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Abstract In contrast to most fishes, salmonids exhibit the unique ability to hold their eggs for several days after ovulation without significant loss of viability. During this period, eggs are held in the body cavity in a biological fluid, the coelomic fluid (CF) that is responsible for preserving egg viability. To identify CF proteins responsible for preserving egg viability, a proteomic comparison was performed using 3 salmonid species and 3 non-salmonid species to identify salmonid-specific highly abundant proteins. In parallel, rainbow trout CF fractions were purified and used in a biological test to estimate their egg viability preservation potential. The most biologically active CF fractions were then subjected to mass spectrometry analysis. We identified 50 proteins overabundant in salmonids and present in analytical fractions with high egg viability preservation potential. The identity of these proteins illuminates the biological processes participating in egg viability preservation. Among identified proteins of interest, the ovarian-specific expression and abundance in CF at ovulation of N-acetylneuraminic acid synthase a (Nansa) suggest a previously unsuspected role. We show that salmonid CF is a complex biological fluid containing a diversity of proteins related to immunity, calcium binding, lipid metabolism, proteolysis, extracellular matrix and sialic acid metabolic pathway that are collectively responsible for preserving egg viability.

Extracellular vesicles (EVs) are mediators of intercellular communication in the tumor microenvironment. Tumor EVs are commonly associated with metastasis, immunosuppression or drug resistance. Viral infections usually increase EV secretion, but little is known about the effect of oncolytic viruses (OVs) on tumor EVs. Here, we investigated the impact of oncolytic vesicular stomatitis virus (VSV) and vaccinia virus on EVs secreted by human melanoma and thoracic cancer cells. We found that OV infection increases the production of EVs by tumor cells. These EVs contain proteins of viral origin, such as VSV-G, thus creating a continuum of particles sharing markers of both canonical EVs and viruses. As such, the presence of VSV-G on EVs improves the transfer of their protein content to cell types commonly found in the tumor microenvironment. A proteomic analysis also revealed that EVs-OV secreted during VSV infection are enriched in immunity-related proteins. Finally, CD8+ T cells incubated with EVs-OV from infected cells display slightly enhanced cytotoxic functions. Taken together, these data suggest that OVs enhance the communication mediated by tumor EVs, which could participate in the therapeutic efficacy of OVs. These results also provide rationale for engineering OVs to exploit EVs and disseminate therapeutic proteins within the tumor microenvironment.

Background/Objectives: Marine collagen peptides (MCPs) and glycosaminoglycans (GAGs) have been described as potential wound-healing (WH) agents. Fish cartilage hydrolysate (FCH) is a natural active food ingredient obtained from enzymatic hydrolysis which combines MCPs and GAGs. Recently, the clinical benefits of FCH supplementation for the skin, as well as its mode of action, have been demonstrated. Some of the highlighted mechanisms are common to the WH process. The aim of the study is therefore to investigate the influence of FCH supplementation on the skin healing processes and the underlying mechanisms. Methods: To this end, an ex vivo clinical approach, which takes into account the clinical digestive course of nutrients, coupled with primary cell culture on human dermal fibroblasts (HDFs) and ultra-deep proteomic analysis, was performed. The effects of human serum enriched in circulating metabolites resulting from FCH ingestion (FCH-enriched serum) were assessed on HDF WH via an in vitro scratch wound assay and on the HDF proteome via diaPASEF (Data Independent Acquisition—Parallel Accumulation Serial Fragmentation) proteomic analysis. Results: Results showed that FCH-enriched human serum accelerated wound closure. In support, proteins with anti-inflammatory and immunomodulatory properties and proteins prone to promote hydration and ECM stability showed increased expression in HDFs after exposure to FCH-enriched serum. Conclusions: Taken together, these data provide valuable new insights into the mechanisms that may contribute to FCH’s beneficial impact on human skin functionality by supporting WH. Further studies are needed to reinforce these preliminary data and investigate the anti-inflammatory and immunomodulatory properties of FCH.

Summary: The advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias.

