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First published online 8 November 2006
doi: 10.1242/dev.02671
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1 Department of Biology, University of Washington, Seattle, Washington 98195,
USA.
2 Department of Genome Sciences and University of Washington, Seattle,
Washington 98195, USA.
3 Center for Developmental Biology, University of Washington, Seattle,
Washington 98195, USA.
* Author for correspondence (e-mail: wakimoto{at}u.washington.edu)
Accepted 3 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fertilization, Male fertility, Acrosome, Paternal effect mutations, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Evans, 2002). The majority of
molecules with confirmed roles in these processes are cell adhesion molecules,
ion channels or signal transduction components with molecular mechanisms of
action that have been studied in multiple cell types (reviewed by
Talbot et al., 2003;
Neill and Vacquier, 2004;
Rubinstein et al., 2006).
There is considerable interest in discovering proteins that act specifically
during fertilization. The characterization of these molecules will contribute
to a better understanding of the specialized properties of gametes and to the
practical goal of identifying new targets for contraceptives.
To identify novel sperm molecules required for fertilization, we have
pursued a genetic approach using Drosophila melanogaster. Previous
studies of male sterile mutants indicated that those affecting fertilization
might be relatively rare. Therefore, we isolated and categorized a large
number of male-sterile mutations to find a subset that disrupt sperm-egg
interactions or induce paternal effect defects
(Fitch et al., 1998;
Wakimoto et al., 2004). We
previously described mutations in a gene called sneaky
(snky), which met our genetic criteria of specifically affecting
fertilization (Fitch and Wakimoto,
1998). Mutations of snky showed detectable effects only
on male fertility. Sperm produced by mutant males were competent to enter the
egg but arrested before sperm nuclear decondensation and aster formation. We
proposed that this sperm activation defect was due to a failure in breakdown
of the sperm plasma membrane, a step that normally occurs immediately after
sperm entry into the egg in Drosophila.
In this study, we provide phenotypic evidence of a role for Snky in affecting the integrity of the sperm plasma membrane during fertilization. We molecularly identify the snky gene and characterize its transcript and predicted protein to investigate how the protein might function. The results indicate that Snky is an acrosomal membrane protein that is contributed to the early embryo. In addition to suggesting a possible molecular role for Snky, these studies have implications for the function and the fate of the acrosome during Drosophila fertilization.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or
FlyBase
(http://flybase.bio.indiana.edu),
except as noted below. The snkyZ0566 and
snkyZ4482 alleles were recovered as ethylmethane sulfonate
(EMS)-induced male sterile mutations from the Zuker collection
(Koundakjian et al., 2004;
Wakimoto et al., 2004). The
strain P{PZ} Syx1301470 ry506/TM3 (Bloomington
Drosophila Stock Center) was used to generate deletions near the
P-element by transposase-induced male recombination
(Preston et al., 1996) and one
snky- chromosome, denoted Df(3L)
Syx1301470R5, was recovered. This chromosome is homozygous
viable but poorly so due to deletion of genes other than
snky+ (Fitch and
Wakimoto, 1998). A transgenic line that expressed the secreted
Green fluorescent protein (GFPsecr) of Pfeiffer et al.
(Pfeiffer et al., 2000;
Pfeiffer et al., 2002) under
the control of the ß2t testis-specific promoter from the tv3 vector
(Wong et al., 2005) was
generously provided by J. Brill.
Molecular characterization of the snky gene, mutations and transcript
Molecular studies were carried out essentially as described by Sambrook and
Russell (Sambrook and Russell,
2001). Genomic fragments flanking P-element insertions in the
P{PZ} Syx1301470 and Df(3L)
Syx1301470R5 chromosomes were isolated and sequenced to
identify the P-element insertion site and locate the deficiency breakpoints
(Preston et al., 1996). These
fragments were used as probes to select hybridizing genomic clones from a
phage library (Tamkun et al.,
1992). Overlapping clones that spanned the deleted region were
used to probe genomic Southern blots containing DNA from
snky1 and its parental mwh red e line to identify
restriction site polymorphisms. The snkyZ0566 and
snkyZ4482 mutations were identified using PCR to amplify
and sequence selected regions from mutant and parental bw; st
chromosomes.
