegans. J. Neurosci. 5,643 -653.[Abstract]
Pasquier, C., Promponas, V. J., Palaios, G. A., Hamodrakas, J. S. and Hamodrakas, S. J. (1999). A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm. Protein Eng. 12,381 -385.[CrossRef][Medline]
Primakoff, P. and Myles, D. G. (2002).
Penetration, adhesion, and fusion in mammalian sperm-egg interaction.
Science 296,2183
-2185.
Putiri, E., Zannoni, S., Kadandale, P. and Singson, A. (2004). Functional domains and temperature-sensitive mutations in SPE-9, an EGF repeat-containing protein required for fertility in Caenorhabditis elegans. Dev. Biol. 272,448 -459.[CrossRef][Medline]
Reinke, V., Gil, I. S., Ward, S. and Kazmer, K.
(2004). Genome-wide germline-enriched and sex-biased expression
profiles in Caenorhabditis elegans. Development
131,311
-323.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Schultz, J., Copley, R. R., Doerks, T., Ponting, C. P. and Bork,
P. (2000). SMART: a web-based tool for the study of
genetically mobile domains. Nucl. Acids Res.
28,231
-234.
Seydoux, G. and Schedl, T. (2001). The germline in C. elegans: origins, proliferation, and silencing. Int. Rev. Cytol. 203,139 -185.[Medline]
Shakes, D. C. and Ward, S. (1989). Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis. Dev. Biol. 134,189 -200.[CrossRef][Medline]
Singson, A. (2001). Every sperm is sacred: fertilization in Caenorhabditis elegans. Dev. Biol. 230,101 -109.[CrossRef][Medline]
Singson, A., Mercer, K. B. and L'Hernault, S. W. (1998). The C. elegans spe-9 gene encodes a sperm transmembrane protein that contains EGF-like repeats and is required for fertilization. Cell 93,71 -79.[CrossRef][Medline]
Singson, A., Hill, K. L. and L'Hernault, S. W.
(1999). Sperm competition in the absence of fertilization in
Caenorhabditis elegans. Genetics
152,201
-208.
Singson, A., Zannoni, S. and Kadandale, P. (2001). Molecules that function in the steps of fertilization. Cytokine Growth Factor Rev. 12,299 -304.[CrossRef][Medline]
Soding, J. (2005). Protein homology detection by HMM-HMM comparison. Bioinformatics (in press).
Strome, S. and Wood, W. B. (1982).
Immunofluorescence visualization of germ-line-specific cytoplasmic granules in
embryos, larvae, and adults of Caenorhabditis elegans. Proc. Natl.
Acad. Sci. USA 79,1558
-1562.
Sulston, J. and Hodgkin, J. (1988). Methods. InThe Nematode Caenorhabditis elegans (ed. W. B. Wood), pp. 587-606. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Swan, K. A., Curtis, D. E., McKusick, K. B., Voinov, A. V.,
Mapa, F. A. and Cancilla, M. R. (2002). High-throughput gene
mapping in Caenorhabditis elegans. Genome Res.
12,1100
-1105.
Todres, E., Nardi, J. B. and Robertson, H. M. (2000). The tetraspanin superfamily in insects. Insect Mol. Biol. 9,581 -590.[CrossRef][Medline]
Tsukita, S. and Furuse, M. (1999). Occludin and claudins in tight-junction strands: leading or supporting players? Trends Cell Biol. 9,268 -273.[CrossRef][Medline]
Turksen, K. and Troy, T. C. (2004). Barriers
built on claudins. J. Cell Sci.
117,2435
-2447.
Vacquier, V. D. (1998). Evolution of gamete
recognition proteins. Science
281,1995
-1998.
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., Argon, Y. and Nelson, G. A. (1981). Sperm morphogenesis in wild-type and fertilization-defective mutants of Caenorhabditis elegans. J. Cell Biol. 91, 26-44.[Abstract]
Wolf, N., Hirsh, D. and McIntosh, J. R. (1978). Spermatogenesis in males of the free-living nematode, Caenorhabditis elegans. J. Ultrastruct. Res. 63,155 -169.[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]
Yanagimachi, R. (1994). Mammalian fertilization. In The Physiology of Reproduction (ed. E. Knobil and J. D. Neill), pp. 189-317. New York: Raven Press.
Yanagimachi, R. (1998). Roles of egg coats in reproduction: an overview. Zygote 6, S1-S3.[Medline]
Yunta, M. and Lazo, P. A. (2003). Tetraspanin proteins as organisers of membrane microdomains and signalling complexes. Cell. Signal. 15,559 -564.[CrossRef][Medline]
Zannoni, S., L'Hernault, S. W. and Singson, A. W. (2003). Dynamic localization of SPE-9 in sperm: a protein required for sperm-oocyte interactions in Caenorhabditis elegans.BMC Dev. Biol. 3,10 .[CrossRef][Medline]