The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary

Mating stimulates the rate of egg-laying by female insects. In Drosophila melanogaster this stimulation is initially caused by seminal fluid molecules transferred from the male (Acps or accessory gland proteins; reviewed in [1] [2] [3]). Egg-laying is a multi-step process. It begins with oocyte release by the ovaries, followed by egg movement down the oviducts and the deposition of eggs onto the substratum. Although two Acps are known to stimulate egg-laying [4] [5], they were detected by assays that do not discriminate between the steps of this process or allow examination of its earliest changes [4] [5] [6] [7]. To determine how egg-laying is regulated, we developed a generally applicable assay to separate the process into quantifiable steps, allowing us to assess the ovulation pattern and rate of egg movement. As the steps are interdependent yet potentially subject to independent controls, we determined the contribution of each step and effector independent of the others. We used a statistical method [8] [9] that separately considers and quantifies each 'path' to a common end. We found that the prohormone-like molecule Acp26Aa [5] [10] stimulates the first step in egg-laying - release of oocytes by the ovary. During mating, Acp26Aa begins to accumulate at the base of the ovaries, a position consistent with action on the ovarian musculature to mediate oocyte release. Understanding how individual Acps regulate egg-laying in fruitflies will help provide a full molecular picture of insects' prodigious fertility, of reproductive hormones, and of the roles of these rapidly evolving proteins [11] [12].     

The Drosophila melanogaster seminal fluid protein Acp62F is a protease inhibitor that is toxic upon ectopic expression.

Drosophila melanogaster seminal fluid proteins stimulate sperm storage and egg laying in the mated female but also cause a reduction in her life span. We report here that of eight Drosophila seminal fluid proteins (Acps) and one non-Acp tested, only Acp62F is toxic when ectopically expressed. Toxicity to preadult male or female Drosophila occurs upon one exposure, whereas multiple exposures are needed for toxicity to adult female flies. Of the Acp62F received by females during mating, approximately 10% enters the circulatory system while approximately 90% remains in the reproductive tract. We show that in the reproductive tract, Acp62F localizes to the lumen of the uterus and the female's sperm storage organs. Analysis of Acp62F's sequence, and biochemical assays, reveals that it encodes a trypsin inhibitor with sequence and structural similarities to extracellular serine protease inhibitors from the nematode Ascaris. In light of previous results demonstrating entry of Acp62F into the mated female's hemolymph, we propose that Acp62F is a candidate for a molecule to contribute to the Acp-dependent decrease in female life span. We propose that Acp62F's protease inhibitor activity exerts positive protective functions in the mated female's reproductive tract but that entry of a small amount of this protein into the female's hemolymph could contribute to the cost of mating.     

Mated Drosophila melanogaster females require a seminal fluid protein, Acp36DE, to store sperm efficiently.

Mated females of many animal species store sperm. Sperm storage profoundly influences the number, timing, and paternity of the female's progeny. To investigate mechanisms for sperm storage in Drosophila melanogaster, we generated and analyzed mutations in Acp36DE. Acp36DE is a male seminal fluid protein whose localization in mated females suggested a role in sperm storage. We report that male-derived Acp36DE is essential for efficient sperm storage by females. Acp36DE(1) (null) mutant males produced and transferred normal amounts of sperm and seminal fluid proteins. However, mates of Acp36DE(1) males stored only 15% as many sperm and produced 10% as many adult progeny as control-mated females. Moreover, without Acp36DE, mated females failed to maintain an elevated egg-laying rate and decreased receptivity, behaviors whose persistence (but not initiation) normally depends on the presence of stored sperm. Previous studies suggested that a barrier in the oviduct confines sperm and Acp36DE to a limited area near the storage organs. We show that Acp36DE is not required for barrier formation, but both Acp36DE and the barrier are required for maximal sperm storage. Acp36DE associates tightly with sperm. Our results indicate that Acp36DE is essential for the initial storage of sperm, and that it may also influence the arrangement and retention of stored sperm.     

