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Investigates protein tyrosine phosphorylation and induction of the acrosome reaction (AR) in non-capacitated human spermatozoa in response to recombinant human zone pellucida glycoprotein. Requirement for the induction of the acrosome reaction with rhZP3; Effect of rhZP3 on tyrosine phosphorylation of a 95 kDa epitope in capacitated spermatozoa; Effect of monoclonal antibody 97.25 on phosphorylation.

Studies the involvement of phosphoinositide 3-kinase (PI 3-kinase) in the human sperm acrosome reaction induced by various agonists known to cause acrosomal exocytosis using the fungal metabolite, wortmannin. Inhibitor effect of wortmannin; Indications of PI 3-kinase operation downstream of tyrosine phosphorylation in the signal transduction cascade.

The sperm head plays a key role in many fertilisation events and determining the precise location of molecules within the head region is important in mechanistically dissecting the fertilisation process. Such molecules may be present in low copy number and many sperm head profiles must be examined to localise them to particular subcellular structures with confidence. Filtration has traditionally been used for the purpose of concentrating biological material, such as free-living cells, spores, and subcellular fractions, and little attempt has been made to extend the procedure to encompass the entire processing schedule, mainly due to the incompatibility of intermediate dehydrating solvents with membrane filters. The novel and simple technique of filtration processing that we describe produced a dense mat of cells, with several sperm heads being visible in coronal orientation in a high-power field at the light microscopic level, and allowed positive immunocytochemical staining to be identified with confidence. This new technique exploits the low viscosity of LR White acrylic resin to allow the entire processing procedure to be undertaken in the filtration apparatus. In contrast, conventional techniques for preparing free-living cells, namely pre-embedding in a supportive matrix prior to processing, and centrifugation at each stage of the processing procedure, proved suboptimal, partly due to the final concentration that could be achieved, but mainly due to the random orientation of cells that these techniques afforded., (© 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.)

Changes within interstitial stromal compartments often accompany carcinogenesis, and this is true of prostate cancer. Typically, the tissue becomes populated by myofibroblasts that can promote progression. Not all myofibroblasts exhibit the same negative influence, however, and identifying the aggressive form of myofibroblast may provide useful information at diagnosis. A means of molecularly defining such myofibroblasts is unknown. We compared protein profiles of normal and diseased stroma isolated from prostate cancer patients to identify discriminating hallmarks of disease-associated stroma. We included the stimulation of normal stromal cells with known myofibroblast inducers namely soluble TGFβ and exosome-associated-TGFβ and compared the function and protein profiles arising. In all 6-patients examined, diseased stroma exhibited a pro-angiogenic influence on endothelial cells, generating large multicellular vessel-like structures. Identical structures were apparent following stimulation of normal stroma with exosomes (5/6 patients), but TGFβ-stimulation generated a non-angiogenic stroma. Proteomics highlighted disease-related cytoskeleton alterations such as elevated Transgelin (TAGLN). Many of these were also changed following TGFβ or exosome stimulation and did not well discriminate the nature of the stimulus. Soluble TGFβ, however triggered differential expression of proteins related to mitochondrial function including voltage dependent ion channels VDAC1 and 2, and this was not found in the other stromal types studied. Surprisingly, Aldehyde Dehydrogenase (ALDH1A1), a stem-cell associated protein was detected in normal stromal cells and found to decrease in disease. In summary, we have discovered a set of proteins that contribute to defining disease-associated myofibroblasts, and emphasise the similarity between exosome-generated myofibroblasts and those naturally arising in situ.

Chronic lymphocytic leukemia (CLL), a malignant B-cell disorder, is characterized by a heterogeneous clinical course. Two-dimensional nano liquid chromatography (2D-nano-LC) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) (LC-MALDI) was used to perform qualitative and quantitative analysis on cellular extracts from 12 primary CLL samples. We identified 728 proteins and quantified 655 proteins using isobaric tag-labeled extracts. Four strategies were used to identify disease-related proteins. First, we integrated our CLL proteome with published gene expression data of normal B-cells and CLL cells to highlight proteins with preferential expression in the transcriptome of CLL. Second, as CLL's outcome is heterogeneous, our quantitative proteomic data were used to indicate heterogeneously expressed proteins. Third, we used the quantitative data to identify proteins with differential abundance in poor prognosis CLL samples. Fourth, hierarchical cluster analysis was applied to identify hidden patterns of protein expression. These strategies identified 63 proteins, and 4 were investigated in a CLL cohort (39 patients). Thyroid hormone receptor-associated protein 3, T-cell leukemia/lymphoma protein 1A, and S100A8 were associated with high-risk CLL. Myosin-9 exhibited reduced expression in CLL samples from high-risk patients. This study shows the usefulness of proteomic approaches, combined with transcriptomics, to identify disease-related proteins.

