Paweł Wysocki

Extracellular Superoxide Dismutase of Boar Seminal Plasma

Superoxide dismutase (SOD) is an enzymatic component of the antioxidant defense system that protects spermatozoa by catalysing the dismutation of superoxide anions to hydrogen peroxide and oxygen. Age and season effects on SOD activity in the seminal plasma were measured in boars at the onset of 8 months through a 35-month period. It was found that age-related changes in SOD activity in the seminal plasma were markedly higher in boars less than 2 years of age. However, it appeared that SOD activity was established at the early sexual maturity age (8-12 months). There were variations in SOD activity throughout the season, being significantly higher in spring and autumn than in summer. A secretory extracellular form of SOD (EC-SOD) was purified to homogeneity (350-fold) from boar seminal plasma, using a three-step purification protocol (affinity chromatography followed by ion exchange and ceramic hydroxyapatite chromatography). The molecular properties and specificity of SOD (molecular mass, isoelectric point, optimum pH, thermostability and susceptibility to inhibitors) confirmed that the purified enzyme is an extracellular form of Cu/Zn-superoxide dismutase occurring in boar seminal plasma. The results of this study indicate that EC-SOD is an important antioxidant enzyme of boar seminal plasma, which plays an important physiological role in counteracting oxidative stress in spermatozoa.

EFFECT OF DEPLETION TESTS (DT) ON THE COMPOSITION OF BOAR SEMEN

We conducted two depletion tests during the summer (DT 1) and winter (DT 2) to study their effect on selected biochemical parameters of boar semen. We subjected three boars to DT for 10 consecutive days. The first 3 days (Period 1) of ejaculate collections represented the reserves of the extragonadal spermatozoa and accessory sex gland secretions, whereas the other seven days (Period 2) represented the daily spermatozoa output and the secretory capacity of the accessory sex glands. We observed noticeable changes in the quantity and quality of the semen in DT 1 and 2. There was an increase in the number of spermatozoa with morphological defects, particularly coiled tails and detached acrosomes. The secretory activity of the accessory sex glands, particularly the vesicular glands, was slightly influenced by season. Depletion tests caused disturbances in the qualitative relations of secretions of the accessory sex glands, which were related to changes in the sperm plasmalemma integrity. These tests can be used to determine the total spermatozoa output, and to assess the secretory capacity of the accessory sex glands of boars.

Superoxide dismutase (SOD) in boar spermatozoa: purification, biochemical properties and changes in activity during semen storage (16°C) in different extenders.

The antioxidant system in semen is composed of enzymes, low-molecular weight antioxidants and seminal plasma proteins. Loss of enzymatic activity of superoxide dismutase (SOD) during semen preservation may cause insufficient antioxidant defense of boar spermatozoa. The aim of this study was to isolate and characterize SOD molecular forms from spermatozoa and to describe changes in SOD activity in boar sperm during preservation at 16°C. Sperm extracts were prepared from fresh or diluted semen and used for SOD purification or activity measurement. Ion-exchange chromatography and gel filtration was used to purify SOD molecular forms. BTS, Dilu Cell, M III and Vitasem were used as diluents for 5-day storage of semen at +16°C. The molecular form of SOD released from spermatozoa after cold shock and homogenization had a molecular weight of approximately 67kDa. The activity of the SOD form was the highest at pH 10 within the temperature range between 20 and 45°C. The enzymatic activity of form released after cold shock was inhibited by H2O2 and diethyldithiocarbamate (DDC; by 65 and 40%, respectively). The SOD form released by homogenization was inhibited by H2O2 and DDC (40%). The molecular form released after urea treatment was a 30kDa protein with maximum activity at 20°C and pH 10. Enzymatic activity of this form was inhibited by H2O2 by 35%, DDC by 80% and 2-mercaptoethanol by 15%. The antigenic determinants of SOD isolated from boar seminal plasma and spermatozoa were similar to each other. Susceptibility of spermatozoa to cold shock increased during storage, but the differences between extenders were statistically non-significant.

