Functional Diversity and Evolution of the Drosophila Sperm Proteome

Spermatozoa are central to fertilization and the evolutionary fitness of sexually reproducing organisms. As such, a deeper understanding of sperm proteomes (and associated reproductive tissues) has proven critical to the advancement of the fields of sexual selection and reproductive biology. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. Here, we provide an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins, and we provide the first label-free quantitation of the sperm proteome for 2125 proteins. The top 20 most abundant proteins included the structural elements α- and β-tubulins and sperm leucyl-aminopeptidases. Both gene content and protein abundance were significantly reduced on the X chromosome, consistent with prior genomic studies of X chromosome evolution. We identified 9 of the 16 Y-linked proteins, including known testis-specific male fertility factors. We also identified almost one-half of known Drosophila ribosomal proteins in the DmSP3. The role of this subset of ribosomal proteins in sperm is unknown. Surprisingly, our expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins originally identified in the accessory glands. We show that a significant fraction of ‘sperm-associated Sfps’ are recalcitrant to concentrated salt and detergent treatments, suggesting this subclass of Sfps are expressed in testes and may have additional functions in sperm, per se. Overall, our results add to a growing landscape of both sperm and seminal fluid protein biology and in particular provides quantitative evidence at the protein level for prior findings supporting the meiotic sex-chromosome inactivation model for male-specific gene and X chromosome evolution.     

Proteins within the seminal fluid are crucial to keep sperm viable in the honeybee Apis mellifera

Seminal fluid is a biochemically complex mixture of glandular secretions that is transferred to the females sexual tract as part of the ejaculate. Seminal fluid has received increasing scientific interest in the fields of evolutionary and reproductive biology, as it seems a major determinant of male fertility/infertility and reproductive success. Here we used the honeybee Apis mellifera, where seminal fluid can be collected as part of a male's ejaculate, and performed a series of experiments to investigate the effects of seminal fluid and its components on sperm viability. We show that honeybee seminal fluid is highly potent in keeping sperm alive and this positive effect is present over a 24 h time span, comparable to the timing of the sperm storage process in the queen. We furthermore show that the presence of proteins within the seminal fluid and their structural integrity are crucial for this effect. Finally, we activated sperm using fructose and provide evidence that the positive effect of seminal fluid proteins on sperm survival cannot be replicated using generic protein substitutes. Our data provide experimental insights into the complex molecular interplay between sperm and seminal fluid defining male fertility and reproductive success.     

Molecular evolutionary analysis of seminal receptacle sperm storage organ genes of Drosophila melanogaster

Sperm storage organs are common and broadly distributed among animal taxa. However, little is known about how these organs function at the molecular level. Additionally, there is a paucity of knowledge about the evolution of genes expressed in these organs. This investigation is an evolutionary expressed sequence tag (EST) study of genes expressed in the seminal receptacle, one of the sperm storage organs in Drosophila. The incidence of positive selection is higher for the seminal receptacle genes than Drosophila reproductive genes as a whole, but lower than genes associated with the spermatheca, a second type of Drosophila sperm storage organ. By identifying overrepresented classes of proteins and classes for which sperm storage function is suggested by the nature of the proteins, candidate genes were discovered. These candidates belong to protein classes such as muscle contraction, odorant binding and odorant receptor, protease inhibitor and immunity.     

Proteome mapping of the Drosophila melanogaster male reproductive system

The fruit fly Drosophila melanogaster is an excellent model organism for studying insect reproductive biology. Although the gene expression profiles of both male and female reproductive organs have been studied in detail, their proteomic profiles and functional characteristics largely remained to be clarified. In this study, we conducted proteome mapping of the male internal reproductive organs using 2‐DE. We identified a total of 440 protein components from gels of the male reproductive organs (testis, seminal vesicle, accessory gland, ejaculatory duct, and ejaculatory bulb). A number of proteins associated with odorant/pheromone‐binding, lipid metabolism, proteolysis, and antioxidation were expressed tissue specifically in the male reproductive system. Based on our proteomic data set, we constructed reference proteome maps of the reproductive organs, which will provide valuable information toward a comprehensive understanding of Drosophila reproduction.     

