Abstract
Protein phosphorylation can induce signal transduction to change sperm motility patterns during sperm capacitation. However, changes in the phosphorylation of sperm proteins in mice are still incompletely understood. Here, capacitation-related phosphorylation in mouse sperms were firstly investigated by label-free quantitative (LFQ) phosphoproteomics coupled with bioinformatics analysis using ingenuity pathway analysis (IPA) methods such as canonical pathway, upstream regulator, and network analysis. Among 1632 phosphopeptides identified at serine, threonine, and tyrosine residues, 1050 novel phosphosites, corresponding to 402 proteins, were reported. Gene heatmaps for IPA canonical pathways showed a novel role for GSK-3 in GP6 signaling pathways associated with capacitation for 60 min. At the same time, the reduction of the abundant isoform-specific GSK-3α expression was shown by western blot (WB) while the LFQ pY of this isoform slightly decreased and then increased. The combined results from WB and LFQ methods explain the less inhibitory phosphorylation of GSK-3α during capacitation and also support the predicted increases in its activity. In addition, pAKAP4 increased at the Y156 site but decreased at the Y811 site in a capacitated state, even though IPA network analysis and WB analysis for overall pAKAP revealed upregulated trends. The potential roles of GSK-3 and AKAP4 in fertility are discussed.
Highlights
Capacitation is an important physiological prerequisite for the sperm cell acrosome reaction and oocyte fertilization [1]
PAKAP4 increased at the Y156 site and decreased at the Y811 site, while pGSK-3 at the Y279 site significantly decreased in Cap 60 and significantly increased in Cap 90, determined by label-free quantitative (LFQ)
Upregulated AKAP4 protein, which directly interacts with downregulated PRKAR1A kinase, and downregulated GSK-3 kinase, which directly interacts with upregulated PPP1R2 kinase and indirectly interacts with reduced PI3K in Cap 60/0 datasets, were identified by network analysis and it was newly found that the GSK-3 pathway interacts with the GP6 signaling canonical pathway during capacitation
Summary
Capacitation is an important physiological prerequisite for the sperm cell acrosome reaction and oocyte fertilization [1]. This principle of capacitation was first introduced by Austin [2] and Chang [3]. Phosphoproteomics workflows have been applied to identify phosphoproteins, localize specific sites of phosphorylation and quantify the extent of modification at particular sites during processes, including sperm capacitation [5]. The first phosphoproteomics study on sperm capacitation, which was performed by Mandal et al [6], identified 18 peptides from a 95 kDa human sperm protein (FSP95) that is tyrosine phosphorylated during sperm capacitation. A newly published study involving proteomics followed by bioinformatics reveals the involvement of proteins in specific biological, molecular, and cellular pathways in male infertility [7]
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