Structure of a mammalian sperm cation channel complex.

The cation channel of sperm (CatSper) is essential for sperm motility and fertility1,2. CatSper comprises the pore-forming proteins CATSPER1-4 and multiple auxiliary subunits, including CATSPERß, γ, δ, ε, ζ, and EFCAB91,3-9. Here we report the cryo-electron microscopy (cryo-EM) structure of the CatSper complex isolated from mouse sperm. In the extracellular view, CATSPER1-4 conform to the conventional domain-swapped voltage-gated ion channel fold10, following a counterclockwise arrangement. The auxiliary subunits CATSPERß, γ, δ and ε-each of which contains a single transmembrane segment and a large extracellular domain-constitute a pavilion-like structure that stabilizes the entire complex through interactions with CATSPER4, 1, 3 and 2, respectively. Our EM map reveals several previously uncharacterized components, exemplified by the organic anion transporter SLCO6C1. We name this channel-transporter ultracomplex the CatSpermasome. The assembly and organization details of the CatSpermasome presented here lay the foundation for the development of CatSpermasome-related treatments for male infertility and non-hormonal contraceptives.

Characterization and biological role of cysteine-rich venom protein belonging to CRISPs from turkey seminal plasma†.

Turkey semen contains cysteine-rich secretory proteins (CRISPs) that belong to the dominant seminal plasma proteins. We aimed to isolate and characterize CRISP from turkey seminal plasma and evaluate its possible involvement in yellow semen syndrome (YSS). YSS, which is well characterized, causes reduced fertility and hatchability. The protein was purified using hydrophobic interaction, gel filtration, and reverse phase chromatography. It then was subjected to identification by mass spectrometry, analysis of physicochemical properties, and specific antibody production. The biological function of the isolated protein was tested and included its effects on sperm motility and migration and sperm-egg interactions. Sperm motility was measured with the CASA system using Hobson Sperm Tracker. The reproductive tract of turkey toms was analyzed for gene expression; immunohistochemistry was used for protein localization in the male reproductive tract, spermatozoa, and inner perivitelline layer. The isolated protein was identified as cysteine-rich venom protein-like isoform X2 (CRVP X2; XP_010706464.1) and contained feature motifs of CRISP family proteins. Turkey CRVP X2 was present in both spermatozoa and seminal plasma. The extensive secretion of CRVP X2 by the epithelial cells of the epididymis and ductus deferens suggests its involvement in post-testicular sperm maturation. The internally localized CRVP X2 in the proximal part of the sperm tail might be responsible for stimulation of sperm motility. CRVP X2 on the sperm head might be involved in several events prior to fusion and may also participate in gamete fusion itself. Although the mechanisms by which CRVP X2 mediates fertilization are still unknown, the involvement of complementary sites cannot be excluded. The disturbance of CRVP X2 expression can serve as an etiologic factor of YSS in the turkey. This study expands the understanding of the detailed mechanism of fertilization in birds by clarifying the specific role of CRVP X2.

Seminal plasma AnnexinA2 protein is a relevant biomarker for stallions which require removal of seminal plasma for sperm survival upon refrigeration†.

Some stallions yield ejaculates that do not tolerate conservation by refrigeration prior to artificial insemination (AI), showing improvement after removal of most of the seminal plasma (SP) by centrifugation. In this study, the SP-proteome of 10 different stallions was defined through high-performance liquid chromatography with tandem mass spectrometry and bioinformatic analysis in relation to the ability of the ejaculates to maintain semen quality when cooled and stored at 5°C. Stallions were classified into three groups, depending on this ability: those maintaining good quality after direct extension in a commercial extender (good), stallions requiring removal of seminal plasma (RSP) to maintain seminal quality (good-RSP), and stallions, unable to maintain good semen quality even after RSP (poor). Pathway enrichment analysis of the proteins identified in whole equine SP using human orthologs was performed using g: profiler showing enriched Reactome and the Kyoto Encyclopedia of Genes and Genomes pathways related to hexose metabolism, vesicle mediated transport, post translational modification of proteins and immune response. Specific proteins overrepresented in stallions tolerating conservation by refrigeration included a peroxiredoxin-6 like protein, and transcobalamin-2, a primary vitamin B12-binding, and transport protein. Also, the protein involved in protein glycosylation, ST3 beta-galactoside alpha-2,3-sialyltransferase 1 was present in good stallions. These proteins were nearly absent in poor stallions. Particularly, annexinA2 appeared as to be the most powerful discriminant variable for identification of stallions needing RSP prior to refrigeration, with a P = 0.002 and a q value = 0.005. Overall this is the first detailed study of the equine SP-proteome, showing the potential value of specific proteins as discriminant bio-markers for clinical classification of stallions for AI.

