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Human Molecular Genetics 2007 16(R2):R124-R133; doi:10.1093/hmg/ddm252
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Fruit flies and the sperm proteome

Timothy L. Karr*

Department of Biology and Biochemistry, University of Bath, Bath, UK

* To whom correspondence should be addressed. Email: t.l.karr{at}bath.ac.uk

Received August 27, 2007; Accepted August 29, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SPERM AS IDEAL CANDIDATES...
 THE DROSOPHILA SPERM PROTEOME
 EVOLUTIONARY ANALYSIS OF THE...
 RELATIONSHIP TO MAMMALIAN...
 CONCLUSION
 REFERENCES
 
Sperm have been studied for their obvious role in fertilization and as a model system for cell–cell interactions and cell signaling. Despite its central and critical role in reproduction, we know surprisingly little about the overall molecular composition of sperm. Interest in sperm function has greatly intensified for two reasons: first, it is becoming increasingly apparent that human infertility can be traced to male factors, including alterations in sperm proteins, and second, there is increasing empirical evidence that sperm provide essential factors, both nucleic acid- and protein-based, to early zygote development possibly beyond their role in fertilization. At the molecular level, study of the sperm proteome has revealed a variety of genetic mechanisms involved in the organization and evolution of sperm form and function. These discoveries are being augmented and expanded by the application of proteomics that directly identifies protein constituents of sperm. In this article I argue that sperm are ideal candidate cell types for proteomic analyses and describe the current state of the field focussing on the recently described sperm proteome in the fruit fly Drosophila melanogaster.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SPERM AS IDEAL CANDIDATES...
 THE DROSOPHILA SPERM PROTEOME
 EVOLUTIONARY ANALYSIS OF THE...
 RELATIONSHIP TO MAMMALIAN...
 CONCLUSION
 REFERENCES
 
‘A Drosophila sperm walks into a bar. Bartender says, "Why the long tail?"’

Anonymous quote.

This joke, in addition to being highly derivative, may not be understood by the wider scientific audience. However, it is instructive as to the degree in variation sperm form and function can, and do, display for Drosophila sperm tails are incredibly, almost absurdly, long structures engulfed by the egg during fertilization (1,2). The purpose of this cellular contortionism is not entirely clear, but is a common feature amongst insects (3). These, and other observations suggest sperm are far more than simple ‘DNA delivery vehicles’ and instead provide additional functions, including egg activation (4), origination of the zygote centrosome (5) and delivery of mRNA (6,7).

It is becoming increasingly apparent that human infertility can be traced to a male factor (8). Indeed, a recent proteomics study found 20 sperm protein spots with altered 2D gel migration patterns in infertile males with otherwise apparently normal amount, motility and morphology of sperm (9,10). The functions of these altered proteins, if any, in male infertility is unknown. These relatively new findings highlight potential new roles for paternally derived protein products and also our limited understanding of the genes and genetic variants that result in more subtle impairments in sperm viability, motility or metabolism. Genetic analyses in the mouse has identified hundreds of genes that influence fertility (11–13), but only recently has proteomics been systematically applied characterize sperm components (14). Otherwise, we have only very limited knowledge as to the molecular genetic basis of infertility.

Drosophila has figured prominently in extending appreciation of the sperm's role in reproduction (as has fertilization studies in the worm, Caenorhabditis elegans, not discussed further due to space constraints). In addition to cell biological studies, mutational analyses has recently identified paternal effect lethal genes in Drosophila providing definitive proof that extragenic paternal factors are essential for zygote formation (15–18). A comprehensive genetic analyses of male infertility has shown as many as 2000 genes in Drosophila underlie male sterility is promising as many may directly involve fertilization (19). It will be of great interest to determine how many of these genes are in fact bona fide paternal effects and how they function during fertilization or early embryogenesis.

The genetics of reproduction is also complicated by rapid evolution and functional diversification of male reproductive genes due to sexual selective pressures (20). Therefore, model systems of infertility will require a combined comparative approach to identify conserved molecular and genetic mechanisms of spermatogenesis. Given its rich genetic heritage, availability of numerous sequenced genomes and a powerful genetic ‘toolbox’ to manipulate gene expression, Drosophila should be an important model systems for a system-wide analysis of sperm structure, and function.