For northern blots (Fig. 2),
the lanes contained 
0.25 µg polyA+ RNA from testes (350 pairs of
testes) or 7.4 µg polyA+ RNA isolated from 40 males after removal of
the testes. The probes were a 32P-labeled 3.3 kb AvaI
genomic fragment isolated from the clone BAC05N10 (UK-HGMP Resource Center)
and a clone containing the ribosomal protein gene rp49.
We sequenced snky cDNA clones, which we generated from polyA+ RNA isolated from testes of bw; st males. Standard protocols were used for reverse transcription (Gibco Superscript II RNAse H-Transcriptase Kit), 5' RACE (Ambion Choice RLM-RACE Kit), and cDNA cloning into the TOPO-TA vector (Invitrogen). Primers (Gibco) for cDNA amplification were: S1(CCTTCCTACTGGGACTCGTG), S2(GTTGTTGAAGGCCGAAAAGA), S3(CGAACTCCACGTCATTGAGA), S4(GTTGAGATCCTCGGCACAAT), S5(TTGTAAACTGCTTGGCCATCAAAT), S6(GGCAAAGGCCACTGCACGTA).
Construction of transgenic lines and assays for male fertility
The rat CD2 coding region was isolated as 1.1 kb
ClaI/XhoI fragment from a plasmid kindly provided by N. H.
Brown (Dunin-Borkowski and Brown,
1995) and cloned downstream of the testis-specific ß2-tubulin
promoter in P-element transformation vector of Hoyle et al.
(Hoyle et al., 1995). The
construct also contained the white+mc gene and was
introduced into y w1118 flies using standard germline
transformation techniques. A line carrying a transposon on chromosome 2 was
used to construct the y w1118; P[w+mc B2t::CD2] 2a;
snky1 e/TM3, y+ Ser e strain.
The genomic region containing the CG11281gene was isolated as a 7.3 kb
SalI fragment from BACN05H10 and cloned into pCasPeR4, which carries
the w+mc gene (Thummel
and Pirrotta, 1992). Three independent insertions, denoted
P[w+mc snky+t7.3]A,
B and C were obtained. Two were on chromosome 3 and were
introduced into a snky- background by recombination onto
Df(3L) Syx1301470R5.
A transgene expressing Snky-GFP fusion protein, denoted P[w+mc snky-GFP] was created by inserting the Enhanced green fluorescent protein (EGFP) coding region from pEGFP-N3 (Clonetec) into the pCasPeR4-7.3 S construct described above. The fusion protein retained the snky+ gene promoter and flanking regions. It extended the snky+ open reading frame (ORF) just before the stop codon to include: a 30 bp linker from pEGPF-N3, the EGFP coding sequences (amino acid 1 to 237) and, as a consequence of the cloning scheme, an in-frame duplication of the last seven codons of the snky ORF. The transgene was used to generate the y w; P[w+mc snky-GFP]; snky1 e/TM3, y+ Ser e lines.
Fertility assays were performed for males carrying P[w+mc
snky+t7.3] or
P[w+mc snky-GFP] in a snky- background
to test for activity of the transgenes. Fertility was monitored in crosses of
single males mated to three wild-type (Canton-S) females. Percentage
of fertile crosses and progeny yield per male were compared to those of
control crosses, which used snky- brothers that lacked the
transgene or snky1/snky+ heterozygotes. Because
snky- males produce one to five progeny on occasion
(Fitch and Wakimoto, 1998),
fertile crosses were defined as those yielding more than five offspring.
Assays for Snky-GFP and CD2 expression
To monitor Snky-GFP in testes, y w; snky-GFP; snky1 e
males were dissected in 2% paraformaldehyde in PBS (130 mmol/l NaCl, 7 mmol/l
Na2HPO4, 3 mmol/l NaH2PO4). Testis
squashes were prepared on poly-L-lysine coated slides, frozen in liquid
nitrogen, submerged in 95% cold ethanol, rinsed in PBS, then stained with 0.1
µg/ml DAPI (4',6-diamidine-2-phenylindole) to label nuclei. To label
the mitochondrial derivative, larval testes were incubated with 400 mmol/l
Mitotracker Dye (MT-CMXRos, Molecular Probes), then processed as described for
adult testes. To assay Snky-GFP in sperm in the reproductive tracts of females
and in eggs, wild-type females were mated to y w; snky-GFP;
snky1 e males. Sperm storage organs were dissected then
prepared as described above. Eggs were collected within 15 minutes of
deposition, dechorionated in 50% bleach, then fixed in 4% paraformaldehyde in
PBS overlaid with heptane. After vitelline envelopes were removed by hand,
eggs were processed without squashing as described above. All preparations
were mounted in Vectashield (Vector Laboratories). Images were acquired at
600x magnification using a Bio-Rad Radiance 2000 confocal microscope
equipped with a 488 nm Kr/Ar laser. Optical sections were typically 0.45
µm. Z-series stacks were assembled using NIH Image J
(http://rsb.info.nih.gov/ij/)
and images were edited using Adobe Photoshop.