Abstract The specific functions and essentiality of type II vitellogenin (Vtg2) in early zebrafish development were investigated in this study. A vtg2-mutant zebrafish line was produced and effects of genomic disturbance were observed in F2 females and F3 offspring. No change in vtg2 transcript has been detected, however, Vtg2 abundance in F2 female liver was 5×, and in 1 hpf F3 vtg2-mutant embryos was 3.8× less than Wt (p

Abstract Background Spermatozoa interact with oviduct secretions before fertilization in vivo but the molecular players of this dialog and underlying dynamics remain largely unknown. Our objectives were to identify an exhaustive list of sperm-interacting proteins (SIPs) in the bovine oviduct fluid and to evaluate the impact of the oviduct anatomical region (isthmus vs. ampulla) and time relative to ovulation (pre-ovulatory vs. post-ovulatory) on SIPs number and abundance. Methods Pools of oviduct fluid (OF) from the pre-ovulatory ampulla, pre-ovulatory isthmus, post-ovulatory ampulla, and post-ovulatory isthmus in the side of ovulation were collected from the slaughterhouse. Frozen-thawed bull sperm were incubated with OF or phosphate-buffered saline (control) for 60 min at 38.5 °C. After protein extraction and digestion, sperm and OF samples were analyzed by nanoLC-MS/MS and label-free protein quantification. Results A quantitative comparison between proteins identified in sperm and OF samples (2333 and 2471 proteins, respectively) allowed for the identification of 245 SIPs. The highest number (187) were found in the pre-ovulatory isthmus, i.e., time and place of the sperm reservoir. In total, 41 SIPs (17%) were differentially abundant between stages in a given region or between regions at a given stage and 76 SIPs (31%) were identified in only one region × stage condition. Functional analysis of SIPs predicted roles in cell response to stress, regulation of cell motility, fertilization, and early embryo development. Conclusion This study provides a comprehensive list of SIPs in the bovine oviduct and evidences dynamic spatio-temporal changes in sperm-oviduct interactions around ovulation time. Moreover, these data provide protein candidates to improve sperm conservation and in vitro fertilization media.

Abstract Background Despite many improvements with in vitro culture systems, the quality and developmental ability of mammalian embryos produced in vitro are still lower than their in vivo counterparts. Though previous studies have evidenced differences in gene expression between in vivo- and in vitro-derived bovine embryos, there is no comparison at the protein expression level. Results A total of 38 pools of grade-1 quality bovine embryos at the 4–6 cell, 8–12 cell, morula, compact morula, and blastocyst stages developed either in vivo or in vitro were analyzed by nano-liquid chromatography coupled with label-free quantitative mass spectrometry, allowing for the identification of 3,028 proteins. Multivariate analysis of quantified proteins showed a clear separation of embryo pools according to their in vivo or in vitro origin at all stages. Three clusters of differentially abundant proteins (DAPs) were evidenced according to embryo origin, including 463 proteins more abundant in vivo than in vitro across development and 314 and 222 proteins more abundant in vitro than in vivo before and after the morula stage, respectively. The functional analysis of proteins found more abundant in vivo showed an enrichment in carbohydrate metabolism and cytoplasmic cellular components. Proteins found more abundant in vitro before the morula stage were mostly localized in mitochondrial matrix and involved in ATP-dependent activity, while those overabundant after the morula stage were mostly localized in the ribonucleoprotein complex and involved in protein synthesis. Oviductin and other oviductal proteins, previously shown to interact with early embryos, were among the most overabundant proteins after in vivo development. Conclusions The maternal environment led to higher degradation of mitochondrial proteins at early developmental stages, lower abundance of proteins involved in protein synthesis at the time of embryonic genome activation, and a global upregulation of carbohydrate metabolic pathways compared to in vitro production. Furthermore, embryos developed in vivo internalized large amounts of oviductin and other proteins probably originated in the oviduct as soon as the 4–6 cell stage. These data provide new insight into the molecular contribution of the mother to the developmental ability of early embryos and will help design better in vitro culture systems.