The activity of N-acetyl-β-hexosaminidase in boar seminal plasma is linked with semen quality and its suitability for cryopreservation

The determination of sperm cryotolerance is an important step in the process of developing optimal techniques for the storage of boar semen. The objective of this study was to determine individual proteome variations in boar seminal plasma and spermatozoa and establish their influence on the cryotolerance of ejaculate. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the presence of protein with estimated molecular weight of 90 kDa in sperm extracts from ejaculates of selected boars. In all cases, dialysis performed at the initial stage of cryopreservation effectively removed the protein from sperm cells. The protein had an affinity for Zn(2+) ions. Mass spectrometry revealed similarities between the discussed protein and the β subunit of N-acetyl-β-hexosaminidase (β-HEX). Seminal plasma β-HEX was purified 252-fold with approximately 27% recovery and specific activity of 1800 U/mg of protein. Enzyme activity in fresh seminal plasma was correlated with superoxide dismutase activity (r = -0.42, P < 0.05), glutathione peroxidase activity (r = -0.42, P < 0.05), mitochondrial function (r = 0.31, P < 0.05), glutathione content (r = 0.34, P < 0.05), total protein content (r = 0.42, P < 0.05), and total oxidant status of seminal plasma (r = 0.37, P < 0.05). After thawing, β-HEX activity in seminal plasma was negatively correlated with the total motile sperm count (r = -0.33, P < 0.05), plasma membrane integrity (r = -0.31, P < 0.05), and lipid peroxidation (r = 0.33, P < 0.05). The observed correlations indicate that lower levels of β-HEX activity in boar seminal plasma are linked with higher quality of sperm after thawing. Based on those observations, the ejaculates were divided into two groups characterized by low (<20,000 U/L) and high (>20,000 U/L) levels of β-HEX activity in seminal plasma. In plasma with high β-HEX activity, spermatozoa were characterized by lower plasma membrane integrity (84.7%, P < 0.05). Higher glutathione levels (1250.3 μM), higher total protein content (50 mg/mL), and higher total oxidant status (6.82-μmol H2O2 Equiv/L) were also observed (P < 0.05). After thawing, lower sperm motility (20.4%), lower plasma membrane integrity (41.7%), and higher lipid peroxidation (30.9-nM malondialdehyde/10(8) spermatozoa/h) were reported in ejaculates with high seminal plasma β-HEX activity. The results of this study indicate that β-HEX activity in seminal plasma is a useful indicator in preliminary evaluations of boar sperm cryotolerance.

Identification and characterization of non-phosphorylcholine-binding and phosphorylcholine-binding proteins of canine seminal plasma

Abstract Seminal plasma (SP) proteins participate in the process of fertilization by binding to the sperm membrane, particularly to the phosphorylcholine-containing lipids. This study aimed to identify and characterize non-phosphorylcholine-binding and phosphorylcholine-binding proteins (nPch- BPs and PchBPs, respectively) of canine SP. The nPchBPs and PchBPs were isolated from canine SP by affinity chromatography. Electrophoretic studies revealed that the nPchBPs and PchBPs occurred in their native state as high-molecular-weight aggregates. Immunofluorescent staining showed preferential binding of nPchBPs to the sperm acrosome membrane, whereas PchBPs coating was uniformly distributed on the sperm post-acrosomal membrane, mid-piece and tail regions. Analysis with mass spectrometry confirmed that canine prostate specific esterase (CPSE) is a component of the nPchBPs and PchBPs, which is implicated in key mechanisms of protein-coating on the sperm plasma membrane surface. In addition, proteins of known binding properties such as prostaglandin-H2 D-isomerase and lipocalin-like 1 protein, identified in canine SP, might have a specific role in the fertilization-associated processes.

Changes in the expression of selected antioxidative proteins in roe deer (Capreolus capreolus) epididymis in different periods of the rutting season

Abstract One of the factors determining the homeostasis of biological systems is the balance between the formation of reactive oxygen species (ROS) and the activity of the antioxidative defence system. Regarding seasonal variations in secretory activity in different parts of roe buck’s (Capreolus capreolus) epididymis, the aim of present study was to compare the expression of selected antioxidative proteins in three periods of the rutting season. Using proteomics methods (2D PAGE , tandem mass spectrometry MS/MS) 5 antioxidative enzymes were identified for the first time in different segments of the epididymis of the roe buck. The highest expression of these enzymes was found in the rutting season. Findings indicated that the antioxidative function of the buck’s epididymis, regardless of the period of the rutting season, but particularly in the rutting season, is maintained mainly because of the presence of three conservative polypeptides: glutathione S-transferase (GST), protein disulfide isomerase A3 (PDIA3), and PDIA3 precursor. Moreover, a protective role against the harmful products of redox reactions is played during the discussed periods by peroxiredoxin-2 (PRDX2), identified in the cauda of the epididymis. In the tissue of the corpus and caput of the epididymis its expression was only found in the rutting season. The expression of biliverdin reductase A (BRVA ) in the epididymis was only observed in the rutting season.

