Proteomic profile of maternal-aged blastocoel fluid suggests a novel role for ubiquitin system in blastocyst quality

Purpose: The etiology of maternal aging, a common cause of female factor infertility and a rate-limiting step in vitro fertilization (IVF) success, remains still unclear. Proteomic changes responsible for the impaired successful pregnancy outcome after IVF with aged blastocysts have not been yet evaluated. The objective of this prospective study was to employ proteomic techniques and bioinformatic tools to enlight differences at the protein level in blastocoel fluid of aged and younger woman. Methods: Protein composition of human blastocoel fluid isolated by micromanipulation from 46 blastocysts of women aged <37 years (group A) and 29 of women aged 6537 years (group B) have been identified by a shotgun proteomic approach based on high-resolution nano-liquid chromatography electrospray-ionization-tandem mass spectrometry (nLC-ESI-MS/MS) using label free for the relative quantification of their expression levels. Results: The proteomic analysis leads to the identification and quantification of 148 proteins; 132 and 116 proteins were identified in groups A and B, respectively. Interestingly, the identified proteins are mainly involved in processes aimed at fine tuning embryo implantation and development. Among the 100 proteins commonly expressed in both groups, 17 proteins are upregulated and 44 downregulated in group B compared to group A. Overall, the analysis identified 33 proteins, which were increased or present only in B while 76 were decreased in B or present only in A. Conclusions: Data revealed that maternal aging mainly affects blastocyst survival and implantation through unbalancing the equilibrium of the ubiquitin system known to play a crucial role in fine-tuning several aspects required to ensure successful pregnancy outcome

PROTEIN TYROSINE NITRATION UNDER PHYSIOLOGICAL CONDITIONS IN CELLULAR AND ANIMAL MODELS

The significance of NO2Tyr in vivo is highlighted by observations that nitrated proteins are markedly elevated in a broad range of human diseases and clinical disorders. The presence of nitrated tyrosine residues was detected in human fluids and pathological tissues such as atherosclerotic plaques of coronary vessels, amyotrophic lateral sclerosis, and Alzheimer\u2019s lesions, among many others. Although the accumulation of nitrated proteins correlates well with many disease states and is considered a marker of oxidative stress under pathological conditions, substantial evidence has accrued that protein tyrosine nitration is a post-translational modification playing a role in physiological processes, including signal transduction, neuronal differentiation, and embryonic development. On this regard, the aim of the research presented in this PhD thesis is to better understand the significance of protein nitration under normal physiological conditions focusing on differentiation and developmental processes. The PhD thesis is divided in four parts. The first introductory part is dedicated to presenting the biological functions of nitric oxide and its cellular effects in biological system. Particular attention has been devoted to protein tyrosine nitration and its pathological and physiological significance. Two different experimental models (cells and organism) have been used to investigate this issue: (a) a cellular model (PC12 cells) to discuss the effects of micro- and nanoscale topography on neuronal proliferation and differentiation and (b) an animal model (Ciona intestinalis) for studying the role of oxidative stress and NO-derived reactive nitrogen species (RNS) during Ciona development and metamorphosis-related events. In particular, as far as the first model is concerned, my studies were directed to the characterization of PC12 cells behavior on nanostructured TiO2 films in the presence and in the absence of the classical inducer of differentiation NGF. In the second part of the thesis I present the experimental procedures used. Proteomics techniques, including mono- and two-dimensional electrophoresis, electroblotting and immunostaining, and mass spectrometry (MALDI-TOF and LTQ-Orbitrap Velos) have been used to study both experimental models. In the third part the results obtained are reported. Our findings suggest that tyrosine nitration is a physiological event not necessary related to pathological processes and that this NO-mediated post-translational modification of proteins may be regarded as a direct way to NO-signaling transduction. Finally, in the last part I resume the main conclusions and present future perspectives