Testes were isolated from adult males carrying the rCD2 transgene and fixed in 4% paraformaldehyde. For immunostaining, we used a 1:100 dilution of the primary OX-34 mouse anti-rat CD2 monoclonal antibody (Harlan Sera-Labs) and Alexa-488 goat anti-mouse antibody (Molecular Probes) as secondary antibody. Nuclei were counterstained with l µg/ml propidium iodide. The tissue was mounted in Vectashield for confocal microscopy. To track CD2 in embryos, wild-type females were mated to snky1 males carrying the rCD2 transgene. Eggs were collected within 20 minutes of deposition, aged for 30 minutes, then dechorionated in 50% bleach and transferred to poly-L-lysine coated slides. Eggs were squashed under a coverslip, and the slide was frozen in liquid nitrogen then submerged in 95% ethanol for 10 minutes. Tissue was fixed and incubated with the OX-34 antibody as described for testes. Secondary and tertiary fluorescent labeling of OX-34 was performed with Alexa 488 Signal-Amplification Kit (Molecular Probes) using the manufacturer's protocol. Nuclei were counterstained with DAPI. Images were obtained using a Nikon fluorescent microscope equipped with a CCD camera and edited with Adobe Photoshop.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We previously reported that the snky
defect is apparent shortly after the sperm enters the egg. The sperm nucleus
remains tightly condensed and located at the egg cortex and no sperm aster is
produced. We also reported that the sperm nucleus stains readily with DAPI, a
membrane permeable with chromatin dye but variably so, or not at all, with
membrane impermeable dyes. We therefore proposed that the defect is due to
persistence of a plasma membrane around the head of snky mutant
sperm, preventing access to activating factors in the egg cytoplasm. To obtain
more direct evidence for this proposal, we expressed an integral membrane
protein, the rat CD2 protein, during spermatogenesis to provide a sperm plasma
membrane marker. Previous studies showed that rCD2, a single pass plasma
membrane protein, can be expressed in Drosophila cells and detected
with a specific monoclonal antibody
(Dunin-Borkowski and Brown,
1995). Our transgenic line expressed the rCD2 gene under the
control of a testis-specific promoter in snky1
homozygotes. Immunostaining showed that rCD2 was a membrane marker in primary
spermatocytes and all subsequent stages of spermatogenesis. Localization was
observed along the entire length of elongating spermatids and mature sperm,
including the head (Fig. 1A,B).
The rCD2 epitope was retained along the sperm after entry into the egg and for
at least 30 minutes after egg deposition
(Fig. 1C,D,E; n=9
eggs), providing evidence that snky mutant sperm fail in plasma
membrane breakdown (PMBD).
The snky gene corresponds to predicted gene CG11281 and encodes a testis transcript
We previously mapped snky to the 69F-70A cytogenetic interval
(Fitch and Wakimoto, 1998). To
refine localization, we generated deficiencies in the region using
P-element-mediated male recombination starting with the P{PZ}
Syx1301470 insertion. The strategy yielded a small deficiency,
denoted Df(3L) Syx1301470R5, that was homozygous viable
and conferred the snky- phenotype to surviving males.
Molecular mapping of the deficiency breakpoints defined a 185 kb genomic
interval within which we identified a restriction site polymorphism between
snky1 and its parent chromosome. The region overlapped
with CG11281, a gene predicted by the Drosophila Genome Project but
for which no expressed sequence tag or cDNAs had been reported.
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), HMMTOP 2.0 (Tusnady and
Simon, 2001), PHOBIUS (Kall et
al., 2004), SOSUI (Hirokawa et
al., 1998) and ConPred II (Arai
et al., 2004). These programs gave conflicting results probably
due to the relatively high content of positively charged residues in Snky.
