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).

Effects of Mutation or Deletion

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).

Reference names in red indicate recommended papers.

Search PubMed for articles about Drosophila Sneaky

Fitch, K. R. and Wakimoto, B. T. (1998). The paternal effect gene ms(3)sneaky is required for sperm activation and the initiation of embryogenesis in Drosophila melanogaster. Dev. Biol. 197: 270-282. Medline abstract: 9630751

Herrick, S. B., Schweissinger, D. L., Kim, S. W., Bayan, K. R., Mann, S. and Cardullo, R. A. (2005). The acrosomal vesicle of mouse sperm is a calcium store. J. Cell Phys. 202: 663-671. Medline abstract: 15389568

Kroft, T., Gleason, E. J. and L'Hernault, S. W. (2005). The spe-42 gene in required for sperm-egg interactions during C. elegans fertilization and encodes a sperm-specific transmembrane protein. Dev. Biol. 286: 169-181. Medline abstract: 16120437

Ohsako, T., Hirai, K. and Yamamoto, M.-T. (2003). The Drosophila misfire gene has an essential role in sperm activation during fertilization. Genes Genet. Syst. 78: 253-266. Medline abstract: 12893967

Perotti, M.-E. (1975). Ultrastructural aspects of fertilization in Drosophila. In The Functional Anatomy of the Spermatozoan, Proceedings of the Second International Symposium (ed. B. A. Afzelins), pp. 57-68. Oxford: Pergamon Press.

Wakimoto, B. T., Lindsley, D. and Herrera, C. (2004). Toward a comprehensive genetic analysis of male fertility in Drosophila melanogaster. Genetics 167: 207-216. Medline abstract: 15166148

Walensky, L. D. and Snyder, S. H. (1995). Inositol 1,4,5-Trisphosphate receptors selectively localized to the acrosomes of mammalian sperm. J. Cell Biol. 130: 857-869. Medline abstract: 7642703

Wilson, K. L., Fitch, K. R., Bafus, B. T. and Wakimoto, B. T. (2006). Sperm plasma membrane breakdown during Drosophila fertilization requires sneaky, an acrosomal membrane protein. Development 133(24): 4871-9. Medline abstract: 17092953

Yagi, M., Miyamoto, T., Sawatani, Y., Iwamoto, K., Hosogane, N., Fujita, N., Morita, K., Ninomiya, K., Suzuki, T., Miyamoto, K. et al. (2005). DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J. Exp. Med. 202: 345-351. Medline abstract: 16061724

date revised: 6 August 2007

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