Sperm ubiquitination in epididymal feline semen

INTRODUCTION. Ubiquitin is a 8.5 kDa peptide that tags other proteins for proteasomal degradation and it is also involved in the regulation of protein function. Ubiquitin has been discovered as a normal component of human blood, ovarian follicular fluid and seminal plasma (1). Ubiquitination might be responsible of the elimination of defective spermatozoa during transit through epididymis in humans and cattle (2, 3). Results indicated that the increase of sperm ubiquitin was inversely associated with spermatic concentration, motility and normal morphology indicating that ubiquitination could be considered as a biomarker of poor semen quality (3). Conversely, other authors (4) found a positive correlation between sperm ubiquitin and good semen parameters suggesting a different role for sperm ubiquitination. These conflicting data indicate that the exact biological function of this peptide in seminal plasma has not yet been clarified. In the domestic cat (Felis catus) no positive or negative correlations between semen quality and ubiquitination have been observed, although no definitive conclusions about its role have been drawn (5). Magnetic cell separation techniques, based on the use of antibodies or proteins-coated magnetic beads as magnetic ubiquitin beads, may allow the removal of ubiquitinated spermatozoa from the sample contributing to the identification of a potential correlation between ubiquitin and abnormal spermatozoa. OBJECTIVES AND METHODS. The present study was designed to investigate whether ubiquitin could be considered a biomarker of quality of epididymal feline semen. Morphology and acrosomal integrity of spermatozoa were correlated with ubiquitination of the protein patterns in semen samples treated with magnetic ubiquitin beads. Moreover, the modification of the two major cytoskeletal proteins (actin and tubulin), and of prohibitin, a protein involved in cell cycle which is ubiquitinated in bovine semen (6), was evaluated in details to investigate the possible correlation of sperm morphological alterations and proteins ubiquitination. Semen samples from 10 healthy and sexually mature cats were collected by squeezing isolated caudae epididymis after routinary orchiectomy. Samples were divided into two aliquots. Magnetic ubiquitin beads (Li Starfish S.r.l., Cernusco S/N, Milan, Italy) were added to one aliquot at the final concentration of 5 particles/spermatozoa. The suspension in the tube was gently mixed for 15 min before placing it on an external laboratory magnet for 15 min. The sample treated with beads (S+B) was collected while the tube was still in the magnetic field, whereas the \u201cubiquitinated\u201d spermatozoa bound to the beads remained attached to the wall of the tube as long as the magnet was in place. The second semen aliquot was used as a control and was treated under the same conditions except for the presence of the beads (S-B). Before and after the treatment, sperm cell concentration was determined with a B\ufcrker chamber and semen morphology was assessed following staining with Bengal Rose and Victoria Blue B on at least 100 spermatozoa per slide. Acrosome integrity was analyzed by staining with Peanut agglutinin (PNA) conjugated with a fluorescein isothiocyanate (FITC) and propidium iodide (PI). Mean\ub1SD of sperm characteristics were analyzed by Student\u2019s t-test (p<0.05). Assessment of the ubiquitination of sperm proteins was carried out by Western Blot analysis using specific antibodies, namely anti-ubiquitin, anti-actin, anti-tubulin and anti-prohibitin. Each sample was solubilized in lysis buffer (50mM Tris HCl pH 8.0) and quantified for its protein content by the Bradford method. Then the samples were separated by sodium dodecyl sulphate gel electrophoresis upon sonication and centrifugation. Following blotting on polivinilidenfluoruro membrane, ubiquitinated proteins, actin, tubulin and prohibitin were detected by a chemiluminescence immunoassay. RESULTS. Proportions of morphologically normal spermatozoa were similar in samples treated with ubiquitin beads compared to the control samples (S+B: 52.9\ub111.9% vs. S-B: 44.5\ub116.4%). Treated samples did not show an increase of spermatozoa with intact acrosome (S+B: 81.3\ub113% vs. S-B: 76.5\ub115.1%), whereas a significant decrease in the total number of spermatozoa (1 x 106sp) was observed after the treatment compared to the control (S+B: 6.7\ub15.2 vs S-B: 12\ub17.5; p=0.002). Western Blot analysis using anti-ubiquitin antibodies clearly showed that the same pattern of protein ubiquitination was present before and after treatment with the beads. Actin and prohibitin, and not tubulin, were target of ubiquitin in cat semen and the extent of this post-translational modification was comparable in samples treated with beads and in the control. CONCLUSIONS. The present data suggest that sperm ubiquitination, morphology and acrosomal integrity of feline epididymal spermatozoa are not related. Sperm anomalies at subcellular level, not revealed by the analyses performed in the present study, should be further investigated before stating that the use of ubiquitin is not a reliable biomarker of semen quality in the domestic cat. REFERENCES. (1) Nandi D, Tahiliani P, Kumar A, Chandu D. The ubiquitin-proteasome system. J Biosci 2006;31:137-155. (2) Baska KM, Manandhar G, Feng D, Agca Y, Tengowski MW, Sutovsky M, Yi YJ, Sutovsky P. Mechanism of extracellular ubiquitination in the mammalian epididymis. J Cell Physio 2008;215:684-696. (3) Sutovsky P, Hauser R, Sutovsky M. Increased levels of sperm ubiquitin correlate with semen quality in men from an andrology laboratory clinic population. Hum Reprod 2004;19(3):628-38. (4) Muratori M, Marchiani S, Forti G, Baldi E. Sperm ubiquitination positively correlates to normal morphology in human semen. Hum Reprod 2005;20:1035-1043. (5) Mota PC, Ramalho-Santos J. Comparison between different markers for sperm quality in the cat: Diff-Quik as a simple optical technique to assess changes in the DNA of feline epididymal sperm. Theriogenology 2006;65:1360\u20131375. (6) Sutovsky P, Thompson W. Ubiquitination of prohibitin in mammalian sperm mitochondria: possible roles in the regulation of mitochondrial inheritance and sperm quality control. Biol Reprod 2003;69:254-260