   SPERM AS IDEAL CANDIDATES FOR WHOLE CELL PROTEOMIC ANALYSES
TOP
 ABSTRACT
 INTRODUCTION
 SPERM AS IDEAL CANDIDATES...
THE DROSOPHILA SPERM PROTEOME
 EVOLUTIONARY ANALYSIS OF THE...
 RELATIONSHIP TO MAMMALIAN...
 CONCLUSION
 REFERENCES
 
It has now well established that the fruit fly Drosophila melanogaster and mammals, including humans, share a great many genes in common. They also share conserved metabolic and signaling pathways at the cellular level, and Drosophila has been an important tool in unraveling many signaling systems including circadian rhythms and some elements of learning and memory. Indeed, there is growing consensus that Drosophila is an appropriate model system for the study of human diseases including Huntington's Disease, Parkinson's Disease and Alzheimer's Disease as recently discussed in this Journal (21,22). It therefore follows that fertilization biology will similarly benefit from knowledge gained in the Drosophila system.

The number of proteins that comprise a cellular proteome is difficult if not impossible to define. As a dynamic process, cellular life changes constantly making the proteome a ‘moving target’. Likewise, proteome complexity rises dramatically when post-translational modifications are taken into account. Diploid cell proteomes can be bewilderingly complex; one recent study identified as many as 20 000 protein–protein interactions (23). The pros and cons of the application of proteomics, particularly mass spectrometry and allied separation techniques, 2D gels and biochemical fractionation/separation, have been well discussed in the literature (24–30). Additional technological and theoretical advances will certainly be forthcoming, but until significant advancements are achieved in these technologies, the general applicability of proteomics to cell structure/function and the loftier goal of systems biology in metazoans will remain limited. In this regard, analysis of sperm proteomes may provide a useful starting point for a systems level analysis of cellular function in eukaryotes. The Drosophila sperm proteome (DmSP), by providing for the first time a broader description of the basic constituents of a sperm should be informative for all areas of sperm research, including humans and mammals. Here, the value, both biological and technological, of using sperm as proteomics targets will be briefly discussed, keeping in mind the limitations of the techniques used.

Sperm, as streamlined haploid cells, are relatively ‘simple’ (Fig. 1) as deduced from 2D gel spot counts. Best estimates from 2D gels suggest that sperm contain anywhere from 1300 + proteins (31) to ~400 (32). There is, in principle, no reason why the number of sperm proteins would be the same in different species and the 2–3-fold difference in estimates may simply reflect taxa specific differences. On the other hand, the measured differences could also reflect differences in sperm purification techniques or how sperm develop and/or are stored in the male. It will be an important issue to resolve as knowledge of a ‘core sperm proteome’ will greatly assist our understanding of sperm structure/function and evolution as more sperm proteomes are unveiled. By any account the number of proteins in the sperm proteome represent a fraction of the predicted repertoire of diploid cells, which range from 5000 to 10 000.


Figure 1
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  		Figure 1. Sperm purification and fertilization in Drosophila. Representative samples (pictures on left) used for high resolution 2D gel electrophoresis (right) show the expected reduction in spot counts during sperm purification. Adult males (top) were dissected and testes + seminal vesicles (second from top) and seminal vesicles further separated (white circle, second from top). Seminal vesicles were then carefully punctured with a needle releasing sperm in a coherent spaghetti-like mass due to their length (fourth photo). The corresponding 2D gels at each stage (right panels) indicate the number and complexity of proteins. Arrowheads indicate spots containing DmSP aminopeptidases as identified by MALDI-TOF MS (U. Gerike, unpublished data). Arrows at bottom indicate fertilization and incorporation of the sperm and DmSP into a Drosophila egg (bottom image). Fertilization was monitored using an anti-sperm antibody and is recognized as the coiled white thread-like structure seen at the anterior end of the egg (2).

