Maternal proteomic profiling reveals alterations in lipid metabolism in late-onset fetal growth restriction.

Fetal growth restriction defined as the failure to achieve the fetal genetic growth potential is a major cause of perinatal morbidity and mortality. The role of maternal adaptations to placental insufficiency in this disorder is still not fully understood. We aimed to investigate the biological processes and protein-protein interactions involved in late-onset fetal growth restriction in particular. We applied 2D nano LC-MS/MS proteomics analysis on maternal blood samples collected at the time of delivery from 5 singleton pregnancies with late-onset fetal growth restriction and 5 uncomplicated pregnancies. Data were analyzed using R package "limma" and Ingenuity Pathway Analysis. 25 proteins showed significant changes in their relative abundance in late-onset fetal growth restriction (p value < 0.05). Direct protein-protein interactions network demonstrated that Neurogenic locus notch homolog protein 1 (NOTCH1) was the most significant putative upstream regulator of the observed profile. Gene ontology analysis of these proteins revealed the involvement of 14 canonical pathways. The most significant biological processes were efflux of cholesterol, efflux of phospholipids, adhesion of blood cells, fatty acid metabolism and dyslipidemia. Future studies are warranted to validate the potential role of the detected altered proteins as potential therapeutic targets in the late-onset form of fetal growth restriction.

GCAP neuronal calcium sensor proteins mediate photoreceptor cell death in the rd3 mouse model of LCA12 congenital blindness by involving endoplasmic reticulum stress.

Loss-of-function mutations in the retinal degeneration 3 (RD3) gene cause inherited retinopathy with impaired rod and cone function and fast retinal degeneration in patients and in the natural strain of rd3 mice. The underlying physiopathology mechanisms are not well understood. We previously proposed that guanylate cyclase-activating proteins (GCAPs) might be key Ca2+-sensors mediating the physiopathology of this disorder, based on the demonstrated toxicity of GCAP2 when blocked in its Ca2+-free form at photoreceptor inner segments. We here show that the retinal degeneration in rd3 mice is substantially delayed by GCAPs ablation. While the number of retinal photoreceptor cells is halved in 6 weeks in rd3 mice, it takes 8 months to halve in rd3/rd3 GCAPs-/- mice. Although this substantial morphological rescue does not correlate with recovery of visual function due to very diminished guanylate cyclase activity in rd3 mice, it is very informative of the mechanisms underlying photoreceptor cell death. By showing that GCAP2 is mostly in its Ca2+-free-phosphorylated state in rd3 mice, we infer that the [Ca2+]i at rod inner segments is permanently low. GCAPs are therefore retained at the inner segment in their Ca2+-free, guanylate cyclase activator state. We show that in this conformational state GCAPs induce endoplasmic reticulum (ER) stress, mitochondrial swelling, and cell death. ER stress and mitochondrial swelling are early hallmarks of rd3 retinas preceding photoreceptor cell death, that are substantially rescued by GCAPs ablation. By revealing the involvement of GCAPs-induced ER stress in the physiopathology of Leber's congenital amaurosis 12 (LCA12), this work will aid to guide novel therapies to preserve retinal integrity in LCA12 patients to expand the window for gene therapy intervention to restore vision.

Proteomic Changes in Human Sperm During Sequential in vitro Capacitation and Acrosome Reaction.

The male gamete is not completely mature after ejaculation and requires further events in the female genital tract to acquire fertilizing ability, including the processes of capacitation and acrosome reaction. In order to shed light on protein changes experienced by the sperm cell in preparation for fertilization, a comprehensive quantitative proteomic profiling based on isotopic peptide labeling and liquid chromatography followed by tandem mass spectrometry was performed on spermatozoa from three donors of proven fertility under three sequential conditions: purification with density gradient centrifugation, incubation with capacitation medium, and induction of acrosome reaction by exposure to the calcium ionophore A23187. After applying strict selection criteria for peptide quantification and for statistical analyses, 36 proteins with significant changes in their relative abundance within sperm protein extracts were detected. Moreover, the presence of peptide residues potentially harboring sites for post-translational modification was revealed, suggesting that protein modification may be an important mechanism in sperm maturation. In this regard, increased levels of proteins mainly involved in motility and signaling, both regulated by protein modifiers, were detected in sperm lysates following incubation with capacitation medium. In contrast, less abundant proteins in acrosome-reacted cell lysates did not contain potentially modifiable residues, suggesting the possibility that all those proteins might be relocated or released during the process. Protein-protein interaction analysis revealed a subset of proteins potentially involved in sperm maturation, including the proteins Erlin-2 (ERLIN2), Gamma-glutamyl hydrolase (GGH) and Transmembrane emp24 domain-containing protein 10 (TMED10). These results contribute to the current knowledge of the molecular basis of human fertilization. It should now be possible to further validate the potential role of the detected altered proteins as modulators of male infertility.

