Proteome Profiling of Canine Epididymal Fluid: In Search of Protein Markers of Epididymal Sperm Motility.

Sperm maturation in the epididymis is based on interactions with proteins from epididymal fluid (EF). The aim of the study was to profile canine EF proteome and investigate correlations between EF protein content and epididymal spermatozoa (ES) motion parameters. Twenty-three male dogs were divided into two groups: good sperm motility (GSM) and poor sperm motility (PSM). The total motility and progressive motility differed significantly (p = 0.031; p < 0.001, respectively) between the GSM group and the PSM group. The semen samples were centrifuged to separate the EF apart from the ES. The canine EF proteins were analyzed using nano-liquid chromatography, which was coupled with quadrupole time-of-flight mass spectrometry (NanoUPLC-Q-TOF/MS) and bioinformatic tools for the first time. A total of 915 proteins were identified (GSM-506; PSM-409, respectively). UniProt identification resulted in six unique proteins (UPs) in the GSM group of dogs and four UPs in the PSM group. A semi-quantitative analysis showed a higher abundance (p < 0.05) of four differentially expressed proteins in the GSM group (ALB, CRISP2, LCNL1, PTGDS). Motility-dependent variations were detected in the EF proteome and were related to important metabolic pathways, which might suggest that several proteins could be potential ES motility biomarkers.

An Analysis of the Risk Factors for Postural Defects among Early School-Aged Children.

A considerable number of problems begin in childhood due to lifestyle changes, which include a transition from a previous period of extensive movement to prolonged hours of staying in a sitting position at school. The aim of this study was to examine the occurrence of back and side view postural defects in the study group of school-aged children and identify risk factors associated with the formation of postural abnormalities in the study group. METHODS: This study was conducted on a group of 141 children aged 7-10, attending the first to third grades at a primary school in Bialystok (northeastern Poland). This study involved measuring the children's height and weight, assessing the children's body posture based on the FITS method (Functional Individual Scoliosis Therapy) by Bialek and M'hango (the authors of this study), and administering a diagnostic survey addressed to parents and guardians of the children (n = 104) using a self-designed questionnaire. RESULTS: Almost all defects were more prevalent in boys, especially in the case of stature triangles (p = 0.0489) and knee alignment in the sagittal plane (p = 0.038). The age of the subjects differentiated the incidence of defects in the scapulae (p = 0.0037) and shoulder (p = 0.0129) alignment, correlating negatively with age. The risk of postural defects for knees (p = 0.0391) and abdominal arching (p = 0.0240) was significant with a higher BMI. The following lifestyle-related factors were significant: the seat for doing homework (stature triangles p = 0.0253), time spent in front of a computer (positioning of the scapulae in relation to each other p = 0.0233; vertical view of the intergluteal cleft p = 0.0324), and snacking between meals (feet p = 0.0003; shoulder positioning p = 0.0013; stature triangles p = 0.0186; positioning of the scapulae in relation to each other p = 0.0404). CONCLUSIONS: The body posture of the examined children was closed with the head pushed forward and drooped, rounded shoulders, hyperlordosis, and pelvic anteversion. Most exhibited various types of abnormalities related to the feet. The recognized risk factors for posture defects are overweight/obesity, the male gender, children who are older, lack of an adjustable work chair, 2 h a day or more spent using the computer, and snacking between meals.

Seminal Plasma Glycoproteins as Potential Ligands of Lectins Engaged in Immunity Regulation.