Boar sperm quality in relation to presence of sp32-like protein in spermatozoa - preliminary studies

Abstract The aim of the study was to analyse sperm proteomes of ejaculates from Polish Large White (PLW) and Polish Landrace (PL) boars and to identify differences which putatively influence semen quality. Spermatozoa protein profiles were analysed by electrophoretic methods followed by selected techniques to evaluate semen quality on the following factors: sperm motility, lipid peroxidation levels (MDA production), ATP content, activities of superoxide dismutase (SOD) and catalase (CAT), total antioxidant status (TAS), and total oxidant status (TOS) of seminal plasma. A protein with an estimated molecular weight of 30 kDa was found in spermatozoa of selected ejaculates. Mass spectrometry demonstrated that this polypeptide is most similar to proacrosin binding protein (sp32). The presence of the protein was more frequently observed in sperm extracts obtained in spring-summer period. Ejaculates containing sp32-like protein demonstrated significantly higher spermatozoa motility, lower inhibition of MDA production by seminal plasma, and higher SOD activity in seminal plasma. Boar semen which included sp32-like protein also demonstrated lower ATP levels in spermatozoa as well as higher TAS and lower TOS of seminal plasma, though the differences were not statistically significant. Ejaculates from PLW boars, with sp32-like protein present in sperm, were characterised by significantly higher sperm motility, lower ATP content in spermatozoa, and higher TAS of seminal plasma. The diminished parameters of semen quality were observed in ejaculates from PL boars that also contained the discussed protein, but the differences were not statistically significant. These findings suggest that the presence of sp32-like protein in boar spermatozoa could influence semen quality

Identification and changes in the seasonal concentrations of proteins regulating the biological functions of spermatozoa and participating in their cytoskeleton organisation in roe deer (Capreolus capreolus) epididymides

Abstract In the case of animals characterized by a strongly marked seasonality of reproduction, extremely significant seem to be periodical fluctuations in the concentration of proteinaceous substances identified in tissues and fluids of epididymides, which take part in the maturation process of sperm cells. The aim of the present study was to compare the seasonal expression of identified proteins present in various regions of the mature roe deer (Capreolus capreolus) epididymides, which are regulating the biological functions of spermatozoa and participating in their cytoskeleton organization during the sperm maturation process. Epididymal tissue homogenates and epididymal fluids were analyzed by two-dimensional electrophoresis (2D-PAGE) and tandem mass spectrometry (MS/MS) to reveal 31 polypeptides with different biochemical functions. Moreover, among the identified polypeptides, twelve of them were similar to: alpha-enolase isoform 3; endoplasmic reticulum resident protein 29; calreticulin, calponin-1 isoform X1; transgelin; vimentin; tubulin; desmin; tropomyosin; actin; alpha-1 antitrypsin isoform X1 and 14-3-3 protein epsilon. Concentrations of the analyzed polypeptides, expressed in optical density units (ODU), differed significantly (P≤0.05) across the examined periods of the reproductive season. The highest ODU values of almost all analyzed proteins were observed during the rutting period. This variation in the identified proteins in the epididymal tissues and fluids of roe deer throughout the reproductive season could be indicative of their important roles in sperm maturation within the epididymis.

Analysis of proapoptotic changes in boar spermatozoa stored at 16 °C in different short-term semen extenders

Abstract The aim of this study was to evaluate the effect of boar semen storage in different short-term extenders (BTS, Kortowo-3, and M III) on the percentage of spermatozoa showing proapoptotic and necrotic changes. For the first time in this study, Annexin V isolated from swine placenta has been used to determine proapoptotic changes in stored boar spermatozoa. The changes were determined using the IN Cell Analyzer 2000. A gradual decrease in motility was observed on successive days of storage. Spermatozoa incubated in the BTS extender were characterised by the highest average motility, which reached 75% on the 1st d and 39% on day 5. Motility of spermatozoa stored in BTS was significantly higher than those stored in Kortowo-3 and M III extenders after 5 d of storage. Diluted semen contained 1.5% to 2.8% spermatozoa with proapoptotic changes. The discussed process was intensified on the 3rd d of storage when the percentage of apoptotic spermatozoa was determined at 8.3% to 14.6%, and the content of dead spermatozoa exceeded 25%. The analysed extenders differed insignificantly in their ability to protect semen against proapoptotic changes during storage. From the methodological point of view, Hoechst 33258 could be used additionally to stain sperm cells regardless of their status.