Fertilization typically involves membrane fusion between sperm and eggs. In Drosophila, however, sperm enter eggs with membranes intact. Consequently, sperm plasma membrane breakdown (PMBD) and subsequent events of sperm activation occur in the egg cytoplasm. It has been proposed that mutations in the sneaky (snky) gene result in male sterility due to failure in PMBD. This study supports this proposal by demonstrating persistence of a plasma membrane protein around the head of snky sperm after entry into the egg. It is further shown that snky is expressed in testes and encodes a predicted integral membrane protein with multiple transmembrane domains, a DC-STAMP-like domain, and a variant RING finger. Using a transgene that expresses an active Snky-Green fluorescent protein fusion (Snky-GFP), it is shown that the protein is localized to the acrosome, a membrane-bound vesicle located at the apical tip of sperm. Snky-GFP also allowed following the fate of the protein and the acrosome during fertilization. In many animals, the acrosome is a secretory vesicle with exocytosis essential for sperm penetration through the egg coats. Surprisingly, the Drosophila acrosome is a paternally inherited structure. Evidence is presented that the acrosome induces changes in sperm plasma membrane, exclusive of exocytosis and through the action of the acrosomal membrane protein Snky. Existence of testis-expressed Snky-like genes in many animals, including humans, suggests conserved protein function. The characteristics of Drosophila Snky, acrosome function and sperm PMBD is related to membrane fusion events that occur in other systems (Wilson, 2006).

Studies of fertilization have revealed the universal importance of membrane events to prepare and coordinate the gametes and ensure their successful union. These events have been most extensively studied in sperm of selected marine invertebrate and mammalian species and include dynamic changes in membrane proteins and lipids during sperm capacitation, acrosome exocytosis, binding, adhesion and membrane fusion with the egg. 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. 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 (Wilson, 2006).

A genetic approach using Drosophila was employed to identify novel sperm molecules required for fertilization. Previous studies of male sterile mutants indicated that those affecting fertilization might be relatively rare. Therefore, a large number of male-sterile mutations were isolated and categorized to find a subset that disrupt sperm-egg interactions or induce paternal effect defects (Fitch, 1998; Wakimoto, 2004). Previously described were mutations in a gene called sneaky (snky), which met genetic criteria of specifically affecting fertilization (Fitch, 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. It was proposed that this sperm activation defect is 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 (Wilson, 2006).

This study provides phenotypic evidence of a role for Snky in affecting the integrity of the sperm plasma membrane during fertilization. The snky gene was molecularly identify and its transcript and predicted protein were characterized 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 (Wilson, 2006).

Fertilization in Drosophila is unorthodox in that it does not involve a membrane fusion event between the sperm and egg. The mechanism used by the sperm to penetrate through the egg plasma membrane remains an interesting mystery. In ultrastructural studies of fertilization in D. melanogaster and D. montana, Perotti (1975) observed that the entire sperm enters the egg, including the excessively long tail surrounded by its plasma membrane. There was no evidence for 'extra' membranes that would support uptake into the egg by endocytosis. While these 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. These 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, 1998). The current studies support this sequence of events and implicate Snky function in an event upstream of PMBD. The 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, 1998). Further support was obtained by Ohsako (2003), who used an antibody to a proposed cell surface proteoglycan to demonstrate retention on the sperm head before and after insemination. In the current study, the rat CD2 protein was ectopically expressed on the sperm plasma membrane and it was 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 (Wilson, 2006).

Molecular studies show that Snky is a member of a family of related membrane proteins that are present in animals but are apparently absent in other lineages. Conservation among Snky family members points to three domains of potential functional significance. The most striking motif is the C4-C4 RING finger, which has been noted in at least four other proteins. 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, 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. 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. 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. 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, 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 (Wilson, 2006).

The predicted structure of Snky, with its multiple transmembrane domains, and 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, 1995; Herrick, 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, 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. Experimental studies of hamster sperm 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 (Wilson, 2006 and references therein).

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. For instance, the absence or reduction of an acrosome in mature sperm of teleosts and certain insect species is well documented and is generally considered a derived condition. 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. 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 (Wilson, 2006).

These 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 GFP^secr, a soluble protein that is secreted into the extracellular space when expressed in other Drosophila cells. Its robust and identical appearance to Snky-GFP argues against exocytosis, at least until after prometaphase of the first embryonic cell cycle (Wilson, 2006).

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, 2005; Kroft, 2005). However, in the case of Snky, the pathway would lead to breakdown of the overlying plasma membrane, rather than fusion between two membranes (Wilson, 2006).

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, these 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. These 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 (Wilson, 2006).

Transgenic flies were generated 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. 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 (Wilson, 2006).

To examine tissue expression of Snky-GFP, larvae and adults that were homozygous for a snky-GFP transgene and the snky¹ mutation were examined. Expression was detected only in the testis. To determine when Snky-GFP is expressed, the different stages of spermatogenesis were distinguished by the characteristic morphology of nuclei and mitochondrial derivatives. 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, 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. The structures at this stage corresponded in size and location to the aggregated Golgi complexes that has been referred to as the acroblast. With continued differentiation, as nuclear condensation began and mitochondrial derivatives and sperm tails elongate, Snky-GFP os present as multiple irregular spots throughout the cytoplasm. However, one spherical Snky-GFP signal, generally larger than the others, is located adjacent to each nucleus. This site of Snky-GFP is the only visible signal retained as spermatids became fully elongated. In individualized mature sperm, the Snky-GFP signal exhibits 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. This pattern of localization and the predicted structure of Snky suggest that it is an integral membrane protein targeted to the acrosome during spermatogenesis (Wilson, 2006).