Abstract Understanding the composition of the oviduct fluid (OF) is crucial to better comprehend the microenvironment in which sperm capacitation, fertilization and early embryo development take place. Therefore, our aim was to determine the spatiotemporal changes in the OF proteome according to the anatomical region of the oviduct (ampulla vs. isthmus), the proximity of the ovulating ovary (ipsilateral vs. contralateral side) and the peri-ovulatory stage (pre-ovulatory or Pre-ov vs. post-ovulatory or Post-ov). Oviducts from adult cyclic cows were collected at a local slaughterhouse and pools of OF were analyzed by nanoLC-MS/MS and label-free protein quantification (n = 32 OF pools for all region × stage × side conditions). A total of 3760 proteins were identified in the OF, of which 65% were predicted to be potentially secreted. The oviduct region was the major source of variation in protein abundance, followed by the proximity of the ovulating ovary and finally the peri-ovulatory stage. Differentially abundant proteins between regions, stages and sides were involved in a broad variety of biological functions, including protein binding, response to stress, cell-to-cell adhesion, calcium homeostasis and the immune system. This work highlights the dynamic regulation of oviduct secretions and provides new protein candidates for interactions between the maternal environment, the gametes and the early embryo.

The major challenge in antigen‐specific immunotherapy of cancer is to select the most relevant tumor antigens to target. To this aim, understanding their mode of expression by tumor cells is critical. We previously identified a melanoma‐specific antigen, melanoma‐overexpressed antigen 1 (MELOE‐1)—coded for by a long noncoding RNA—whose internal ribosomal entry sequence (IRES)‐dependent translation is restricted to tumor cells. This restricted expression is associated with the presence of a broad‐specific T‐cell repertoire that is involved in tumor immunosurveillance in melanoma patients. In the present work, we explored the translation control of MELOE‐1 and provide evidence that heterogeneous nuclear ribonucleoprotein A1 (hnRNP‐A1) binds to the MELOE‐1 IRES and acts as an IRES trans‐activating factor (ITAF) to promote the translation of MELOE‐1 in melanoma cells. In addition, we showed that endoplasmic reticulum (ER) stress induced by thapsigargin, which promotes hnRNP‐A1 cytoplasmic translocation, enhances MELOE‐1 translation and recognition of melanoma cells by a MELOE‐1‐specific T‐cell clone. These findings suggest that pharmacological stimulation of stress pathways may enhance the efficacy of immunotherapies targeting stress‐induced tumor antigens such as MELOE‐1.

Abstract Background We previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence on essentiality and disparate functioning of Vtgs at different stages during early development. However, the specific contributions of different types of Vtg to major cellular processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to meet this deficit. Proteomic profiles of zebrafish eggs lacking three type-I Vtgs simultaneously (vtg1-KO), or lacking only type III Vtg (vtg3-KO) were compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts. Results The vtg-KO caused severe changes in the proteome of 1-cell stage zebrafish eggs. These changes were disclosed by molecular signatures that highly resembled the proteomic phenotype of poor quality zebrafish eggs reported in our prior studies. Proteomic profiles of vtg-KO eggs and perturbations in abundances of hundreds of proteins revealed unique, noncompensable contributions of multiple Vtgs to protein and in energy homeostasis. The lack of this contribution appears to have a significant impact on endoplasmic reticulum and mitochondrial functions, and thus embryonic development, even after zygotic genome activation. Increased endoplasmic reticulum stress, Redox/Detox activities, glycolysis/gluconeogenesis, enrichment in cellular proliferation and in human neurodegenerative disease related activities in both vtg1- and vtg3-KO eggs were found to be indicators of the aforementioned conditions. Distinctive increase in apoptosis and Parkinson disease pathways, as well as the decrease in lipid metabolism related activities in vtg3-KO eggs implies compelling roles of Vtg3, the least abundant form of Vtgs in vertebrate eggs, in mitochondrial activities. Several differentially abundant proteins representing the altered molecular mechanisms have been identified as strong candidate markers for studying the details of these mechanisms during early embryonic development in zebrafish and possibly other vertebrates. Conclusions These findings indicate that the global egg proteome is subject to extensive modification depending on the presence or absence of specific Vtgs and that these modifications can have a major impact on developmental competence.