ADS-J1 disaggregates semen-derived amyloid fibrils.

Semen-derived amyloid fibrils, comprising SEVI (semen-derived enhancer of viral infection) fibrils and SEM1 fibrils, could remarkably enhance HIV-1 sexual transmission and thus are potential targets for the development of an effective microbicide. Previously, we found that ADS-J1, apart from being an HIV-1 entry inhibitor, could also potently inhibit seminal amyloid fibrillization and block fibril-mediated enhancement of viral infection. However, the remodeling effects of ADS-J1 on mature seminal fibrils were unexplored. Herein, we investigated the capacity of ADS-J1 to disassemble seminal fibrils and the potential mode of action by applying several biophysical and biochemical measurements, combined with molecular dynamic (MD) simulations. We found that ADS-J1 effectively remodeled SEVI, SEM186-107 fibrils and endogenous seminal fibrils. Unlike epigallocatechin gallate (EGCG), a universal amyloid fibril breaker, ADS-J1 disaggregated SEVI fibrils into monomeric peptides, which was independent of oxidation reaction. MD simulations revealed that ADS-J1 displayed strong binding potency to the full-length PAP248-286 via electrostatic interactions, hydrophobic interactions and hydrogen bonds. ADS-J1 might initially bind to the fibrillar surface and then occupy the amyloid core, which eventually lead to fibril disassembly. Furthermore, the binding of ADS-J1 with PAP248-286 might induce conformational changes of PAP248-286 Disassembled PAP248-286 might not be favorable to re-aggregate into fibrils. ADS-J1 also exerts abilities to remodel a panel of amyloid fibrils, including Aß1-42, hIAPP1-37 and EP2 fibrils. ADS-J1 displays promising potential to be a combination microbicide and an effective lead-product to treat amyloidogenic diseases.

Wbp2nl has a developmental role in establishing neural and non-neural ectodermal fates.

In many animals, maternally synthesized mRNAs are critical for primary germ layer formation. In Xenopus, several maternal mRNAs are enriched in the animal blastomere progenitors of the embryonic ectoderm. We previously identified one of these, WW-domain binding protein 2 N-terminal like (wbp2nl), that others previously characterized as a sperm protein (PAWP) that promotes meiotic resumption. Herein we demonstrate that it has an additional developmental role in regionalizing the embryonic ectoderm. Knock-down of Wbp2nl in the dorsal ectoderm reduced cranial placode and neural crest gene expression domains and expanded neural plate domains; knock-down in ventral ectoderm reduced epidermal gene expression. Conversely, increasing levels of Wbp2nl in the neural plate induced ectopic epidermal and neural crest gene expression and repressed many neural plate and cranial placode genes. The effects in the neural plate appear to be mediated, at least in part, by down-regulating chd, a BMP antagonist. Because the cellular function of Wbp2nl is not known, we mutated several predicted motifs. Expressing mutated proteins in embryos showed that a putative phosphorylation site at Thr45 and an α-helix in the PH-G domain are required to ectopically induce epidermal and neural crest genes in the neural plate. An intact YAP-binding motif also is required for ectopic epidermal gene expression as well as for down-regulating chd. This work reveals novel developmental roles for a cytoplasmic protein that promotes epidermal and neural crest formation at the expense of neural ectoderm.

Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage.

Cryopreservation causes sub-lethal damage which limits the fertility of frozen thawed spermatozoa. Seminal plasma has been investigated as a cryoprotectant, but has yielded inconsistent results due to considerable variation in its constituents. Individual seminal plasma proteins offer an ideal alternative to whole seminal plasma, and several have been correlated with freezing success. Binder of Sperm Proteins (BSPs) are abundant ram seminal plasma proteins which have been suggested to have significant protective effects on ram spermatozoa during cold shock. This is in direct opposition to bull spermatozoa, where BSPs cause sperm deterioration during in vitro handling. We investigated the potential of BSP1 and BSP5 to prevent freezing associated damage to important functional parameters of ram spermatozoa. BSPs purified by size exclusion chromatography improved post thaw motility and penetration through artificial mucus. Highly purified BSP1 and BSP5, isolated by gelatin affinity and RP-HPLC, improved motility and membrane integrity, and reduced post thaw protein tyrosine phosphorylation. Exposure to BSP5 before freezing increased the amount of phosphatidylethanolamine on the sperm surface after thawing. Neither BSP1 nor BSP5 prevented freezing associated changes in membrane lipid disorder. These results suggest that BSPs may significantly improve freezing outcomes of ram spermatozoa.

