Project description:Honeybee semen was collected by gently squeezing the male abdomens. Samples were pooled and then centrifuged. Pelleted sperm was collected and analysed with both 2D PAGE and gel-free methods. For the 2D PAGE, protein spots were digested using trypsin. Extracted peptides, resolved on a C18 column, were analysed by an Agilent LC/MSD Trap XCT Ultra 6330 mass spectrometer. Spectra were searched against the honeybee protein sequences (RefSeq release 48) using Mascot algorithm (with 1 missed cleavage, Cys-carbamidomethylation as fixed modification, and Met-oxidation and N/Q-deamidation as variable modifications). For the MudPit analysis the sperm sample was digested by trypsin. Peptides were resolved using strong cation exchange chromatography followed by reverse phase (C18) HPLC and finally analysed by an Agilent QTOF mass spectrometer. Resulting spectrum files were converted to mzXML and merged using ProteoWizard msconvert. These were then searched against honeybee protein sequences using Mascot, Omssa and X!tandem (all with 1 missed cleavage, and Met-oxidation and N/Q-deamidation as variable modifications). Results of the three search engines were pooled using TPP.
Project description:Aphid adaptation to harsh winter conditions is illustrated by an alternation of their reproductive mode. Aphids detect photoperiod shortening by sensing the length of the night and switch from viviparous parthenogenesis in spring and summer, to oviparous sexual reproduction in autumn. The photoperiodic signal is transduced from the head to the reproductive tract to change the fate of the future oocytes from mitotic diploid embryogenesis to haploid formation of gametes. Because of viviparous parthenogenesis, the whole process takes place in three consecutive generations. To understand the molecular basis of the switch in the reproductive mode, a transcriptomic approach was used to detect significantly regulated transcripts in the heads of the pea aphid Acyrthosiphon pisum. The transcriptomic profiles of the heads of the first generation were slightly affected by photoperiod shortening. This suggests that trans-generation signaling does not occur between the grand-mothers and the viviparous embryos they contain. By analogy, many of the genes regulated in the heads of the second generation are implicated in visual functions, photoreception and cuticle structure. The modification of the cuticle could decrease the storage of N-β-alanyldopamine and provoke an increase in free dopamine concentration. Based in results in Drosophila, modification of the insulin pathway could cause a decrease of juvenile hormones in short-day reared aphids. Biological material for microarray experiments was prepared under two dayly photoperiodic regimes both at constant temperature of 18°C: i) âShort Nightâ (SN) at 16h of light and ii) âLong Nightâ (LN) at 12h of light to induce the production of sexual morphs. To initiate the experiment, two groups of 105 L3 larvae were placed either under SN or LN condition. This corresponds to generation G0. At the middle of the photophase, 25 individual were frozen when they had reached both the L4 and the wingless adult (WA) stages, in the two photoperiod conditions. The 55 remaining WA individuals (still divided in two groups) were left on 55 plants to lay their offspring: one larva of the 1st stage (L1) was kept per WA. This larva was selected among the 20 first born larvae. This is the generation G1. At the middle of the photophase, 25 individual were frozen when they had reached both the L2 and the L4 stages, in the two photoperiodic conditions. Thus, 25 individuals from 4 different stages (L4-G0, WA-G0, L2-G1 and L4-G1) were collected in the two photoperiod conditions with 3 biological replicates, forming the 24 samples used for microarray experiments. RNAs from heads of aphids from the two photoperiodic conditions were hybridized one against the other for each stage with a dye-swap.The experimental design is thus 24 arrays which corresponds to the described samples of that series.
Project description:New RNA-seq data was generated to create a large-scale Seminavis robusta gene expression atlas profiling a wide-range of experimental conditions including life cycle stages and abiotic stressors. These stresses encompass among others changes in temperature and salt concentration, silica depletion, high light exposure, H202 and decadienal treatment.