We have used a novel affinity-based proteomics technology to examine the protein signature of small secreted extracellular vesicles called exosomes. The technology uses a new class of protein binding reagents called SOMAmers® (slow off-rate modified aptamers) and allows the simultaneous precise measurement of over 1000 proteins. Exosomes were highly purified from the Du145 prostate cancer cell line, by pooling selected fractions from a continuous sucrose gradient (within the density range of 1.1 to 1.2 g/ml), and examined under standard conditions or with additional detergent treatment by the SOMAscan™ array (version 3.0). Lysates of Du145 cells were also prepared, and the profiles were compared. Housekeeping proteins such as cyclophilin-A, LDH, and Hsp70 were present in exosomes, and we identified almost 100 proteins that were enriched in exosomes relative to cells. These included proteins of known association with cancer exosomes such as MFG-E8, integrins, and MET, and also those less widely reported as exosomally associated, such as ROR1 and ITIH4. Several proteins with no previously known exosomal association were confirmed as exosomally expressed in experiments using individual SOMAmer® reagents or antibodies in micro-plate assays. Western blotting confirmed the SOMAscan™-identified enrichment of exosomal NOTCH-3, L1CAM, RAC1, and ADAM9. In conclusion, we describe here over 300 proteins of hitherto unknown association with prostate cancer exosomes and suggest that the SOMAmer®-based assay technology is an effective proteomics platform for exosome-associated biomarker discovery in diverse clinical settings.

Context: Exposure to ambient particulate air pollution is associated with increased cardiovascular and respiratory morbidity and mortality. It is necessary to understand causal pathways driving the observed health effects, particularly if they are differentially associated with particle size., Objectives: To investigate the effect of different size ranges of ambient particulate matter (PM) on gene and protein expression in an in vitro model., Materials and Methods: Normal human tracheobronchial epithelium (NHTBE) three-dimensional cell constructs were exposed for 24 h to washed ambient PM of different sizes (size 1: 7-615 nm; size 2: 616 nm-2.39 µm; size 3: 2.4-10 µm) collected from a residential street. A human stress and toxicity PCR array was used to investigate gene expression and iTRAQ was used to perform quantitative proteomics., Results: Eighteen different genes of the 84 on the PCR array were significantly dysregulated. Treatment with size 2 PM resulted in the greatest number of genes with altered expression, followed by size 1 and lastly size 3. ITRAQ identified 317 proteins, revealing 20 that were differentially expressed. Enrichment for gene ontology classification revealed potential changes to various pathways., Discussion and Conclusions: Different size fractions of ambient PM are associated with dysregulatory effects on the cellular proteome and on stress and toxicity genes of NHTBE cells. This approach not only provides an investigative tool to identify possible causal pathways but also permits the relationship between particle size and responses to be explored.

Acrosomal exocytosis (AE) is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM) with the outer acrosomal membrane (OAM). This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of OAM to the PM brought about by the formation of the trans-SNARE complex (syntaxin 1B, SNAP 23 and VAMP 3). By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes. These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2. Our data indicate that the earlier reported raft and capacitation-dependent docking phenomenon between the PM and OAM allows a specific rearrangement of molecules between the two docked membranes and is involved in (1) recruiting SNAREs and complexin 2 in the newly formed lipid-ordered microdomains, (2) the assembly of a fusion-driving SNARE complex which executes Ca(2+)-dependent AE, (3) the disassembly of the earlier reported docking SNARE complex, (4) the recruitment of secondary zona binding proteins at the zona interacting sperm surface. The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca(2+) which are all involved in AE make sperm an ideal model for studying exocytosis.

Historically, it has been challenging to go beyond epidemiology to investigate the pathogenic changes caused by tobacco smoking. The EpiAirway-100 (MatTek Corp., Ashland, MA) was employed to investigate the effects of cigarette smoke components. Exposure at the air-liquid-interface represented particle and vapour phase components of cigarette smoke. A proteomic study utilising iTRAQ labelling compared expression profiles. The correlative histopathology revealed focal regions of hyperplasia, hypertrophy, cytolysis and necrosis. We identified 466 proteins, 250 with a parameter of two or more peptides. Four of these proteins are potential markers of lung injury and three are related to mechanistic pathways of disease.

