Jerzy Strzeżek

Purification and partial characterization of a 5700 Da sperm motility inhibiting factor from seminal plasma of boar

Abstract Sperm motility inhibiting factor (SMIF) has been isolated from boar seminal plasma by acetone fractionation and thin-layer chromatography on silica gel. Low molecular weight (5700 Da), maximal absorbance at 220 nm, significant thermostability, positive ninhidrin reaction and dominating acidic amino acid residues indicate the SMIF is a peptide and anionic. SMIF showed non-species-specific inhibition of sperm motility in microscopic studies. An antigenic species-specificity of SMIF was demonstrated. Immunofluorescence confirmed that the peptide was secreted by epithelial cells of the vesicle glands. No fluorescence was observed in other tissues of the boars reproductive system. SMIF was associated with macromolecular protein fractions of seminal plasma and vesicle fluid. Whole plasma of boar semen showed a modulating effect on the inhibiting activity of SMIF. Adenosine receptor antagonist, in 10 mM concentration, as well as caffeine and theophylline, decreased the inhibitory activity of the peptide on sperm motility. SMIF significantly decreased the adenosine 5′,-triphosphate content of spermatozoa and inhibited growth of Gram-positive bacterial species. The lytic activity of SMIF against Micrococcus lysodeikticus (luteus) , observed by a turbidimetric method, suggests that SMIF could interact with the spermatozoa membrane. The results suggest that SMIF is a regulating peptide of boar seminal plasma.

Low stability of aspartate aminotransferase activity in boar semen

Abstract This study was conducted to evaluate and characterize aspartate aminotransferase (AAT) activity in boar semen and to determine the effect of dilution on the activity. In the first experiment it was found that the activity of AAT which had leaked from spermatozoa increased gradually during semen storage in various diluents at 16–18°C. In contrast, AAT activity was decreased after exposure of spermatozoa to cold-shock (1 hour, 4°C). Correlation between semen quality and the enzyme activity was greater after cold-shock than after storage at above-zero temperatures. Low stability of extracellular AAT pool was observed. In the second experiment, inactivation by dilution of AAT released after cold-shock was found. This phenomenon was prevented by the presence of 5% BSA. The AAT activity measured after cold-shock of spermatozoa by physiological saline with 5% BSA was 65% higher, than in samples without albumin. A high correlation between sperm concentration and amounts of the enzyme released in the presence of albumin was obtained (r = 0.88).

Seasonal changes in antioxidant defence systems in seminal plasma and fluids of the boar reproductive tract

This study aimed to analyze seasonal variations in the antioxidant defence systems of the seminal plasma and fluids of the cauda epididymis and vesicular glands of the boar. The analyzed antioxidants included superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total L-glutathione (GSH+GSSG). Seasonal changes in total protein content and total antioxidant status (TAS) of the seminal plasma and reproductive fluids were also analyzed. Compared with the spring-summer period, total protein content in the seminal plasma was significantly higher during the autumn-winter period. Among the antioxidants analyzed, only SOD activity showed marked seasonal variations, being significantly higher during the spring-summer period. Likewise, the fluid of the cauda epididymis exhibited greater SOD and CAT activity during the spring-summer period, whereas TAS levels were markedly higher during the autumn-winter period. Neither GPx activity nor total GSH+GSSG content in the cauda epididymal fluid was significantly affected by the seasonal periods. The vesicular gland fluid exhibited an approximately 4-fold greater level of SOD activity during the autumn-winter period, as compared with the spring-summer period. By contrast, greater CAT and GPx activity, and a higher level of total GSH+GSSG were observed in the vesicular gland fluid during the spring-summer period. In conclusion, the findings of this study indicate that seasonal variations could have varying effects on the antioxidant defence systems in the seminal plasma and fluids of the boar reproductive tract.

Cloning of Complementary DNA encoding the pB1 component of the 54-kilodalton glycoprotein of boar seminal plasma

Complementary DNA (cDNA) encoding a protein component pB1 (also pAIF‐1 and DQH) of the 54‐kilodalton glycoprotein of boar seminal plasma was cloned and its nucleotide sequence was determined (Gene Bank accession no. AF047026). The pB1 precursor protein is a 130‐amino‐acid‐long polypeptide containing a 25‐amino‐acid‐long signal peptide. The amino acid sequence of the pB1 is homologous to that of SFP1_BOVIN (named also BSP‐A1/A2, PDC‐109/major protein and SVSp109), SFP3_BOVIN (BSP‐A3), SFP4_BOVIN (BSP‐30 KD), and SP1_HORSE (HSP‐1) seminal plasma proteins. The homology extends also for the signal peptide of SFP1_BOVIN protein. All these seminal plasma proteins contain two fibronectin type‐II domains that differ from those found in other proteins such as colagenases, fibronectins, and mannose receptors. The first domain located in the N‐terminal region of pB1 is four amino acids shorter than those present in other proteins. High homology is also observed between 3′ noncoding regions of the nucleotide sequences of cDNAs of pB1_PIG and SFP1_BOVIN (Gene Bank accession nos. AF047026 and P02784, respectively). Mol. Reprod. Dev. 52:303–309, 1999.

