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

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.

Keywords: canine; epididymal spermatozoa; proteomic; semen quality; sonication.

Figure 1

Morphological changes in dog (Canis lupus familiaris) cauda epididymal spermatozoa (n = 15) after sonication stained with the Spermac stain (1000× magnification in light microscope). (A)—normal epididymal sperm; (B)—damaged epididymal sperm, with detached head and detached tail; (C)—damaged epididymal sperm showing acrosome loss; (D)—epididymal sperm showing damaged acrosomal membrane. Arrows show the exact sperm structures.

Figure 2

The evaluation of the morphological changes in dog (Canis lupus familiaris) cauda epididymal spermatozoa (n = 15) after the treatment with different sonication variants. C—the control sample (without sonication), S1—5 min, 50 W; S2—10 min, 50 W; S3—30 min, 50 W; S4—10 min, 150 W. Values are presented as the mean ± SE. Different sonication variants were compared with their respective control each by each. Different letters indicate significant difference (p ≤ 0.05).

Figure 3

Average amounts of intracellular proteins (SIPs) and membrane-associated proteins (SMAPs) (mg/mL) of dog (Canis lupus familiaris) cauda epididymal spermatozoa (n = 15) obtained using different sonication variants. C—control sample (without sonication), the S1—5 min, 50 W; S2—10 min, 50 W; S3—30 min, 50 W; S4—10 min, 150 W. Values are presented as the mean ± SE. Different sonication variants were compared with their respective control each by each. Different letters indicate significant difference (p ≤ 0.05).

Figure 4

One-dimensional SDS-PAGE (12%). Line A: sperm intracellular proteins (SIPs) of dog (Canis lupus familiaris) cauda epididymal spermatozoa (n = 15); Line B: sperm membrane-associated proteins (SMAPs) of the dog cauda epididymal spermatozoa. Proteins were identified using mass spectrometry in ranges 1–8 (Range 1 to 8) for SIPs and SMAPs. Differentially abundant protein fractions were marked with letters from a to j. STD—molecular weight markers.

Figure 5

Venn diagram showing the number of proteins identified in the sperm intracellular proteins (SIPs) and the sperm membrane-associated proteins (SMAPs) of the dog (Canis lupus familiaris) cauda epididymal spermatozoa.

Figure 6

Gene ontology (GO) enrichment of (A) sperm intracellular proteins (SIPs) and (B) sperm membrane-associated proteins (SMAPs) of the dog (Canis lupus familiaris) cauda epididymal spermatozoa. Significant GO terms for molecular function (orange) and biological process (blue) are presented. The analyses were made by the PANTHER Classification System (v. 16).

Figure 7

Protein classes in (A) sperm intracellular proteins (SIPs) and (B) sperm membrane-associated proteins (SMAPs) of the dog (Canis lupus familiaris) cauda epididymal spermatozoa. The protein class was analyzed by the PANTHER Classification System (v. 16). Each color represents percentage of protein participation in each protein class.

Figure 8

Pathways according to PANTHER Classification System (v. 16) for (A) sperm intracellular proteins (SIPs) and (B) sperm membrane-associated proteins (SMAPs) of dog (Canis lupus familiaris) cauda epididymal spermatozoa. Each color represents percentage of protein participation in each protein class.