Proteomic Analysis of Intracellular and Membrane-Associated Fractions of Canine (Canis lupus familiaris) Epididymal Spermatozoa and Sperm Structure Separation

SIMPLE SUMMARY: Epididymal spermatozoa have great potential in current dog reproductive technologies. In the case of azoospermia or when the male dies, the recovery of epididymal spermatozoa opens new possibilities for reproduction. It is of great importance to analyze the quality of the sperm in such cases. Proteomic studies contribute to explaining the role of proteins at various stages of epididymal sperm maturation and offer potential opportunities to use them as markers of sperm quality. The present study showed, for the first time, mass spectrometry and bioinformatic analysis of intracellular and membrane-associated proteins of canine epididymal spermatozoa. Additionally, sonication was used for the separation of dog epididymal sperm morphological elements (heads, tails and acrosomes). The results revealed the presence of differentially abundant proteins in both sperm protein fractions significant for sperm function and fertilizing ability. It was also shown that these proteins participate in important sperm metabolic pathways, which may suggest their potential as sperm quality biomarkers. ABSTRACT: This study was provided for proteomic analysis of intracellular and membrane-associated fractions of canine (Canis lupus familiaris) epididymal spermatozoa and additionally to find optimal sonication parameters for the epididymal sperm morphological structure separation and sperm protein isolation. Sperm samples were collected from 15 dogs. Sperm protein fractions: intracellular (SIPs) and membrane-associated (SMAPs) were isolated. After sonication, sperm morphology was evaluated using Spermac Stain™. The sperm protein fractions were analyzed using gel electrophoresis (SDS-PAGE) and nanoliquid chromatography coupled to quadrupole time-of-flight mass spectrometry (NanoLC-Q-TOF/MS). UniProt database-supported identification resulted in 42 proteins identified in the SIPs and 153 proteins in the SMAPs. Differentially abundant proteins (DAPs) were found in SIPs and SMAPs. Based on a gene ontology analysis, the dominant molecular functions of SIPs were catalytic activity (50%) and binding (28%). Hydrolase activity (33%) and transferase activity (21%) functions were dominant for SMAPs. Bioinformatic analysis of SIPs and SMAPs showed their participation in important metabolic pathways in epididymal sperm, which may suggest their potential as sperm quality biomarkers. The use of sonication 150 W, 10 min, may be recommended for the separation of dog epididymal sperm heads, tails, acrosomes and the protein isolation.