Nanostructured titanium oxide as substrate for PC12 cell growth and differentiation

Neuronal differentiation is a crucial event in the regeneration of nervous tissues and adhesion on a substrate is critical for neurite extenction. The initiation and guidance of a neurite rely on extra-cellular signals, such as substrate energy of adhesion. There fore it is of great interest to unveil the substrates characteristics that are sensed by the growth cone and translated into neurite extention. In tehe present work a proteomic approach is used to characterize at the molecular level PC12 cells cultured on nanostructured TiO2 in order to evaluate how the nanoscale topography may modulate the dynamic of adhesion and control the shape and functions of cells as well as of tissues. Protein patterns and post-translational modifications are evaluated by immunochemistry and mass-spectrometry with particular emphasis on nitration, which is involved in neuronal proliferation and differentiation

ADSA&#174; -EAAP PhD Student Travel Award Presentation: Bioactivities of milk proteins evaluated after in vitro digestion and peptidomic/proteomic profile

The health-promoting functions of milk peptides are related to their structures, which depend on the parent protein composition. A crucial issue in this field is the demonstration of a cause-effect relationship, from the ingested form to the bioactive form. Thus, the aim our study was to in vitro digest intact whey proteins (WPI) and casein proteins (CP), mimicking in vivo digestion, to investigate their bioactive effects. WPI and CP were digested using a pepsin/pancreatin protocol and ultrafiltered (3kDa cut-off membrane). A permeate (mimicking absorbed fraction) and a retentate (mimicking intestinal fraction) were obtained. Soya protein was included as control (CTR). Antioxidant (AOX) and angiotensin-I-converting enzyme inhibitory activity (ACEI-i) of the WPI and CP fractions were assessed and compared with undigested sample (UND) and CTR. Further, the effects of WPI and CP retentate on proliferation and on mucus production (MUC5AC gene expression and mucin secretion) were assessed in intestinal goblet HT29-MTX cells. Finally, permeate has been characterized by LC-nano ESI MS/ MS using a shotgun-peptidomic approach while retentate has been further digested with trypsin and analyzed by mass spectrometry with a shotgun-proteomic approach and the potentially bioactive peptides and polypeptides have been identified. WPI and CP permeate showed a significant (P 0.1) by WPI retentate. CP retentate evoked mucinsecretory effect. Our results confirm that milk proteins may be rich sources of bioactive compounds with a greatest beneficial potential of CP at intestinal goblet cell level

Influence of subclinical mastitis and intramammary infection by coagulase-negative staphylococci on the cow milk peptidome

Coagulase-negative staphylococci (CNS) are the most prevalent microorganisms isolated from cow milk and are associated with subclinical mastitis and persistent increases in the bulk milk somatic cell count (BMSCC) of low BMSCC herds. By combining peptide enrichment, LC-ESI-MS/MS, and statistical analysis, we investigated the influence of subclinical mastitis and CNS infection on the milk peptidome. Quarter milk samples from clinically healthy Holstein cows were subjected to bacteriological culture (BC) and somatic cell counting (SCC) for two consecutive samplings and 28 (including 11 negatives and 17 positives) were selected for peptidomic analysis. The study identified 1363 different endogenous peptides and highlighted a significant increase of peptides in CNS-positive milk, mainly represented by casein fragments. Milk peptidome changes increased with the SCC, as also demonstrated by protein electrophoresis and densitometry. Peptides significantly different in CNS or CONTROL samples were identified and characterized. Our results indicate that subclinical mastitis by CNS can induce significant changes in the milk peptidome, opening the way to future studies for the identification of a biomarker panel as well as for the understanding of their consequences for the technological and sensorial characteristics of cow milk and dairy products

The Neuroprotective Role of the GM1 Oligosaccharide, II3Neu5Ac-Gg4, in Neuroblastoma Cells

Recently, we demonstrated that the GM1 oligosaccharide, II3Neu5Ac-Gg4 (OligoGM1), administered to cultured murine Neuro2a neuroblastoma cells interacts with the NGF receptor TrkA, leading to the activation of the ERK1/2 downstream pathway and to cell differentiation. To understand how the activation of the TrkA pathway is able to trigger key biochemical signaling, we performed a proteomic analysis on Neuro2a cells treated with 50&nbsp;\u3bcM OligoGM1 for 24&nbsp;h. Over 3000 proteins were identified. Among these, 324 proteins were exclusively expressed in OligoGM1-treated cells. Interestingly, several proteins expressed only in OligoGM1-treated cells are involved in biochemical mechanisms with a neuroprotective potential, reflecting the GM1 neuroprotective effect. In addition, we found that the exogenous administration of OligoGM1 reduced the cellular oxidative stress in Neuro2a cells and conferred protection against MPTP neurotoxicity. These results confirm and reinforce the idea that the molecular mechanisms underlying the GM1 neurotrophic and neuroprotective effects depend on its oligosaccharide chain, suggesting the activation of a positive signaling starting at plasma membrane level