Zinc ion-dependent protein in boar semen. II. Effects on sperm motility and antibacterial properties

Abstract New functions of a zinc ion-dependent protein isolated from boar seminal plasma are presented. The results of studies suggest that plasma proteins in boar semen are arranged specifically and control the motility of the spermatozoa. The zinc ion-dependent protein seems to be a factor inactivating the plasmatic inhibitor of spermatozoa motility. The protein exhibited a regulating activity in the pH range 7.3–8.2, and at a quantitative protein ratio of 13:1, in favour of the inhibitor. This protein also inhibited the growth of bacteria, especially Gram-positive species. At a concentration of 4 mg/ml of the medium, the protein or its fractions totally inhibited the growth of bacteria of the genus Micrococcus after 6 h of incubation. As regards other strains of bacteria, total inhibition of growth was observed after 24–48 h. Antibacterial activity of the protein did not change after a short period of heating at 100°C, nor after freezing/thawing.

Zinc ion-dependent protein in boar semen. I. Egg yolk precipitating activity and some biochemical properties

Abstract A Zn 2+ -dependent protein with a special affinity for egg yolk was isolated from boar seminal plasma. It was electrophoretically homogeneous after separation on chelating Sepharose 6B, and had a subunit structure on SDS-gel electrophoresis with three fractions of molecular weights 25 000, 38 000 and 64 000. Precipitating activity toward egg yolk (optimal at pH 6.5–7.0) was stimulated by chloride ions and inhibited by a high concentration of zinc ions. The protein maintained its precipitating activity after incubation at 100°C and −196°C as well as after treatment with proteolytic enzymes. Indirect immunofluorescence showed that the Zn 2+ -dependent protein was secreted by epithelial cells of the seminal vesicle glands. The protein enveloped the spermatozoa after ejaculation, especially in the middle-piece area.

Post-thaw sperm characteristics following long-term storage of boar semen in liquid nitrogen

This study investigated the effect of long-term liquid nitrogen storage of semen from individual boars on post-thaw sperm characteristics. Ejaculates, collected from five Polish large white (PLW) and five Polish landrace (PLR) boars, were frozen using a standard cryopreservation protocol. Post-thaw analysis was performed within a week (Period 1) and 42-48 months (Period 2) of semen storage in liquid nitrogen. Post-thaw sperm assessments included total motility, mitochondrial function (JC-1/PI assay), plasma membrane integrity (SYBR-14/PI assay), osmotic resistance test (ORT), lipid peroxidation (LPO) status and DNA fragmentation, analysed by the neutral Comet assay. Individual boar variability within breed and cryostorage periods had significant effects on the analysed parameters of frozen-thawed spermatozoa. Prolonged semen storage in liquid nitrogen (Period 2) induced a marked reduction in post-thaw sperm motility, mitochondrial function and plasma membrane integrity in most of the boars. Post-thaw semen of eight boars exhibited a marked decrease in osmotic resistance of the sperm acrosomal membrane, whereas a significant increase in the sperm cryo-susceptibility to induced LPO and DNA fragmentation was observed only in three boars after long-term semen storage. Additionally, frozen-thawed spermatozoa of PLR boars exhibited significantly lower osmotic resistance of the acrosomal membrane than PLW boars following prolonged semen storage in liquid nitrogen. The results of this study provide evidence of ageing processes in frozen-thawed boar spermatozoa following prolonged cryostorage. It seems that, even though cryopreservation allows long-term semen storage in liquid nitrogen, spermatozoa from individual boars are more susceptible to cryo-induced damage.

Stimulation of aspartate aminotransferase from farm animal semen by pyridoxal 5′-phosphate

Abstract The incorporation of pyridoxal 5′-phosphate (PLP) into assay mixtures for estimating aspartate aminotransferase (AAT) activity in human semen was used in 1967. However, in the literature published since then, activity measurements of the enzyme in semen have not included the exogenous coenzyme. Our data for farm animal semen indicate that higher AAT activities are recorded when PLP is present during assay. The maximal potential catalytic activity of AAT is not achieved without exogenous PLP and the relation between the activity and the enzyme concentration can therefore be lost. We preincubated semen samples for 1 h with 0.1 mM PLP before assaying AAT activity, to check the effect of PLP. We found that the seminal phosphatases hydrolyse pyridoxal 5′-phosphate (a potential physiological substrate for these enzymes) very efficiently; EDTA was added to the preincubation mixture to maintain the coenzyme active. The activity of AAT measured with PLP in boar, ram, and bull semen was significantly higher than in assay without PLP. The highest stimulation of the enzyme by PLP was noticed for bull and ram seminal plasma (approximately 100%). AAT activity in ram and boar semen estimated by the new procedure correlated better with sperm characteristics than the activity measured without exogenous PLP. The high amount of AAT apoenzyme in farm animal semen can be the result of easy dissociation of the coenzyme from holoenzyme. It could be caused in vitro by heating or gel filtration on Sephadex G-150 (data for ram seminal plasma). Stabilization of AAT by PLP was also observed.

Isolation and characteristics of aspartate aminotransferase from boar spermatozoa.

1. Gel electrophoresis of aspartate aminotransferase released from boar spermatozoa after cold shock showed one band migrating towards anode. 2. Physico-chemical and kinetic properties of isolated enzyme were similar to cytoplasmic isoenzyme of AAT from somatic tissues.

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.