Differences in sperm protein abundance and carbonylation level in bull ejaculates of low and high quality

Abstract

In breeding and insemination centres, significant variation in bull ejaculate quality is often observed between individuals and also within the same individual. Low-quality semen does not qualify for cryopreservation and is rejected, generating economic loss. The mechanisms underlying the formation of low-quality ejaculates are poorly understood; therefore, the aim of the present study was to investigate the proteomic differences and oxidative modifications (measured as changes in protein carbonylation level) of bull ejaculates of low and high quality. Flow cytometry and computer-assisted sperm analysis were used to assess differences in viability, reactive oxygen species (ROS) level, and sperm motility. To analyse changes in protein abundance, two-dimensional difference gel electrophoresis (2D-DIGE) was performed. Western blotting in conjunction with two-dimensional electrophoresis (2D-oxyblot) was used to quantitate carbonylated sperm proteins. Proteins were identified using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight spectrometry. High quality ejaculates were characterised by higher sperm motility, viability, concentration, and a lower number of ROS-positive cells (ROS+). We found significant differences in the protein profile between high- and low-quality ejaculates, and identified 14 protein spots corresponding to 10 proteins with differences in abundance. The identified sperm proteins were mainly associated with energetic metabolism, capacitation, fertilisation, motility, and cellular detoxification. High-quality ejaculates were characterised by a high abundance of extracellular sperm surface proteins, likely due to more efficient secretion from accessory sex glands and/or epididymis, and a low abundance of intracellular proteins. Our results show that sperm proteins in low-quality ejaculates are characterised by a high carbonylation level. Moreover, we identified, for the first time, 14 protein spots corresponding to 12 proteins with differences in carbonylation level between low- and high-quality ejaculates. The carbonylated proteins were localised mainly in mitochondria or their immediate surroundings. Oxidative damage to proteins in low-quality semen may be associated with phosphorylation/dephosphorylation disturbances, mitochondrial dysfunction, and motility apparatus disorders. Our results contribute to research regarding the mechanism by which low- and high-quality ejaculates are formed and to the identification of sperm proteins that are particularly sensitive to oxidative damage.

Fig 1

Sperm concentration (A), motility (B), viability (C), and ROS⁺ cells (D) of low- and high-quality bull ejaculates (n = 6 in each group). Results are expressed as the mean ±SD. Different letters indicate significant differences between low- and high-quality ejaculates (p < 0.05).

Fig 2. Two-dimensional differential electrophoresis gels showing the proteomic profiles of sperm from HQ and LQ bull ejaculates.

Blue protein spots show a significant (p < 0.05) increase in abundance in HQ ejaculates and red protein spots show an increase in abundance in LQ ejaculates. The marked protein spots are numbered and listed in Table 2. Single channel representative image (A); and fluorescent overlay view (B).

Fig 3. Representative two-dimensional (2D) gels and oxyblots of LQ and HQ ejaculates.

2D oxyblot showing carbonylated proteins in HQ (A) and LQ ejaculates (C). 2D gel stained with Coomassie Brilliant Blue showing the total proteins in HQ (B) and LQ ejaculates (D). Differentially carbonylated protein spots are numbered and listed in Table 3. The protein spots marked in red indicate higher carbonylation in LQ ejaculates while protein spots marked in blue indicate higher carbonylation in HQ ejaculates.