Marzena Mogielnicka-Brzozowska

Proteomic characterization of zinc-binding proteins of canine seminal plasma.

The zinc-binding proteins (ZnBPs) of the seminal plasma are implicated in different processes related to sperm-egg fusion. The aim of this study was to characterize the ZnBPs of canine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. The ZnBPs were isolated from the ejaculates of five dogs by affinity chromatography and subjected to 2D-PAGE analysis. The acquired spots, detected across the gels, were analysed by mass spectrometry. Using 2D-PAGE analysis, it was shown that canine seminal plasma comprised about 46-57 zinc-binding polypeptides, with molecular mass ranging from 9.3 to 138.7 kDa and pI at pH 5.2-10.0. It was found that zinc-binding polypeptides of low molecular masses (9.3-19.0 kDa and pI at pH 6.1-10.0) were predominant in the seminal plasma, and seven polypeptides, with molecular masses ranging from 11.7 to 15.4 kDa and pI at pH 6.8-8.7, were characterized by high optical density values. In addition, analysis with mass spectrometry (LC-MS-MS/MS) revealed that the identified seven polypeptides are canine prostate-specific esterase (CPSE), which is the main proteolytic enzyme of the seminal plasma. The findings of this study indicate an important regulatory role of seminal plasma zinc ions in the functional activity of CPSE, which is of great significance for maintaining the normal function of canine prostate and the spermatozoa functions.

Effect of seminal plasma zinc–binding proteins on motility and membrane integrity of canine spermatozoa stored at 5°C

Abstract Sperm surface binding sites for non-zinc-binding proteins (nZnBPs) and zinc-binding proteins (ZnBPs) were studied by the fluorescence technique with biotin-labelled proteins. The nZnBPs binding pattern was unspecific, no characteristic sites on plasmalemma were found. ZnBPs were attached mainly to the acrosomal region of sperm head and to the sperm flagellum. ZnBPs added to the incubation mixture of the canine spermatozoa allowed the preservation of higher values for total motility, progressive motility, curvilinear line velocity, straight line velocity, and beat cross frequency (P < 0.05), both at time 0 and after 1 h incubation at 5ºC. The addition of nZnBPs to the incubation mixture caused only weak positive effects when compared with control sample (PBS). A higher percentage of canine-ejaculated spermatozoa with intact membranes were observed when ejaculate was incubated with ZnBPs in comparison to control sample stored with PBS (P < 0.05) or nZnBPs (P < 0.05). Spermatozoa diluted with ZnBPs and nZnBPs exhibited a higher percentage of cells with active mitochondria when compared with control, both at time 0 and after 1 h; however, no statistical differences were observed. Our results emphasise the role of seminal plasma protein in securing the correct quantity and availability of zinc ions as a component regulating the motility of canine spermatozoa. The protective effect of ZnBPs against the cooling effect may be due to their ability of preventing sperm membrane damage.

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.

Effects of storage in different semen extenders on the pre-freezing and post-thawing quality of boar spermatozoa

The aim of this study was to investigate the effects of storage of semen in different commercial extenders on the pre-freezing and post-thawing quality of boar spermatozoa. Semen was diluted in BTS, Androhep (AH) and Gedil (GD), stored for 24 h at 17°C, and then frozen in accordance with the cryopreservation protocol. Analyses of the quality of spermatozoa included: motility, normal apical ridge (NAR) acrosome, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), measurements of ATP content and activity of superoxidase dismutase (SOD) and glutathione peroxidase (GPx). Prior to the freezing process, no significant effect of the extender on the sperm quality parameters was noted. After thawing the spermatozoa it was demonstrated that the type of extender used influenced PMI, MMP, ATP content and activity of GPx. In the AH extender the percentage of spermatozoa with PMI and ATP content in spermatozoa was significantly higher (P<0.05) as compared to the BTS or GD extenders. In addition, semen stored in the AH was characterised by a statistically higher (P<0.05) percentage of spermatozoa with MMP and increased activity of GPx as compared with the BTS. The results obtained indicate that for the cryopreservation process, boar spermatozoa stored for 24 hours in liquid state can be used. However, the type of extender used prior to freezing may have a significant effect on the post-thawing quality of the spermatozoa. The AH extender better secured the quality of thawed boar spermatozoa as compared with the BTS or GD.