However, all programs predict a minimum of four transmembrane domains and one,
ConPred II, predicts eight transmembrane domains. The topology shown in
Fig. 3A is based on the SOSUI
prediction, which identifies a signal sequence within the first 18 residues
and predicts the 6 hydrophobic membranespanning domain most commonly predicted
by the suite of programs we used.
Snky has two regions with noteworthy Cysteine residue patterns
(Fig. 3A,B). One region is
located in the largest predicted extramembrane loop and contains six Cys that
are separated by eight to 12 amino acids. The functional significance of this
region, which we refer to as the patterned 6 Cys motif, is indicated by the
snkyZ0566 mutation, which changes the second of these Cys
to a Ser. The second region is located near the carboxyl end and consists of
four pairs of C-X2-Cs (where X is any amino acid) that are linked by four to
ten residues. This second pattern corresponds to a variant of the Pfam C3HC4
RING finger (zf-PF00097) (Finn et
al., 2006). In Snky, a Cys is substituted for the His resulting in
a C4-C4 variant. Additionally, a coiled-coil domain is predicted in Snky from
residue 597 to 624.
Snky is a member of a protein family
BLAST searches (blastp)
(Altschul et al., 1997) of
non-redundant sequences in NCBI identified a series of Snky-related proteins
present in invertebrates and vertebrates. Of these, a set of proteins, which
we refer to as the Snky family, not only have the highest level of amino acid
identity across their entire length, but also share additional features. All
are predicted to have multiple transmembrane domains. Each also has the
patterned 6 Cys motif in a predicted extramembrane loop that follows the
pattern: C-(10X)-C-(10X)-C-(12-13X)-C-(8X)-C-(9-12X)-C. These proteins also
contain a RING finger with the consensus pattern:
C-(2X)-C-(10-11X)-C-(2-4X)-C-(4X)-C-(2X)-C-(7X)-C-(2X)-C. The Snky proteins
include: a mosquito protein (Aedes aegypti, EAT46485, conceptual
translation) with 30% amino acid identity; a zebrafish protein (XP683798,
conceptual translation) with 22.2% identity; a human protein (BAB71440) with
22% identity; and a mouse protein (XP994048) with 21.8% identity. The human
and mouse protein sequences are supported by full-length testis cDNAs.
Although expressed sequence tags have also been isolated from other tissues,
comparison of available sequence indicates the possibility of testis-specific
isoforms in humans. CLUSTAL W alignment
(Thompson et al., 1994)
revealed three regions, which are the most highly conserved among Snky family
members. These regions are denoted a, b and c in Figs
3 and
4. Regions b and c overlap with
the ends of the region that defines the DC-STAMP sequence family (Pfam
PF07782). In Snky, the DC-STAMP-like region extends 192 amino acids, from
amino acid 406-597.
Snky is more distantly related to other proteins that had been previously
recognized as containing DC-STAMP-like regions. They include the human and
mouse DC-STAMP-Domain Containing-2 Proteins, the Caenorhabditis
elegans sperm protein SPE-42 (Kroft
et al., 2005) and a Drosophila protein encoded by the
CG6845 gene. These proteins contain a RING finger and a Cysteine-containing
region spaced similar to the patterned 6 Cys motif in Snky. However, compared
with proteins described above, they show a reduced level of identity with Snky
at the amino acid level (10-17% identity), particularly in regions a, b and c.
Included in this group is a predicted sea urchin protein (XP795449) with 15.1%
identity to Snky. Even more distantly related are the human and mouse
DC-STAMP, which contain the DC-STAMP domain but lack a RING finger and a
patterned 6 Cys motif.
Localization of a Snky-Green fluorescent protein fusion during spermatogenesis
We generated transgenic flies that contained a snky-Green fluorescent
protein fusion gene expressed from the snky promoter. The fusion
protein contained the entire Snky protein and Enhanced GFP at the carboxyl
terminus. Four independent transgenic lines were recovered and in each line,
the transgene significantly rescued the snky- male sterile
phenotype in a single dose. The transgene used in all subsequent studies
rescued fertility to a level comparable to that conferred by the
snky+ transgene (Table
1). Hence, the Snky-GFP fusion protein was highly active and could
be used as a tool to deduce the localization of the normal Snky protein.
To examine tissue expression of Snky-GFP, we examined larvae and adults
that were homozygous for a snky-GFP transgene and the
snky1 mutation. We detected expression only in the testis.