This study investigates the dynamics of serine/threonine (S/T) protein phosphorylation in sperm incubated under capacitating (C) conditions using the boar as a model system. For the first time, this approach has identified multiple dephosphorylation events that occur in a bicarbonate-dependent fashion. Different phospho-(S/T) kinase substrate antibodies were used, and dephosphorylation of 5 S/T phosphoproteins was observed in C sperm compared with noncapacitated (N) cells. Specifically, dephosphorylation of 96-, 90-, 64-, and 55-kd proteins was detected by immunoblotting using 2 phospho-Akt substrate antibodies and a phosphoprotein kinase A substrate antibody. In addition, dephosphorylation of a 105-kd protein was detected using a phospho-ATM/ATR substrate antibody. In contrast, no dephosphorylation was observed using a phosphoprotein kinase C substrate antibody, and increased tyrosine phosphorylation of 32- and 20-kd proteins was detected in C compared with N sperm. Immunolocalization experiments revealed subtle changes in the pattern expression as well as a reduction of phosphorylation in C sperm. Whereas sperm incubated in N medium containing dibutyryl cAMP (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) did not show protein dephosphorylation, incubation in C medium with dbcAMP/IBMX showed dephosphorylation as well as increased phosphorylation of other proteins (p68, p51, and p29). Finally, calyculin A, a phosphatase inhibitor, prevented dephosphorylation of p96, p90, p64, and p55 but not p105. Based on these data, we propose 2 pathways of protein dephosphorylation that are active during capacitation and independent of cAMP. Together, this provides direct evidence for more complex S/T phosphorylation dynamics than has been previously described for sperm undergoing capacitation.

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function.

Proteomics technologies have matured significantly in recent years and proteomics driven research articles in reproductive biology and medicine are increasingly common. The key challenge is to move from lists of identified proteins to informed understanding of biological function. This review introduces the range of proteomics workflows most commonly used for protein identification before focusing on the mammalian sperm cell at fertilization as an exemplar for proteomic studies. We review the work of others on entire cells but then argue that proper subcellular fractionation and proper solubilization strategies offers critical advantages to achieving increased biological understanding. In relation to understanding initial gamete recognition events at fertilization (capacitation, zona binding and acrosomal exocytosis) it is imperative to study the sperm surface proteome by using purified plasma membrane fractions. Although this task is challenging there are now strategies at our disposal to achieve comprehensive coverage of the proteins at the sperm surface. Within this context it is also important to understand the milieu of the sperm cell during transit from the testis to the oviduct as proteins (or other entities) from the genital tract epithelia and fluids may also affect the composition and organization of proteins on the sperm surface. Finally the arguments presented for studying the cell plasma membrane proteome to understand the role of the cell surface equally apply to all cell types with important roles in reproductive function.

This review provides an introduction for the nonspecialist to proteomics and in particular the major approaches available for global protein identification and quantification. Proteomics technologies offer considerable opportunities for improved biological understanding and biomarker discovery. The central platform for proteomics is tandem mass spectrometry (MS) but a number of other technologies, resources, and expertise are absolutely required to perform meaningful experiments. These include protein separation science (and protein biochemistry in general), genomics, and bioinformatics. There are a range of workflows available for protein (or peptide) separation prior to tandem MS and subsequent bioinformatics analysis to achieve protein identifications. The predominant approaches are 2D electrophoresis (2DE) and subsequent MS, liquid chromatography-MS (LC-MS), and GeLC-MS. Beyond protein identification, there are a number of well-established options available for protein quantification. Difference gel electrophoresis (DIGE) following 2DE is one option but MS-based methods (most commonly iTRAQ-Isobaric Tags for Relative and Absolute Quantification or SILAC-Stable Isotope Labeling by Amino Acids) are now the preferred options. Sample preparation is critical to performing good experiments and subcellular fractionation can additionally provide protein localization information compared with whole cell lysates. Differential detergent solubilization is another valid option. With biological fluids, it is possible to remove the most abundant proteins by immunodepletion. Sample enrichment is also used extensively in certain analyses and most commonly in phosphoproteomics with the initial purification of phosphopeptides. Proteomics produces considerable datasets and resources to facilitate the necessary extended analysis of this data are improving all the time. Beyond the opportunities afforded by proteomics there are definite challenges to achieving full proteomic coverage. Proteomes are highly complex and identifying and quantifying low abundance proteins is a significant issue. Additionally, the analysis of poorly soluble proteins, such as membrane proteins and multiprotein complexes, is difficult. However, it is without doubt that proteomics has already provided significant insights into biological function and this will continue as the technology continues to improve. We also anticipate that the promise of proteomics in terms of biomarker discovery will increasingly be realized., (Copyright © 2010 Elsevier Inc. All rights reserved.)