Honey bee males and queens use glandular secretions to enhance sperm viability before and after storage

Internal fertilization requires live sperm to be transferred from male to female before egg fertilization. Both males and females assist the insemination process by providing sperm with glandular secretions, which have been inferred to contain subsets of proteins that maintain sperm viability. Here we show that in the honeybee (Apis mellifera) secretions of the male accessory glands, the major contributors towards seminal fluid, enhance sperm survival. We further demonstrate that the protein fraction of the male accessory gland secretion is indeed important for achieving the maximal effect on sperm survival. After sperm storage, the queens also provide sperm with secretions from spermathecal glands and we show that these secretions have a comparable positive effect on sperm viability. SDS gels show that the proteomic profiles of accessory gland secretion and spermathecal fluid secretion hardly overlap, which suggests that males and females use different proteins to enhance sperm viability during, respectively, ejaculation and final sperm storage.     

Proteomic analysis of Drosophila mojavensis male accessory glands suggests novel classes of seminal fluid proteins

Fruit-flies of the genus Drosophila are characterized by overwhelming variation in fertilization traits such as copulatory plug formation, sperm storage organ use, and nutritional ejaculatory donation. Despite extensive research on the genetic model Drosophila melanogaster, little is known about the molecular underpinnings of these interspecific differences. This study employs a proteomic approach to pin-point candidate seminal fluid proteins in Drosophila mojavensis, a cactophilic fruit-fly that exhibits divergent reproductive biology when compared to D. melanogaster. We identify several classes of candidate seminal fluid proteins not previously documented in the D. melanogaster male ejaculate, including metabolic enzymes, nutrient transport proteins, and clotting factors. Conversely, we also define 29 SFPs that are conserved despite >40 million years of Drosophila evolution. We discuss our results in terms of universal processes in insect reproduction, as well as the specialized reproductive biology of D. mojavensis.     

Identification of major royal jelly proteins in the brain of the honeybee Apis mellifera

The consumption of royal jelly (RJ) determines the differences between castes and behavioral development in the honeybee Apis mellifera. However, it is not known whether the proteins of RJ are related to these differences, or which proteins are responsible for the changes. To understand the functions of RJ proteins that are present in other tissues of the bee, in addition to hypopharyngeal gland, we used a polyclonal antibody anti-MRJP1 to investigate the presence of this protein in nervous system of honeybee. This study showed the presence of three polypeptides (p57, p70 and p128) in specific tissues of bee brain. Mushroom body, optic lobe and antennal lobe neuropils all contained proteins recognized by anti-MRJP1. Proteomic analysis showed that the three polypeptides are correlated with proteins of the MRJP family. p57 is correlated with MRJP1, p70 with MRJP3, while p128 may be an oligomeric form or a new polypeptide. Immunostaining of the brain and hypopharyngeal gland revealed differential expression of MRJPs in various brain regions and in different honeybee castes and subcastes. The identification and localization of these MRJPs contribute to the elucidation of the biological roles of this protein family.     

Proteome and phosphoproteome of Africanized and European honeybee venoms

Honey bee venom toxins trigger immunological, physiological, and neurological responses within victims. The high occurrence of bee attacks involving potentially fatal toxic and allergic reactions in humans and the prospect of developing novel pharmaceuticals make honey bee venom an attractive target for proteomic studies. Using label‐free quantification, we compared the proteome and phosphoproteome of the venom of Africanized honeybees with that of two European subspecies, namely Apis mellifera ligustica and A. m. carnica. From the total of 51 proteins, 42 were common to all three subspecies. Remarkably, the toxins melittin and icarapin were phosphorylated. In all venoms, icarapin was phosphorylated at the ²⁰⁵Ser residue, which is located in close proximity to its known antigenic site. Melittin, the major toxin of honeybee venoms, was phosphorylated in all venoms at the ¹⁰Thr and ¹⁸Ser residues. ¹⁸Ser phosphorylated melittin—the major of its two phosphorylated forms—was less toxic compared to the native peptide.     