Environmental pollution, chronic stress, and unhealthy lifestyle are factors that negatively affect reproductive potential. Currently, 15-20% of couples in industrialized countries face the problem of infertility. This growing health and social problem prompts researchers to explore the regulatory mechanisms that may be important for successful fertilization. In recent years, more attention has been paid to male infertility factors, including the impact of seminal plasma components on regulation of the female immune response to allogenic sperm, embryo and fetal antigens. Directing this response to the tolerogenic pathway is crucial to achieve a healthy pregnancy. According to the fetoembryonic defense hypothesis, the regulatory mechanism may be associated with the interaction of lectins and immunomodulatory glycoepitopes. Such interactions may involve lectins of dendritic cells and macrophages, recruited to the cervical region immediately after intercourse. Carbohydrate binding receptors include C type lectins, such as DC-SIGN and MGL, as well as galectins and siglecs among others. In this article we discuss the expression of the possible lectin ligands, highly fucosylated and high mannose structures, which may be recognized by DC-SIGN, glycans of varying degrees of sialylation, which may differ in their interaction with siglecs, as well as T and Tn antigens in O-glycans.

Age-Dependent Variations in Functional Quality and Proteomic Characteristics of Canine (Canis lupus familiaris) Epididymal Spermatozoa.

Increased male age is associated with a significant reduction in semen quality. Little is known about the sperm proteome changes resulting from the aging process. This study aimed to investigate the relationship between the functional quality and proteome of epididymal spermatozoa of dogs that were differing in age. The study was conducted on 30 male dogs that were divided into three age groups. G1-12 to 41 months old, G2-42 to 77 months old, and G3-78 to 132 months old. The sperm samples were assessed using a computer-assisted semen analysis (CASA). The epididymal sperm proteins were analyzed using gel electrophoresis (SDS-PAGE), nano-liquid chromatography coupled to quadrupole time of flight mass spectrometry (NanoUPLC-Q-TOF/MS) and bioinformatic tools. The sperm quality parameters were significantly lower in older dogs. NanoUPLC-Q-TOF/MS identification resulted in 865 proteins that were found in the G1, 472 in G2, and 435 in G3. There were seven proteins that were present in all three age groups, and four of them (ACTB, CE10, NPC2, CRISP2) showed significant changes among the studied groups. Age-dependent variations were detected in the sperm proteome composition and were related to important metabolite pathways, which might suggest that several proteins are implicated in sperm maturation and could be potential aging biomarkers.

Proteomic Analysis of Intracellular and Membrane-Associated Fractions of Canine (Canis lupus familiaris) Epididymal Spermatozoa and Sperm Structure Separation.

This study was provided for proteomic analysis of intracellular and membrane-associated fractions of canine (Canis lupus familiaris) epididymal spermatozoa and additionally to find optimal sonication parameters for the epididymal sperm morphological structure separation and sperm protein isolation. Sperm samples were collected from 15 dogs. Sperm protein fractions: intracellular (SIPs) and membrane-associated (SMAPs) were isolated. After sonication, sperm morphology was evaluated using Spermac Stain™. The sperm protein fractions were analyzed using gel electrophoresis (SDS-PAGE) and nanoliquid chromatography coupled to quadrupole time-of-flight mass spectrometry (NanoLC-Q-TOF/MS). UniProt database-supported identification resulted in 42 proteins identified in the SIPs and 153 proteins in the SMAPs. Differentially abundant proteins (DAPs) were found in SIPs and SMAPs. Based on a gene ontology analysis, the dominant molecular functions of SIPs were catalytic activity (50%) and binding (28%). Hydrolase activity (33%) and transferase activity (21%) functions were dominant for SMAPs. Bioinformatic analysis of SIPs and SMAPs showed their participation in important metabolic pathways in epididymal sperm, which may suggest their potential as sperm quality biomarkers. The use of sonication 150 W, 10 min, may be recommended for the separation of dog epididymal sperm heads, tails, acrosomes and the protein isolation.

Application of selected biosensor techniques in clinical diagnostics.

INTRODUCTION: Examination of disease biomarkers mostly performed on crude materials, such as serum, meets some obstacles, resulting from sample complexity and the wide range of concentrations and sizes of the components. Techniques currently used in clinical diagnostics are usually time-consuming and expensive. The more sensitive and portable devices are needed for early diagnostics. Chemical sensors are devices that convert chemical information into parameters suitable for fast and precise processing and measurement. AREA COVERED: We review the use of biosensors and their possible application in early diagnostics of some diseases like cancer or viral infections. We focus on different types of biorecognition and some technical modifications, lowering the limit of detection potentially attractive to medical practitioners. EXPERT OPINION: Among the new diagnostic strategies, the use of biosensors is of increasing interest. In these techniques, the capture ligand interacts with the analyte of interest. Measuring interactions between partners in real time by surface plasmon resonance yields valuable information about kinetics and affinity in a short time and without labels. Importantly, the tendency in such techniques is to make biosensor devices smaller and the test results apparent with the naked eye, so they can be used in point-of-care medicine.