Snky-GFP made possible examination of the fate of Snky and the acrosome during normal fertilization. In Drosophila, females store sperm after mating. Seminal receptacles (sperm storage organs) were examined from wild-type females mated to snky¹ males that carried two copes of the Snky-GFP transgene. The Snky-GFP signal observed in stored sperm appeared identical to that seen in mature sperm from the testis (Wilson, 2006).

Fertilization occurs within the reproductive tract of females. Sperm are released from the seminal receptacle and enter the egg as it passes down the uterus. The events from sperm entry into the egg to the completion of the first embryonic cell cycle occur rapidly (estimated <17 minutes) and are often completed before the egg is laid. To capture the earliest stages, eggs fertilized by sperm from Snky-GFP males were collected and fixed within 15 minutes of egg deposition, counterstained with DAPI and examined by confocal microscopy. Even with this rapid processing, the majority of embryos examined had progressed beyond cycle 2. Of the 17 fertilized eggs and embryos captured before metaphase of cycle 1, a Snky-GFP signal was observed in 16. The signal was located in close proximity to the sperm nucleus before visible evidence of nuclear decondensation, and more distantly as decondensation progressed. During pronuclear migration and pronuclear apposition, Snky-GFP was seen peripheral to what was judged to be the male pronucleus based on the state of chromatin condensation or position in the egg. During prometaphase of cycle 1, the Snky-GFP signal was observed near the condensing chromosomes. Although the position of the Snky-GFP signal relative to the sperm chromatin changed during these stages of fertilization, neither the shape nor the intensity of the signal appeared to change. Five embryos were found at stages between metaphase of cycle 1 and 2 and only one, which was at anaphase of cycle 1, exhibited a GFP signal (Wilson, 2006).

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. Two possibilities were considered 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, a transgenic line was obtained that expressed a soluble form of GFP in the acrosome. This line expresses the GFP^secr protein under the control of a testis-specific promoter, and it contains the signal peptide from the Wingless protein fused to GFP. When expressed in embryonic or imaginal disc epithelial cells, GFP^secr is targeted to the secretory pathway, packaged in secretory vesicles and released into the extracellular space upon secretion. These properties predict that if GFP^secr is present in the acrosome, the GFP signal will dissipate upon acrosome exocytosis (Wilson, 2006).

Eggs fertilized by sperm expressing GFP^secr weew examined. Of the 22 eggs captured between earliest stages of sperm nuclear decondensation to prometaphase of cycle 1, 21 showed a GFP^secr signal. In each case, the appearance was similar to that observed with Snky-GFP. 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 (Wilson, 2006).

Although a large number of maternal factors are known to be essential for fertilization or the earliest stages of embryogenesis in Drosophila melanogaster, the role of paternally supplied products is not clearly understood. Paternal effect mutations provide a means to identify factors specifically required by the sperm after its entry into the egg. This study describes the third strict paternal effect gene to be identified in Drosophila ms³sneaky^snky, which defines the earliest developmental arrest phenotype so far described. Characterization of two independently isolated snky mutations showed that they affect male fertility, but not viability or female fertility. Cytological analyses showed that spermatogenesis proceeds normally in snky males. However, the snky defect is evident after sperm entry into the egg; snky sperm did not undergo nuclear decondensation, form a functional male pronucleus, or initiate mitotic divisions in the egg. Immunolocalization of tubulin and Drosophila Centrosomin, a known centrosomal component, showed that snky-inseminated eggs fail to reconstitute a microtubule-organizing center. In addition, snky sperm chromatin retain the histochemical properties of mature sperm chromatin for several hours after sperm entry, show reduced staining with membrane-impermeant nuclear dyes, and fail to replicate. It is concluded that the snky+ product is required for the initial response of the sperm to cytoplasmic cues in the egg and for the subsequent initiation of embryogenesis in Drosophila. It is suggested that all of the snky defects can be explained by the failure of the sperm plasma membrane to break down after entry into the egg (Fitch, 1998).

Males mutant for the snky gene are sterile despite normal spermatogenesis, successful transfer of normal quantities of sperm to females and efficient sperm entry into eggs (Fitch, 1998). It has been 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. The sperm nucleus stains readily with DAPI, a membrane permeable with chromatin dye but variably so, or not at all, with membrane impermeable dyes. It has therefore been 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, an integral membrane protein, the rat CD2 protein, was expressed 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. The 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. The rCD2 epitope was retained along the sperm after entry into the egg and for at least 30 minutes after egg deposition, providing evidence that snky mutant sperm fail in plasma membrane breakdown (PMBD) (Wilson, 2006).

snky has been mapped to the 69F-70A cytogenetic interval (Fitch, 1998). To refine localization, deficiencies in the region were generated using P-element-mediated male recombination starting with the P{PZ} Syx13⁰¹⁴⁷⁰ insertion. The strategy yielded a small deficiency, denoted Df(3L) Syx13^01470R5, 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 a restriction site polymorphism was identified 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 (Wilson, 2006).

Two additional observations showed that CG11281 corresponds to the snky gene. Northern analysis revealed a CG11281 transcript with an expression profile consistent with the specific effect of snky mutations on male fertility. A genomic fragment containing CG11281 hybridized to a rare 2.7 kb polyadenylated RNA that was found in testes, but not in carcasses (bodies lacking testes) of wild-type adult males. A corresponding transcript that was reduced in size and abundance was detected in testes of snky¹ males. A hybridizing transcript was absent in females. Definitive evidence to assign CG11281 as the snky gene was achieved by transgenic rescue of the snky^- male sterility by a 7.3 kb genomic SalI fragment that included CG11281 but no other predicted genes. Three independent insertions of the transgene were obtained and each one conferred high levels of male fertility in a single dose, indicating that the SalI fragment contains most, if not all, of the necessary sequences for the snky⁺ gene (Wilson, 2006).