Drosophila sperm swim backwards in the female reproductive tract and are activated via TRPP2 ion channels

Background

Sperm have but one purpose, to fertilize an egg. In various species including Drosophila melanogaster female sperm storage is a necessary step in the reproductive process. Amo is a homolog of the human transient receptor potential channel TRPP2 (also known as PKD2), which is mutated in autosomal dominant polycystic kidney disease. In flies Amo is required for sperm storage. Drosophila males with Amo mutations produce motile sperm that are transferred to the uterus but they do not reach the female storage organs. Therefore Amo appears to be a mediator of directed sperm motility in the female reproductive tract but the underlying mechanism is unknown.

Methodology/Principal Findings

Amo exhibits a unique expression pattern during spermatogenesis. In spermatocytes, Amo is restricted to the endoplasmic reticulum (ER) whereas in mature sperm, Amo clusters at the distal tip of the sperm tail. Here we show that flagellar localization of Amo is required for sperm storage. This raised the question of how Amo at the rear end of sperm regulates forward movement into the storage organs. In order to address this question, we used in vivo imaging of dual labelled sperm to demonstrate that Drosophila sperm navigate backwards in the female reproductive tract. In addition, we show that sperm exhibit hyperactivation upon transfer to the uterus. Amo mutant sperm remain capable of reverse motility but fail to display hyperactivation and directed movement, suggesting that these functions are required for sperm storage in flies.

Conclusions/Significance

Amo is part of a signalling complex at the leading edge of the sperm tail that modulates flagellar beating and that guides a backwards path into the storage organs. Our data support an evolutionarily conserved role for TRPP2 channels in cilia.     

A flagellar polycystin-2 homolog required for male fertility in Drosophila

A common inherited cause of renal failure, autosomal dominant polycystic kidney disease results from mutations in either of two genes, PKD1 and PKD2, which encode polycystin-1 and polycystin-2, respectively. Polycystin-2 has distant homology to TRP cation channels and associates directly with polycystin-1. The normal functions of polycystins are poorly understood, although recent studies indicate that they are concentrated in the primary cilia of a variety of cell types. In this report we identified a polycystin-2 homolog in Drosophila melanogaster; this homolog localized to the distal tip of the sperm flagella. A targeted mutation in this gene, almost there (amo), caused nearly complete male sterility. The amo males produced and transferred normal amounts of motile sperm to females, but mutant sperm failed to enter the female sperm storage organs, a prerequisite for fertilization. The finding that Amo functions in sperm flagella supports a common and evolutionarily conserved role for polycystin-2 proteins in both motile and nonmotile axonemal-containing structures.     

Reduction of endoplasmic reticulum stress attenuates the defects caused by Drosophila mitofusin depletion

Ablation of the mitochondrial fusion and endoplasmic reticulum (ER)–tethering protein Mfn2 causes ER stress, but whether this is just an epiphenomenon of mitochondrial dysfunction or a contributor to the phenotypes in mitofusin (Mfn)-depleted Drosophila melanogaster is unclear. In this paper, we show that reduction of ER dysfunction ameliorates the functional and developmental defects of flies lacking the single Mfn mitochondrial assembly regulatory factor (Marf). Ubiquitous or neuron- and muscle-specific Marf ablation was lethal, altering mitochondrial and ER morphology and triggering ER stress that was conversely absent in flies lacking the fusion protein optic atrophy 1. Expression of Mfn2 and ER stress reduction in flies lacking Marf corrected ER shape, attenuating the developmental and motor defects. Thus, ER stress is a targetable pathogenetic component of the phenotypes caused by Drosophila Mfn ablation.     

