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Resume Objectif Au cours des fecondations in vitro (FIV) classiques, 5 % des tentatives se soldent par un echec de fecondation imprevisible, du fait d’une absence d’anomalies des parametres spermatiques. Dans 56 % des cas, aucune cause ovocytaire evidente n’est retrouvee. On observe, par contre, un defaut ou une absence de fixation des spermatozoides a la zone pellucide (ZP). Cette etude a pour objectif de preciser les causes moleculaires d’origine spermatique des echecs de FIV, qui sont a ce jour imprevisibles d’apres les donnees de la spermiologie classique. Patientes et methodes Le profil d’expression proteique des spermatozoides de sujets, presentant un echec complet de fecondation et un defaut de fixation des spermatozoides a la ZP, est compare a celui de temoins (hommes a sperme normal et fecondant, participant a une FIV d’indication tubaire). Pour cela, les echantillons de sperme sont analyses par la technologie d’electrophorese 2 Dimensional Electrophoresis-Differential In Gel Electrophoresis (2D-DIGE) avec au prealable une etape de pre-fractionnement des extraits proteiques selon leur point isoelectrique (fraction acide, intermediaire et basique). Resultats Sept proteines differentiellement exprimees chez tous les cas et chez tous les temoins ont ete mises en evidence dans la fraction acide et sept dans la fraction basique. Douze d’entre elles ont ete identifiees par spectrometrie de masse, dont deux (le recepteur a la laminine LR67 et la L-xylulose reductase) ont deja fait la preuve de leur implication dans l’interaction gametique. Discussion et conclusion Ces proteines meritent des investigations complementaires visant a determiner si certains echecs de fecondation en FIV sont causes par des anomalies au niveau de leur expression.

International audience; Objective: To identify the male molecular causes of failures of IVF (with a deficient binding of spermatozoa to the zona pellucida, without any obvious oocyte anomaly), which are undetected by classical sperm analysis. Design: Case-control prospective study. Setting: University hospital. Patient(s): Proteomic profiles of spermatozoa in patients with a complete failure of fertilization and no spermatozoa bound to the zona pellucida were compared with those of controls (men with normal fertilization and cleavage rates after classical IVF for tubal indication). Intervention(s): All samples were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) after being divided into three fractions according to their isoelectric point. Main Outcome Measure(s): Differentially expressed proteins between infertile men and controls were identified by mass spectrometry. Result(s): Seventeen proteins differentially expressed between cases and controls were found. Twelve of these proteins were identified by mass spectrometry, and two may influence gametes interaction: laminin receptor LR67 and L-xylulose reductase (P34H). Conclusion(s): This study shows that 2D-DIGE might be useful in finding potential targets for diagnosis and prognosis of idiopathic infertility in IVF.

Waldenström's macroglobulinemia (WM) is a clonal B-cell lymphoproliferative disorder (LPD) of post-germinal center nature. Despite the fact that the precise molecular pathway(s) leading to WM remain(s) to be elucidated, a hallmark of the disease is the absence of the immunoglobulin heavy chain class switch recombination. Using two-dimensional gel electrophoresis, we compared proteomic profiles of WM cells with that of other LPDs. We were able to demonstrate that WM constitutes a unique proteomic entity as compared with chronic lymphocytic leukemia and marginal zone lymphoma. Statistical comparisons of protein expression levels revealed that a few proteins are distinctly expressed in WM in comparison with other LPDs. In particular we observed a major downregulation of the double strand repair protein Ku70 (XRCC6); confirmed at both the protein and RNA levels in an independent cohort of patients. Hence, we define a distinctive proteomic profile for WM where the downregulation of Ku70-a component of the non homologous end-joining pathway-might be relevant in disease pathophysiology. journal article 2012 Sep 07 2012 09 07 imported

Unraveling the molecular basis for the different functional consequences of BCR cross-linking is essential to understand the leukemogenesis process of CLL. There is increasing evidence that an antigen driven process is crucial in CLL proliferation, based on restriction of the IgVH repertory as well as shared antigen-binding motifs used by mutated or unmutated CLL B-cells. BCR activation leads to a signaling cascade reinforced in the more aggressive CLL form by the ZAP70 protein tyrosine kinase and constitutive phosphorylation of HS1 protein. We have previously shown, using gene expression profiling over time, that crosslinking of the BCR induces a specific temporal gene expression program in the leukemic cells, leading to a complex balance disorder between proliferation and cell death. These results demonstrated a core BCR gene expression shared among all B cell (and across species). Furthermore, each cell category (healthy B-cells, indolent and aggressive CLL cells) also showed a specific temporal gene expression after BCR cross-linking. This analysis has revealed complex expression disorder of multiple genes coding proteins involved in proliferation and death regulation (Vallat et al. Blood 07). To further investigate the functional consequences of BCR cross-linking we also examined modulation of the BCR proteome, since a dynamic description of the BCR functional consequences in CLL cells would not be complete without parallel temporal proteomic analysis. We now have cross-linked the BCR in freshly isolated B-cells from CLL patients. We have isolated total proteins at 2 early, one intermediate and a later time point 30 min later than the time points selected in the previous mRNA experiments. At each analysis time point both stimulated and control unstimulated cells were examined. A 2D electrophoresis analysis (pH 3–10, 150–20 kDa) revealed between 1100 and 1800 (mean 1400) polypetidic spots at each time point after BCR engagement. A total of 600 different polypeptides show a specific pattern of expression over time after stimulation, up- (450 polypeptides) or down-regulated (150 polypeptides). We are currently identifying these different proteins of interest by mass spectrometry (Maldi-TOF and LC-MS/MS) and learning these results in parallel of the previous genomic results. Preliminary results show the complementarity of the two analyses. Many of the proteins identified correspond to changes in mRNA belonging to the temporal gene program previously described after BCR engagement, thereby validating the gene expression data at the protein level, but some proteins show a specific temporal pattern of expression whereas their corresponding coding mRNA were not present in the temporal gene expression. Such complementarities in this dynamical approach allow us to refine the molecular basis for the functional BCR cross-linking consequences in CLL and to identify putative therapeutic targets for intervention in this disease. Ongoing experiments are examining the consequences of intervention to alter expression of critical components within the functional consequences of BCR cross-linking in CLL, and potentially the consequences of antigen driven leukemogenesis.