Early embryo development is a dynamic process involving important molecular and structural changes leading to the embryonic genome activation (EGA) and early cell lineage differentiation. Our aim was to elucidate proteomic changes in bovine embryos developed in vivo. Eleven females were used as embryo donors and pools of embryos at the 4–6 cell, 8–12 cell, morula, compact morula and blastocyst stages were analyzed by nanoliquid chromatography coupled with label free quantitative mass spectrometry. A total of 2,757 proteins were identified, of which 1,950 were quantitatively analyzed. Principal component analysis of data showed a clear separation of embryo pools according to their developmental stage. The hierarchical clustering of differentially abundant proteins evidenced a first cluster of 626 proteins that increased in abundance during development and a second cluster of 400 proteins that decreased in abundance during development, with most significant changes at the time of EGA and blastocyst formation. The main pathways and processes overrepresented among upregulated proteins were RNA metabolism, protein translation and ribosome biogenesis, whereas Golgi vesicle transport and protein processing in endoplasmic reticulum were overrepresented among downregulated proteins. The pairwise comparison between stages allowed us to identify specific protein interaction networks and metabolic pathways at the time of EGA, morula compaction and blastocyst formation. This is the first comprehensive study of proteome dynamics in non-rodent mammalian embryos developed in vivo. These data provide a number of protein candidates that will be useful for further mechanistic and functional studies.

The Human Proteome Project (HPP) was launched in 2010 to enhance accurate annotation of the genome-encoded proteome. Ten years later, the HPP releases its first blueprint of the human proteome, annotating 90% of all known proteins at high-stringency and discussing the implications of proteomics for precision medicine.

Ribosome biogenesis is a complex and multistep process that depends on various assembly factors. To understand this process and identify the ribosome assembly intermediates, most studies have set out to delete or deplete these assembly factors. Instead, we took advantage of the impact of heat stress (45 °C) on the late stages of the biogenesis of the 30S ribosomal subunit to explore authentic precursors. Under these conditions, reduced levels of the DnaK chaperone proteins devoted to ribosome assembly lead to the transient accumulation of 21S ribosomal particles, which are 30S precursors. We constructed strains with different affinity tags on one early and one late 30S ribosomal protein and purified the 21S particles that form under heat shock. A combination of relative quantification using mass spectrometry-based proteomics and cryo-electron microscopy (cryo-EM) was then used to determine their protein contents and structures.

Abstract Background Accurate structural annotation of genomes is still a challenge, despite the progress made over the past decade. The prediction of gene structure remains difficult, especially for eukaryotic species, and is often erroneous and incomplete. We used a proteogenomics strategy, taking advantage of the combination of proteomics datasets and bioinformatics tools, to identify novel protein coding-genes and splice isoforms, assign correct start sites, and validate predicted exons and genes. Results Our proteogenomics workflow, Peptimapper, was applied to the genome annotation of Ectocarpus sp., a key reference genome for both the brown algal lineage and stramenopiles. We generated proteomics data from various life cycle stages of Ectocarpus sp. strains and sub-cellular fractions using a shotgun approach. First, we directly generated peptide sequence tags (PSTs) from the proteomics data. Second, we mapped PSTs onto the translated genomic sequence. Closely located hits (i.e., PSTs locations on the genome) were then clustered to detect potential coding regions based on parameters optimized for the organism. Third, we evaluated each cluster and compared it to gene predictions from existing conventional genome annotation approaches. Finally, we integrated cluster locations into GFF files to use a genome viewer. We identified two potential novel genes, a ribosomal protein L22 and an aryl sulfotransferase and corrected the gene structure of a dihydrolipoamide acetyltransferase. We experimentally validated the results by RT-PCR and using transcriptomics data. Conclusions Peptimapper is a complementary tool for the expert annotation of genomes. It is suitable for any organism and is distributed through a Docker image available on two public bioinformatics docker repositories: Docker Hub and BioShaDock. This workflow is also accessible through the Galaxy framework and for use by non-computer scientists at https://galaxy.protim.eu. Data are available via ProteomeXchange under identifier PXD010618.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a well-known apoptosis inducer and a potential anticancer agent. When caspases and inhibitors of apoptosis proteins (IAPs) are inhibited, TRAIL induces necroptosis. Molecular mechanisms of necroptosis rely on kinase activation, and on the formation of a necrosome complex, bringing together the receptor-interacting protein kinases 1 and 3 (RIPK1, RIPK3), and the mixed lineage kinase domain-like protein (MLKL). In this study, mass spectrometry approach allowed to identify the tripartite motif containing 21 (TRIM21), an E3 ubiquitin-protein ligase as a new partner of the endogenous TRAIL-induced necrosome. Alteration of TRIM21 expression level, obtained by transient transfection of HT29 or HaCat cells with TRIM21-targeted siRNAs or cDNA plasmids coding for TRIM21 demonstrated that TRIM21 is a positive regulator of TRAIL-induced necroptosis. Furthermore, the invalidation of TRIM21 expression in HT29 cells by CRISPR-Cas9 technology also decreased cell sensitivity to TRAIL-induced necroptosis, a shortcoming associated with a reduction in MLKL phosphorylation, the necroptosis executioner. Thus, TRIM21 emerged as a new partner of the TRAIL-induced necrosome that positively regulates the necroptosis process.