B-lymphocytes are essential for the production of antibodies to fight pathogens and are the cells of origin in 95% of human lymphomas. During their activation, and immortalisation by Epstein-Barr virus (EBV) which contributes to human cancers, B-lymphocytes undergo dramatic changes in cell size and protein content. This study was initiated to compare the proteome of two B-cell lines, from the same individual, that reflect different patterns of activation, one is EBV negative and the other is EBV positive. Using isobaric tags, LC-MALDI TOF-TOF and subcellular fractionation, we quantified 499 proteins from B-cells. From a detergent lysed protein extract, we identified 34 proteins that were differentially expressed in EBV-immortalised B-cells. By analysing a nuclear extract, we identified a further 29 differentially expressed proteins with only four proteins shared between the two extracts, illustrating the benefit of subcellular fractionation. This analysis has identified proteins involved in the cytoskeletal phenotype of activated B-cells and the increased antigen recognition in EBV-immortalised cells. Importantly, we have also identified new regulators of transcription and changes in ribonuclear proteins that may contribute to the increased cell size and immortalisation of lymphoblastoid cells., (Copyright © 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

For sperm to successfully fertilize an oocyte, it needs to pass through certain steps prior to, during and after initial recognition of the zona pellucida (ZP). During capacitation, the surface of the sperm head becomes remodelled, priming it to bind to the ZP and subsequently to undergo the ZP-induced acrosome reaction. During capacitation, sperm ZP-binding proteins are ordered in functional protein complexes that only emerge at the apical tip of the sperm head plasma membrane; this is also functionally the exclusive sperm surface area involved in primary ZP binding. After primary ZP binding, the same area is probably involved in the induction of the acrosome reaction. A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane. This clustering of these rafts was dependent on the presence of bicarbonate (involved in protein kinase A activation) and on the presence of albumin (involved in cholesterol removal). Remarkably, cholesterol removal was restricted to the non-raft membrane fraction of the sperm plasma membrane, but did not cause any depletion of cholesterol in the raft membrane fraction. Interestingly, sperm SNARE proteins (both VAMP from the outer acrosomal membrane, as well syntaxin from the apical sperm head plasma membrane) shared lateral redistribution properties, along with the ZP-binding protein complex and raft marker proteins. All of these were recovered after capacitation in detergent-resistant membrane preparations from sperm thought to represent membrane lipid rafts. We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.

The sperm cell has a characteristic polarized morphology and its surface is also highly differentiated into different membrane domains. Junctional protein ring structures seal the surface of the mid-piece from the head and the tail respectively and probably prevent random diffusion of membrane molecules over the protein rings. Despite the absence of such lateral diffusion-preventing structures, the sperm head surface is also highly heterogeneous. Furthermore, lipid and membrane protein ordering is subjected to changes when sperm become capacitated. The forces that maintain the lateral polarity of membrane molecules over the sperm surface, as well as those that cause their dynamic redistribution, are only poorly understood. Nevertheless, it is known that each of the sperm head surface regions has specific roles to allow sperm to fertilize the oocyte: a specific region is devoted to zona pellucida binding, a larger area of the sperm head surface is involved in the acrosome reaction (intracellular fusion), while yet another region is involved in egg plasma membrane binding and fertilization fusion (intercellular membrane fusion). All three events occur in the area of the sperm head where the plasma membrane covers the acrosome. Recently, lipid ordered microdomains (lipid rafts) were discovered in membranes of many biological specimens including sperm. In this review, we cover the latest insights about sperm lipid raft research and discuss how sperm lipid raft dynamics may relate to sperm-zona binding and the zona-induced acrosome reaction.

An important step in fertilization is the recognition and primary binding of the sperm cell to the zona pellucida (ZP). Primary ZP binding proteins are located at the apical plasma membrane of the sperm head. In order to exclusively study primary zona binding proteins, plasma membranes of sperm heads were isolated, highly purified and subsequently solubilized with a mild or a strong solubilization procedure. Native, highly purified ZP ghosts were used as the binding substrate for solubilized sperm plasma membrane proteins, and a proteomic approach was employed to identify ZP binding proteins. Two-dimensional gel electrophoresis of ZP fragments with bound sperm proteins showed very reproducibly 24 sperm protein spots to be associated to the zona ghosts after mild plasma membrane solubilization whereas only three protein spots were detected after strong plasma membrane solubilization. This indicates the involvement of multiple sperm proteins in ZP binding. The three persistently bound proteins were identified by a tandem mass spectrometry as isoforms of AQN-3 and probably represent the main sperm protein involved in ZP binding. P47, fertilin beta and peroxiredoxin 5 were also conclusively identified. None of the identified proteins has a known acrosomal origin, which further indicated that there was no sample contamination with secondary ZP binding proteins from the acrosomal matrix. In this study, we showed and identified multiple zona binding proteins involved in primary sperm-zona binding. Although we were not able to identify all of the proteins involved, this is a first step in understanding the event of primary sperm-zona interactions and the relevance of this for fertilization is discussed.