Polycystin-1 distribution is modulated by polycystin-2 expression in mammalian cells

Mutations in PKD1 and PKD2, the genes that encode polycystin-1 and polycystin-2 respectively, account for almost all cases of autosomal dominant polycystic kidney disease. Although the polycystins are believed to interact in vivo, the two proteins often display dissimilar patterns and gradients of expression during development. In an effort to understand this apparent discrepancy, we investigated how changes in polycystin-2 expression can affect the subcellular localization of polycystin-1. We show that, when polycystin-1 is expressed alone in a PKD2 null cell line, it localizes to the cell surface, as well as to the endoplasmic reticulum. When co-expressed with polycystin-2, however, polycystin-1 is not seen at the cell surface and co-localizes completely with polycystin-2 in the endoplasmic reticulum. The localization of a polycystin-1 fusion protein was similarly affected by changes in its level of expression relative to that of polycystin-2. This phenomenon was observed in populations as well as in individual COS-7 cells. Our data suggest that the localization of polycystin-1 can be regulated via the relative expression level of polycystin-2 in mammalian cells. This mechanism may help to explain the divergent patterns and levels of expression observed for the polycystins, and may provide clues as to how the function of these two proteins are regulated during development.     

Spermatogenic cyst and organ culture in <Emphasis Type="Italic">Drosophila pseudoobscura</Emphasis>

Spermatogenesis is a complex series of processes that involves (1) the maintenance of a renewable pool of diploid stem cells within a niche, (2) the mitotic expansion of a subpopulation of stem cells committed to the spermatogenic pathway, and (3) the differentiation of diploid cells into highly specialized, haploid spermatozoa through meiotic and post-meiotic cellular transformations. Drosophila melanogaster is a desirable model for studying spermatogenesis, as similarities exist between mammalian and fly spermatogenesis. Like mammals, flies maintain a spermatogenic stem cell niche; the steps involved in mammalian spermatogenesis are mimicked in flies, with the main difference being that fly sperm develop within cysts rather than an epithelial cell layer. Here, we report a reliable robust system for culturing whole testes and individual spermatogenic cysts obtained from mid- to late-pupal stages of Drosophila pseudoobscura. D. pseudoobscura testes can be easily distinguished in later pupal stages because of their intense red pigmentation and are easily handled because of their simple ellipsoidal morphology. Cultured cysts are comparable in length to cysts obtained from adult flies, and motility is consistently achieved in vitro. This system not only offers a method for dissecting the mechanisms involved in meiotic and post-meiotic cellular transformations, but also can be used for the study of signaling during spermatogenesis.     

Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development

Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be substantially reduced by RNAi-mediated silencing of cathepsin L. In contrast to mammalian cells, lysosomal delivery of dCREG does not depend on its carbohydrate moiety. Furthermore, depletion of the putative D. melanogaster lysosomal sorting receptor lysosomal enzyme receptor protein did not compromise cellular retention of dCREG. We also investigated the developmental consequences of dCREG ablation in whole D. melanogaster flies. Ubiquitous depletion of dCREG proved lethal at the late pupal stage once a knock-down efficiency of > 95% was achieved. These results demonstrate that dCREG is essential for proper completion of fly development.     

Polycystins: what polycystic kidney disease tells us about sperm

Experimental evidence indicates that the membrane-associated proteins polycystin-1 and polycystin-2 operate as a receptor-calcium channel complex that regulates signaling pathways essential for modulation of renal tubulogenesis. Polycystic kidney disease is characterized by defective renal tubular structure and results from mutations in either PKD1 or PKD2 genes. Recent data suggest that polycystin-1 and polycystin-2 might localize to primary cilium in principal cells of renal collecting tubules and are thought to act as mechanosensors of fluid flow and contents. Ciliary bending by fluid flow or mechanical stimulation induce Ca(2+) release from intracellular stores, presumably to modulate ion influx in response to tubular fluid flow. Polycystins are also emerging as playing a significant role in sperm development and function. Drosophila polycystin-2 is associated with the head and tail of mature sperm. Targeted disruption of the PKD2 homolog results in nearly complete male sterility without disrupting spermatogenesis. Mutant sperm are motile but are unable to reach the female storage organs (seminal receptacles and spermathecae). The sea urchin polycystin-1-equivalent suPC2 colocalizes with the polycystin-1 homolog REJ3 to the plasma membrane over the acrosomal vesicle. This localization site suggests that the suPC2-REJ3 complex may function as a cation channel mediating acrosome reaction when sperm contact the jelly layer surrounding the egg at fertilization. Future studies leading to the identification of specific ligands for polycystins, including the signaling pathways, might define the puzzling relationship between renal tubular morphogenesis and sperm development and function.     