Project description:Given the continued advances in mass spectrometry technology and methods for database searching since the previous characterization of the Drosophila melanogaster sperm proteome, a new proteomic analysis of sperm samples was conducted to expand the size and coverage of the sperm proteome. This dataset is part of a larger project examining the molecular life history of Drosophila sperm.
Project description:PKA signaling plays important functions during mouse spermatogenesis and for the regulation of sperm motility. The efficiency of PKA signaling is regulated partly by its binding protein A-kinase anchoring protein (AKAP) family. Mature mouse sperm contain several AKAPs, of which AKAP3 and AKAP4 are localized in the principal piece of sperm tail and they are sperm-specific. Using gene knock-out mouse models, it was found that both AKAP3 and AKAP4 are essential for the formation of a complete fibrous sheath, the electron-dense sub-cellular structure within sperm flagellum that is important for the motility of sperm and male fertility. Lack of AKAP3 or AKAP4 caused aberrant sperm morphology and inmotility, leading to eventual male sterility. In the present study, we investigated the changes of cellular proteome in the absence of AKAP3 or AKAP4 in mature sperm, using quantitative mass spectrometry. It was found that global changes of sperm proteome occur in the absence of AKAP3 or AKAP4. In Akap3 null sperm, proteins related to flagella structure, PKA signaling and motility regulations are down-regulated, whereas proteins involved in RNA metabolism, protein translation and actin filaments formation are accumulated. Akap4 null sperm also displayed changes of sperm proteome at lesser extends. These results suggest that both AKAP3 and AKAP4 are essential for the formation of sperm sub-cellular structures, proper regulation of PKA activity and motility of sperm, providing new clues to elucidate the molecular pathways that underlie the coordinated synthesis and organization of sperm proteome and sperm morphology
Project description:Identification of bovine sperm surface carbohydrate-binding proteins relevant in two important aspects of fertilization: (i) formation of the sperm reservoir in the oviductal epithelium, and (ii) gamete recognition (oocyte-sperm interaction). Using whole sperm cells and a novel affinity capture method that combines proteolysis of protein-glycan complexes and mass spectrometry (i.e., CREDEX-MS), several lectins were enriched, identified by MS/MS proteomics, and mapped within the fertilization events in bovine species.
Project description:Sperm contains essential proteins for interaction with eggs, however, there are only several sperm proteins reported with important role in fertilization, and gamete proteomics are limited in marine invertebrate species. We present here a sperm proteomic profile of marine mussel Mytilus galloprovincialis. There are 816 proteins were successfully identified by LC-MS/MS based on 1-DE SDS-PAGE. Many of the identifications are relevant to sperm cell physiology and mtDNA functioning. The results will contribute to better understand the proteins involved in fertilization in M. galloprovincialis, as well as the other marine invertebrate species.
Project description:Interactions between sperm and the female reproductive tract (FRT) are critical to fertility, but knowledge of the molecular mechanisms by which the FRT interacts with sperm and influences sperm fate remains limited. Here, we used whole-cell quantitative proteomics to track the protein composition of sperm across three female reproductive tissues (bursa, seminal receptacle, and spermatheca) and three post-mating timepoints (30 minutes, 2 hours, and 4 days). In combination with an update and expansion of the seminal vesicle sperm proteome and sex-specific isotopic labeling to identify female-contributed sperm proteins, our data provide a comprehensive, quantitative analysis of the molecular life history of Drosophila sperm.
Project description:All but the most basal Lepidopteran species produce two sperm morphs. Only one of these morphs is capable of completing karyogamy and producing offspring, this morph contains the correct genetic complement and is termed eupyrene sperm. Apyrene sperm, on the other hand, is completely devoid of nuclear DNA and fertilisation incompetent. Despite the fact apyrene sperm is believed to be functional, the function of this sperm type is largely unknown. Here we apply tandem mass spectrometry based proteomics to the two sperm types independently in the monarch butterfly (Danaus plexippus) and the carolina sphinx moth (Manduca sexta). Comparative analysis between sperm morphs and species shows a reduced complexity and greater divergence in apyrene sperm relative to eupyrene consistent across the two species.