Sperm dysfunction is the single most common defined cause of infertility. One in 15 men is sub-fertile and the condition is increasing in frequency. However, the diagnosis is poor and, excluding assisted conception, there is no treatment. The reason for this is our limited understanding of the biochemical, molecular and genetic functions of the spermatozoon. The underlying premise of our research programme is to establish a rudimentary understanding of the processes necessary for successful fertilisation. In this manuscript, we detail advances in our understanding of calcium signalling in the cell and outline genetic and proteomic technologies that are being used to improve the diagnosis of the condition.

Lipid architecture of the plasma membrane plays an important role in the capacitation process of the sperm cell. During this process, an increase in membrane fluidity takes place, which coincides with a redistribution of cholesterol to the apical region of the head plasma membrane and subsequently an efflux of cholesterol. Cholesterol is also a major player in the formation of lipid rafts or microdomains in the membrane. Lipid rafts favour specific protein-protein interactions by concentrating certain proteins in these microdomains while excluding others. In this study, we investigated the organization of lipid rafts during in vitro capacitation of boar sperm cells. We report on the presence of the lipid raft-specific proteins caveolin-1 and flotillin-1 in sperm cells. Capacitation induced a change in membrane distribution of these proteins. Lipid analysis on detergent-resistant membranes (DRMs) of sperm cells indicated that capacitation induces a lipid raft concentration rather than a disintegration of lipid rafts, because the total amount of lipid in the DRM fraction remained unaltered. Using a proteomic approach, we identified several major DRM proteins, including proteins involved in capacitation-dependent processes and zona pellucida binding. Our data indicate that sperm raft reorganization may facilitate capacitation-specific signalling events and binding to the zona pellucida.

Sperm capacitation is a prerequisite for successful in vitro fertilization (IVF) and therefore a focus of sperm preparation in IVF laboratories. The technology of IVF is, therefore, potentially valuable in advancing our understanding of the molecular processes that occur during sperm capacitation. We have investigated sperm capacitation induced by a commercial IVF medium compared to that occurring in standard capacitating medium (CM) typically used in a nonclinical setting. Percoll-washed spermatozoa were resuspended in Cook Sydney IVF medium, Cook Sydney IVF sperm buffer, Earle's balanced salt medium (capacitating medium) or a modified Earle's balanced salt medium [non-capacitating medium (NCM)] for up to 120 min at 37 degrees C and, if applicable, in the presence of 5% CO2 in air. Sperm protein kinase A (PKA) activity, PKA-dependent serine/threonine phosphorylation, tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction were evaluated. IVF medium was shown to accelerate sperm capacitation (compared with capacitating medium) as determined by tyrosine phosphorylation, sperm hyperactivation and progesterone-induced acrosome reaction. This effect was not associated with enhanced activation of PKA or increased levels of serine/threonine phosphorylation. In contrast, IVF sperm buffer (used for sperm preparation) did not stimulate sperm capacitation when incubated for up to 90 min. We have shown that different capacitating media vary strikingly in their efficacy and that this difference reflects activation of a pathway other than the well-characterized activation of soluble adenylyl cyclase/cAMP/PKA.

Background: The zona pellucida (ZP) is an extracellular glycoprotein matrix which surrounds all mammalian oocytes. Recent data have shown the presence of four human zona genes (ZP1, ZP2, ZP3 and ZPB). The aim of the study was to determine if all four ZP proteins are expressed and present in the human., Methods: cDNA derived from human oocytes were used to amplify by PCR the four ZP genes. In addition, isolated native human ZP were heat-solubilized, trypsin-digested and subjected to tandem mass spectrometry (MS/MS)., Results: All four genes were expressed and the respective proteins present in the human ZP. Moreover, a bioinformatics approach showed that the mouse ZPB gene, although present, is likely to encode a non-functional protein., Conclusions: Four ZP genes are expressed in human oocytes (ZP1, ZP2, ZP3 and ZPB) and preliminary data show that the four corresponding ZP proteins are present in the human ZP. Therefore, this is a fundamental difference with the mouse model

The aim of this study was to compare the sperm protein expression profile (proteome map) from a patient who experienced failed fertilization at IVF with fertile controls. One patient and three fertile donor sperm samples were characterized using two-dimensional electrophoresis. Differences in protein expression were established using gel analysis software before attempted protein identification. Gel analysis of the fertile donor proteome maps revealed excellent reproducibility as well as very low intra-donor and inter-donor variability in the presence of protein spots. In the patient samples, we have noted 20 consistent differences in protein expression (six spots missing, three additional spots, four less abundant, seven more abundant) compared with the controls. Two proteins that were more intense in the patient have been conclusively identified as secretory actin-binding protein and outer dense fibre protein 2/2. In conclusion proteome variation between different fertile donors was very low. In contrast, the patient proteome exhibited 20 differences compared with controls, which we believe is an underestimate. These proteins merit further investigation to determine whether failed fertilization at IVF might be caused by abnormalities in their expression. This case report represents a proof of principle that proteomics may be useful to study defects in sperm function.