The seminal vesicle of the African catfish,Clarias gariepinus

Summary The seminal vesicle of the African catfish, Clarias gariepinus, consists of 36–44 fingerlike lobes built up of tubules in which a fluid is secreted containing acid polysaccharides, acid-, neutral- and basic proteins, and phospholipids. In this fluid sperm cells are stored. The seminal vesicle fluid immobilizes the sperm cells. After ejaculation, it prolongs the period of sperm activity. The seminal vesicle fluid is secreted by the epithelium lining the tubules. The tubules in the proximal part of the lobes are predominantly lined by a simple cylindrical and those of the distal part by a simple squamous epithelium. These epithelial cells contain enzymes involved in energy-liberating processes, the enzyme activites being proportional to the height of the cells. Interstitial cells between the tubules have enzyme-histochemical and ultrastructural features indicative of steroid biosynthesis. Similar characteristics are found in testicular interstitial cells. The most rostral seminal vesicle lobes and the most caudal testicular efferent tubules form a network of tubules that opens at the point where the paired parts of the sperm ducts fuse with each other. The tubules of most seminal vesicle lobes, however, form a complex system that fuses with the unpaired part of the sperm duct.     

Stored sperm differs from ejaculated sperm by proteome alterations associated with energy metabolism in the honeybee Apis mellifera

Sperm are exposed to substantially different environments during their life history, such as seminal fluid or the female sexual tract, but remarkably little information is currently available about whether and how much sperm composition and function alters in these different environments. Here, we used the honeybee Apis mellifera and quantified differences in the abundance and activity of sperm proteins sampled either from ejaculates or from the female’s sperm storage organ. We find that stored and ejaculated sperm contain the same set of proteins but that the abundance of specific proteins differed substantially between ejaculated and stored sperm. Most proteins with a significant change in abundance are related to sperm energy metabolism. Enzymatic assays performed for a subset of these proteins indicate that specific protein activities differ between stored and ejaculated sperm and are typically higher in ejaculated compared to stored sperm. We provide evidence that the cellular machinery of sperm is plastic and differs between sperm within the ejaculate and within the female’s storage organ. Future work will be required to test whether these changes are a consequence of active adaptation or sperm senescence and whether they alter sperm performance indifferent chemical environments or impact on the cost of sperm storage by the female.However, these changes can be expected to influence sperm performance and therefore determine sperm viability or sperm competitiveness for storage or egg fertilization.     

Yolk protein is expressed in the insect testis and interacts with sperm

Background  

Male and female gametes follow diverse developmental pathways dictated by their distinct roles in fertilization. While oocytes of oviparous animals accumulate yolk in the cytoplasm, spermatozoa slough off most of their cytoplasm in the process of individualization. Mammalian spermatozoa released from the testis undergo extensive modifications in the seminal ducts involving a variety of glycoproteins. Ultrastructural studies suggest that glycoproteins are involved in sperm maturation in insects; however, their characterization at the molecular level is lacking. We reported previously that the circadian clock controls sperm release and maturation in several insect species. In the moth, Spodoptera littoralis, the secretion of glycoproteins into the seminal fluid occurs in a daily rhythmic pattern. The purpose of this study was to characterize seminal fluid glycoproteins in this species and elucidate their role in the process of sperm maturation.     

The Hemolymph Proteome of Fed and Starved Drosophila Larvae

The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.     

