Cell type-specific gene expression in the Drosophila melanogaster male accessory gland.

The accessory gland of the male Drosophila melanogaster plays a vital role in reproduction. This secretory organ synthesizes products that are transferred to the female and are necessary to elicit the proper physiological and behavioral responses in the female. The accessory gland is composed of two morphologically distinct secretory cell types, the main cells and the secondary cells. Previous studies identified some genes expressed in main cells or in all accessory gland cells. In this paper we use P-element mediated enhancer traps to examine gene expression in the accessory gland. We show that, in addition to genes expressed in main cells only or in all accessory gland secretory cells, there are genes expressed specifically in secondary cells. Each cell type is uniform in the expression of its genes. Our results demonstrate that the two cell types are not only morphologically distinct but also biochemically distinct. We also show that the two cell types differ in their regulation of gene expression in response to mating activity.     

Mating and hormonal triggers regulate accessory gland gene expression in male Drosophila

Drosophila melanogaster males transfer accessory gland proteins, as part of their seminal fluid, to females during each mating. Since accessory gland proteins are important for male reproductive success, it is important that the male replenish the proteins he transferred during mating. Previous studies had shown that mating induces the resynthesis of accessory gland proteins, but since mating includes a set of stereotyped behavior patterns as well as the act of copulation, it was not known which aspect of the mating process induces accessory gland protein synthesis. By exposing males to females whose ovipositors had been sealed shut, we have shown that resynthesis of accessory gland proteins occurs only when seminal fluid is transferred to females. By applying juvenile hormone or 20-hydroxyecdysone topically to the cuticle of male flies, we showed that these hormones can act in vivo to stimulate the synthesis of accessory gland proteins to levels similar to those observed after mating.     

Fates and targets of male accessory gland proteins in mated female Drosophila melanogaster

Male accessory gland proteins (Acps) in Drosophila are components of the seminal fluid and are transferred to females during copulation. In mated females, Acps enhance egg production, augment sperm storage, induce refractory mating behaviors, and affect the female's longevity. To address the functions of eight previously uncharacterized Acps and further analyze five others, we determined the tissues to which they target after transfer to females. Each Acp has multiple targets and is unique in its pattern of localization. Within the reproductive tract, Acps target to the uterus, oviduct, sperm storage organs, ovary and oocytes. Some Acps also leave the reproductive tract, to enter the hemolymph. Some Acps are detected on the surface of eggs laid by mated females but were not detectable within those eggs. Our results can help to identify the likely functions of these Acps as well as to create models for the mechanism of action of Acps.     

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.     

Cross-species comparison of Drosophila male accessory gland protein genes

Drosophila melanogaster males transfer seminal fluid proteins along with sperm during mating. Among these proteins, ACPs (Accessory gland proteins) from the male's accessory gland induce behavioral, physiological, and life span reduction in mated females and mediate sperm storage and utilization. A previous evolutionary EST screen in D. simulans identified partial cDNAs for 57 new candidate ACPs. Here we report the annotation and confirmation of the corresponding Acp genes in D. melanogaster. Of 57 new candidate Acp genes previously reported in D. melanogaster, 34 conform to our more stringent criteria for encoding putative male accessory gland extracellular proteins, thus bringing the total number of ACPs identified to 52 (34 plus 18 previously identified). This comprehensive set of Acp genes allows us to dissect the patterns of evolutionary change in a suite of proteins from a single male-specific reproductive tissue. We used sequence-based analysis to examine codon bias, gene duplications, and levels of divergence (via dN/dS values and ortholog detection) of the 52 D. melanogaster ACPs in D. simulans, D. yakuba, and D. pseudoobscura. We show that 58% of the 52 D. melanogaster Acp genes are detectable in D. pseudoobscura. Sequence comparisons of ACPs shared and not shared between D. melanogaster and D. pseudoobscura show that there are separate classes undergoing distinctly dissimilar evolutionary dynamics.     