SL15: A seminal plasma-derived lectin from the sperm of llama (Lama glama).

The oviductal sperm reservoir of South American camelids is formed when sperm bind to N-acetylgalactosamine (GalNAc) on the surface of oviductal epithelium. The aim of this study was to characterize the GalNAc-binding proteins on llama sperm, and to establish their origin. Sperm-adsorbed proteins were extracted with 0.5 M KCl in Hepes-balanced salts. Sperm-adsorbed and seminal plasma proteins were then subjected to ligand blotting for their GalNAc affinity, and the labeled bands were identified by mass spectrometry. Three proteins were identified in seminal plasma versus only one in the sperm-adsorbed population; SL15, a seminal lectin, was common to both. SL15 is a homologue of Zymogen granule protein 16, homolog B-like, which belongs to the Jacalin-related lectin family. This lectin is likely presented to sperm via seminal plasma since epididymal sperm are not capable of binding GalNAc, whereas ejaculated sperm does, and its transcript was enriched predominantly in the prostate and bulbourethral glands. This is the first report of a seminal lectin in South American camelids that originates in the male reproductive tract, and is probably involved in sperm reservoir formation.

Identification and characterisation of putative seminal fluid proteins from male reproductive tissue EST libraries in tiger beetles.

BACKGROUND: The study of proteins transferred through semen can provide important information for biological questions such as adaptive evolution, the origin of new species and species richness. The objective of this study was to identify seminal fluid proteins (SFPs) that may contribute to the study of the reproductive system of tiger beetles (cicindelids), a group of more than 2,500 species distributed worldwide that occupy a great diversity of habitats. RESULTS: Two cDNA libraries were constructed from the male gonads of Calomera littoralis and Cephalota litorea. Expressed sequence tags (ESTs) were analysed by bioinformatics approaches and 14 unigenes were selected as candidate SFPs, which were submitted to Reverse Transcription Polymerase Chain Reaction (RT-PCR) to identify patterns of tissue-specific expression. We have identified four novel putative SFPs of cicindelids, of which similarity searches did not show homologues with known function. However, two of the protein classes (immune response and hormone) predicted by Protfun are similar to SFPs reported in other insects. Searches for homology in other cicindelids showed one lineage specific SFPs (rapidly evolving proteins), only present in the closely related species C. littoralis and Lophyra flexuosa and two conserved SFP present in other tiger beetles species tested. CONCLUSIONS: This work represents the first characterisation of putative SFPs in Adephagan species of the order Coleoptera. The results will serve as a foundation for further studies aimed to understand gene (and protein) functions and their evolutionary implications in this group of ecologically relevant beetles.

Equine sperm-neutrophil binding.

When mares are inseminated repeatedly, protein molecules from the seminal plasma (SP) prevent sperm-neutrophil binding and reduced fertility. The molecule(s) responsible for sperm-neutrophil binding is not known and the identification of beneficial SP proteins is complicated by their large numbers and abundant variation. We examined several important aspects of sperm-neutrophil binding to ultimately facilitate the identification and isolation of the molecule(s) responsible. First, we raised anti-equine P-selectin antibodies to determine the involvement of this adhesion molecule in sperm-neutrophil binding. While these antibodies identified equine P-selectin, they did not inhibit sperm-neutrophil binding. However, acrosome-reacted equine sperm expressed a molecule similar to the ligand recognition unit of P-selectin. Second, we attempted to characterize SP protein binding to equine sperm and gauge their affinity. Biotinylated SP proteins were incubated with fresh sperm, washed over a viscous medium, electrophoresed, and probed with avidin. Several SP proteins bound to sperm with a strong affinity to withstand these treatments. This finding may prove valuable for future attempts to identify and characterize specific SP molecules. Lastly, we compared the secretions from male sex organs/glands on sperm motility, sperm-neutrophil binding, and their protein profile. We expected fewer proteins from individual organs/glands, which would facilitate isolation and identification of target molecules. While each secretion had a varying effect on motility and sperm-neutrophil binding, the protein profile was as complex as that seen in whole SP, indicating that collection of proteins from individual sources will not facilitate this work. Together, these experiments answer several important questions related to sperm-neutrophil binding, sperm-SP proteins interaction, and the complexity of the SP proteome.