Stable-protein Pair Analysis as A Novel Strategy to Identify Proteomic Signatures: Application To Seminal Plasma From Infertile Patients.

Our aim was to define seminal plasma proteome signatures of infertile patients categorized according to their seminal parameters using TMT-LC-MS/MS. To that extent, quantitative proteomic data was analyzed following two complementary strategies: (1) the conventional approach based on standard statistical analyses of relative protein quantification values; and (2) a novel strategy focused on establishing stable-protein pairs. By conventional analyses, the abundance of some seminal plasma proteins was found to be positively correlated with sperm concentration. However, this correlation was not found for all the peptides within a specific protein, bringing to light the high heterogeneity existing in the seminal plasma proteome because of both the proteolytic fragments and/or the post-translational modifications. This issue was overcome by conducting the novel stable-protein pairs analysis proposed herein. A total of 182 correlations comprising 24 different proteins were identified in the normozoospermic-control population, whereas this proportion was drastically reduced in infertile patients with altered seminal parameters (18 in patients with reduced sperm motility, 0 in patients with low sperm concentration and 3 in patients with no sperm in the ejaculate). These results suggest the existence of multiple etiologies causing the same alteration in seminal parameters. Additionally, the repetition of the stable-protein pair analysis in the control group by adding the data from a single patient at a time enabled to identify alterations in the stable-protein pairs profile of individual patients with altered seminal parameters. These results suggest potential underlying pathogenic mechanisms in individual infertile patients, and might open up a window to its application in the personalized diagnostic of male infertility.

Identification of a complex population of chromatin-associated proteins in the European sea bass (Dicentrarchus labrax) sperm.

A very common conception about the function of the spermatozoon is that its unique role is to transmit the paternal genome to the next generation. Most of the sperm genome is known to be condensed in many species by protamines, which are small and extremely positively charged proteins (50-70% arginine) with the functions of streamlining the sperm cell and protecting its DNA. However, more recently, it has been shown in mammals that 2-10% of its mature sperm chromatin is also associated to a complex population of histones and chromatin-associated proteins differentially distributed in the genome. These proteins are transferred to the oocyte upon fertilization and may be involved in the epigenetic marking of the paternal genome. However, little information is so far available on the additional potential sperm chromatin proteins present in other protamine-containing non-mammalian vertebrates detected through high-throughput mass spectrometry. Thus, we started the present work with the goal of characterizing the mature sperm proteome of the European sea bass, with a particular focus on the sperm chromatin, chosen as a representative of non-mammalian vertebrate protamine-containing species. Proteins were isolated by acidic extraction from purified sperm cells and from purified sperm nuclei, digested with trypsin, and subsequently the peptides were separated using liquid chromatography and identified through tandem mass spectrometry. A total of 296 proteins were identified. Of interest, the presence of 94 histones and other chromatin-associated proteins was detected, in addition to the protamines. These results provide phylogenetically strategic information, indicating that the coexistence of histones, additional chromatin proteins, and protamines in sperm is not exclusive of mammals, but is also present in other protamine-containing vertebrates. Thus, it indicates that the epigenetic marking of the sperm chromatin, first demonstrated in mammals, could be more fundamental and conserved than previously thought. Abbreviations: AU-PAGE: acetic acid-urea polyacrylamide gel electrophoresis; CPC: chromosomal passenger complex; DTT: dithiothreitol; EGA: embryonic genome activation; FDR: false discovery rate; GO: Gene Ontology; IAA: iodoacetamide; LC: liquid chromatography; LC-MS/MS: liquid chromatography coupled to tandem mass spectrometry; MS: mass spectrometry; MS/MS: tandem mass spectrometry; MW: molecular weight; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate buffered saline; SDS: sodium dodecyl sulfate; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCA: trichloroacetic acid.

Identification of lysosomal Npc1-binding proteins: Cathepsin D activity is regulated by NPC1.

Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disorder, characterized by severe neurodegeneration. It is mostly produced by mutations in the NPC1 gene, encoding for a protein of the late endosomes/lysosomes membrane, involved in cholesterol metabolism. However, the specific role of this protein in NPC disease still remains unknown. We aimed to identify Npc1-binding proteins in order to define new putative NPC1 lysosomal functions. By affinity chromatography using an Npc1 peptide (amino acids 1032-1066 of loop I), as bait, we fished 31 lysosomal proteins subsequently identified by LC-MS/MS. Most of them were involved in proteolysis and lipid catabolism and included the protease cathepsin D. Cathepsin D and NPC1 interaction was validated by immunoprecipitation and the functional relevance of this interaction was studied. We found that fibroblasts from NPC patients with low levels of NPC1 protein have high amounts of procathepsin D but reduced quantities of the mature protein, thus showing a diminished cathepsin D activity. The increase of NPC1 protein levels in NPC cells by treatment with the proteasome inhibitor bortezomib, induced an elevation of cathepsin D activity. All these results suggest a new lysosomal function of NPC1 as a regulator of cathepsin D processing and activity.

Human sperm chromatin epigenetic potential: genomics, proteomics, and male infertility.

The classical idea about the function of the mammalian sperm chromatin is that it serves to transmit a highly protected and transcriptionally inactive paternal genome, largely condensed by protamines, to the next generation. In addition, recent sperm chromatin genome-wide dissection studies indicate the presence of a differential distribution of the genes and repetitive sequences in the protamine-condensed and histone-condensed sperm chromatin domains, which could be potentially involved in regulatory roles after fertilization. Interestingly, recent proteomic studies have shown that sperm chromatin contains many additional proteins, in addition to the abundant histones and protamines, with specific modifications and chromatin affinity features which are also delivered to the oocyte. Both gene and protein signatures seem to be altered in infertile patients and, as such, are consistent with the potential involvement of the sperm chromatin landscape in early embryo development. This present work reviews the available information on the composition of the human sperm chromatin and its epigenetic potential, with a particular focus on recent results derived from high-throughput genomic and proteomic studies. As a complement, we provide experimental evidence for the detection of phosphorylations and acetylations in human protamine 1 using a mass spectrometry approach. The available data indicate that the sperm chromatin is much more complex than what it was previously thought, raising the possibility that it could also serve to transmit crucial paternal epigenetic information to the embryo.

Advances in sperm proteomics: best-practise methodology and clinical potential.

The recent application of mass spectrometry to the study of the sperm cell has led to an unprecedented capacity for identification of sperm proteins in a variety of species. Knowledge of the proteins that make up the sperm cell represents the first step towards understanding its normal function and the molecular anomalies associated with male infertility. The present review starts with an introduction of the sperm cell biology and is followed by the consideration of the methodological key aspects to be aware of during sample sourcing and preparation, including data interpretation. It then overviews the initiatives developed so far towards the completion of the sperm proteome, with a particular focus in human but with the inclusion of some comments on different model species. Finally, all studies performing differential proteomics in infertile patients are reviewed, pointing to future potential applications.

Identification of proteins involved in human sperm motility using high-throughput differential proteomics.

Mammalian sperm motility is a prerequisite for in vivo fertilization, and alterations in this parameter are commonly observed in infertile males. However, we still do not have a complete understanding of the molecular mechanisms controlling it. The aim of this study was to identify proteins involved in human sperm motility deficiency by using TMT protein labeling and LC-MS/MS. Two complementary approaches were used: comparison between sperm samples differing in motility (asthenozoospermic versus normozoospermic) and comparison between sperm subpopulations of fractionated normozoospermic samples differing in motility (non-migrated versus migrated). LC-MS/MS resulted in the identification of 1157 and 887 proteins in the first and second approaches, respectively. Remarkably, similar proteomic alterations were detected in the two experiments, with 80 proteins differentially expressed in the two groups of samples and 93 differentially expressed in the two groups of subpopulations. The differential proteins were analyzed by GO, cellular pathways, and clustering analyses and resulted in the identification of core deregulated proteins and pathways associated with sperm motility dysfunction. These included proteins associated with energetic metabolism, protein folding/degradation, vesicle trafficking, and the cytoskeleton. Contrary to what is usually accepted, the outcomes support the hypothesis that several metabolic pathways (notably, mitochondrial-related ones) contribute toward regulating sperm motility.

Genomic and proteomic dissection and characterization of the human sperm chromatin.

