Age-Associated Proteomic Changes in Human Spermatozoa

Abstract

Advancing age in men significantly contributes to declining sperm fertility. Information on age-related proteomic changes in spermatozoa is limited. This study involved normal fertile Arab men in three age groups: young adult (21-30 years; n = 6), late adult (31-40 years; n = 7), and advanced age (40-51 years; n = 5). Gradient-purified spermatozoa were analyzed using LC-MS/MS and proteomic data were processed using Progenesis QI (QIfp) v3.0 and UniProt/SwissProt. Significantly enriched annotations and clustering of proteins in the proteomic datasets were identified (2-fold change; p < 0.05). A total of 588 proteins were identified, with 93% shared across the three groups. Unique proteins were MYLK4 for the young adult group, PRSS57 for the late adult group, and HMGB4, KRT4, LPGAT1, OXCT2, and MGRN1 for the advanced age group. Furthermore, 261 (44%) proteins were differentially expressed (p < 0.05) across the three groups. Functional enrichment analysis suggested an aging-related significant increase in pathways associated with neurodegenerative diseases and protein folding, alongside decreases in glycolysis/gluconeogenesis, flagellated sperm motility, acetylation, phosphoprotein modifications, oxidation processes, and Ubl conjugation. Cluster analysis highlighted significantly upregulated proteins in young adults (e.g., H2BC1, LAP3, SQLE, LTF, PDIA4, DYNLT2) and late adults (e.g., ATP5F1B, ODF2, TUBA3C, ENO1, SPO11, TEX45, TEKT3), whereas most proteins in the advanced age group exhibited downregulation (e.g., SPESP1, RAB10, SEPTIN4, RAB15, PTPN7, USP5, ANXA1, PRDX1). In conclusion, this study revealed aging-associated proteomic changes in spermatozoa that impact critical processes, including spermatogenesis, motility, metabolism, and fertilization, potentially contributing to fertility decline. These changes provide a molecular framework for developing therapies to preserve sperm proteostasis and enhance fertility in older men.

Keywords: LC-MS/MS; aging; human; proteomics; spermatozoa.

Figure 1

Venn diagram of total (A), top 20 abundant (B), and top 20 sparse (C) protein distribution of spermatozoa across the age groups. The age groups of men are young adult (21–30 years), late adult (31–40 years), and advanced age (41–51 years).

Figure 2

Principal component analysis (A) and hierarchical heat map (B) of sperm proteome during aging. The age groups are young adult (Y1–Y3; 21–30 years), late adult (L1–L3; 31–40 years), and advanced age (A1–A3; 41–51 years).

Figure 3

Functional enrichment analysis of the sperm proteome during aging. The top 10 significantly enriched GO terms (p < 0.05) in the Cellular component and Molecular function categories are shown. The fold enrichment of each GO term is indicated in parentheses. The age groups of men are young adult (21–30 years), late adult (31–40 years), and advanced age (41–51 years).

Figure 4

Functional enrichment analysis of the sperm proteome during aging. The top 10 significantly enriched GO terms (p < 0.05) in the Biological category and post-translational modification terms or PTMs are shown. The fold enrichment of each GO term is indicated in parentheses. The age groups of men are young adult (21–30 years), late adult (31–40 years), and advanced age (41–51 years).

Figure 5

KEGG pathway analyses of sperm proteome during aging. The top 10 significantly (p < 0.05) enriched pathways are shown. The fold enrichment of each pathway term is indicated in parentheses. The age groups of men are young adult (21–30 years), late adult (31–40 years), and advanced age (41–51 years).

Figure 6

Clustering analysis (fuzzy C-means) of the sperm proteome during aging. Nine different protein expression profiles were detected (A), and substantial numbers of proteins were distributed across clusters (B). Each line in the cluster represents one protein. Blue lines correspond to proteins with a strong fit to the cluster centroid, while green and yellow lines represent proteins with progressively weaker associations peripheral to the cluster centroid. The age groups of men are young adult (21–30 years), late adult (31–40 years), and advanced age (41–51 years).