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).

Mapping of the binding sites involved in PSP94-CRISP-3 interaction by molecular dissection of the complex.

BACKGROUND: Human Prostate Secretory Protein of 94 amino acids (PSP94) has been shown to bind human CRISP-3 (cysteine-rich secretory protein 3) with very high affinity. CRISP-3 belongs to the CRISP family of proteins having a PR-1 (pathogenesis related protein 1) domain at its N-terminal and ion channel regulatory (ICR) domain at its C-terminal connected by a hinge region. Functional significance of this complex is not yet known. METHODS: In order to identify the residues and/or regions involved in PSP94-CRISP-3 interaction, site-directed mutagenesis was employed. Effect of the mutations on the interaction was studied by co-immunoprecipitation (Co-IP). RESULTS: For PSP94, amino acids Y(3), F(4), P(56) and the C-terminal ß-strand were found to be crucial for interacting with CRISP-3. A disulfide bond between the two domains of PSP94 (C(37)A-C(73)A) was also important for this interaction. In case of CRISP-3, the N-terminal domain alone could not maintain a strong interaction with PSP94 but it required presence of the hinge region and not the C-terminal domain. Apart from CRISP-3, CRISP-2 was also found to interact with human PSP94. Based on our findings the most likely model of PSP94-CRISP-3 complex has been proposed. CONCLUSION: The terminal ß-strands of PSP94 contact the first α-helix and the hinge region of CRISP-3. GENERAL SIGNIFICANCE: Involvement of the hinge region of CRISPs in interaction with PSP94 may affect the domain movement of CRISPs essential for the ion-channel regulatory activity resulting in inhibition of this activity.

Isolation and characterization of an ovoinhibitor, a multidomain Kazal-like inhibitor from Turkey (Meleagris gallopavo) seminal plasma.

Turkey seminal plasma contains three serine proteinase inhibitors. Two of them, with low molecular masses (6 kDa), were identified as single-domain Kazal-type inhibitors responsible for regulating acrosin activity. Our experimental objective was to isolate and characterize the inhibitor with the high molecular weight from turkey seminal plasma. The inhibitor was purified using hydrophobic interaction and affinity chromatography. Pure preparations of the inhibitor were used for identification by mass spectrometry, for determination of physicochemical properties (molecular weight, pI, and content and composition of the carbohydrate component), for kinetic studies, and for antibacterial tests. Gene expression and immunohistochemical detection of the inhibitor were analyzed in the testis, epididymis, and ductus deferens. The inhibitor with a high molecular weight from turkey seminal plasma was identified as an ovoinhibitor, which was found in avian semen for the first time. The turkey seminal plasma ovoinhibitor was a six-tandem homologous Kazal-type domain serine proteinase inhibitor that targeted multiple proteases, including subtilisin, trypsin, and elastase, but not acrosin. Our results suggested that hepatocyte growth factor activator was a potential target proteinase for the ovoinhibitor in turkey seminal plasma. The presence of the ovoinhibitor within the turkey reproductive tract suggested that its role was to maintain a microenvironment for sperm in the epididymis and ductus deferens. The turkey seminal plasma ovoinhibitor appeared to play a significant role in an antibacterial semen defense against Bacillus subtilis and Staphylococcus aureus.

Human prostasomes express glycolytic enzymes with capacity for ATP production.

Prostasomes are prostate-derived, exosome-like microvesicles that transmit signaling complexes between the acinar epithelial cells of the prostate and sperm cells. The vast majority of prostasomes have a diameter of 30-200 nm, and they are generally surrounded by a classical membrane bilayer. Using a selected proteomic approach, it became increasingly clear that prostasomes harbor distinct subsets of proteins that may be linked to adenosine triphosphate (ATP) metabolic turnover that in turn might be of importance in the role of prostasomes as auxiliary instruments in the fertilization process. Among the 21 proteins identified, most of the enzymes of anaerobic glycolysis were represented, and three of the glycolytic enzymes present are among the top 10 proteins found in most exosomes, once again linking prostasomes to the exosome family. Other prostasomal enzymes involved in ATP turnover were adenylate kinase, ATPase, 5'-nucleotidase, and hexose transporters. The identified enzymes in their prostasomal context were operational for ATP formation when supplied with substrates. The net ATP production was low due to a high prostasomal ATPase activity that could be partially inhibited by vanadate that was utilized to profile the ATP-forming ability of prostasomes. Glucose and fructose were equivalent as glycolytic substrates for prostasomal ATP formation, and the enzymes involved were apparently surface located on prostasomes, since an alternative substrate not being membrane permeable (glyceraldehyde 3-phosphate) was operative, too. There is no clear-cut function linked to this subset of prostasomal proteins, but some possible roles are discussed.