To determine when Snky-GFP is expressed, we distinguished the different stages
of spermatogenesis by the characteristic morphology of nuclei and
mitochondrial derivatives (A. D. Tates, PhD thesis, University of Leiden,
1971) (Fuller, 1993). Nuclei
were monitored by DAPI staining and mitochondrial derivatives were visualized
by MitoTracker Red-CMXRos staining. Snky-GFP was first detected in primary
spermatocytes as a diffuse cytoplasmic signal
(Fig. 5A), consistent with
localization to the endoplasmic reticulum and as expected for an integral
membrane protein. Following meiosis, at the onion stage, which is
characterized by round spermatid nuclei and comparably sized mitochondrial
derivatives, a prominent cluster of Snky-GFP spots was seen just adjacent each
nucleus (Fig. 5B). The
structures at this stage corresponded in size and location to the aggregated
Golgi complexes that Tates (A. D. Tates, PhD thesis, University of Leiden,
1971) referred to as the acroblast. With continued differentiation, as nuclear
condensation began and mitochondrial derivatives and sperm tails elongated,
Snky-GFP was present as multiple irregular spots throughout the cytoplasm.
However, one spherical Snky-GFP signal, generally larger than the others, was
located adjacent to each nucleus (Fig.
5C). This site of Snky-GFP was the only visible signal retained as
spermatids became fully elongated (Fig.
5D,E). In individualized mature sperm, the Snky-GFP signal
exhibited a thin oval shape located at the tip of the sperm, with its distal
end slightly overlapping with apical end of the needle-shaped nucleus
(Fig. 5F). This pattern of
localization and the predicted structure of Snky suggest that it is an
integral membrane protein targeted to the acrosome during spermatogenesis.
The fate of Snky-GFP and the acrosome during fertilization
Snky-GFP allowed us to examine the fate of Snky and the acrosome during
normal fertilization. In Drosophila, females store sperm after
mating. We examined seminal receptacles (sperm storage organs) from wild-type
females mated to snky1 males that carried two copes of the
Snky-GFP transgene. The Snky-GFP signal we observed in stored sperm appeared
identical to that seen in mature sperm from the testis (data not shown).
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These studies revealed two surprising discoveries. The first of these was
that sperm contributed Snky-GFP to the egg. The second remarkable finding was
that the GFP signals observed in the egg and early cycle 1 embryos
consistently appeared similar to those observed in mature sperm. We considered
two possibilities to account for the retention of bright GFP signals in the
egg cytoplasm. The paternally inherited structure could be an acrosomal
vesicle, with the Snky-GFP signal marking the boundaries of an intact
acrosome. Alternatively, the GFP signal could reflect retention of a large
membrane remnant from a spent acrosome. To distinguish between the two
possibilities, we obtained a transgenic line that expressed a soluble form of
GFP in the acrosome. This line (gift of J. Brill) expresses the
GFPsecr protein under the control of a testis-specific promoter.
GFPsecr was designed and characterized by Pfeiffer et al.
(Pfeiffer et al., 2000;
Pfeiffer et al., 2002), and it
contains the signal peptide from the Wingless protein fused to GFP. When
expressed in embryonic or imaginal disc epithelial cells, GFPsecr
is targeted to the secretory pathway, packaged in secretory vesicles and
released into the extracellular space upon secretion
(Pfeiffer et al., 2002). These
properties predict that if GFPsecr is present in the acrosome, the
GFP signal will dissipate upon acrosome exocytosis.
We examined eggs fertilized by sperm expressing GFPsecr. Of the 22 eggs captured between earliest stages of sperm nuclear decondensation (Fig. 6E) to prometaphase of cycle 1 (Fig. 6H), 21 showed a GFPsecr signal. In each case, the appearance was similar to that observed with Snky-GFP (compare higher magnification insets of Fig. 6A-H). These data are consistent with the retention of an intact acrosomal vesicle by the sperm during fertilization, the release of the vesicle into the egg cytoplasm after PMBD, and the persistence of the acrosome at least as late as prometaphase of the first embryonic cycle.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
observed that the entire sperm enters the egg, including the excessively long
tail surrounded by its plasma membrane. She noted that there was no evidence
for `extra' membranes that would support uptake into the egg by endocytosis.