The mammalian spermatozoon has a unique chromatin structure where the majority of DNA is packaged by protamines, while a small fraction (∼8%) remains associated with nucleosomes. However, the chromatin affinity and repertoire of the additional proteins constituting the different sperm chromatin fractions have not yet been explored. To address this we have carried out a genomic and proteomic characterization of human sperm samples subjected to chromatin fractionation using either 0.65 M NaCl extraction followed by EcoRI/BamHI DNA restriction enzyme digestion, or micrococcal nuclease digestion. DNA fractions corresponding to the nucleosome-packaged DNA were sequenced, confirming an appropriate dissection of the sperm chromatin. In addition we detected and sequenced a subnucleosomal particle. Although both fractions were highly enriched at gene promoters, some sequences were found to be exclusively associated with one of those. The results of the proteomic analyses demonstrate that there are two distinct sets of sperm proteins which differ in chromatin affinity. Histone variants, transcription factors, chromatin-associated and modifying proteins involved in regulatory roles were identified as weakly attached to the sperm DNA, whereas proteins with structural roles were identified in the condensed fraction. Many factors, such as the histone lysine demethylase PHF8 identified for the first time in the human sperm cell in this study, were identified exclusively in soluble fraction. Our results provide additional support to the possibility that all of these factors may constitute additional layers of sperm epigenetic information or have structural or regulatory roles transmitted by the sperm cell to the oocyte at fertilization.

High-throughput sperm differential proteomics suggests that epigenetic alterations contribute to failed assisted reproduction.

STUDY QUESTION: Are there quantitative alterations in the proteome of normozoospermic sperm samples that are able to complete IVF but whose female partner does not achieve pregnancy? SUMMARY ANSWER: Normozoospermic sperm samples with different IVF outcomes (pregnancy versus no pregnancy) differed in the levels of at least 66 proteins. WHAT IS KNOWN ALREADY: The analysis of the proteome of sperm samples with distinct fertilization capacity using low-throughput proteomic techniques resulted in the detection of a few differential proteins. Current high-throughput mass spectrometry approaches allow the identification and quantification of a substantially higher number of proteins. STUDY DESIGN, SIZE, DURATION: This was a case-control study including 31 men with normozoospermic sperm and their partners who underwent IVF with successful fertilization recruited between 2007 and 2008. PARTICIPANTS/MATERIALS, SETTING, METHODS: Normozoospermic sperm samples from 15 men whose female partners did not achieve pregnancy after IVF (no pregnancy) and 16 men from couples that did achieve pregnancy after IVF (pregnancy) were included in this study. To perform the differential proteomic experiments, 10 no pregnancy samples and 10 pregnancy samples were separately pooled and subsequently used for tandem mass tags (TMT) protein labelling, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry (LC-MS/MS) identification and peak intensity relative protein quantification. Bioinformatic analyses were performed using UniProt Knowledgebase, DAVID and Reactome. Individual samples (n = 5 no pregnancy samples; n = 6 pregnancy samples) and aliquots from the above TMT pools were used for western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: By using TMT labelling and LC-MS/MS, we have detected 31 proteins present at lower abundance (ratio no pregnancy/pregnancy < 0.67) and 35 at higher abundance (ratio no pregnancy/pregnancy > 1.5) in the no pregnancy group. Bioinformatic analyses showed that the proteins with differing abundance are involved in chromatin assembly and lipoprotein metabolism (P values < 0.05). In addition, the differential abundance of one of the proteins (SRSF protein kinase 1) was further validated by western blotting using independent samples (P value < 0.01). LIMITATIONS, REASONS FOR CAUTION: For individual samples the amount of recovered sperm not used for IVF was low and in most of the cases insufficient for MS analysis, therefore pools of samples had to be used to this end. WIDER IMPLICATIONS OF THE FINDINGS: Alterations in the proteins involved in chromatin assembly and metabolism may result in epigenetic errors during spermatogenesis, leading to inaccurate sperm epigenetic signatures, which could ultimately prevent embryonic development. These sperm proteins may thus possibly have clinical relevance. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economia y Competividad; FEDER BFU 2009-07118 and PI13/00699) and Fundación Salud 2000 SERONO13-015. There are no competing interests to declare.

Methods for the analysis of the sperm proteome.