A role for Acp29AB, a predicted seminal fluid lectin, in female sperm storage in Drosophila melanogaster

Females of many animal species store sperm for taxon-specific periods of time, ranging from a few hours to years. Female sperm storage has important reproductive and evolutionary consequences, yet relatively little is known of its molecular basis. Here, we report the isolation of a loss-of-function mutation of the Drosophila melanogaster Acp29AB gene, which encodes a seminal fluid protein that is transferred from males to females during mating. Using this mutant, we show that Acp29AB is required for the normal maintenance of sperm in storage. Consistent with this role, Acp29AB localizes to female sperm storage organs following mating, although it does not appear to associate tightly with sperm. Acp29AB is a predicted lectin, suggesting that sugar–protein interactions may be important for D. melanogaster sperm storage, much as they are in many mammals. Previous association studies have found an effect of Acp29AB genotype on a male's sperm competitive ability; our findings suggest that effects on sperm storage may underlie these differences in sperm competition. Moreover, Acp29AB's effects on sperm storage and sperm competition may explain previously documented evidence for positive selection on the Acp29AB locus.     

Targeted gene deletion and phenotypic analysis of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F

Internally fertilizing organisms transfer a complex assortment of seminal fluid proteins, a substantial fraction of which are proteolysis regulators. In mammals, some seminal protease inhibitors have been implicated in male infertility and these same molecular classes of protease inhibitors are also found in Drosophila seminal fluid. Here, we tested the reproductive functions of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F by generating a precise deletion of the Acp62F gene. We did not detect a nonredundant function for Acp62F in modulating the egg laying, fertility, remating frequency, or life span of mated females. However, loss of Acp62F did alter a male's defensive sperm competitive ability, consistent with the localization of Acp62F to sperm storage organs. In addition, the processing of at least one seminal protein, the ovulation hormone ovulin, is slower in the absence of Acp62F.     

Sex in Drosophila mauritiana: a very high level of amino acid polymorphism in a male reproductive protein gene, Acp26Aa

Many genes pertaining to male reproductive functions have been shown to evolve rapidly between species, and evidence increasingly suggest the influence of positive Darwinian selection. The accessory gland protein gene (Acp26Aa) of Drosophila is one such example. In order to understand the mechanism of selection, it is often helpful to examine the pattern of polymorphism. We report here that the level of amino acid polymorphism in the N-terminal quarter of Acp26Aa is high in Drosophila melanogaster and is unprecedented in its sibling species Drosophila mauritiana. We postulate that (1) this N-terminal segment may play a role in sperm competition, and (2) D. mauritiana may have been under much more intense sexual selection than other species. Both postulates have important ramifications and deserve to be tested rigorously.     

Positive selection and the molecular evolution of a gene of male reproduction, Acp26Aa of Drosophila

The gene for a male ejaculatory protein, Acp26Aa, in four sibling species of the Drosophila melanogaster subgroup has previously been shown to have a nonsynonymous rate (Ka) of nucleotide substitution that is indistinguishable from the synonymous rate (Ks). By examining this gene in two other species of this subgroup, we found that Ka is generally large and can sometimes be more than twice as large as Ks. This suggests that positive selection may be operating at this locus of male reproduction.      

The role of male accessory gland protein Acp36DE in sperm competition in Drosophila melanogaster

A crucial factor determining sperm fertilization success in multiply mated Drosophila melanogaster females is the efficiency with which sperm are stored. This process is modulated by the accessory gland protein Acp36DE. In this study, we show that the effect of Acp36DE on sperm storage itself alters the outcome of sperm competition. As second-mating males, Acp36DE1 (null) males had significantly lower P2-values than Acp36DE2 (truncation) or Acp36DE+ (control) males, as might be expected as the null males' sperm are poorly stored. We used spermless males, which are null for Acp36DE, to show that, in the absence of sperm co-transfer, Acp36DE itself could not displace first-male sperm. The results therefore suggest that males null for Acp36DE suffer in sperm displacement because fewer sperm are stored or retained, not because Acp36DE itself displaces sperm. Acp36DE1 (null) males also gained significantly fewer fertilizations than controls when they were the first males to mate. Using spermless males, we also showed that significantly more second-male offspring were produced following the transfer of Acp36DE by spermless first-mating males. This implies that the transfer of Acp36DE itself by the first male facilitated the storage or use of the second male's sperm and that co-transfer with sperm is not necessary for Acp36DE effects on second-male sperm storage. Acp36DE may persist in the reproductive tract and aid the storage of any sperm including those of later-mating males or prime the female for future efficient sperm storage. Our results indicate that mutations in genes that affect sperm storage can drastically affect the outcome of sperm competition.     