Proteome of cat semen obtained after urethral catheterization.

The binding of seminal plasma (SP) proteins by spermatozoa plays an important role in the regulation of sperm epididymal maturation, motility gaining in female reproductive tracts and sperm-egg interaction. The aim of the study was to analyze the SP and sperm extracts proteome of cat (Felis catus) semen. The seminal plasma and spermatozoa were obtained by urethra catheterization from 10 male cats. Proteins were extracted using RIPA buffer and separated by electrophoresis (SDS-PAGE). The gels were analyzed using MultiAnalyst software. The proteins were subsequently analyzed using NanoUPLC-Q-TOF/MS. UniProt database-supported identification resulted in 106 proteins identified in the cat SP and 98 proteins in the extracts of spermatozoa. Based on a gene ontology analysis, dominant molecular functions of feline SP proteins were binding, catalytic, and antioxidant activity (56%, 33%, and 11% of cases, respectively). The molecular functions of sperm extracts proteins were mainly involved in catalytic activity (41%) and binding (23%). The proteins present in both, the SP and spermatozoa's extracts, were: serum albumin (ALB), semenogelin 2 (SEMG 2), clusterin (CLU), lactoferrin (LTF), prostatic acid phosphatase (ACPP), prolactin inducible protein (PIP), negative elongation factor E (NELF-E) and ectonucleotide pyrophosphatase (ENPP3). Protein-protein interactions analysis showed significant connection for 12 proteins in the cat semen. The seminal plasma proteins which, with high probability score, participate in important metabolic pathways are: glutathione peroxidases (GPx5 and 6), prostatic acid phosphatase (ACPP), ß-hexosaminidase (HEXB), polymeric immunoglobulin receptor (pIgR) and serpin family F member 1 (SERPINF1). For sperm protein extracts it were: pyruvate dehydrogenase (PDHB), succinate-CoA-ligase (SUCLA2), malate dehydrogenase (MDH2), ATP synthase F1 subunit alpha (ATP5F1A) and tubulin beta (TUBB).

The content of immunomodulatory glycoepitopes in seminal plasma glycoproteins of fertile and infertile men.

According to a concept of fetoembryonic defence, protein-carbohydrate interaction may be involved in the regulation of maternal immunity that prevents rejection of allograft spermatozoa, embryo and fetus. In the present study we focussed on the evaluation of the expression of glycoepitopes that may be of crucial importance in this process: LewisY (LeY) and LewisX (LeX) as well as terminal sialylation. Polyacrylamide gel electrophoresis with sodium dodecyl sulphate was used to separate seminal plasma samples of fertile (n=10) and infertile (n=103) men; these were then probed with lectins specific to fucose (Lotus tetragonolobus agglutinin and Ulex europaeus agglutinin) and sialic acid (Sambucus nigra agglutinin and Maackia amurensis agglutinin). Differential expression of α2,3-bound sialic acid was found in six out of seven analysed bands, whereas differences in the other analysed glycoepitopes were found in fewer numbers of bands. Mass spectrometry analysis focussed on the identification of proteins carrying glycans with immunomodulatory epitopes, including fibronectin, lactoferrin, clusterin, zinc-α2-glycoprotein, prostate acid phosphatase and prostate-specific antigen; these should be submitted to further detailed analysis.

EGFR and Bcl-2 in gastric mucosa of children infected with Helicobacter pylori.