Unbiased label-free quantitative proteomic profiling and enriched proteomic pathways in seminal plasma of adult men before and after varicocelectomy.

STUDY QUESTION: Does the seminal plasma proteomic profile and functional enrichment of gene ontology terms change after microsurgical varicocelectomy? Are there any potential targets for diagnosis or therapeutic intervention in varicocele? SUMMARY ANSWER: A shift in state from a responsive-to-stress condition before varicocele correction to a responsive-to-environment condition after varicocelectomy was observed in enriched proteomic pathways. WHAT IS KNOWN ALREADY: Varicocele may lead to many adverse effects, including failure of testicular growth and development, and is associated with decreased semen quality and increased semen oxidative stress. Varicocelectomy is the treatment of choice, and is associated with improved semen quality, but little is known regarding the underlying molecular mechanisms and post-genomic pathways following intervention. STUDY DESIGN, SIZE, DURATION: A prospective study was carried out including 18 adult men with varicocele. These patients provided one semen sample before they were submitted for bilateral varicocele repair through microsurgical varicocelectomy, and one other semen sample 90 days after the surgery. PARTICIPANTS/MATERIALS, SETTING, METHODS: An aliquot of each semen sample was used for unbiased proteomics analysis by a label-free quantitative approach (2D nanoUPLC-ESI-MS(E)). Samples were pooled according to group (normalized to protein content) and run in quadruplicate. These quadruplicate runs provided degrees of freedom in order to compare groups using a non-parametric Mann-Whitney test for quantified proteins. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 316 proteins were quantified or identified, of which 91 were exclusively identified or quantified in one of the groups (53 in the pre- and 38 in the post-varicocelectomy group), and 68 were quantified in both groups and submitted to statistical analysis, of which 5 were overrepresented in the pre-varicocelectomy group (P < 0.05). In enriched functional analysis, binding and response to stimulus functions were enriched in a common cluster (present in both groups), nitric oxide metabolism and tetratricopeptide repeat domain-binding functions were enriched in the pre-varicocelectomy group, and response to reactive oxygen species, gluconeogenesis, nicotinamide adenine dinucleotide-binding and protein stabilization were enriched in the post-varicocelectomy. LIMITATIONS, REASONS FOR CAUTION: Because a shotgun proteomics analysis was chosen in order to generate a list of putative biomarkers, a targeted follow-up study should be performed to confirm these biomarkers. WIDER IMPLICATIONS OF THE FINDINGS: The proteins found in both groups possess functions usually found in human semen. The enriched function analysis demonstrated a shift back to homeostasis after varicocelectomy, suggesting that varicocele correction promotes return of semen to a physiological state. STUDY FUNDING/COMPETING INTEREST(S): The funding for this project was received from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) as a scholarship for Ms Camargo. There was no conflict of interest.

Identification of genital tract markers in the human seminal plasma using an integrative genomics approach.