Summary: The adaptor SHARPIN composes, together with the E3 ligases HOIP and HOIL1, the linear ubiquitin chain assembly complex (LUBAC). This enzymatic complex catalyzes and stamps atypical linear ubiquitin chains onto substrates to modify their fate and has been linked to the regulation of the NF-κB pathway downstream of most immunoreceptors, inflammation, and cell death. However, how this signaling complex is regulated is not fully understood. Here, we report that a portion of SHARPIN is constitutively phosphorylated on the serine at position 165 in lymphoblastoid cells and can be further induced following T cell receptor stimulation. Analysis of a phosphorylation-resistant mutant of SHARPIN revealed that this mark controls the linear ubiquitination of the NF-κB regulator NEMO and allows the optimal activation of NF-κB in response to TNFα. These results identify an additional layer of regulation of the LUBAC and unveil potential strategies to modulate its action.

Over the past several decades, stress has dramatically increased in occidental societies. The use of natural resources, such as fish hydrolysates, may be an attractive strategy to improve stress management. Our previous study demonstrated the anxiolytic effects of fish hydrolysate supplementation in mice exposed to acute mild stress by limiting stress-induced corticosterone release and modulating the expression of a number of stress-responsive genes. Here, we explore hippocampal protein modulation induced by fish hydrolysate supplementation in mice submitted to acute mild stress, with the aim of better elucidating the underlying mechanisms. Hippocampi from the same cohort of Balb/c mice supplemented with fish hydrolysate (300 mg·kg−1 body weight) or vehicle daily for seven days before being submitted or not to an acute mild stress protocol (four groups, n = 8/group) were subjected to label-free quantitative proteomics analysis combined with gene ontology data mining. Our results show that fish hydrolysate supplementation prevented the observed stress-induced dysregulation of proteins relative to mitochondrial pathways and the neuronal network. These findings suggest that fish hydrolysate represents an innovative strategy to prevent the adverse effects of stress and participate in stress management.

Cerebrospinal fluid (CSF) is a biofluid in direct contact with the brain and as such constitutes a sample of choice in neurological disorder research, including neurodegenerative diseases such as Alzheimer or Parkinson. Human CSF has still been less studied using proteomic technologies compared to other biological fluids such as blood plasma or serum. In this work, a pool of “normal” human CSF samples was analysed using a shotgun proteomic workflow that combined removal of highly abundant proteins by immunoaffinity depletion and isoelectric focussing fractionation of tryptic peptides to alleviate the complexity of the biofluid. The resulting 24 fractions were analysed using liquid chromatography coupled to a high-resolution and high-accuracy timsTOF Pro mass spectrometer. This state-of-the-art mass spectrometry-based proteomic workflow allowed the identification of 3’174 proteins in CSF. The dataset reported herein completes the pool of the most comprehensive human CSF proteomes obtained so far. An overview of the identified proteins is provided based on gene ontology annotation. Mass and tandem mass spectra are made available as a possible starting point for further studies exploring the human CSF proteome.

