Search

Résumé Contexte Les limites des analyses conventionnelles du sperme ont mis en évidence la nécessité de moyens supplémentaires d’évaluation de la qualité du sperme. Matériel et Méthodes La méthode d’électrophorèse unidimensionnelle a été utilisée pour évaluer la teneur et la qualité protéique dans des échantillons d’éjaculats individuels de 245 hommes avec des paramètres spermatiques connus. Résultats La teneur en protéines du sperme variait d’un échantillon à l’autre, en particulier dans la gamme des poids moléculaires élevés. L’intensité des bandes de 80–110 kDa était corrélée à la mobilité progressive (r = 0,15, p = 0,015) et était significativement plus élevée (p = 0,0367) dans le groupe des hommes avec des paramètres conventionnels spermatiques supérieurs aux valeurs de référence (OMS, 2010) comparé au groupe d’hommes dont l’éjaculat présentait au moins un paramètre spermatique (volume de sperme, concentration de spermatozoïdes, numération des spermatozoïdes, motilité progressive, formes normales et indice d’anomalies multiples) en deça de la valeur de référence. Les bandes dans la gamme de 80–110 kDa ont été identifiées en spectrométrie de masse et se sont révélées être des protéines de la gaine fibreuse du flagelle: la protéine d’ancrage A-kinase 4, la protéine d’ancrage A-kinase 3 et l’hexokinase de type 1. Conclusion La méthode d’électrophorèse unidimensionnelle constitue une méthode simple, rapide, fiable et peu coûteuse pour analyser la qualité des protéines du sperme dans les éjaculats humains individuels.

Abstract Background Brain capillary endothelial cells (BCECs) form the physiological basis of the blood-brain barrier (BBB). The barrier function is (at least in part) due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein). Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the well-established in vitro BBB model developed in our laboratory. Results A total of 215 protein spots (corresponding to 130 distinct proteins) were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin) and constitutes valuable evidence for predictions based on genome annotation. Conclusions Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets.

Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.

Low birth weight is associated with an increased risk for developing type 2 diabetes and metabolic diseases. The placental capacity to supply nutrients and oxygen to the fetus represents the main determiner of fetal growth. However, few studies have investigated the effects of maternal diet on the placenta. We explored placental adaptive proteomic processes implicated in response to maternal undernutrition. Rat term placentas from 70% food-restricted (FR30) mothers were used for a proteomic screen. Placental mitochondrial functions were evaluated using molecular and functional approaches, and ATP production was measured. FR30 drastically reduced placental and fetal weights. FR30 placentas displayed 14 proteins that were differentially expressed, including several mitochondrial proteins. FR30 induced a marked increase in placental mtDNA content and changes in mitochondrial functions, including modulation of the expression of genes implicated in biogenesis and bioenergetic pathways. FR30 mitochondria showed higher oxygen consumption but failed to maintain their ATP production. Maternal undernutrition induces placental mitochondrial abnormalities. Although an increase in biogenesis and bioenergetic efficiency was noted, placental ATP level was reduced. Our data suggest that placental mitochondrial defects may be implicated in fetoplacental pathologies.