STUDY QUESTION: Can protein biomarkers of the male genital tract be identified in human seminal plasma? SUMMARY ANSWER: We identified potential biomarkers for each of the organs participating in the secretions of the human seminal plasma. WHAT IS KNOWN ALREADY: The seminal plasma fulfills critical functions for fertility by providing spermatozoa with a protective milieu, promoting their final maturation and modulating the immune responsiveness of the female reproductive tract. It is also considered to be a promising source of biomarkers of male infertility and/or pathologies of the male genital tract. STUDY DESIGN, SIZE, DURATION: This study combines proteomic analyses of normal seminal plasma together with transcriptomic gene expression profiling of human healthy tissues. MATERIALS, SETTING, METHODS: Non-liquefied seminal plasma proteins from a healthy donor were prefractionated using two sequential Proteominer™ libraries. Eight subproteome fractions were collected, trypsin digested and subjected to three successive mass spectrometry analyses for peptide characterization. The list of identified proteins was compared with and merged with other available data sets of the human seminal plasma proteome. The expression of corresponding genes was then investigated using tissue transcriptome profiles to determine where, along the male reproductive tract, these proteins were produced. Finally, tissue specificity of a selected subset of biomarker candidates was validated on human tissues. MAIN RESULTS AND THE ROLE OF CHANCE: We first performed a proteomic analysis of the human seminal plasma and identified 699 proteins. By comparing our protein list with other previous proteomic data sets, we found that 2545 unique proteins have been described so far in the human seminal plasma. We then profiled their expression at the gene level and identified 83 testis, 42 epididymis, 7 seminal vesicle and 17 prostate candidate protein markers. For a subset of testis-specific candidates, i.e. TKTL1, LDHC and PGK2, we further validated their germ cell expression and demonstrated that such markers could distinguish between semen from fertile and infertile men. LIMITATIONS, REASONS FOR CAUTION: While some of the markers we identified are well-known tissue-specific products, further dedicated studies to validate the biomarker status of new candidates will be required. Additionally, whether or not the abundance of these proteins is indeed decreased in some specific pathological situations remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS: Using an integrative genomics approach, we identified biomarker candidates for each of the organs participating in the seminal plasma production. In this study, we essentially focused on germ cell markers and their potential application for the diagnosis of male infertility. Other types of markers also deserve a focused attention given their potential predictive value for various reproductive disorders, notably for prostate cancers. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Proteomics Core Facility at Biogenouest and was funded by Conseil Régional de Bretagne, IBiSA and Agence de la Biomédecine grants. The authors declare that there exists a competing financial interest in this work that is related to a patent application on the use of identified germ cell-specific proteins in an antibody-based assay (Fertichip™) to predict the successful testicular biopsy outcomes in human non-obstructive azoospermia.

Interlaboratory reproducibility of selective reaction monitoring assays using multiple upfront analyte enrichment strategies.

Over the past few years, mass spectrometry has emerged as a technology to complement and potentially replace standard immunoassays in routine clinical core laboratories. Application of mass spectrometry to protein and peptide measurement can provide advantages including high sensitivity, the ability to multiplex analytes, and high specificity at the amino acid sequence level. In our previous study, we demonstrated excellent reproducibility of mass spectrometry-selective reaction monitoring (MS-SRM) assays when applying standardized standard operating procedures (SOPs) to measure synthetic peptides in a complex sample, as lack of reproducibility has been a frequent criticism leveled at the use of mass spectrometers in the clinical laboratory compared to immunoassays. Furthermore, an important caveat of SRM-based assays for proteins is that many low-abundance analytes require some type of enrichment before detection with MS. This adds a level of complexity to the procedure and the potential for irreproducibility increases, especially across different laboratories with different operators. The purpose of this study was to test the interlaboratory reproducibility of SRM assays with various upfront enrichment strategies and different types of clinical samples (representing real-world body fluids commonly encountered in routine clinical laboratories). Three different, previously published enrichment strategies for low-abundance analytes and a no-enrichment strategy for high-abundance analytes were tested across four different laboratories using different liquid chromatography-SRM (LC-SRM) platforms and previously developed SOPs. The results demonstrated that these assays were indeed reproducible with coefficients of variation of less than 30% for the measurement of important clinical proteins across all four laboratories in real world samples.

The C-terminal α-helix of SPAS-1, a Caenorhabditis elegans spastin homologue, is crucial for microtubule severing.

Spastin belongs to the meiotic subfamily, together with Vps4/SKD1, fidgetin and katanin, of AAA (ATPases associated with diverse cellular activities) proteins, and functions in microtubule severing. Interestingly, all members of this subgroup specifically contain an additional α-helix at the very C-terminal end. To understand the function of the C-terminal α-helix, we characterised its deletion mutants of SPAS-1, a Caenorhabditis elegans spastin homologue, in vitro and in vivo. We found that the C-terminal α-helix plays essential roles in ATP binding, ATP hydrolysing and microtubule severing activities. It is likely that the C-terminal α-helix is required for cellular functions of members of meiotic subgroup of AAA proteins, since the C-terminal α-helix of Vps4 is also important for assembly, ATPase activity and in vivo function mediated by ESCRT-III complexes.

HSP-1/2, a major protein of equine seminal plasma, exhibits chaperone-like activity.