Endometriosis is a common chronic gynaecological disease causing various symptoms, such as infertility and chronic pain. The gold standard for its diagnosis is still laparoscopy and the biopsy of endometriotic lesions. Here, we aimed to compare the eutopic endometrium from women with or without endometriosis to identify proteins that may be considered as potential biomarker candidates. Eutopic endometrium was collected from patients with endometriosis (n = 4) and women without endometriosis (n = 5) during a laparoscopy surgery during the mid-secretory phase of their menstrual cycle. Total proteins from tissues were extracted and digested before LC-MS-MS analysis. Among the 5301 proteins identified, 543 were differentially expressed and enriched in two specific KEGG pathways: focal adhesion and PI3K/AKT signaling. Integration of our data with a large-scale proteomics dataset allowed us to highlight 11 proteins that share the same trend of dysregulation in eutopic endometrium, regardless of the phase of the menstrual cycle. Our results constitute the first step towards the identification of potential promising endometrial diagnostic biomarkers. They provide new insights into the mechanisms underlying endometriosis and its etiology. Our results await further confirmation on a larger sample cohort.

Abstract The regulatory mechanisms involved in the acquisition of thermal tolerance are unknown in insects. Reversible phosphorylation is a widespread post-translational modification that can rapidly alter proteins function(s). Here, we conducted a large-scale comparative screening of phosphorylation networks in adult Drosophila flies that were cold-acclimated versus control. Using a modified SIMAC method followed by a multiple MS analysis strategy, we identified a large collection of phosphopeptides (about 1600) and phosphoproteins (about 500) in both groups, with good enrichment efficacy (80%). The saturation curves from the four biological replicates revealed that the phosphoproteome was rather well covered under our experimental conditions. Acclimation evoked a strong phosphoproteomic signal characterized by large sets of unique and differential phosphoproteins. These were involved in several major GO superclusters of which cytoskeleton organization, positive regulation of transport, cell cycle, and RNA processing were particularly enriched. Data suggest that phosphoproteomic changes in response to acclimation were mainly localized within cytoskeletal network, and particularly within microtubule associated complexes. This study opens up novel research avenues for exploring the complex regulatory networks that lead to acquired thermal tolerance.

Neuropeptides exert essential functions in animal physiology by controlling e.g., reproduction, development, growth, energy homeostasis, cardiovascular activity and stress response. Thus, identification of neuropeptides has been a very active field of research over the last decades. This review article presents the various methods used to discover novel bioactive peptides in vertebrates. Initially identified on the basis of their biological activity, some neuropeptides have also been discovered for their ability to bind/activate a specific receptor or based on their biochemical characteristics such as C-terminal amidation which concerns half of the known neuropeptides. More recently, sequencing of the genome of many representative species has facilitated peptidomic approaches using mass spectrometry and in silico screening of genomic libraries. Through these different approaches, more than a hundred of bioactive neuropeptides have already been identified in vertebrates. Nevertheless, researchers continue to find new neuropeptides or to identify novel functions of neuropeptides that had not been detected previously, as it was recently the case for nociceptin.

The bovine embryo develops in contact with the oviductal fluid (OF) during the first 4−5 days of pregnancy. The aim of this study was to decipher the protein interactions occurring between the developing embryo and surrounding OF. In-vitro produced 4−6 cell and morula embryos were incubated or not (controls) in post-ovulatory OF (OF-treated embryos) and proteins were then analyzed and quantified by high resolution mass spectrometry (MS) in both embryo groups and in OF. A comparative analysis of MS data allowed the identification and quantification of 56 embryo-interacting proteins originated from the OF, including oviductin (OVGP1) and several annexins (ANXA1, ANXA2, ANXA4) as the most abundant ones. Some embryo-interacting proteins were developmental stage-specific, showing a modulating role of the embryo in protein interactions. Three interacting proteins (OVGP1, ANXA1 and PYGL) were immunolocalized in the perivitelline space and in blastomeres, showing that OF proteins were able to cross the zona pellucida and be taken up by the embryo. Interacting proteins were involved in a wide range of functions, among which metabolism and cellular processes were predominant. This study identified for the first time a high number of oviductal embryo-interacting proteins, paving the way for further targeted studies of proteins potentially involved in the establishment of pregnancy in cattle.

Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine.