 

   THE DROSOPHILA SPERM PROTEOME
TOP
 ABSTRACT
 INTRODUCTION
 SPERM AS IDEAL CANDIDATES...
 THE DROSOPHILA SPERM PROTEOME
EVOLUTIONARY ANALYSIS OF THE...
 RELATIONSHIP TO MAMMALIAN...
 CONCLUSION
 REFERENCES
 
As shown in Figure 1, Drosophila sperm can be purified by simple methods to a highly purified level (32). As shown also in Figure 1, something perhaps not generally appreciated, is that the presence of this rather large sperm cell means that a correspondingly complete sperm proteome also resides in the egg both during, and following, fertilization. Sperm as large as 16 000 µm in length have been visualized inside Drosophila eggs (2) suggesting that some rather elaborate mechanisms have evolved to carry out this task. Perhaps even more remarkable is the stereotypical nature of the structures formed (1,2) suggesting that a high degree of sperm–egg interactions mediate sperm entrance. A natural extension of these observations was to ask what proteins were in the egg, something proteomics could answer once high throughput mass spectrometry technologies were developed.

The DmSP was studied using a combination of MS/MS mass spectrometry and 2D gels to identify 381 proteins (32). This included 37 ambiguous assignments, usually proteins in highly conserved gene families or recently duplicated genes. GO annotation identified six broad functional categories as shown in Figure 2. A majority of DmSP genes fall into categories involved in energy production and utilization (energetics and central metabolism and by genes encoding cytoskeletal proteins). This distribution is consistent with a cell type substantially composed of a mitochondrial derivative and a microtubule-based axoneme. However, as found in other genomics and proteomic studies, a large category representing genes with no annotated function was also present in the data set. Many central metabolic enzymes were identified, including enzymes of the lower half of glycolysis, e.g. enolase, phosphoglyceromutase, and pyruvate kinase involved in the generation of ATP directly from phophorlyated intermediate compounds. Their presence is consistent with a recent biochemical study that identified similar enzymatic activities in the Chlamydomonas reinhardtii flagellum (33). The key metabolic enzyme, pyruvate dehydrogenase, and members of the oxidative phosphorylation pathway were also found.


Figure 2
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  		Figure 2. Functional and molecular evolution of the DmSP. Functional categories taken from the GO ontology (FlyBase) were binned by the indicated molecular function. Associated with each color coded category are the computed pairwise Ka/Ks ratios between the DmSP and corresponding D. simulans orthologues (adapted from 32). Numbers in parentheses indicate percent representation of each category. The overall Ka/Ks for all combined categories is indicated at the bottom.

 
The DmSP contains a diverse repertoire of categories representing a wide spectrum of functional capacities including some unexpected ones (Table 1). These include seven annotated leucyl aminopeptidases (Laps) among a total of 13 peptidases/proteases. Many of the Laps are members of a gene family that has recently expanded and diversified (S. Dorus and T.L.K., unpublished data). Two Laps reside in the meta-cluster of spermatogenesis genes at cytological band 53C (discussed later), and CG32351, is a major constituent of sperm as estimated on 2D gels (Fig. 1). Only dPsa, a puromycin-sensitive aminopeptidase, has been studied at the molecular level (34). Although these studies failed to reveal a clear function in Drosophila fertilization, a related aminopeptidase in C. elegans is required for anteroposterior polarity in the early embryo (35). Aminopeptidases and endoproteases have long been known associated with seminal fluid secretions and involved in fertilization competence in a variety of organisms (36). However, other than Drosophila, there appears to be only one other, a GPI-anchored aminopeptidase identified biochemically from the surfaces of sperm of the mussel, Mytilus (37). MALDI-TOF MS (matrix absorbed laser dissociation–ionization time-of-flight mass spectrometry) analysis identified five spots on 2D gels containing annotated leucylaminopeptidases (Fig. 1; (32), U. Gerike and T.L.K., unpublished data). Inspection of these spots indicate that these aminopeptidases represent a substantial proportion of the total protein in Drosophila sperm. While at present this family of aminopeptidases appear unique to Drosophila, future studies will focus on the identification of related enzymes in mammalian and human sperm. Understanding the function of this enzyme family in sperm, and its relationship to other peptidases/proteases found in seminal fluids should provide a deeper understanding of the fertilization process in Drosophila and other taxa.