Partial ablation of adult Drosophila insulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance

In Drosophila melanogaster (D. melanogaster), neurosecretory insulin-like peptide-producing cells (IPCs), analogous to mammalian pancreatic β cells are involved in glucose homeostasis. Extending those findings, we have developed in the adult fly an oral glucose tolerance test and demonstrated that IPCs indeed are responsible for executing an acute glucose clearance response. To further develop D. melanogaster as a relevant system for studying age-associated metabolic disorders, we set out to determine the impact of adult-specific partial ablation of IPCs (IPC knockdown) on insulin-like peptide (ILP) action, metabolic outcomes and longevity. Interestingly, while IPC knockdown flies are hyperglycemic and glucose intolerant, these flies remain insulin sensitive as measured by peripheral glucose disposal upon insulin injection and serine phosphorylation of a key insulin-signaling molecule, Akt. Significant increases in stored glycogen and triglyceride levels as well as an elevated level of circulating lipid measured in adult IPC knockdown flies suggest profound modulation in energy metabolism. Additional physiological outcomes measured in those flies include increased resistance to starvation and impaired female fecundity. Finally, increased life span and decreased mortality rates measured in IPC knockdown flies demonstrate that it is possible to modulate ILP action in adult flies to achieve life span extension without insulin resistance. Taken together, we have established and validated an invertebrate genetic system to further investigate insulin action, metabolic homeostasis and regulation of aging regulated by adult IPCs.Key words: Drosophila melanogaster, insulin-producing cells (IPCs), drosophila insulin-like peptides (DILPs), type 2 diabetes, oral glucose tolerance test (OGTT), insulin sensitivity, energy metabolism, life span     

Drosophila Adiponectin Receptor in Insulin Producing Cells Regulates Glucose and Lipid Metabolism by Controlling Insulin Secretion

Adipokines secreted from adipose tissue are key regulators of metabolism in animals. Adiponectin, one of the adipokines, modulates pancreatic beta cell function to maintain energy homeostasis. Recently, significant conservation between Drosophila melanogaster and mammalian metabolism has been discovered. Drosophila insulin like peptides (Dilps) regulate energy metabolism similarly to mammalian insulin. However, in Drosophila, the regulatory mechanism of insulin producing cells (IPCs) by adipokine signaling is largely unknown. Here, we describe the discovery of the Drosophila adiponectin receptor and its function in IPCs. Drosophila adiponectin receptor (dAdipoR) has high homology with the human adiponectin receptor 1. The dAdipoR antibody staining revealed that dAdipoR was expressed in IPCs of larval and adult brains. IPC- specific dAdipoR inhibition (Dilp2>dAdipoR-Ri) showed the increased sugar level in the hemolymph and the elevated triglyceride level in whole body. Dilps mRNA levels in the Dilp2>dAdipoR-Ri flies were similar with those of controls. However, in the Dilp2>dAdipoR-Ri flies, Dilp2 protein was accumulated in IPCs, the level of circulating Dilp2 was decreased, and insulin signaling was reduced in the fat body. In ex vivo fly brain culture with the human adiponectin, Dilp2 was secreted from IPCs. These results indicate that adiponectin receptor in insulin producing cells regulates insulin secretion and controls glucose and lipid metabolism in Drosophila melanogaster. This study demonstrates a new adipokine signaling in Drosophila and provides insights for the mammalian adiponectin receptor function in pancreatic beta cells, which could be useful for therapeutic application.     

