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).