Few studies have been undertaken to assess the possible effects of bisphenol A (BPA) on the reproductive hormone balance in animals or humans with often contradictory results. We investigated possible direct endocrine disruption by BPA of the fetal testes of 2 rat strains (14.5-17.5 days post-coitum) and humans (8-12 gestational weeks) and under different culture conditions. BPA concentrations of 10(-8)M and 10(-5)M for 72 h reduced testosterone production by the Sprague-Dawley fetal rat testes, while only 10-5M suppressed it in the Wistar strain. The suppressive effects at 10-5M were seen as early as 24h and 48 h in both strains. BPA at 10(-7)-10(-5)M for 72 h suppressed the levels of fetal rat Leydig cell insulin-like factor 3 (INSL3). BPA exposure at 10(-8)M, 10(-7)M, and 10(-5)M for 72 h inhibited testosterone production in fetal human testes. For the lowest doses, the effects observed occurred only when no gonadotrophin was added to the culture media and were associated with a poorly preserved testicular morphology. We concluded that (i) BPA can display anti-androgenic effects both in rat and human fetal testes; (ii) it is essential to ascertain that the divergent effects of endocrine disruptors between species in vitro do not result from the culture conditions used, and/or the rodent strain selected; (iii) the optimization of each in vitro assay for a given species should be a major objective rather than the search of an hypothetical trans-species consensual model-system, as the organization of the testis is intrinsically different between mammalian species; (iv) due to the uncertainty existing on the internal exposure of the human fetal testis to BPA, and the insufficient number of epidemiological studies on the endocrine disruptive effects of BPA, caution should be taken in the extrapolation of our present results to the human reproductive health after fetal exposure to BPA.

Mammalian spermatogenesis, which takes place in complex testicular structures called seminiferous tubules, is a highly specialized process controlled by the integration of juxtacrine, paracrine and endocrine information. Within the seminiferous tubules, the germ cells and Sertoli cells are surrounded by testicular fluid (TF), which probably contains most of the secreted proteins involved in crosstalk between these cells. It has already been established that germ cells can modulate somatic Sertoli cell function through the secretion of diffusible factors. We studied the germ cell secretome, which was previously considered inaccessible, by analyzing the TF collected by microsurgery in an "integrative omics" strategy combining proteomics, transcriptomics, genomics and interactomics data. This approach identified a set of proteins preferentially secreted by Sertoli cells or germ cells. An interaction network analysis revealed complex, interlaced cell-cell dialog between the secretome and membranome of seminiferous cells, mediated via the TF. We then focused on germ cell-secreted candidate proteins, and we identified several potential interacting partners located on the surface of Sertoli cells. Two interactions, APOH/CDC42 and APP/NGFR, were validated in situ, in a proximity ligation assay (PLA). Our results provide new insight into the crosstalk between germ cells and Sertoli cells occurring during spermatogenesis. Our findings also demonstrate that this "integrative omics" strategy is powerful enough for data mining and highlighting meaningful cell-cell communication events between different types of cells in a complex tissue, via a biological fluid. This integrative strategy could be applied more widely, to gain access to secretomes that have proved difficult to study whilst avoiding the limitations of in vitro culture.

Vertebrate genomes contain around 20,000 protein-encoding genes, of which a large fraction is still not associated with specific functions. A major task in future genomics will thus be to assign physiological roles to all open reading frames revealed by genome sequencing. Here we show that C2orf62, a highly conserved protein with little homology to characterized proteins, is strongly expressed in testis in zebrafish and mammals, and in various types of ciliated cells during zebrafish development. By yeast two hybrid and GST pull-down, C2orf62 was shown to interact with TTC17, another uncharacterized protein. Depletion of either C2orf62 or TTC17 in human ciliated cells interferes with actin polymerization and reduces the number of primary cilia without changing their length. Zebrafish embryos injected with morpholinos against C2orf62 or TTC17, or with mRNA coding for the C2orf62 C-terminal part containing a RII dimerization/docking (R2D2) - like domain show morphological defects consistent with imperfect ciliogenesis. We provide here the first evidence for a C2orf62-TTC17 axis that would regulate actin polymerization and ciliogenesis.