Drosophila Oocyte Polarity and Cytoskeleton Organization Require Regulation of Ik2 Activity by Spn-F and Javelin-Like

The Drosophila melanogaster Spn-F, Ik2, and Javelin-like (Jvl) proteins interact to regulate oocyte mRNA localization and cytoskeleton organization. However, the mechanism by which these proteins interact remains unclear. Using antibodies to activated Ik2, we showed that this protein is found at the region of oocyte and follicle cell where microtubule minus ends are enriched. We demonstrate that germ line Ik2 activation is diminished both in jvl and in spn-F mutant ovaries. Structure-function analysis of Spn-F revealed that the C-terminal end is critical for protein function, since it alone was able to rescue spn-F sterility. On the other hand, germ line expression of Spn-F lacking its conserved C-terminal region (Spn-FΔC) phenocopied ik2, leading to production of ventralized eggshell and bicaudal embryos. In Spn-FΔC-expressing oocytes, Gurken protein is mislocalized and oskar mRNA and protein localization is disrupted. Expression of Ik2 rescued Spn-FΔC ovarian phenotypes. We found that whereas Spn-F physically interacts with Ik2 and Jvl, Spn-FΔC physically interacts with Ik2 but not with Jvl. Thus, expression of Spn-FΔC, which lacks the Jvl-interacting domain, probably interferes with interaction of Ik2 and Jvl. In summary, our results demonstrate that Spn-F mediates the interaction between Ik2 and Jvl to control Ik2 activity.     

Polycystin-2 associates with the polycystin-1 homolog, suREJ3, and localizes to the acrosomal region of sea urchin spermatozoa

Polycystin-2, the protein mutated in type 2 autosomal dominant polycystic kidney disease, is an integral transmembrane protein with nonselective cation channel activity. Here we report on the sea urchin sperm homolog of polycystin-2 (suPC2). Like other polycystin-2 family members, suPC2 is a six-pass transmembrane protein containing C-terminal cytoplasmic EF hand and coiled-coil domains. The protein localizes exclusively to the plasma membrane over the sperm acrosomal vesicle. This localization coincides with the previously reported localization of the sea urchin PC1 homolog, suREJ3. Co-immunoprecipitation shows that suPC2 and suREJ3 are associated in the membrane. The location of suPC2 suggests that it may function as a cation channel mediating the sperm acrosome reaction. The low cation selectivity of PC2 channels would explain data indicating that Na(+) and Ca(2+) may enter sea urchin sperm through the same channel during the acrosome reaction.     

Separating the spindle,checkpoint, and timer functions of BubR1

BubR1 performs several roles during mitosis, affecting the spindle assembly checkpoint (SAC), mitotic timing, and spindle function, but the interdependence of these functions is unclear. We have analyzed in Drosophila melanogaster the mitotic phenotypes of kinase-dead (KD) BubR1 and BubR1 lacking the N-terminal KEN box. bubR1-KD individuals have a robust SAC but abnormal spindles with thin kinetochore fibers, suggesting that the kinase activity modulates microtubule capture and/or dynamics but is relatively dispensable for SAC function. In contrast, bubR1-KEN flies have normal spindles but no SAC. Nevertheless, mitotic timing is normal as long as Mad2 is present. Thus, the SAC, timer, and spindle functions of BubR1 are substantially separable. Timing is shorter in bubR1-KEN mad2 double mutants, yet in these flies, lacking both critical SAC components, chromosomes still segregate accurately, reconfirming that in Drosophila, reliable mitosis does not need the SAC.     

dTULP,the Drosophila melanogaster Homolog of Tubby,Regulates Transient Receptor Potential Channel Localization in Cilia

Mechanically gated ion channels convert sound into an electrical signal for the sense of hearing. In Drosophila melanogaster, several transient receptor potential (TRP) channels have been implicated to be involved in this process. TRPN (NompC) and TRPV (Inactive) channels are localized in the distal and proximal ciliary zones of auditory receptor neurons, respectively. This segregated ciliary localization suggests distinct roles in auditory transduction. However, the regulation of this localization is not fully understood. Here we show that the Drosophila Tubby homolog, King tubby (hereafter called dTULP) regulates ciliary localization of TRPs. dTULP-deficient flies show uncoordinated movement and complete loss of sound-evoked action potentials. Inactive and NompC are mislocalized in the cilia of auditory receptor neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and suggests that dTULP is a protein that regulates ciliary neurosensory functions.     