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  		Table 1. Survey of DmSP genes

 
Table 1 also contains a number of other DmSP genes, many previously identified by genetic analyses or electronic annotation. These include genes shown previously to affect sperm development, several oxidoreductases and transport proteins presumably involved in energy generation and transduction (e.g. sesB and Ant2), ion channel proteins (e.g. Porin 2) and cell–cell signaling (e.g. Plkk1). Amongst the neural genes are a number of genes that interact with the cytoskeleton (mir and Glued), ion-channel proteins involved in behavioral responses such as the detection of sound (nompC) and larval feeding behavior (Pkd2). Recognition of these as members of the DmSP may identify new sperm functions for these genes. Given the extensive interactions sperm undergo with the female environment, such genes may have novel new functions related to the functions already recognized in other tissues.

The extensive cytological mapping of Drosophila genes was utilized to map all 381 DmSP genes onto the polytene chromosomes (Fig. 3) representing the first such genetic and evolutionary analysis of a proteome (32). This genome-wide view of DmSP gene distributions revealed interesting features including a significant under representation of sperm genes on the X-chromosome. This is consistent with the theory of sexually antagonistic selection upon genes functioning during spermatogenesis (38). Another insightful finding was a highly localized physical clustering of DmSP genes. A total of 24 adjacent gene pairs were observed (Fig. 3) that deviated significantly from normal distribution of genes along the chromosome. Additionally, 27 larger ‘loose’ gene clusters were identified separated by an average of less than two non-DmSP genes (Fig. 3). A striking six gene cluster in a 173 kb region of cytological band 53C that correlated with a high incidence of spermatogenesis genes, as previously shown (39). Thus the DmSP genes display a mosaic-like structure with regional concentrations of genes. Interestingly, DmSP gene clusters are not correlated with functional properties (except for instances where clustered genes are the product of recent localized duplications) suggesting that coregulation involving higher order chromatin domains may coordinate expression during spermatogenesis. Several hypotheses might account for this observation, one involving chromatin condensation in a hierarchical fashion beginning with non-sperm-specific regions of the genome followed by housekeeping genes and finally genes encoding sperm specific proteins necessary for the final stages of sperm development. These genes would represent ‘last-orders-at-the-bar’ regions of the genome and, in an ever increasingly crowded environment as chromatin compaction progresses, would favor coregulation of gene expression and genome organization. Genome-wide questions such as these can be addressed effectively using comparative genomic and evolutionary studies for which Drosophila is particularly well suited.


Figure 3
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  		Figure 3. DmSP genome distribution. All genes of the DmSP were mapped onto the D. melanogaster polytene chromosomes and color-coded based on their annotated functions. Horizontal bars above each chromosome indicate DmSP gene clusters, and the asterisk above the bar indicates a spermatogenesis gene metacluster in cytological region 53C. Triangles below each chromosome indicate loci that were not unambiguously identified by mass spectroscopy (adapted from 32).

 

   EVOLUTIONARY ANALYSIS OF THE DmSP
TOP
 ABSTRACT
 INTRODUCTION
 SPERM AS IDEAL CANDIDATES...
 THE DROSOPHILA SPERM PROTEOME
 EVOLUTIONARY ANALYSIS OF THE...
RELATIONSHIP TO MAMMALIAN...
 CONCLUSION
 REFERENCES
 
Evolutionary analyses are greatly accelerated by the availability of twelve Drosophila genomes in the database (40). As an initial step orthologous Drosophila simulans sequences representing > 500 kb were analyzed. Pairwise comparison of the synonymous and non-synonymous averages suggested that overall the DmSP is evolving quite conservatively presumably due to functional constraints (32). However, there was considerable variation in divergence rates of proteins in various categories of the DmSP (Fig. 2). Genes encoding DNA/RNA binding factors appear to be evolving at a rapid rate, although a thorough and systematic analysis must be performed to confirm this supposition. High levels of selective constraint on the DmSP overall is expected as many perform critical cellular functions of motility and primary metabolism. The ability to exploit the genomics of the twelve sequenced Drosophila genomes, coupled with knowledge of the sperm proteome will provide a unique perspective of the forces that have shaped the evolutionary landscape of sperm form and function in this clade.