The major bovine seminal plasma protein, PDC-109 exhibits chaperone-like activity (CLA) against a variety of target proteins. The present studies show that the homologous protein from equine seminal plasma, HSP-1/2 also exhibits CLA and inhibits the thermal aggregation of target proteins such as lactate dehydrogenase, and DTT-induced aggregation of insulin in a concentration-dependent manner. Phosphorylcholine binding inhibited the CLA of HSP-1/2, suggesting that aggregation state of the protein is important for this activity. These results demonstrate that HSP-1/2 functions as a molecular chaperone in vitro, and suggest that it may protect other proteins of equine seminal plasma from unfolding/misfolding or aggregation. These results suggest that homologous proteins from the seminal plasma of other mammals also exhibit CLA, which will be physiologically relevant.

Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele.

STUDY QUESTION: What are the effects of smoking on the functional aspects of the sperm, the levels of lipid peroxidation and the protein profile of seminal plasma in patients with varicocele? SUMMARY ANSWER: In men with varicocele, smoking is associated with altered semen quality, decreased sperm functional integrity and seminal oxidative stress. Alterations in seminal plasma protein profiles are also present and may explain the altered semen phenotype. WHAT IS KNOWN ALREADY: Varicocele is a major cause of male infertility. It reduces testicular blood renewal with a consequent accumulation of toxic substances. Thus, it can potentiate the toxic effects of environmental exposure to genotoxic substances such as those found in cigarette smoke. STUDY DESIGN, SIZE AND DURATION: A cross-sectional study was performed in 110 patients presenting with variococele to the Human Reproduction Section of the Sao Paulo Federal University (2006-2010). The patients were divided into a control group of non-smokers, a moderate smokers group and a heavy smokers group. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Semen parameters were analysed by standard methods. Sperm DNA integrity and mitochondrial activity were assessed by Comet assays and by 3,3'-diaminobenzidine deposition, respectively. The level of lipid peroxidation in semen was determined by malondialdehyde quantification. Proteomic studies were performed by 2D-electrophoresis and mass spectrometry. MAIN RESULTS AND THE ROLE OF CHANCE: Both groups of smokers showed reduced semen quality in comparison with the control group. In the groups of smokers, sperm DNA integrity and mitochondrial activity were also decreased and lipid peroxidation levels were increased. Proteomic analyses revealed 20 proteins differentially expressed between the study groups. LIMITATIONS AND REASONS FOR CAUTION: A study including smokers without varicocele is still warranted as these results apply only to smokers who present varicocele. WIDER IMPLICATIONS OF THE FINDINGS: Patients with varicocele who are exposed to tobacco smoking present more important alterations to semen quality and sperm functional integrity and show changes in the seminal plasma proteome. This suggests testicular, and possibly systemic, adverse effects of smoking. STUDY FUNDING/COMPETING INTEREST(S): Funding for the study was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) (2007/59423-7) and by the Division of Urology, Human Reproduction Section at the São Paulo Federal University.

Expression, purification and structural analysis of recombinant rBdh-2His6, a spermadhesin from buck (Capra hircus) seminal plasma.

Spermadhesins, a family of secretory proteins from the male genital tract of ungulate species, belong to the group of animal lectins. Spermadhesins have a prominent role in different aspects of fertilisation, such as spermatozoid capacitation, acrosomal stabilisation, sperm-oviduct interaction and during sperm-oocyte fusion. Proteins (spermadhesins) in buck seminal plasma were described. In the present study, bodhesin Bdh-2 cDNA present in buck seminal plasma was subcloned with the expression plasmid pTrcHis TOPO used to transform Escherichia coli Top10 One shot cells. The recombinant clones were selected by growth in 50 µg mL⁻¹ ampicillin-containing LB broth and polymerase chain reaction amplification. Recombinant rBdh-2His6 synthesis was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by immunoblotting using monoclonal anti-His antibody. Production of rBdh-2 using low temperatures was not satisfactory. Greater production of rBdh-2 occurred with 1.5mM isopropyl ßd-thiogalactoside after 2h of induction. The method used to purify rBdh-2 was affinity chromatography on a His-Trap column following ion-exchange chromatography on a DEAE-Sephacel column. The secondary structure of the rBdh-2His6 was evaluated by spectral profile circular dichroism (CD). The prevalence of secondary structures like ß-sheets, with fewer unfolded structures and α-helices, was confirmed. The structure of rBdh-2His6 remained stable up to 35°C. However, significant structural changes were observed at temperatures higher than 40 °C related to a distortion of the CD spectrum.