Sustained post-mating response in Drosophila melanogaster requires multiple seminal fluid proteins

Successful reproduction is critical to pass genes to the next generation. Seminal proteins contribute to important reproductive processes that lead to fertilization in species ranging from insects to mammals. In Drosophila, the male's accessory gland is a source of seminal fluid proteins that affect the reproductive output of males and females by altering female post-mating behavior and physiology. Protein classes found in the seminal fluid of Drosophila are similar to those of other organisms, including mammals. By using RNA interference (RNAi) to knock down levels of individual accessory gland proteins (Acps), we investigated the role of 25 Acps in mediating three post-mating female responses: egg production, receptivity to remating and storage of sperm. We detected roles for five Acps in these post-mating responses. CG33943 is required for full stimulation of egg production on the first day after mating. Four other Acps (CG1652, CG1656, CG17575, and CG9997) appear to modulate the long-term response, which is the maintenance of post-mating behavior and physiological changes. The long-term post-mating response requires presence of sperm in storage and, until now, had been known to require only a single Acp. Here, we discovered several novel Acps together are required which together are required for sustained egg production, reduction in receptivity to remating of the mated female and for promotion of stored sperm release from the seminal receptacle. Our results also show that members of conserved protein classes found in seminal plasma from insects to mammals are essential for important reproductive processes.     

Male seminal fluid proteins are essential for sperm storage in Drosophila melanogaster.

The seminal fluid that is transferred along with sperm during mating acts in many ways to maximize a male's reproductive success. Here, we use transgenic Drosophila melanogaster males deficient in the seminal fluid proteins derived from the accessory gland (Acps) to investigate the role of these proteins in the fate of sperm transferred to females during mating. Competitive PCR assays were used to show that while Acps contribute to the efficiency of sperm transfer, they are not essential for the transfer of sperm to the female. In contrast, we found that Acps are essential for storage of sperm by females. Direct counts of stored sperm showed that 10% of normal levels are stored by females whose mates transfer little or no Acps along with sperm.     

Positive selection driving the evolution of a gene of male reproduction, Acp26Aa, of Drosophila: II. Divergence versus polymorphism

The evolution of the gene for a male ejaculatory protein, Acp26Aa, has been shown to be driven by positive selection when nonsibling species in the Drosophila melanogaster subgroup are compared. To know if selection has been operating in the recent past and to understand the details of its dynamics, we obtained DNA sequences of Acp26Aa and the nearby Acp26Ab gene from 39 D. melanogaster chromosomes. Together with the 10 published sequences, we analyzed 49 sequences from five populations in four continents. The southern African population is somewhat differentiated from all other populations, but its nucleotide diversity is lower at these two loci. We find the following results for Acp26Aa: (1) The R: S (replacement : silent changes) ratio is significantly higher in the between-species comparisons than in the within-species data by the McDonald and Kreitman test. Positive selection is probably responsible for the excess of amino acid replacements between species. (2) However, within-species nucleotide diversity is high. Neither the Tajima test nor the Fu and Li test indicates a reduction in nucleotide diversity due to positive selection in the recent past. (3) The newly derived nucleotides in D. melanogaster are at high frequency significantly more often than predicted by the neutral equilibrium. Since the nearby Acp26Ab gene does not show these patterns, these observations cannot be attributed to the characteristics of this chromosomal region. We suggest that positive selection is active, but may be weak, for each amino acid change in the Acp26Aa gene.      

The Drosophila melanogaster brainiac protein is a glycolipid-specific beta 1,3N-acetylglucosaminyltransferase

Mutations at the Drosophila melanogaster brainiac locus lead to defective formation of the follicular epithelium during oogenesis and to neural hyperplasia. The brainiac gene encodes a type II transmembrane protein structurally similar to mammalian beta1,3-glycosyltransferases. We have cloned the brainiac gene from D. melanogaster genomic DNA and expressed it as a FLAG-tagged recombinant protein in Sf9 insect cells. Glycosyltransferase assays showed that brainiac is capable of transferring N-acetylglucosamine (GlcNAc) to beta-linked mannose (Man), with a marked preference for the disaccharide Man(beta1,4)Glc, the core of arthro-series glycolipids. The activity of brainiac toward arthro-series glycolipids was confirmed by showing that the enzyme efficiently utilized glycolipids from insects as acceptors whereas it did not with glycolipids from mammalian cells. Methylation analysis of the brainiac reaction product revealed a beta1,3 linkage between GlcNAc and Man, proving that brainiac is a beta1,3GlcNAc-transferase. Human beta1,3GlcNAc-transferases structurally related to brainiac were unable to transfer GlcNAc to Man(beta1,4)Glc-based acceptor substrates and failed to rescue a homozygous lethal brainiac allele, indicating that these proteins are paralogous and not orthologous to brainiac.     

The Drosophila melanogaster seminal fluid protease "seminase" regulates proteolytic and post-mating reproductive processes

Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females.     

