Proteomic Characterization of Zinc‐Binding Proteins of Canine Seminal Plasma

Abstract

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.

Canine prostate specific esterase (CPSE) as an useful biomarker in preventive screening programme of canine prostate: CPSE threshold value assessment and its correlation with ultrasonographic prostatic abnormalities in asymptomatic dogs

“…Recently, it was suggested that the CPSE is secreted in the form of prostasomes (prostate granules) to semen during ejaculation. The activity of CPSE is regulated by the level of available zinc that is of great importance for maintaining the normal functions of the prostate and the spermatozoa (Mogielnicka-Brzozowska, Kowalska, Fraser, & Kordan, 2015). The production of CPSE is regulated by androgen hormone (testosterone); it can be inhibited by the surgical castration or antiandrogen treatment (Frenette, Dube, Marcotre, & Tremblay, 1983;Isaacs & Sharper, 1985;Juniewicz et al, 1990) or promoted by the exogenous androgen administration following the surgical castration (Frenette et al, 1983;Isaacs & Sharper, 1985).…”

Highlights on the Canine Prostatic Specific Esterase (CPSE): A diagnostic and screening tool in veterinary andrology

“…Conversely, the epididymal spermatozoa do not show the enzyme (Isaacs & Coffey, 1984). As a sperm‐binding protein, the CPSE seems to be involved in events related to sperm fertilization (Mogielnicka‐Brzozowska, Kowalska, Fraser, & Kordan, 2015) and, given to its ability to bind to zinc ions, it turns out to be a multifunctional molecule (Acquino‐Cortez et al., 2017; Chapdelaine et al., 1984; Dube et al., 1986). Studies are warranted to better clarify the specific role of the CPSE in the fusion process between oocyte and spermatozoa (Mogielnicka‐Brzozowska, Sowinska, & Fraser, 2017).…”

Evaluation of spermatozoal function—useful tools or just science

“…There were positive correlations between two particular protein bands, B4 (67 kDa) and B5 (58.6 kDa), and sperm motility, sperm vigour, percentage of morphologically normal spermatozoa, the hypo‐osmotic swelling test and fluorescent staining. Using 2D‐PAGE analysis, it was shown that canine seminal plasma contains approximately 46–57 zinc‐binding polypeptides (Mogielnicka‐Brzozowska, Kowalska, Fraser, & Kordan, ). Their molecular mass ranging from 9.3 to 138.7 kDa.…”