Unraveling hallmark suitability for staging pre- and post-implantation stem cell models.

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.

Evolutionarily conserved ovarian fluid proteins are responsible for extending egg viability in salmonid fish.

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.

Inactivation of Exosc10 in the oocyte impairs oocyte development and maturation, leading to a depletion of the ovarian reserve in mice.

EXOSC10 is a catalytic subunit of the nuclear RNA exosome, and possesses a 3'-5' exoribonuclease activity. The enzyme processes and degrades different classes of RNAs. To delineate the role of EXOSC10 during oocyte growth, specific Exosc10 inactivation was performed in oocytes from the primordial follicle stage onward using the Gdf9-iCre; Exosc10 f/- mouse model (Exosc10 cKO(Gdf9)). Exosc10 cKO(Gdf9) female mice are infertile. The onset of puberty and the estrus cycle in mutants are initially normal and ovaries contain all follicle classes. By the age of eight weeks, vaginal smears reveal irregular estrus cycles and mutant ovaries are completely depleted of follicles. Mutant oocytes retrieved from the oviduct are degenerated, and occasionally show an enlarged polar body, which may reflect a defective first meiotic division. Under fertilization conditions, the mutant oocytes do not enter into an embryonic development process. Furthermore, we conducted a comparative proteome analysis of wild type and Exosc10 knockout mouse ovaries, and identified EXOSC10-dependent proteins involved in many biological processes, such as meiotic cell cycle progression and oocyte maturation. Our results unambiguously demonstrate an essential role for EXOSC10 in oogenesis and may serve as a model for primary ovarian insufficiency in humans. Data are available via ProteomeXchange with identifier PXD039417.

Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock.

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.

The sperm-interacting proteome in the bovine isthmus and ampulla during the periovulatory period.

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.

Metalloprotease-mediated cleavage of CD95 ligand.

CD95 is a member of the TNF receptor superfamily that is ubiquitously expressed in healthy and pathological tissues. Stimulation of CD95 by its physiological ligand CD95L induces its oligomerization leading in turn to the transduction of either apoptotic or nonapoptotic signals. CD95L can exist as both membrane-anchored and soluble forms (sCD95L), the latter resulting from the proteolytic cleavage of the former. Candidate proteases able to achieve CD95L cleavage were identified as matrix metalloproteases (MMP) due to their demonstrated ability to cleave other TNF superfamily ligands. The main goal of this study was to systematically identify the MMP family members capable of cleaving CD95L and subsequently determine the corresponding cleavage sites. By using different orthogonal biochemical approaches and combining them with molecular modelling, we confirmed data from the literature regarding CD95L cleavage by MMP-3 and MMP-7. Moreover, we found that MMP-2 and MMP-12 can cleave CD95L and characterized their resulting cleavage sites. This study provides a systematic approach to analyse the cleavage of CD95L, which until now had only been poorly described.

Naive Pluripotent and Trophoblastic Stem Cell Lines as a Model for Detecting Missing Proteins in the Context of the Chromosome-Centric Human Proteome Project.

The Chromosome-centric Human Proteome Project (C-HPP) aims at identifying the proteins as gene products encoded by the human genome, characterizing their isoforms and functions. The existence of products has now been confirmed for 93.2% of the genes at the protein level. The remaining mostly correspond to proteins of low abundance or difficult to access. Over the past years, we have significantly contributed to the identification of missing proteins in the human spermatozoa. We pursue our search in the reproductive sphere with a focus on early human embryonic development. Pluripotent cells, developing into the fetus, and trophoblast cells, giving rise to the placenta, emerge during the first weeks. This emergence is a focus of scientists working in the field of reproduction, placentation and regenerative medicine. Most knowledge has been harnessed by transcriptomic analysis. Interestingly, some genes are uniquely expressed in those cells, giving the opportunity to uncover new proteins that might play a crucial role in setting up the molecular events underlying early embryonic development. Here, we analyzed naive pluripotent and trophoblastic stem cells and discovered 4 new missing proteins, thus contributing to the C-HPP. The mass spectrometry proteomics data was deposited on ProteomeXchange under the data set identifier PXD035768.

The proteomic analysis of bovine embryos developed in vivo or in vitro reveals the contribution of the maternal environment to early embryo.

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.

Fish Hydrolysate Supplementation Prevents Stress-Induced Dysregulation of Hippocampal Proteins Relative to Mitochondrial Metabolism and the Neuronal Network in Mice.

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.

Dynamic Changes in the Proteome of Early Bovine Embryos Developed In Vivo.

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.

Spatiotemporal profiling of the bovine oviduct fluid proteome around the time of ovulation.

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 Eutopic Endometrium Proteome in Endometriosis Reveals Candidate Markers and Molecular Mechanisms of Physiopathology.

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.