Proteomics is the study of the proteins of cells or tissues. Sperm proteomics aims at the identification of the proteins that compose the sperm cell and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput for the identification and study of the sperm proteins. Catalogues of spermatozoal proteins in human and in model species are becoming available laying the groundwork for subsequent research, diagnostic applications, and the development of patient-specific treatments. A wide range of MS techniques is also rapidly becoming available for researchers. This chapter describes a methodological option to study the sperm cell using MS and provides a detailed protocol to identify the proteins extracted from a Percoll-purified human sperm population and separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using LC-MS/MS.

Human sperm tail proteome suggests new endogenous metabolic pathways.

Proteomic studies are contributing greatly to our understanding of the sperm cell, and more detailed descriptions are expected to clarify additional cellular and molecular sperm attributes. The aim of this study was to characterize the subcellular proteome of the human sperm tail and, hopefully, identify less concentrated proteins (not found in whole cell proteome studies). Specifically, we were interested in characterizing the sperm metabolic proteome and gaining new insights into the sperm metabolism issue. Sperm were isolated from normozoospermic semen samples and depleted of any contaminating leukocytes. Tail fractions were obtained by means of sonication followed by sucrose-gradient ultracentrifugation, and their purity was confirmed via various techniques. Liquid chromatography and tandem mass spectrometry of isolated sperm tail peptides resulted in the identification of 1049 proteins, more than half of which had not been previously described in human sperm. The categorization of proteins according to their function revealed two main groups: proteins related to metabolism and energy production (26%), and proteins related to sperm tail structure and motility (11%). Interestingly, a great proportion of the metabolic proteome (24%) comprised enzymes involved in lipid metabolism, including enzymes for mitochondrial beta-oxidation. Unexpectedly, we also identified various peroxisomal proteins, some of which are known to be involved in the oxidation of very long chain fatty acids. Analysis of our data using Reactome suggests that both mitochondrial and peroxisomal pathways might indeed be active in sperm, and that the use of fatty acids as fuel might be more preponderant than previously thought. In addition, incubation of sperm with the fatty acid oxidation inhibitor etomoxir resulted in a significant decrease in sperm motility. Contradicting a common concept in the literature, we suggest that the male gamete might have the capacity to obtain energy from endogenous pools, and thus to adapt to putative exogenous fluctuations.

Proteomic characterization of the human sperm nucleus.

Generating a catalogue of sperm nuclear proteins is an important first step towards the clarification of the function of the paternal chromatin transmitted to the oocyte upon fertilization. With this goal, sperm nuclei were obtained through CTAB treatment and isolated to over 99.9% purity without any tail fragments, acrosome or mitochondria as assessed by optical microscopy and transmission electron microscopy. The nuclear proteins were extracted and separated in 2-D and 1-D gels and the 2-D spots and 1-D bands were excised and analysed to identify the proteins through LC-MS/MS. With this approach, 403 different proteins have been identified from the isolated sperm nuclei. The most abundant family of proteins identified are the histones, for which several novel members had not been reported previously as present in the spermatogenic cell line or in the human mature spermatozoa. More than half (52.6%) of the proteins had not been detected in the previous human whole sperm cell proteome reports. Of relevance, several chromatin-related proteins, such as zinc fingers and transcription factors, so far not known to be associated with the sperm chromatin, have also been detected. This provides additional information about the nuclear proteins that are potentially relevant for epigenetic marking, proper fertilization and embryo development.

Sperm cell proteomics.

The spermatozoon is an accessible cell which can be easily purified and therefore it is particularly well suited for proteomic analysis. It is also an extremely differentiated cell with very marked genetic, cellular, functional and chromatin changes as compared to other cells, and has profound implications for fertility, embryo development and heredity. The recent developments in MS have boosted the potential for identification and study of the sperm proteins. Catalogues of hundreds to thousands of spermatozoan proteins in human and in model species are becoming available setting up the basis for subsequent research, diagnostic applications and the development of specific treatments. The present article reviews the available scientific publications dealing with the composition and function of the sperm cell using an MS proteomic approach.

Proteomics in the study of the sperm cell composition, differentiation and function.

A first step in the characterization of cellular functions is the identification of the proteins involved. The spermatozoon is an accessible cell that is particularly suited for analysis and indeed it was one of the first cells from which proteins were identified. An important advance in the identification of the protein composition of the spermatozoa was accomplished in the past using electrophoresis separation methods and protein sequencing with the Edmman procedure. However the recent developments in mass spectrometry have boosted the potential for the identification and study of sperm proteins. Catalogs of thousands of spermatozoan proteins in human and in model species are becoming available setting up the basis for subsequent research, diagnostic applications and development of specific treatments. The present article reviews the available scientific publications dealing with the composition and function of the sperm cell using a mass spectrometry proteomic approach.