Overall, the DmSP did not show evidence of positive selection (as indicated by a Ka/Ks > 1) in stark contrast to another reproductive tissue, the accessory gland. The primary secretory products of accessory glands are accessory proteins, ACPs, which then bind to interact with sperm and/or the female (41–43) and many are under positive directional selection (44,45). Thus, differential selection appears to operate on these two reproductive tissues and led to the suggestion that ‘compartmentalization of adaptation’ is a response to sexual selection (32). This idea is consistent with the fact that proteins such as ACPs interact directly with both sperm and with the female reproductive tract whereas sperm proteins may undergo less direct interaction. Thus, rapid molecular changes can occur in one without directly altering the regulation and developmental processes within the other. Mammals also produce similar secretions, semen, that contain a variety of proteins, which interact with sperm and many of these also show evidence of positive selection (46,47). Adaptive responses to sexual selection are an important element of the evolution of sexual systems and further analysis of the relationships between these two systems will provide a framework for how adaptive responses are realized within other complex biological structures and systems.


   RELATIONSHIP TO MAMMALIAN PROTEOMES
TOP
 ABSTRACT
 INTRODUCTION
 SPERM AS IDEAL CANDIDATES...
 THE DROSOPHILA SPERM PROTEOME
 EVOLUTIONARY ANALYSIS OF THE...
 RELATIONSHIP TO MAMMALIAN...
CONCLUSION
 REFERENCES
 
Identification of genes orthologous to the DmSP will be useful for studies on sperm function in mammals and other taxa. These comparative proteomic analyses will provide deeper understanding of sperm function by providing parallel genetic systems. For example, Steve Dorus at the University of Bath is currently analyzing a number of genes from the mouse sperm proteome based in part on orthologous sequences in the DmSP. These studies will identify target genes for genetic and functional analysis in both Drosophila and the mouse. A longer term goal will be to extend these approaches to the study of human fertility and the identification of sperm factors responsible for male infertility. The DmSP has already provided one candidate for which orthologues exist in both the mouse and human, the gene Growth arrest-specific 8 (Gas8), which has been implicated in spermatid growth arrest and sperm motility in mammals (48). Drosophila Gas8 may serve analogous functions in Drosophila sperm motility and analysis of Gas8 may provide relevant functional data concerning its role in mammals and other taxa.

The utility of a comparative approach was further demonstrated by comparison of the DmSP to the recently described mouse axoneme accessory structure (49). Significant homology was found in greater than 40% of the comparisons between DmSP genes and genes that comprise this structure (Table 2). High levels of similarity were observed for various structural proteins, including the tektins, and a diverse set of metabolic proteins. It seems reasonable to expect similar relationships will be found as more extensive comparative work progresses with an ever expanding diversity of sperm proteomes.


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  		Table 2. Homologous mouse sperm flagellum accessory structure proteins

 

   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 SPERM AS IDEAL CANDIDATES...
 THE DROSOPHILA SPERM PROTEOME
 EVOLUTIONARY ANALYSIS OF THE...
 RELATIONSHIP TO MAMMALIAN...
 CONCLUSION
REFERENCES
 
Clearly a universal feature of sperm function is to deliver the paternal genetic heritage to the egg. But as shown dramatically in Figure 1 in D. melanogaster and throughout the genus Drosophila (1,50,51) even sperm of gigantic proportions can completely enter the egg at fertilization. This appears now a general rule in animal fertilization as incorporation of sperm tails has been observed in the mouse (52) and other insects including beetles (3) and has led to renewed appreciation of the relevance of sperm–egg interactions both at the surface of gametes and within the egg during and following fertilization. These observations raise the obvious question – why incorporate such a huge structure into the egg? Such enormous products and their by-products must be managed and eliminated properly and could place a considerable burden on the developing egg. Indeed Drosophila sperm by-products are present in larval midguts as intact remnants of the sperm axoneme (53). These findings challenge our concepts about the overall role of sperm in fertilization, but also points to exciting new discoveries that await developmental, evolutionary and reproductive biologists interested sperm function.

Whole sperm proteomics should be applicable to other systems where pure sperm can be obtained, free from contaminating seminal fluid products and modifications brought on by physiological activation of sperm. Although clearly in its infancy, future studies of sperm proteomes in related mammalian and human systems will determine how useful the DmSP will be in furthering our understanding of fertility.