Huntingtin’s Function in Axonal Transport Is Conserved in Drosophila melanogaster

Huntington’s disease (HD) is a devastating dominantly inherited neurodegenerative disorder caused by an abnormal polyglutamine expansion in the N-terminal part of the huntingtin (HTT) protein. HTT is a large scaffold protein that interacts with more than a hundred proteins and is probably involved in several cellular functions. The mutation is dominant, and is thought to confer new and toxic functions to the protein. However, there is emerging evidence that the mutation also alters HTT’s normal functions. Therefore, HD models need to recapitulate this duality if they are to be relevant. Drosophila melanogaster is a useful in vivo model, widely used to study HD through the overexpression of full-length or N-terminal fragments of mutant human HTT. However, it is unclear whether Drosophila huntingtin (DmHTT) shares functions similar to the mammalian HTT. Here, we used various complementary approaches to analyze the function of DmHTT in fast axonal transport. We show that DmHTT interacts with the molecular motor dynein, associates with vesicles and co-sediments with microtubules. DmHTT co-localizes with Brain-derived neurotrophic factor (BDNF)-containing vesicles in rat cortical neurons and partially replaces mammalian HTT in a fast axonal transport assay. DmHTT-KO flies show a reduced fast axonal transport of synaptotagmin vesicles in motoneurons in vivo. These results suggest that the function of HTT in axonal transport is conserved between flies and mammals. Our study therefore validates Drosophila melanogaster as a model to study HTT function, and its dysfunction associated with HD.     

Ancient Origins of RGK Protein Function: Modulation of Voltage-Gated Calcium Channels Preceded the Protostome and Deuterostome Split

RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca²⁺ channel β subunits to modify voltage-gated Ca²⁺ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein–Ca²⁺ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca²⁺ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein – Ca_vβ subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca²⁺ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago.     

Functional characterization of PKDREJ, a male germ cell-restricted polycystin

Polycystin-1 regulates a number of cellular processes through the formation of complexes with the polycystin-2 ion channel or with other signal transduction proteins. Polycystin-1 is expressed in many tissues but other members of this gene family are distributed in a more restricted fashion. PKDREJ expression has been detected only in the mammalian testis, where it is restricted to the spermatogenic lineage and retained in mature sperm. However, the functional characteristics of this protein and its role in sperm biology are not well understood. In this study it is shown that PKDREJ can modulate G protein signaling and associates with several members of the polycystin-2 family. These interactions, as well as polycystin-2 association with TRPC channels, are consistent with a role of this protein in the regulation of the acrosome reaction and in other aspects of sperm physiology.     

Visual signals in the courtship of Drosophila melanogaster: Mutant analysis

Visual cues are necessary for optimal mating success in Drosophila melanogaster. The male's most important visually guided behaviour is tracking. It is shown here that tracking requires intact visual receptor cells R1–6 and the presence of screening pigments in the eye. Thus flies carrying the mutation ebony as well as wild type flies affected in receptor cell R1–6 are unable to use visual cues when they track females. A similar defect was obseved in white-eyed flies lacking screening pigments. Female receptivity depends on visual signals provided by the male flies. Most important cues are the light reflection from and the shape of the male's eyes. No influence of the light reflected from the thorax could be seen. Absence of eyes in the male, however, does not depress female receptivity as much as white eyes. Some evidence is provided that male courtship behaviour is evaluated visually by the female.     

BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing

Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.