Proteomics analysis of asthenozoospermia and identification of glucose-6-phosphate isomerase as an important enzyme for sperm motility.

Asthenozoospermia, in which sperm motility is affected, is one of the primary causes of male infertility. However, the exact mechanism responsible for the defective motility remains unknown. It is important to identify the precise proteins or pathways involved in sperm motility. The present study analyzed five asthenozoospermic sperm samples and five healthy controls using TMT-based quantitative method and identified 152 differentially expressed proteins, with 84 upregulated and 68 downregulated in asthenozoospermia. Four proteins (GPI, MDH1, PGAM1 and PGAM2) were found in several over-represented energy metabolism pathways using bioinformatics analysis. Glucose-6-phosphate isomerase (GPI), a rate-limiting enzyme converting glucose-6-phosphate to fructose-6-phosphate, was found to be significantly decreased in asthenozoospermia by Western blotting and ELISA on an extended sample size. Furthermore, substitution of glucose with fructose-6-phosphate significantly promoted asthenozoospermic sperm motility in vitro. Taken together, our results suggest that the poor motility of sperm in asthenozoospermia may partly result from defects in GPI-associated energy metabolism. SIGNIFICANCE: To identify the key proteins or pathways involved in sperm motility, the accurate TMT-based quantitative method was applied to characterize protein profiles of asthenozoospermic sperm. GPI, an enzyme involved in energy metabolism, was found to be differentially abundant, and validated by extended sample analysis. The supplement of the product of GPI, fructose-6-phosphate, could significantly improve sperm motility. Our study could provide new insights into the molecular basis of sperm motility and the improvement of motility in asthenozoospermia.

Proteomic analysis of N-glycosylation of human seminal plasma.

Seminal plasma is a mixture of secretions from several male accessory glands. The seminal plasma contains many secreted proteins which are important for sperm function and male fertility. In this study, we employed N-linked glycosylated peptide enrichment, combined with LC-MS/MS analysis, and establish the first large scale N-linked glycoproteome of human seminal plasma. Combined with the results of five biological replicates, a total of 720 N-glycosylated sites on 372 proteins were identified. Analysis of variations among five individuals revealed similar compositions of N-glycosylated proteins in seminal plasma. The N-linked glycoproteome could help us understanding the biological functions of human seminal plasma. The data set could also be a resource for further screening of biomarkers for male diseases including cancer and infertility at the level of N-glycosylation. For example, N-glycosylated prostate-specific antigen is known to be an efficient biomarker that can distinguish benign prostate hyperplasia from prostate cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD000959 (http://proteomecentral.proteomexchange.org/dataset/PXD000959).

Mapping of the N-linked glycoproteome of human spermatozoa.

N-Linked glycosylation, a type of post-translational modification, plays important roles in cell-cell recognition, adhesion, and interactions. Although N-linked glycosylated proteins in sperm are known to be important for gamete binding, little is known about the composition of these proteins, particularly glycosylation sites, in humans. In the present study, the use of glyco-FASP, coupled with the tandem mass spectrometry (MS/MS) method, led to the identification of 554 N-glycosylation sites and 297 N-glycosylated proteins in human sperm. Bioinformatics analysis revealed enrichment of proteins with functions in cell recognition and fertilization. Overall, about 91% of the human sperm N-glycosylated proteins were classified into "membrane", "extracellular region", and "lysosome" groups, based on subcellular localization annotation. Furthermore, glutathione peroxidase 4 (GPX4), a membrane glycoprotein identified in our glycoproteome, was shown to play a significant role in gamete interactions using the in vitro fertilization assay. Accordingly, we propose that characterization of the human sperm glycoproteome should effectively aid in clarifying the mechanisms of fertilization and provide a valuable resource for the future development of male contraceptives and diagnosis of male infertility.