   ACKNOWLEDGEMENTS
 
I am very grateful to S. Dorus for collaboration on the sperm proteome project. Appreciation is also extended to U. Gerike for proteomic analyses and to K. Steeds for expert technical support. The contributions and discussions with C. Bergman, particularly in regard to the ‘last orders at the bar’ ruminations, are particularly appreciated. Funding for this work has been supported by the BBSRC, Royal Society and the NIH.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SPERM AS IDEAL CANDIDATES...
 THE DROSOPHILA SPERM PROTEOME
 EVOLUTIONARY ANALYSIS OF THE...
 RELATIONSHIP TO MAMMALIAN...
 CONCLUSION
 REFERENCES
 

  1. Karr T.L. Intracellular sperm/egg interactions in Drosophila: a three-dimensional structural analysis of a paternal product in the developing egg. Mech. Dev. (1991) 34:101–111.[CrossRef][Web of Science][Medline]

  2. Karr T.L., Pitnick S. The ins and outs of fertilization. Nature (1996) 379:405–406.[CrossRef][Medline]

  3. Loppin B., Karr T.L. Molecular genetics of insect fertilization. In: Comprehensive Insect Molecular Science—Gilbert L.B., Iatrou K, eds. (2004) Elsevier: Oxford.

  4. Gilbert S. Developmental Biology (2006) Sunderland: Sinauer Associates, Inc.

  5. Sutovsky P., Schatten G. Paternal contributions to the mammalian zygote: fertilization after sperm–egg fusion. Int. Rev. Cytol. (2000) 195:1–65.[Web of Science][Medline]

  6. Ostermeier G.C., Goodrich R.J., Moldenhauer J.S., Diamond M.P., Krawetz S.A. A suite of novel human spermatozoal RNAs. J. Androl. (2005) 26:70–74.[Abstract/Free Full Text]

  7. Ostermeier G.C., Miller D., Huntriss J.D., Diamond M.P., Krawetz S.A. Reproductive biology: delivering spermatozoan RNA to the oocyte. Nature (2004) 429:154.[Medline]

  8. Chow V., Cheung A.P. Male infertility. J. Reprod. Med. (2006) 51:149–156.[Web of Science][Medline]

  9. Lefievre L., Barratt C.L., Harper C.V., Conner S.J., Flesch F.M., Deeks E., Moseley F.L., Pixton K.L., Brewis I.A., Publicover S.J. Physiological and proteomic approaches to studying prefertilization events in the human. Reprod. Biomed. Online (2003) 7:419–427.[Medline]

  10. Pixton K.L., Deeks E.D., Flesch F.M., Moseley F.L., Bjorndahl L., Ashton P.R., Barratt C.L., Brewis I.A. Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: case report. Hum. Reprod. (2004) 19:1438–1447.[Abstract/Free Full Text]

  11. Ferlin A., Raicu F., Gatta V., Zuccarello D., Palka G., Foresta C. Male infertility: role of genetic background. Reprod. Biomed. Online (2007) 14:734–745.[Web of Science][Medline]

  12. Matzuk M.M., Lamb D.J. Genetic dissection of mammalian fertility pathways. Nat. Cell. Biol. (2002) 4(suppl.):s41–s49.[Web of Science][Medline]

  13. Rajkovic A., Matzuk M.M. Functional analysis of oocyte-expressed genes using transgenic models. Mol. Cell. Endocrinol. (2002) 187:5–9.[CrossRef][Web of Science][Medline]

  14. Chu D.S., Liu H., Nix P., Wu T.F., Ralston E.J., Yates J.R., Meyer B.J. Sperm chromatin proteomics identifies evolutionarily conserved fertility factors. Nature (2006) 443:101–105.[CrossRef][Medline]

  15. Loppin B., Lepetit D., Dorus S., Couble P., Karr T.L. Origin and neofunctionalization of a Drosophila paternal effect gene essential for zygote viability. Curr. Biol. (2005) 15:87–93.[CrossRef][Web of Science][Medline]

  16. Ohsako T., Hirai K., Yamamoto M.T. The Drosophila misfire gene has an essential role in sperm activation during fertilization. Genes Genet. Syst. (2003) 78:253–266.[CrossRef][Web of Science][Medline]

  17. Smith M.K., Wakimoto B.T. Complex regulation and multiple developmental functions of misfire, the Drosophila melanogaster ferlin gene. BMC Dev. Biol. (2007) 7:21.[CrossRef][Medline]

  18. Wilson K.L., Fitch K.R., Bafus B.T., Wakimoto B.T. Sperm plasma membrane breakdown during Drosophila fertilization requires Sneaky, an acrosomal membrane protein. Development (2006) 133:4871–4879.[Abstract/Free Full Text]