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 dorsal protein is distributed in a gradient in early Drosophila embryos

dorsal is one of the maternally active dorsal-ventral polarity genes of Drosophila and is closely related to the vertebrate proto-oncogene c-rel. Genetic experiments suggest that dorsal represents one of the last (if not the last) steps in the maternal pathway involved in establishing dorsal-ventral polarity in the early embryo. Even though the dorsal RNA is uniformly distributed in the embryo, we have found that the dorsal protein is specifically localized in peripheral nuclei of syncytial and cellular blastoderm stage embryos, and it is distributed in a ventral-to-dorsal gradient. These findings suggest possible mechanisms for how the dorsal protein may communicate maternal positional information to the zygotic genome.     

Dietary protein:carbohydrate balance is a critical modulator of lifespan and reproduction in Drosophila melanogaster: A test using a chemically defined diet

Macronutrient balance is an important determinant of fitness in many animals, including insects. Previous studies have shown that altering the concentrations of yeast and sugar in the semi-synthetic media has a profound impact on lifespan in Drosophila melanogaster, suggesting that dietary protein:carbohydrate (P:C) balance is the main driver of lifespan and ageing processes. However, since yeast is rich in multiple nutrients other than proteins, this lifespan-determining role of dietary P:C balance needs to be further substantiated through trials using a chemically-defined, synthetic diet. In the present study, the effects of dietary P:C balance on lifespan and fecundity were investigated in female D. melanogaster flies fed on one of eight isocaloric synthetic diets differing in P:C ratio (0:1, 1:16, 1:8, 1:4, 1:2, 1:1, 2:1 or 4:1). Lifespan and dietary P:C ratio were related in a convex manner, with lifespan increasing to a peak at the two intermediate P:C ratios (1:2 and 1:4) and falling at the imbalanced ratios (0:1 and 4:1). Ingesting nutritionally imbalanced diets not only caused an earlier onset of senescence but also accelerated the age-dependent increase in mortality. Egg production was suppressed when flies were fed on a protein-deficient food (0:1), but increased with increasing dietary P:C ratio. Long-lived flies at the intermediate P:C ratios (1:2 and 1:4) stored a greater amount of lipids than those short-lived ones at the two imbalanced ratios (0:1 and 4:1). These findings provide a strong support to the notion that adequate dietary P:C balance is crucial for extending lifespan in D. melanogaster and offer new insights into how dietary P:C balance affects lifespan and ageing through its impacts on body composition.     

The inturned protein of Drosophila melanogaster is a cytoplasmic protein located at the cell periphery in wing cells

The inturned (in) gene is a component of the frizzled (fz) signaling pathway that controls the polarity of hairs and bristles in the epidermis of Drosophila. It appears to act downstream of fz, which encodes a putative receptor for a tissue polarity signal. The in gene encodes a novel protein that had been suggested to contain two potential transmembrane domains. It has been suggested that the In protein interacts with the actin cytoskeleton to regulate the formation of the pupal wing prehairs that become adult hairs. The initiation of prehairs is normally restricted to the vicinity of the distal most vertex along the apical surface of the pupal wing cells. In an in mutant, prehairs initate at a variety of locations along the apical cell periphery. We have used immunofluorescence to study the subcellular localization of the In protein. When expressed in cultured cells, we found that In is a cytoplasmic protein. However, we found that it is localized in the vicinity of plasma membrane and the cortical actin cytoskeleton of Drosophila wing disc and pupal wing cells. Thus, in wing cells the In protein is localized to the region of the cell where it appears to function. This subcellular localization presumably requires the function of other proteins and may represent a regulatory mechanism. Our data suggest that fz does not play a major role in the subcellular localization of In. The In protein is notably insoluble in buffers containing high salt and nonionic detergents. This lack of solubility is significantly reduced in fz and mwh mutants, implying that it may be related to the mechanism of in function.     

The molecular genetics of early neurogenesis in Drosophila melanogaster

The extent of neurogenesis in Drosophila is under the control of the so-called neurogenic genes, named for their mutant phenotype of causing neural hyperplasia. Their wild-type products appear to be responsible for a signal chain that decides the fate of ectodermal cells in the embryo. Various kinds of data, from cell transplantation experiments as well as from genetic and molecular analyses, suggest that the proteins encoded by the genes Notch and Delta may act at the membrane of the signal-transmitting cells to provide a ligand to a still unknown receptor molecule; in contrast, the locus of Enhancer of split codes for several functions related to the transduction and further processing of the signal.