Molecular population genetics of male accessory gland proteins in Drosophila

Drosophila seminal proteins have an unusually high rate of molecular sequence evolution, suggesting either a high rate of neutral substitution or rapid adaptive evolution. To further quantify patterns of polymorphism and divergence in genes encoding seminal proteins, also called accessory gland proteins (Acp's), we conducted a sequencing survey of 10 Acp genes in samples of Drosophila melanogaster and D. simulans (Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp76A, Acp95EF, and Acp98AB). Mean heterozygosity at replacement sites in D. simulans was 0.0074 for Acp genes and 0.0013 for a set of 19 non-Acp genes, and mean melanogaster-simulans divergence at replacement sites was 0.0497 for Acp genes and 0.0107 at non-Acp genes. The elevated divergence of Acp genes is thus accompanied by elevated within-species polymorphism. In addition to the already-reported departures of Acp26A, Acp29AB, and Acp70A from neutrality, our data reject neutrality at Acp29AB and Acp36DE in the direction of excess replacements in interspecific comparisons.     

Structure and regulated expression of the delta-aminolevulinate synthase gene from Drosophila melanogaster

The structure of the single copy gene encoding the putative housekeeping isoform of Drosophila melanogaster delta-aminolevulinate synthase (ALAS) has been determined. Southern and immunoblot analyses suggest that only the housekeeping isoform of the enzyme exists in Drosophila. We have localized a critical region for promoter activity to a sequence of 121 base pairs that contains a motif that is potentially recognized by factors of the nuclear respiratory factor-1 (NRF-1)/P3A2 family, flanked by two AP4 sites. Heme inhibits the expression of the gene by blocking the interaction of putative regulatory proteins to its 5' proximal region, a mechanism different from those proposed for other hemin-regulated promoters. Northern and in situ RNA hybridization experiments show that maternal alas mRNA is stored in the egg; its steady-state level decreases rapidly during the first hours of development and increases again after gastrulation in a period where the synthesis of several mRNAs encoding metabolic enzymes is activated. In the syncytial blastoderm, the alas mRNA is ubiquitously distributed and decreases in abundance substantially through cellular blastoderm. Late in embryonic development alas shows a specific pattern of expression, with an elevated mRNA level in oenocytes, suggesting an important role of these cells in the biosynthesis of hemoproteins in Drosophila.     

Gene expression changes in male accessory glands during ageing are accompanied by reproductive decline in Drosophila melanogaster

Senescence is accompanied by loss of reproductive functions. Here, we studied reproductive ageing in Drosophila melanogaster males and asked whether the expected decline in male reproductive success is due to diminished functionality of the male accessory gland (AG). The male AG produces the majority of seminal fluid proteins (SFPs) transferred to the female at mating. SFPs induce female postmating changes and are key to male reproductive success. We measured age‐dependent gene expression changes for five representative SFP genes in males from four different age groups ranging from 1 to 6 weeks after eclosion. Simultaneously, we also measured male reproductive success in postmating traits mediated by transfer of these five SFPs. We found a decreased in male SFP gene expression with advancing age and an accompanying decline in male postmating success. Hence, male reproductive senescence is associated with a decline in functionality of the male AG. While overall individual SFP genes decreased in expression, our results point towards the idea that the composition of an ejaculate might change with male age as the rate of change was variable for those five genes.     

Quantitative evolutionary genomics: differential gene expression and male reproductive success in Drosophila melanogaster

We combined traditional quantitative genetics and oligonucleotide microarrays to examine within-population genetic variation in a trait closely related to fitness. The trait, male reproductive success under competitive conditions (MCRS), is of central importance to both life-history and sexual-selection theory. We identified 27 candidate genes whose expression levels were associated with within-population variation in MCRS. "High" MCRS was associated with low expression of a cytochrome P450 that causes pesticide resistance, suggesting a fitness cost to resistance. Two groups of metabolic proteins (glutathione transferases and phosphatases) were significantly over-represented, and a large portion of the candidates are genes involved in oxidative stress resistance, energy acquisition or energy storage. Genes expressed in accessory glands and testes were not over-represented among differentially expressed genes, but testis-expressed genes were significantly more likely to be upregulated in high MCRS genotypes. Finally, nine candidate genes that we identified had no previous functional annotation, and this experiment suggests that they play a role in male reproductive success.