Marked correlations in protein expression identified by proteomic analysis of human spermatozoa.

The present work was started to explore whether a correlation could be detected among proteomic expression, protamine content and DNA integrity in human sperm cells. Towards this goal, we extracted the proteins present in the sperm cells from 47 sperm samples from infertile patients and from ten semen donors, analysed each sample by 2-D gel electrophoresis, and quantified the expression of 101 spots identified by MALDI-TOF analysis. Additionally, the protamine content and DNA integrity were also determined. Several interesting proteins such as transcription factors, prohibitin, heat shock and proteasome proteins have been identified. We have found that the expression of an important number of proteins (58 different 2-D spots) is correlated in independent sperm samples at high statistical significance (p<0.001 and r>0.5). Additionally, eight proteins have also been found to correlate with DNA integrity and seven with protamine content (p<0.05). To our knowledge, this is the first report describing the correlation between proteomics, DNA integrity and protamine content. It also sheds new light into the fundamental aspects of the human sperm and points to new potential proteins involved in male infertility.

Proteomics of human spermatozoa, protamine content and assisted reproduction outcome.

It is well known that alterations in the expression of the major sperm nuclear proteins (protamines) are related to infertility in man. In addition, other minor proteins extracted from human spermatozoa are being analysed by 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and identified by MALDI-TOF MS analysis. The function of the identified proteins turns out to be energy production, transcription, protein synthesis, transport, folding and turnover, cell cycle, apoptosis and oxidative stress, signal transduction, cytoskeleton, flagella and cell movement, cell recognition, metabolism and unknown function. Many of the proteins identified using MALDI-TOF had not been yet been described as being expressed in human spermatozoa. Substantial differences have been detected in the levels of some of the newly identified human sperm proteins in the different groups of infertile patients. Present research efforts are targeting the potential correlations among changes in the proteomic composition, protamine content, DNA integrity and assisted reproduction outcome.

Peripherin is a relevant neuroendocrine autoantigen recognized by islet-infiltrating B lymphocytes.

Most of our knowledge of the antigenic repertoire of autoreactive B lymphocytes in type 1 diabetes (T1D) comes from studies on the antigenic specificity of both circulating islet-reactive autoantibodies and peripheral B lymphocyte hybridomas generated from human blood or rodent spleen. In a recent study, we generated hybridoma cell lines of infiltrating B lymphocytes from different mouse strains developing insulitis, but with different degrees of susceptibility to T1D, to characterize the antigenic specificity of islet-infiltrating B lymphocytes during progression of the disease. We found that many hybridomas produced mAbs restricted to the peripheral nervous system (PNS), thus indicating an active B lymphocyte response against PNS elements in the pancreatic islet during disease development. The aim of this study was to identify the autoantigen recognized by these anti-PNS mAbs. Our results showed that peripherin is the autoantigen recognized by all anti-PNS mAbs, and, therefore, a relevant neuroendocrine autoantigen targeted by islet-infiltrating B lymphocytes. Moreover, we discovered that the immune dominant epitope of this B lymphocyte immune response is found at the C-terminal end of Per58 and Per61 isoforms. In conclusion, our study strongly suggests that peripherin is a major autoantigen targeted during T1D development and poses a new question on why peripherin-specific B lymphocytes are mainly attracted to the islet during disease.

Proteomic identification of human sperm proteins.

Conventional 1-DE has in the past provided a wealth of information concerning the major sperm proteins. However, so far there are relatively few reports exploiting the potential of the present proteomic tools to identify and to study additional yet-unidentified important proteins present in human spermatozoa. In the present work, 2-DE of proteins extracted from human normozoospermic spermatozoa led to the resolution of over 1000 spots. Subsequent excision from the gels of 145 spots and MALDI-TOF MS analysis allowed the identification of 98 different proteins. The function of these proteins turned out to be energy production (23%), transcription, protein synthesis, transport, folding and turnover (23%), cell cycle, apoptosis and oxidative stress (10%), signal transduction (8%), cytoskeleton, flagella and cell movement (10%), cell recognition (7%), metabolism (6%) and unknown function (11%). As many as 23% of the proteins identified have not been previously described as being expressed in human spermatozoa. The present data provide an important clue towards determining the function of these proteins and opens up the possibility to perform additional experiments.