Proteomic identification of rainbow trout seminal plasma proteins

In the study, the combination of protein fractionation by 1DE and HPLC‐ESI‐MS/MS was used to characterize the rainbow trout seminal plasma proteome. Our results led to the creation of a catalogue of rainbow trout seminal plasma proteins (152 proteins) and significantly contributed to the current knowledge regarding the protein composition of fish seminal plasma. The major proteins of rainbow trout seminal plasma, such as transferrin, apolipoproteins, complement C3, serum albumin, and hemopexin‐, alpha‐1‐antiproteinase‐, and precerebellin‐like protein, were recognized as acute‐phase proteins (proteins that plasma concentration changes in response to inflammation). This study provides the basis for further functional studies of fish seminal plasma proteins, as well as for the identification of novel biomarkers for sperm quality. The MS data have been deposited in the ProteomeXchange with identifier PXD000306 ( http://proteomecentral.proteomexchange.org/dataset/PXD000306 ).     

Social behavior and the evolution of neuropeptide genes: lessons from the honeybee genome

Honeybees display a fascinating social behavior. The structural basis for this behavior, which made the bee a model organism for the study of communication, learning and memory formation, is the tiny insect brain. Neurons of the brain communicate via messenger molecules. Among these molecules, neuropeptides represent the structurally most‐diverse group and occupy a high hierarchic position in the modulation of behavior. A recent analysis of the honeybee genome revealed a considerable number of predicted (200) and confirmed (100) neuropeptides in this insect. 1 Is this quantity merely the result of advanced mass spectrometric techniques and bioinformatic tools or does it reflect the expression of more of these important messenger molecules, more than known from other insects studied so far? Our analysis of the data suggests that the social behavior is by no means correlated with a specific increase in the number of neuropeptides. Indeed, the honeybee genome is likely to contain fewer neuropeptide genes, neuropeptide paralogues and neuropeptide receptor genes than the solitary fruitfly Drosophila. BioEssays 29:416–421, 2007. © 2007 Wiley Periodicals, Inc.     

Low molecular weight factor in bovine caudal epididymal fluid that stimulates calcium uptake in caput spermatozoa

Secretions from the mammalian epididymis contain proteins that bind to developing sperm and are presumed to play a role in sperm maturation. The biochemical functions in sperm of most of these proteins are not known. In this report we describe the presence of a low molecular weight compound in bovine caudal epididymal luminal fluid (CF) that has a potent stimulatory effect on calcium (⁴⁵Ca²⁺) uptake in immature caput epididymal spermatozoa. The studies were initially undertaken to characterize the effect of the protein caltrin, present in bovine seminal plasma (BSP), on calcium uptake into caput spermatozoa. Caltrin is known to block calcium influx into mature bovine sperm. Unexpectedly, the kinetics of calcium uptake into caput sperm showed a biphasic response when treated with BSP, namely, a stimulation of uptake at 1 to 5 min and inhibition of uptake after this time. Since caudal sperm do not show this biphasic response, we reasoned that BSP contained a factor derived from CF that must interact with developing sperm before the binding of caltrin to sperm can prevent further calcium uptake. We first demonstrated that preincubation of caput sperm with CF eliminated the biphasic calcium uptake effect induced in caput sperm by BSP and that caudal fluid alone had a potent stimulatory effect on calcium uptake in caput sperm. Half-maximal stimulation (fivefold over control) occurred at a caudal fluid protein concentration of 0.27 mg/ml. Partial purification of the factor indicates that it is of low molecular weight (MW ～ 1,000), but further chemical characterization has not been carried out and its epididymal site of origin is not known. The results indicate that the regulation of intracellular calcium levels in sperm differs in immature and mature bovine sperm in that an epididymal factor promotes calcium uptake during epididymal maturation, and the seminal fluid protein caltrin prevents it at ejaculation.     

