Transwell isolation and difference analysis of capacitated boar sperm proteins based on the iTRAQ technique

Abstract

During capacitation, proteins in boar sperm are released to maintain the stability of their own state and membrane structure. No studies have analyzed the differences between retained proteins and released proteins during sperm capacitation. In the present study, a Transwell chamber and polycarbonate membrane were used to separate the proteins of boar sperm and their released proteins. Isotopically labeled relative and absolute quantification (iTRAQ) was used to analyze each compartment protein. A total of 108 differential proteins were identified in the upper and lower chambers of the Transwell, among which 27 were significantly upregulated (p-value≤0.05 and |log2 (fold change)|≥1) and 81 were significantly downregulated (p-value≤0.05 and |log2 (fold change)|≤1). These differential proteins were mainly involved in biological processes (e.g., the regulation of cysteine peptidase activity, transmembrane transportation, ion transportation and ATP synthesis) and major signaling pathways (e.g., glutathione/galactose metabolism, cellular adhesion and PI3K-Akt), and most of them interacted with each other to some extent. In conclusion, retained proteins and released proteins of capacitated sperm were effectively separated using a Transwell chamber, and differential proteins were successfully identified from among the proteins. Bioinformatics analysis suggested that these differential proteins affect sperm capacitation mainly by adjusting sperm energy metabolism, motion characteristics and acrosome membrane status.

Keywords: Capacitation; Differential proteins; Transwell chambers; iTRAQ.