While her studies did not capture the sperm before nuclear decondensation,
Perotti described one case in which a decondensing sperm nucleus was
surrounded by what appeared to be remnants of the sperm plasma membrane. Her
studies support the idea that the Drosophila sperm head must undergo
PMBD in the egg cytoplasm. This step is required for the sperm nucleus to gain
access to activating cytoplasmic factors that promote nuclear decondensation
and replication, and for the centrosome to elaborate the sperm aster
(Fitch et al., 1998). Our
studies support this sequence of events and implicate Snky function in an
event upstream of PMBD. Our original proposal that snky mutant sperm
fail in sperm activation because they arrest before PMBD was based on their
poor accessibility to membrane impermeant chromatin dyes
(Fitch and Wakimoto, 1998).
Further support was obtained by Ohsako et al.
(Ohsako et al., 2003), who
used an antibody to a proposed cell surface proteoglycan to demonstrate
retention on the sperm head before and after insemination. In our current
study, we ectopically expressed the rat CD2 protein on the sperm plasma
membrane and showed that immunoreactivity to this specific integral membrane
protein was retained on the head of snky1 sperm for at
least 30 minutes after entry into the egg. These observations provide strong
support for the proposal that snky sperm do not shed the plasma
membrane surrounding the head. They also suggest that understanding the
function of the Snky protein should provide clues about how PMBD is initiated
or accomplished at the molecular level.
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;
Kellenberger et al., 2005;
Pugh et al., 2006;
Arioli et al., 1998). In one of
these, hNOT4, the C4-C4 RING has been shown to fold and bind zinc in a
cross-brace fashion similar to the more common C3HC4 RING finger
(Hanzawa et al., 2001),
suggesting structural and functional similarity between these variants.
Although RING finger proteins have been implicated in a broad variety of
cellular functions, recent studies suggest that their unifying role is in
mediating protein-protein interactions in large multiprotein assemblages
(Borden, 2000). Thus, Snky may
play a role in organizing and holding together macromolecular complexes at the
membrane via its C4-C4 RING. The second Cys-containing region is the patterned
6 Cys motif, the functional significance of which is indicated by conservation
and by the recovery of a male sterile snky allele that mutates the
second Cys to a Ser. Cysteines in this region may form disulfide bonds to
create a binding pocket or otherwise allow interactions with additional
proteins. The third region is the DC-STAMP-like region, named after Dendritic
cell specific transmembrane protein, a plasma membrane protein that was first
described for its expression in human dendritic cells
(Hartgers et al., 2000). Thus
far, the function of only a few DC-STAMP-domain-containing proteins have been
examined through the analysis of mutations or other knockdown strategies. The
general classification of these proteins has been as members of a new class of
putative receptors. Mouse DC-STAMP is upregulated in differentiating
osteoclasts, and required for osteoclast fusion to form multinucleate cells
(Kukita et al., 2004;
Yagi et al., 2005). The C.
elegans SPE-42 is required in sperm and is proposed to act at a step in
fertilization just before or during sperm-egg membrane fusion
(Kroft et al., 2005). Snky is
the third DC-STAMP domain-containing protein to have its function studied and,
like Spe-42, it has a role in sperm function. Although the precise molecular
function of the DC-STAMP domain remains unknown, these three examples support
a role in mediating membrane-membrane interactions.
The predicted structure of Snky, with its multiple transmembrane domains,
and our Snky-GFP localization studies provide evidence that Snky is an
acrosomal membrane protein. This localization presents the intriguing question
of how an acrosomal membrane protein is able to influence the integrity of the
sperm plasma membrane. The findings also have broader implications for
acrosome function. The acrosome is a Golgi-derived membrane-bound organelle
found at the apical end of sperm, and its function has been extensively
studied in marine invertebrates and mammals. In these organisms, the acrosome
is best known as a specialized secretory vesicle that undergoes exocytosis.
Like many secretory events, a rise in intracellular Ca2+ is
required for acrosome exocytosis. This increase in Ca2+ requires
influx into the sperm as well as efflux from internal stores. The acrosome has
been identified as an internal source of Ca2+ and is believed to
release Ca2+ to contribute to its own exocytosis
(Walensky and Snyder, 1995;
Herrick et al., 2005).