This research aims firstly to understand, in cellular and molecular terms, how a mature human spermatozoon is prepared for fertilization, and secondly, to identify what factors are involved in the initial signalling interactions between the egg and spermatozoon. In order to achieve these objectives, a combination of approaches is being used, including single-cell imaging, patch clamping and proteomics. Single-cell imaging reveals hidden complexity and heterogeneity in signalling responses in spermatozoa. Characterization of cell physiology at the single-cell level must be a future aim, including the study of ion channel expression and function by patch clamping. Proteomic experiments are aimed at identifying defects in protein expression in specific subgroups of men, e.g. those with globozoospermia. A better understanding of prefertilization events will allow the development of non-assisted reproductive therapy, drug-based treatments for male infertility.

The British Fertility Society organised a workshop on Functional Genomics in Reproductive Medicine at the University of Birmingham on 13-14 September 2001. The primary aim was to inform delegates about the power of the technology that has been made available after completion of the sequencing of the human genome, and to stimulate debate about using functional genomics to address both clinical and scientific questions in reproductive medicine. Three specific areas were addressed: proteomics, gene expression and bioinformatics. Although the sophistication and plethora of techniques available were obvious, major limitations in the technology were also discussed. The future promises to be very challenging indeed.

Background: The aim of this study was to evaluate the ability of human spermatozoa to penetrate methylcellulose (MC) and to compare this with penetration in hyaluronic acid., Methods: Spermatozoa from normal (>or=20 x 10(6) sperm/ml, >or=50% progressive motility, >or=5% normal forms) and oligozoospermic (<20 x 10(6) sperm/ml) semen samples were allowed to swim into glass capillary tubes containing methylcellulose with a viscosity of 15 centipoise (cp) (MC15) and 4000 cp (MC4000), hyaluronic acid (rooster comb) or Sperm Select. Penetration of the spermatozoa at 1, 2, 3 and 4 cm were correlated with basic semen parameters (concentration, motility and morphology). The effects of temperature on penetration into MC4000 were explored at 17-37 degrees C., Results: Higher numbers of spermatozoa penetrated MC4000 (10 mg/ml) compared with MC15 and the hyaluronic acid preparations. There was a highly significant correlation between the number of spermatozoa at all migration distances in MC4000 (10 mg/ml) and semen parameters. Increases in temperature from 17-37 degrees C were accompanied by significantly higher numbers of spermatozoa at each penetration distance. MC4000 at 10 mg/ml was at least as favourable to sperm penetration as human cervical mucus. Effective discrimination between normal and abnormal samples was achieved using MC4000 (10 mg/ml)., Conclusion: Our results suggest the potential use of methylcellulose (MC4000, 10 mg/ml) as a reproducible and effective alternative to hyaluronic acid in sperm migration tests.

Spermatozoa from the sperm-rich fractions of the semen of 6 beagle dogs were capacitated and the effect of both zona pellucida (ZP) proteins and progesterone on calcium flux and the acrosome reaction measured. Sperm calcium flux was determined using the dual wavelength calcium probe indo-1/AM (6 microM) in a flow cytometric assay (one ejaculate from each dog examined; n = 6). No calcium flux was observed in the negative control treatments (RPMI medium or DMSO). Both heat-solubilized bitch ZP proteins and progesterone caused a similar response characterized by a gradual but marked influx of calcium ions which was sustained over 2 min. Acrosomal status was assessed by indirect immunofluorescence using a specific monoclonal antibody following 1 hr incubation for each treatment (four ejaculates from each dog examined; n = 24). The level of acrosomal exocytosis was very high for samples treated with ZP proteins (70.3 +/- 2.1%) and progesterone (84.6 +/- 1.5%) and was significantly different from the respective controls (P < 0.001). Interestingly the patterns of calcium flux in response to both ZP proteins and progesterone were in contrast to the situation in other species studied to date raising the possibility that the mechanism for triggering the acrosome reaction may be different in dog spermatozoa. In addition the high degree of progesterone-induced acrosomal exocytosis compared to other species raises the probability that the majority of dog spermatozoa are already undergoing the acrosome reaction before they reach the egg ZP., (Copyright 2001 Wiley-Liss, Inc.)

