The proteome of frozen-thawed pig spermatozoa is dependent on the ejaculate fraction source

Abstract

The preservation of sperm functional parameters and fertility post-cryopreservation largely varies in the porcine, a species with a fractionated ejaculate. Although intrinsic individual differences have primarily been linked to this variation, differences in protein abundance among frozen-thawed (FT)-spermatozoa are far more relevant. This study, performed in two experiments, looked for proteomic quantitative differences between FT-sperm samples differing in post-thaw viability, motility, apoptosis, membrane lipid peroxidation and nuclear DNA fragmentation. The spermatozoa were either derived from the sperm-rich ejaculate fraction (SRF) or the entire ejaculate (Experiment 1) or from the first 10 mL of the SRF, the remaining SRF and the post-SRF (Experiment 2). Quantitative sperm proteomic differences were analysed using a LC-ESI-MS/MS-based SWATH approach. In Experiment 1, FT-spermatozoa from the SRF showed better preservation parameters than those from the entire ejaculate, with 26 Sus scrofa proteins with functional sperm relevance showing relative quantitative differences (FC ≥ 1.5) between sperm sources. In Experiment 2, FT-spermatozoa from the first 10 mL of the SRF and the remaining SRF were qualitatively better than those from the post-SRF, and 187 proteins showed relative quantitative differences among the three ejaculate sources. The results indicate that quantitative proteome differences are linked to sperm cryosurvival.

Cryopreservation Differentially Alters the Proteome of Epididymal and Ejaculated Pig Spermatozoa

“…Protein extraction of sperm samples was conducted as described previously by Perez-Patiño et al [50]. Briefly, sperm samples were centrifuged (14,000× g for 10 min; Eppendorf 5424R, Eppendorf AG, Hamburg, Germany) and the total protein of the resulting sperm pellets were extracted using UTC buffer (7 M Urea, 2 M thiourea, 4% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS)), supplemented with protease inhibitor cocktail (1%, v / v ).…”

“…A SWATH analysis was performed by liquid chromatography (LC) using a NanoLC Ultra 1D plus Eksigent (Eksigent Technologies, Dublin, CA, USA) connected to an AB SCIEX TripleTOF 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA) in direct injection mode following the procedure described by Perez-Patiño et al [50]. Firstly, for the spectral library acquisition, 5 µL of a mixture of the twelve digested samples (2 µL per sample) was loaded on a trap NanoLC precolumn (3-μm particle size C18-CL, 350 µm diameter × 0.5 mm long; Eksigent Technologies, Dublin, CA, USA) and desalted with 0.1% TFA at 3 µL/min for 5 min.…”

Comparative Proteomics and Phosphoproteomics Analysis Reveal the Possible Breed Difference in Yorkshire and Duroc Boar Spermatozoa

“…Previous studies have shown that absence or weak expression of AKAP3 and AKAP4 has been described over the years as being related to sperm dysfunctions with motility impairments (Miki et al, 2002;Hillman et al, 2013;Xu et al, 2020). In contrast, the upregulation of either AKAP4 or AKAP3 in frozen-thawed boar sperm might be associated with their premature capacitation (Chen et al, 2014;Perez-Patino et al, 2019b). A higher expression of AKAP3 and AKAP4 in Duroc boar sperm might promote the sperm function; they play a crucial role in sperm motility.…”

iTRAQ-based proteomic analysis of sperm reveals candidate proteins that affect the quality of spermatozoa from boars on plateaus

“…In-depth proteomic analyses of boar spermatozoa have been performed using shotgun and gel-based methods [6]. The proteome of pig spermatozoa is remodeled during ejaculation [7]. The comparative proteomes of prenatal muscle tissues among TP, Wujin pigs and large White pigs have been analyzed using the isobaric tag for relative and absolute quantification (iTRAQ) [8].…”