Ultrastructural studies show that exocytosis involves vesiculation of the
outer acrosomal membrane and overlying plasma membrane. This results in the
release of contents of the acrosome, which include hydrolytic enzymes and
other components that facilitate binding and penetration through the egg
coats. Exocytosis also exposes the inner acrosomal membrane, resulting in a
new membrane patch and associated acrosomal molecules on the surface of the
sperm. In marine invertebrates, this newly exposed region is the site of
binding and membrane fusion with the egg
(Colwin and Colwin, 1967;
Lopo et al., 1982), providing
a direct physical link between the requirement for acrosome exocytosis and
membrane fusion. In mammals, acrosome exocytosis is also a prerequisite for
sperm to bind to and fuse with the egg. However, the plasma membrane that lies
over the equatorial segment of the acrosome, and not the acrosome membrane
itself, is believed to be the point of membrane fusion with the egg
(Yanagimachi, 1994).
Experimental studies of hamster sperm by Takano et al.
(Takano et al., 1998) suggest
that fusion competency of this specialized region of mammalian sperm requires
changes that occur immediately before or during acrosome exocytosis, as well
as the contents of the acrosome.
Considering these known acrosome functions, it is interesting that
acrosomes are not universal features of animal sperm. Acrosomes are not
present in the amoeboid sperm of nematodes, and they are occasionally absent
or reduced in species that otherwise possess the flagellated sperm typical of
animals (Baccetti, 1979). For
instance, the absence or reduction of an acrosome in mature sperm of teleosts
(Coward et al., 1996) and
certain insect species (Baccetti,
1972; Dallai et al.,
2003) is well documented and is generally considered a derived
condition (Baccetti, 1979).
Hence the requirement for the acrosome during fertilization has been
eliminated or bypassed in some lineages during the course of evolution.
Ultrastructural studies show that an acrosome is typical of insect sperm
(Baccetti, 1972). However, few
studies have examined the role of the insect acrosome during fertilization.
Studies of fertilization in the house fly Musca domestica suggested
`loosening or loss' of the sperm plasma membrane before entry into the
micropyle of the egg, followed by exocytosis of acrosomal contents during
passage of the sperm through the micropyle
(Degrugillier and Leopold,
1976).
Our studies revealed a different fate for the Drosophila acrosome.
The observation that Snky-GFP was a paternally contributed molecule to the
early egg was a surprising finding. The persistence of a single prominent
Snky-GFP structure, with an intensity and shape in eggs that appeared similar
to those seen in mature sperm, suggests the possibility that the inherited
structure was an intact acrosome. This possibility was further supported by
studies tracking GFPsecr, a soluble protein that is secreted into
the extracellular space when expressed in other Drosophila cells
(Pfeiffer et al., 2002). Its
robust and identical appearance to Snky-GFP argues against exocytosis, at
least until after prometaphase of the first embryonic cell cycle.
These cytological observations of the fate of the acrosome during normal
fertilization, combined with the defect in PMBD observed in
snky- mutant sperm, suggest that the acrosome may be
acting primarily as a signaling vesicle to elicit changes in the overlying
sperm plasma membrane. This activity requires the Snky acrosomal membrane
protein, but occurs without acrosome exocytosis. Snky may be serving as a
receptor that permits communication between the acrosome and plasma membrane.
Alternatively, Snky or its associated proteins may serve to initiate or
maintain contact between the membranes, or modify membrane lipids or proteins
in preparation for PMBD. In this sense, Snky, DC-STAMP and SPE-42 may share a
common mechanism in promoting membrane interactions
(Yagi et al., 2005;
Kroft et al., 2005). However,
in the case of Snky, the pathway would lead to breakdown of the overlying
plasma membrane, rather than fusion between two membranes.
In Drosophila, Snky is required specifically for male fertility. It will be interesting to determine whether Snky family members are required for sperm function in zebrafish, which have sperm that lack acrosomes, and for acrosome function in marine invertebrate and mammalian sperm. If human Snky-like proteins are specifically required for sperm function, then they may be potential targets for male contraceptives. More immediately, our studies of Snky provide tools for further investigating how the membrane events that occur during Drosophila fertilization compare to conventional views of membrane dynamics during sperm activation and fertilization in animals. Research on the acrosome has focused primarily on its function as a specialized secretory or Ca2+ storage vesicle. Our studies suggest a primary role as signaling vesicle in Drosophila, with a newly identified acrosomal membrane protein communicating directly or indirectly with the plasma membrane to affect changes in membrane integrity. Comparisons among species should continue to shed light on the intriguing ways in which sperm structures and fertilization molecules, such as Snky, may be selected for conservation or diversification during the course of evolution.
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