UNLABELLED: The aim of the study was to evaluate the expression of EGFR and Bcl-2 proteins as inhibitory markers of apoptosis in surface epithelial cells and gland cells of antral gastric mucosa in children infected with Helicobacter pylori according to the severity and activity of antral gastritis and to assess the correlation between the number of cells expressing EGFR and the number of cells expressing Bcl-2 in H. pylori infected children. MATERIALS AND METHODS: The study included 44 children: 68.2% with chronic gastritis and positive IgG against H. pylori, and 31.8% with functional disorders of the gastrointestinal tract and with normal IgG against H. pylori. The evaluation of EGFR expression in gastric mucosa was performed immunohistochemically using monoclonal mouse anti-EGFR antibody. The polyclonal antibody was used to determine the expression of anti-Bcl-2. RESULTS: A significant increase in the number of cells expressing EGFR and Bcl-2 protein was found in the epithelial cells in severe as well as mild and moderate gastritis in the group of children infected with H. pylori. An increase in the number of cells expressing EGFR and Bcl-2 protein was also found in the epithelial cells in group I according to the activity of gastritis. There was a statistically significant positive correlation between the numbers of cells expressing EGFR and Bcl-2 in H. pylori infected children. CONCLUSION: Increased expression of EGFR and Bcl-2 proteins in the epithelial cells and a statistically significant positive correlation between the numbers of cells expressing EGFR and Bcl-2 in H. pylori infected children could suggest increased regeneration abilities of gastric mucosa.

Gelatinases and their tissue inhibitors are associated with oxidative stress: a potential set of markers connected with male infertility.

The expression and activity of matrix metalloproteinases (MMPs) may be regulated by oxidative stress in various pathophysiological processes; therefore, the aim of the present study was to analyse the associations between the expression of the gelatinases MMP-9 and MMP-2 and their tissue inhibitors TIMP-1, TIMP-2 and levels of total antioxidant capacity (TAC) and advanced oxidation protein products (AOPP) in seminal plasma prepared for artificial insemination. Levels of MMPs and TIMPs were evaluated using ELISA, whereas TAC and AOPP in the seminal plasma of 131 childless men and 38 fertile volunteers were determined spectrophotometrically. Seminal MMP-9 expression was higher in childless men than in fertile subjects, whereas there was no significant differences in MMP-2 expression between the analysed seminal groups. TIMP-1 and TIMP-2 expression was similar in all groups. However, TAC expression was significantly higher in infertile normozoospermic and oligozoospermic men and AOPP expression was higher in astheno-, oligo- and normozoospermic infertile patients than in fertile men. High AOPP, together with an increased MMP-9:TIMP-1 ratio alters the oxidative-antioxidative balance of the ejaculate, thereby reducing male fertility, and therefore these parameters may serve as additional diagnostic markers of semen quality and male reproductive potential.

Terminal Mannose Residues in Seminal Plasma Glycoproteins of Infertile Men Compared to Fertile Donors.

The impact of seminal plasma components on the fertilization outcomes in humans is still under question. The increasing number of couples facing problems with conception raises the need for predictive biomarkers. Detailed understanding of the molecular mechanisms accompanying fertilization remains another challenge. Carbohydrate-protein recognition may be of key importance in this complex field. In this study, we analyzed the unique glycosylation pattern of seminal plasma proteins, the display of high-mannose and hybrid-type oligosaccharides, by means of their reactivity with mannose-specific Galanthus nivalis lectin. Normozoospermic infertile subjects presented decreased amounts of lectin-reactive glycoepitopes compared to fertile donors and infertile patients with abnormal semen parameters. Glycoproteins containing unveiled mannose were isolated in affinity chromatography, and 17 glycoproteins were identified in liquid chromatography-tandem mass spectrometry with electrospray ionization. The N-glycome of the isolated glycoproteins was examined in matrix-assisted laser desorption ionization mass spectrometry. Eleven out of 27 identified oligosaccharides expressed terminal mannose residues, responsible for lectin binding. We suggest that lowered content of high-mannose and hybrid type glycans in normozoospermic infertile patients may be associated with impaired sperm protection from preterm capacitation and should be considered in the search for new infertility markers.