  19. Wakimoto B.T., Lindsley D.L., Herrera C. Toward a comprehensive genetic analysis of male fertility in Drosophila melanogaster. Genetics (2004) 167:207–216.[Abstract/Free Full Text]

  20. Swanson W.J., Vacquier V.D. The rapid evolution of reproductive proteins. Nat. Rev. Genet. (2002) 3:137–144.[Web of Science][Medline]

  21. Bier E. Drosophila, the golden bug, emerges as a tool for human genetics. Nat. Rev. Genet. (2005) 6:9–23.[Web of Science][Medline]

  22. Greenspan R.J., Dierick H.A. Am not I a fly like thee?’ From genes in fruit flies to behavior in humans. Hum. Mol. Genet. (2004) 13(Spec No. 2):R267–R273.[Abstract/Free Full Text]

  23. Grigoriev A. On the number of protein–protein interactions in the yeast proteome. Nucleic Acids Res. (2003) 31:4157–4161.[Abstract/Free Full Text]

  24. Ahn N.G., Shabb J.B., Old W.M., Resing K.A. Achieving in-depth proteomics profiling by mass spectrometry. ACS Chem. Biol. (2007) 2:39–52.[CrossRef][Medline]

  25. Bantscheff M., Schirle M., Sweetman G., Rick J., Kuster B. Quantitative mass spectrometry in proteomics: a critical review. Anal. Bioanal. Chem (2007) doi:10.1007/s00216-007-1486-6.

  26. Bradshaw R.A., Burlingame A.L. From proteins to proteomics. IUBMB Life (2005) 57:267–272.[Web of Science][Medline]

  27. Koomen J.M., Li D., Xiao L.C., Liu T.C., Coombes K.R., Abbruzzese J., Kobayashi R. Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery. J. Proteome Res. (2005) 4:972–981.[CrossRef][Web of Science][Medline]

  28. Rabilloud T. Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics (2002) 2:3–10.[CrossRef][Web of Science][Medline]

  29. Rifai N., Gillette M.A., Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat. Biotechnol. (2006) 24:971–983.[CrossRef][Web of Science][Medline]

  30. Tchabo N.E., Liel M.S., Kohn E.C. Applying proteomics in clinical trials: assessing the potential and practical limitations in ovarian cancer. Am. J. Pharmacogenomics (2005) 5:141–148.[CrossRef][Web of Science][Medline]

  31. Naaby-Hansen S., Flickinger C.J., Herr J.C. Two-dimensional gel electrophoretic analysis of vectorially labeled surface proteins of human spermatozoa. Biol. Reprod. (1997) 56:771–787.[Abstract]

  32. Dorus S., Busby S.A., Gerike U., Shabanowitz J., Hunt D.F., Karr T.L. Genomic and functional evolution of the Drosophila melanogaster sperm proteome. Nat. Genet. (2006) 38:1440–1445.[CrossRef][Web of Science][Medline]

  33. Mitchell B.F., Pedersen L.B., Feely M., Rosenbaum J.L., Mitchell D.R. ATP production in Chlamydomonas reinhardtii flagella by glycolytic enzymes. Mol. Biol. Cell. (2005) 16:4509–4518.[Abstract/Free Full Text]

  34. Schulz C., Perezgasga L., Fuller M.T. Genetic analysis of dPsa, the Drosophila orthologue of puromycin-sensitive aminopeptidase, suggests redundancy of aminopeptidases. Dev. Genes Evol. (2001) 211:581–588.[CrossRef][Web of Science][Medline]

  35. Lyczak R., Zweier L., Group T., Murrow M.A., Snyder C., Kulovitz L., Beatty A., Smith K., Bowerman B. The puromycin-sensitive aminopeptidase PAM-1 is required for meiotic exit and anteroposterior polarity in the one-cell Caenorhabditis elegans embryo. Development (2006) 133:4281–4292.[Abstract/Free Full Text]

  36. Fernandez D., Valdivia A., Irazusta J., Ochoa C., Casis L. Peptidase activities in human semen. Peptides (2002) 23:461–468.[CrossRef][Web of Science][Medline]