Functions and analysis of the seminal fluid proteins of male Drosophila melanogaster fruit flies

The study of insect seminal fluid proteins provides a unique window upon adaptive evolution in action. The seminal fluid of Drosophila melanogaster contains over 80 proteins and peptides, which are transferred together with sperm by mating males. The functions of many of these substances are not yet known. However, those that have been characterized have marked effects on the reproductive success of males and females. For example, seminal fluid proteins and peptides can decrease female receptivity, can increase egg production and can increase sperm storage, and are necessary for sperm transfer and success in sperm competition. In this review we focus on the currently known functions of seminal fluid molecules and on new technologies and approaches that are enabling novel questions about their form and function to be addressed. We discuss how techniques for disrupting the production of seminal fluid proteins, such as homologous recombination and RNA interference, along with the use of microarrays and yeast two hybrid systems, should allow us to address ever more sophisticated questions about seminal fluid protein function. These and similar techniques promise to reveal the function of naturally-occurring variants of these proteins and hence the evolutionary significance of genetic variation for them.     

The Sperm Proteome of the Echiuran Urechis unicinctus (Annelida,Echiura)

Sperm proteins presumably play critical roles in reproduction, but in many non‐model animals their identities are unknown. A total of 147 sperm proteins from the echiuran worm Urechis unicinctus, the first sperm proteome in the phylum Annelida, are reported. The echiuran sperm proteome can be classified into diverse functional groups: energy metabolism (31%), protein synthesis and degradation (18%), spermatogenesis and sperm motility (12%), signal pathway (11%), ion channel and transport proteins (6%), cytoskeleton (4%), immunity and stress responses (3%), and fertilization (1%). These results will facilitate studies of mechanisms of fertilization in echiurans, as well as comparative studies of reproduction and evolution across lophotrochozoans. Data are available via ProteomeXchange with identifier PXD009176.     

In vitro evidence for the participation of Drosophila melanogaster sperm β‐N‐acetylglucosaminidases in the interactions with glycans carrying terminal N‐acetylglucosamine residues on the egg's envelopes

Fertilization is a complex and multiphasic process, consisting of several steps, where egg‐coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm‐associated β‐N‐acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid‐phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well‐defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β‐N‐acetylglucosamine, but not core β‐N‐acetylglucosamine residues. The capacity of sperm β‐N‐acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β‐N‐acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β‐N‐acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm–egg interactions in Drosophila.     

Comparative proteomics reveals evidence for evolutionary diversification of rodent seminal fluid and its functional significance in sperm competition

During insemination, males of internally fertilizing speciestransfer a complex array of seminal fluid proteins to the femalereproductive tract. These proteins can have profound effectson female reproductive physiology and behavior and are thoughtto mediate postcopulatory sexual selection and intersexual conflict.Such selection may cause seminal fluid to evolve rapidly, withpotentially important consequences for speciation. Here we investigatethe evolution of seminal fluid proteins in a major mammalianradiation, the muroid rodents, by quantifying diversity in seminalfluid proteome composition for the first time across a broadrange of closely related species. Using comparative proteomicstechniques to identify and cross-match proteins, we demonstratethat rodent seminal fluid is highly diverse at the level ofboth proteomes and individual proteins. The striking interspecificheterogeneity in seminal fluid composition revealed by our surveyfar exceeds that seen in a second proteome of comparable complexity,skeletal muscle, indicating that the complement of proteinsexpressed in seminal fluid may be subject to rapid diversification.We further show that orthologous seminal fluid proteins exhibitsubstantial interspecific variation in molecular mass. Becausethis variation cannot be attributed to differential glycosylationor radical differences in termination sites, it is stronglysuggestive of rapid amino acid divergence. Sperm competitionis implicated in generating such divergence for at least onemajor seminal fluid protein in our study, SVS II, which is responsiblefor copulatory plug formation via transglutaminase-catalyzedcross-linking after insemination. We show that the molecularmass of SVS II is positively correlated with relative testissize across species, which could be explained by selection foran increased number of cross-linking sites involved in the formationof the copulatory plug under sperm competition.