Identification of Proteins Involved in Human Sperm Motility Using High-Throughput Differential Proteomics

Abstract

Mammalian sperm motility is a prerequisite for in vivo fertilization, and alterations in this parameter are commonly observed in infertile males. However, we still do not have a complete understanding of the molecular mechanisms controlling it. The aim of this study was to identify proteins involved in human sperm motility deficiency by using TMT protein labeling and LC-MS/MS. Two complementary approaches were used: comparison between sperm samples differing in motility (asthenozoospermic versus normozoospermic) and comparison between sperm subpopulations of fractionated normozoospermic samples differing in motility (non-migrated versus migrated). LC-MS/MS resulted in the identification of 1157 and 887 proteins in the first and second approaches, respectively. Remarkably, similar proteomic alterations were detected in the two experiments, with 80 proteins differentially expressed in the two groups of samples and 93 differentially expressed in the two groups of subpopulations. The differential proteins were analyzed by GO, cellular pathways, and clustering analyses and resulted in the identification of core deregulated proteins and pathways associated with sperm motility dysfunction. These included proteins associated with energetic metabolism, protein folding/degradation, vesicle trafficking, and the cytoskeleton. Contrary to what is usually accepted, the outcomes support the hypothesis that several metabolic pathways (notably, mitochondrial-related ones) contribute toward regulating sperm motility.

Identification of endogenous metabolites in human sperm cells using proton nuclear magnetic resonance (1H-NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS)

“…Although clearly still in its infancy, the application of metabolomics to the study of human spermatozoa has a huge potential, and its application as a phenotypic readout can be an important key to understand fundamental biological processes acting in human sperm cells and also to better comprehend distinct male reproductive pathologies, and perhaps to discover new (in)fertility biomarkers. Particularly interesting should be the future application of these techniques to study different types of sperm phenotypes and to determine whether significant differences in the abundance of specific metabolites can be identified, such as those that have been recently detected at the protein level Amaral et al, 2014b;Azpiazu et al, 2014).…”

Human Spermatozoa Quantitative Proteomic Signature Classifies Normo- and Asthenozoospermia

“…Cluster 2, 3 and 5 on the other hand, had majority of the pathways common with AZS samples and cluster 4 had overlapping pathways with NZS samples. 4. DISCUSSION Twenty-four percent of infertile men present with isolated AZS (14) caused by various factors including varicocele, infection or genetic causes (15)(16)(17).…”

Integrated Transcriptomic and Proteomic Analysis of the Global Response of Synechococcus to High Light Stress*

“…With the NextGeneration Sequencing (NGS) technology, RNA-Seq has become a powerful tool for transcriptomic profiling (19,20). Tandem mass tags (TMT)-based quantitative proteomics has been widely used and proven to be a reliable method for determining protein expression levels (21)(22)(23). Here, we demonstrated that an integrated study coupling NGS-based RNASeq transcriptomics and quantitative TMT-LC-MS/MS proteomics has gained a system level understanding of the functional components involved in acclimation to HL.…”