The aim of this work was to investigate the effect of elementary bodies (EB) of Chlamydia trachomatis serovars E and LGV on sperm motility, viability and acrosomal status. Highly motile preparations of spermatozoa from normozoospermic patients were co-incubated for 6 h with 0.54x10(6) EB per ml. At 1, 3 and 6 h of incubation, sperm motility was determined by computer-assisted semen analysis (CASA) and the proportion of dead cells determined by the hypo-osmotic swelling (HOS) test. Acrosomal status was also examined using a standard monoclonal antibody assay. In the absence of EB, the percentage of motile spermatozoa remained >69% over the 6h incubation and the proportion of dead spermatozoa at <12%. However, during the incubation with EB of serovar E there was a significant decline in the percentage of motile spermatozoa (P < 0.05), and a corresponding increase in the proportion of dead spermatozoa (P < 0.05) at all time-points. However, following incubation with serovar LGV, only the percentage of dead spermatozoa after 6 h incubation was significantly different from the control (P < 0.05). The amount of acrosome-reacted spermatozoa remained unchanged (<16%) in all incubations at all time-points. Dose-response experiments indicated that increasing the concentration of EB to 2.5x10(6) per ml did not significantly alter the results. Furthermore, co-incubation of spermatozoa with dead EB (killed by heat treatment) abolished the chlamydia-mediated response, indicating that the effect is a result of the live organism and not soluble components or membrane elements. These data suggest that a detrimental effect on sperm function by some serovars may be an as yet unrecognized component of infertility problems.

Elementary bodies (EBs) of the obligate intracellular bacterium Chlamydia trachomatis are responsible for the first step of attachment to host cells. We have studied the effects of EBs on human sperm protein tyrosine phosphorylation, which is important to sperm function. Indirect immunofluorescence using antiphosphotyrosine antibodies showed that serovar E, but not LGV, caused increased tyrosine phosphorylation which was localized to the sperm tail region. Immunoblotting revealed that serovar E caused a marked increase in tyrosine phosphorylation of 80- and 95-kDa sperm proteins, whereas serovar LGV caused increased phosphorylation of only the 80-kDa moiety. Considering the importance of tyrosine phosphorylation for sperm capacitation and other aspects of sperm function, we conclude that EBs may affect these events.

We report 2 novel approaches using flow cytometry to measure intracellular calcium concentration and plasma membrane potential in human spermatozoa. Both approaches have the potential to measure different responses in subpopulations of cells, which is particularly useful when studying heterogeneous populations such as human spermatozoa. Intracellular calcium concentration ([Ca2+]i) was measured using the probe indo-1/AM. This allowed measurements to be made that were independent of variation in cell size and dye loading. It also enabled dead cells to be directly identified and excluded from the analyses without the need for counterstaining. Mean basal [Ca2+]i was determined as 50 nM (25-75 nM range) and, in response to the agonist progesterone (20 microM), this increased transiently to 195 nM (125-285 nM range) before declining to approximately half the maximal level within 2 minutes (values in parentheses correspond to the range of values typically found within a sperm population from 1 sample). These results are comparable with previously published data on whole sperm populations. Sperm membrane potential (VM) was assayed using the probe DiOC6(3). In carefully controlled experiments, a marked depolarization of the plasma membrane potential of capacitated spermatozoa was observed in response to progesterone (20 microM). Following in vitro capacitation, the sperm plasma membrane potential became hyperpolarized compared with the noncapacitated state. Therefore, this technique may be used to assay for sperm capacitation in vitro.

The gamete recognition and initial binding processes that are crucial for the success of mammalian fertilization are mediated by moieties associated with the extracellular matrix of the egg (the zona pellucida) and the head of the fertilizing spermatozoon. The zona proteins involved have been characterized in some detail, with ZP3 and ZP2 generally acknowledged to be responsible for the initial (primary) and secondary interactions, respectively. However, the identity of the complementary molecules on the sperm surface is highly contentious and remains unresolved. This review summarizes the current knowledge and controversies in this research area. The credentials of some of the major candidates and the probability of the involvement of multiple sperm receptors with different binding characteristics are assessed. Resolving this very important gap in our understanding is an essential prerequisite to understanding fully the molecular and signal transduction events that cause sperm acrosomal exocytosis. Such fundamental information is also imperative for the development of novel forms of contraception (or sterilization) targeted against specific sperm epitopes. Moreover, this information may contribute to our understanding of certain types of male infertility.

Sperm maturation involves an intimate interaction between spermatozoa and the epididymal epithelium. Aspects of this relationship can be examined by co-incubating epididymal spermatozoa with epididymal epithelium in vitro. Plaques of epididymal epithelium from a variety of species (for example rodents, dogs, humans) can be maintained in culture medium supplemented with growth factors and androgens. When co-incubated with these epithelial cultures, immature epididymal spermatozoa undergo maturation changes that lead to the acquisition of progressive motility, zona binding and, in some instances, fertilizing capacity in vitro. The use of such co-culture techniques for the understanding of sperm maturation in vitro and in vivo is reviewed with reference to recent experiments.