Liquid Chromatography and Tandem Mass Spectrometry in Label-Free Protein Quantification of Zebrafish (Danio rerio) Eggs.

Rapid innovations in core proteomic technologies and proteome-based bioinformatics fortified by recent genome sequencing allow the characterization and quantification of proteins on a global scale. These capabilities empower research to develop a more comprehensive understanding of how changes in protein expression and modification can affect complex signaling and regulatory networks. The consequences of these studies have significant implications for understanding how myriad activities are regulated in biological systems.Proteomic approaches have been applied to investigate the physiology, developmental biology, and impact of contaminants in fishes as model organisms. Here, we describe the use of label-free protein quantification and global proteome profiling to characterize eggs of different quality grades in the zebrafish .

Serine 165 phosphorylation of SHARPIN regulates the activation of NF-κB.

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.

Spatial segmentation and metabolite annotation involved in sperm maturation in the rat epididymis by MALDI imaging mass spectrometry.

Spermatozoa acquire their fertilizing capacity during a complex maturation process that occurs in the epididymis. This process involves a substantial molecular remodeling at the surface of the gamete. Epididymis is divided into three regions (the caput, corpus, and cauda) or into 19 intraregional segments based on histology. Most studies carried out on epididymal maturation have been performed on sperm samples or tissue extracts. Here, we used MALDI imaging mass spectrometry in the positive and negative ion modes combined with spatial segmentation and automated metabolite annotation to study the precise localization of metabolites directly in the rat epididymis. The spatial segmentation revealed that the rat epididymis could be divided into several molecular clusters different from the 19 intraregional segments. The discriminative m/z values that contributed the most to each molecular cluster were then annotated and corresponded mainly to phosphatidylcholines, sphingolipids, glycerophosphates, triacylglycerols, plasmalogens, phosphatidylethanolamines, and lysophosphatidylcholines. A substantial remodeling of lipid composition during epididymal maturation was observed. It was characterized in particular by an increase in the number of sphingolipids and plasmalogens and a decrease in the proportion of triacylglycerols annotated from caput to cauda. Ion images reveal that molecules belonging to the same family can have very different localizations along the epididymis. For some of them, annotation was confirmed by on-tissue MS/MS experiments. A 3D model of the epididymis head was reconstructed from 61 sections analyzed with a lateral resolution of 50 µm and can be used to obtain information on the localization of a given analyte in the whole volume of the tissue.

Exploration of human cerebrospinal fluid: A large proteome dataset revealed by trapped ion mobility time-of-flight mass spectrometry.

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.

Identification of 56 Proteins Involved in Embryo-Maternal Interactions in the Bovine Oviduct.

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.

Numerous cultivated and uncultivated viruses encode ribosomal proteins.

Viruses modulate ecosystems by directly altering host metabolisms through auxiliary metabolic genes. However, viral genomes are not known to encode the core components of translation machinery, such as ribosomal proteins (RPs). Here, using reference genomes and global-scale viral metagenomic datasets, we identify 14 different RPs across viral genomes arising from cultivated viral isolates and metagenome-assembled viruses. Viruses tend to encode dynamic RPs, easily exchangeable between ribosomes, suggesting these proteins can replace cellular versions in host ribosomes. Functional assays confirm that the two most common virus-encoded RPs, bS21 and bL12, are incorporated into 70S ribosomes when expressed in Escherichia coli. Ecological distribution of virus-encoded RPs suggests some level of ecosystem adaptations as aquatic viruses and viruses of animal-associated bacteria are enriched for different subsets of RPs. Finally, RP genes are under purifying selection and thus likely retained an important function after being horizontally transferred into virus genomes.

Peptimapper: proteogenomics workflow for the expert annotation of eukaryotic genomes.

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.

Deciphering the Dark Proteome: Use of the Testis and Characterization of Two Dark Proteins.

For the C-HPP consortium, dark proteins include not only uPE1, but also missing proteins (MPs, PE2-4), smORFs, proteins from lncRNAs, and products from uncharacterized transcripts. Here, we investigated the expression of dark proteins in the human testis by combining public mRNA and protein expression data for several tissues and performing LC-MS/MS analysis of testis protein extracts. Most uncharacterized proteins are highly expressed in the testis. Thirty could be identified in our data set, of which two were selected for further analyses: (1) A0AOU1RQG5, a putative cancer/testis antigen specifically expressed in the testis, where it accumulates in the cytoplasm of elongated spermatids; and (2) PNMA6E, which is enriched in the testis, where it is found in the germ cell nuclei during most stages of spermatogenesis. Both proteins are coded on Chromosome X. Finally, we studied the expression of other dark proteins, uPE1 and MPs, in a series of human tissues. Most were highly expressed in the testis at both the mRNA and protein levels. The testis appears to be a relevant organ to study the dark proteome, which may have a function related to spermatogenesis and germ cell differentiation. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD009598.