  37. Togo T., Morisawa M. GPI-anchored aminopeptidase is involved in the acrosome reaction in sperm of the mussel mytilusedulis. Mol. Reprod. Dev. (2004) 67:465–471.[CrossRef][Web of Science][Medline]

  38. Parisi M., Nuttall R., Naiman D., Bouffard G., Malley J., Andrews J., Eastman S., Oliver B. Paucity of genes on the Drosophila X chromosome showing male-biased expression. Science (2003) 299:697–700.[Abstract/Free Full Text]

  39. Boutanaev A.M., Kalmykova A.I., Shevelyov Y.Y., Nurminsky D.I. Large clusters of co-expressed genes in the Drosophila genome. Nature (2002) 420:666–669.[CrossRef][Medline]

  40. Crosby M.A., Goodman J.L., Strelets V.B., Zhang P., Gelbart W.M. FlyBase: genomes by the dozen. Nucleic Acids Res. (2007) 35:D486–D491.[Abstract/Free Full Text]

  41. Heifetz Y., Wolfner M.F. Mating, seminal fluid components, and sperm cause changes in vesicle release in the Drosophila female reproductive tract. Proc. Natl Acad. Sci. USA (2004) 101:6261–6266.[Abstract/Free Full Text]

  42. Kubli E. Sex-peptides: seminal peptides of the Drosophila male. Cell. Mol. Life Sci. (2003) 60:1689–1704.[CrossRef][Web of Science][Medline]

  43. Peng J., Chen S., Busser S., Liu H., Honegger T., Kubli E. Gradual release of sperm bound sex-peptide controls female postmating behavior in Drosophila. Curr. Biol. (2005) 15:207–213.[CrossRef][Web of Science][Medline]

  44. Mueller J.L., Ravi Ram K., McGraw L.A., Bloch Qazi M.C., Siggia E.D., Clark A.G., Aquadro C.F., Wolfner M.F. Cross-species comparison of Drosophila male accessory gland protein genes. Genetics (2005) 171:131–143.[Abstract/Free Full Text]

  45. Swanson W.J., Clark A.G., Waldrip-Dail H.M., Wolfner M.F., Aquadro C.F. Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila. Proc. Natl Acad. Sci. USA (2001) 98:7375–7379.[Abstract/Free Full Text]

  46. Dorus S., Evans P.D., Wyckoff G.J., Choi S.S., Lahn B.T. Rate of molecular evolution of the seminal protein gene SEMG2 correlates with levels of female promiscuity. Nat. Genet. (2004) 36:1326–1329.[CrossRef][Web of Science][Medline]

  47. Swanson W.J., Nielsen R., Yang Q. Pervasive adaptive evolution in mammalian fertilization proteins. Mol. Biol. Evol. (2003) 20:18–20.[Abstract/Free Full Text]

  48. Yeh S.D., Chen Y.J., Chang A.C., Ray R., She B.R., Lee W.S., Chiang H.S., Cohen S.N., Lin-Chao S. Isolation and properties of Gas8, a growth arrest-specific gene regulated during male gametogenesis to produce a protein associated with the sperm motility apparatus. J. Biol. Chem. (2002) 277:6311–6317.[Abstract/Free Full Text]

  49. Cao W., Gerton G.L., Moss S.B. Proteomic profiling of accessory structures from the mouse sperm flagellum. Mol. Cell. Proteomics (2006) 5:801–810.[Abstract/Free Full Text]

  50. Karr T.L. Paternal investment and intracellular sperm–egg interactions during and following fertilization in Drosophila. Curr. Top. Dev. Biol. (1996) 34:89–115.[Web of Science][Medline]

  51. Snook R.R., Karr T.L. Only long sperm are fertilization-competent in six sperm-heteromorphic Drosophila species. Curr. Biol. (1998) 8:291–294.[CrossRef][Web of Science][Medline]

  52. Simerly C.R., Hecht N.B., Goldberg E., Schatten G. Tracing the incorporation of the sperm tail in the mouse zygote and early embryo using an anti-testicular alpha-tubulin antibody. Dev. Biol. (1993) 158:536–548.[CrossRef][Web of Science][Medline]

  53. Pitnick S., Karr T.L. Paternal products and by-products in Drosophila development. Proc. Biol. Sci. (1998) 265:821–826.[Abstract/Free Full Text]


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