Glycoprotein fucosylation is increased in seminal plasma of subfertile men.

Fucose, the monosaccharide frequent in N- and O-glycans, is a part of Lewis-type antigens that are known to mediate direct sperm binding to the zona pellucida. Such interaction was found to be inhibited in vitroby fucose-containing oligo- and polysaccharides, as well as neoglycoproteins. The objective of this study was to screen seminal plasma proteins of infertile/subfertile men for the content and density of fucosylated glycoepitopes, and compare them to samples of fertile normozoospermic subjects. Seminal proteins were separated in polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane and probed with fucose-specific Aleuria aurantia lectin (AAL). Twelve electrophoretic bands were selected for quantitative densitometric analysis. It was found that the content, and especially the density of fucosylated glycans, were higher in glycoproteins present in seminal plasma of subfertile men. No profound differences in fucosylation density were found among the groups of normozoospermic, oligozoospermic, asthenozoospermic, and oligoasthenozoospermic subfertile men. According to the antibody probing, AAL-reactive bands can be attributed to male reproductive tract glycoproteins, including prostate-specific antigen, prostatic acid phosphatase, glycodelin and chorionic gonadotropin. Fibronectin, α1 -acid glycoprotein, α1 -antitrypsin, immunoglobulin G and antithrombin III may also contribute to this high fucosylation. It is suggested that the abundant fucosylated glycans in the sperm environment could interfere with the sperm surface and disturb the normal course of the fertilization cascade.

[Selected eating habits and caries occurrence in adolescents]. / Wybrane zachowania zywieniowe a wystepowanie próchnicy U Mlodziezy.

INTRODUCTION: Normal nutrition is an important element of caries prophylaxis. The aim of the study was to evaluate the influence of chosen bad eating habits on caries occurrence in adolescents. MATERIAL AND METHODS: The examination was carried out in the group of 367 (68.5% girls and 31.5% boys) pupils of the 3rd year of high schools in Bialystok in 2011. Own questionnaire was used for the evaluation of eating habits. Caries intensity (PUW) was assessed by dental examination. To verify statistic hypotheses, the level of significance was estimated at p < 0.05. All calculation was performed with the use of the Statistic programme Statistica 7.0 (StatSoft). RESULTS: It was stated that 5 meals a day were consumed by 29.4% of examined pupils, 4 meals were eaten by 33.2%, 3 meals--by 31.3%, 2 meals--by 5.4%, and one meal was consumed by 0.5% of pupils. There were 55.3% of pupils who had irregular meals while 24.3% did not eat breakfast and 62.9%--did not have elevenses. Eating between meals was stated in 96.7% of pupils. Sweets consumption was revealed as follows: 16.8% several times a day, 19%--once a day, 40.5%--several times a week, 18.5%--several times a month, 5.2%--rarely or never. "Fast food" was not consumed by 34% of examined pupils while 54.6% consumed such meals several times a month, 10.9%--several times a week, and 0.5%--once a day. Higher level of caries intensity was observed in girls (mean coefficient PUW = 11.3) than in boys (mean coefficient PUW = 10.8). Mean coefficient PUW = 11.5 concerned pupils with consumption of fast food several times a month while PUW = 11.0 in those who consumed it several times a week, and PUW = 10.7 in pupils who eat rarely or never such food. As far as eating sweets, PUW was higher than mean (12.4) in pupils who eat sweets several times a day. Persons who eat between meals were characterized by higher coefficient (PUW = 13.0) as compared to those who eat between meals rarely (PUW = 11.3) and those who do not eat between meals (PUW = 8.9). CONCLUSIONS: (1) Bad eating habits (irregular meals, skipping breakfast, eating between meals as well as overeating sweets) in the developmental age can be a significant caries coefficient in adolescents. 2. High mean coefficient PUW in the examined group of 18-year-old pupils, in relation to abnormal eating habits in the significant percentage of those pupils, indicates the necessity of health education as far as caries prophylaxis is concerned.