Recombinant human ZP3 (rhuZP3) generated by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA was used to study the acrosome reaction (AR) and intracellular calcium fluxes in capacitated human spermatozoa. Conditioned medium containing rhuZP3 significantly induced the AR (P < or = 0.005) in 59.4 +/- 4.7% of spermatozoa (control = 8.5 +/- 3.1%) and caused complete acrosomal loss in a further 17.2 +/- 3.8% of cells (control = 3.7 +/- 0.7%; mean +/- SEM, n = 5). Sperm motility was not affected and acrosomal exocytosis in response to rhuZP3 was also shown to be time-dependent. Basal concentrations of sperm intracellular calcium were measured (82 +/- 7 nM; mean +/- SEM, n = 9). A transient increase in intracellular calcium (typically up to 400-450 nM) occurred within 1 min of rhuZP3 addition and was followed by sustained lower values of calcium (200-400 nM). These responses were dependent on the amount of rhuZP3. This is the first report of zona protein-induced changes in intracellular calcium levels in human spermatozoa. The results support the premise that ZP3 is an agonist of the human sperm AR and that rhuZP3 generated in a eukaryotic cell is effective in this respect.

The glycan core structures of the glycosyl-phosphatidylinositol (GPI) anchors on porcine and human renal membrane dipeptidase (EC 3.4.13.19) were determined following deamination and reduction by a combination of liquid chromatography, exoglycosidase digestions, and methylation analysis. The glycan core was found to exhibit microheterogeneity with three structures observed for the porcine GPI anchor: Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN (29% of the total population), Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN (33%), and Man alpha 1-2Man alpha 1-6(Gal beta 1-3GalNAc beta 1-4)Man alpha 1-4GlcN (38%). The same glycan core structures were also found in the human anchor but in slightly different proportions (25, 52, and 17%, respectively). Additionally, a small amount (6%) of the second structure with an extra mannose alpha (1-2)-linked to the non-reducing terminal mannose was also observed in the human membrane dipeptidase GPI anchor. A small proportion (maximally 9%) of the porcine GPI anchor structures was found to contain sialic acid, probably linked to the GalNAc residue. The porcine GPI anchor was found to contain 2.5 mol of ethanolamine/mol of anchor. Negative-ion electrospray-mass spectrometry revealed the presence of exclusively diacyl-phosphatidylinositol (predominantly distearoyl-phosphatidylinositol with a minor amount of stearoyl-palmitoyl-phosphatidylinositol) in the porcine membrane dipeptidase anchor. Porcine membrane dipeptidase was digested with trypsin and the C-terminal peptide attached to the GPI anchor isolated by removal of the other tryptic peptides on anhydrotrypsin-Sepharose. The sequence of this peptide was determined as Thr-Asn-Tyr-Gly-Tyr-Ser, thereby identifying the site of attachment of the GPI anchor as Ser368. This work represents a comprehensive study of the GPI anchor structure of porcine membrane dipeptidase and the first interspecies comparison of mammalian GPI anchor structures on the same protein.

Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5'-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.

The cell-surface expression of endopeptidase-24.11 (EC 3.4.24.11) on Caco-2 cells cultured to confluency is markedly heterogeneous unlike that of dipeptidylpeptidase IV (EC 3.4.14.5). Here we have investigated the cell-surface expression of three other ectopeptidases: angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase N (EC 3.4.11.2) and aminopeptidase W (EC 3.4.11.16). We show by indirect immunofluorescent staining that these three enzymes are present on the surface of some cells but not on others. However, these enzymes were detected in the majority of detergent-permeabilised Caco-2 cells indicating the presence of intracellular pools of these enzymes. This suggests that there may either be differential regulation of apical transport for these peptidases or that they recycle at different rates.

The consumption of oxygen (QO2), the production of lactate and the profile of four key metabolic enzymes were measured in small samples of human oviductal mucosa (endosalpinx) removed at surgery. The QO2 in the absence of substrate was 3.4 microliters O2 (mg dry wt)-1 h-1, a value typical of quiescent tissue. The QO2 was stimulated by glucose, but diminished by glutamine and acetoacetate. Tissue lactate production was low and not increased by glucose. Hexokinase had the highest activity of the enzymes measured, followed by 2-oxoglutarate dehydrogenase; 6-phosphofructokinase and glycogen phosphorylase had low activities. The data are consistent with the proposition that glucose is a major metabolic fuel for human endosalpinx.