iTRAQ-based proteomics of testicular interstitial fluid during aging in mice

Advancing age is associated with a decline in fertility and testicular function in males. The testicular interstitial fluid (TIF) bathing the seminiferous tubules and testicular interstitium is considered an important part of the testicular microenvironment. However, the TIF proteome in mice and whether it changes with age remain unclear. This study aimed to map the TIF proteome and identify differentially abundant proteins (DEPs) among young, middle-aged, and old mice using isobaric tags for relative and absolute quantification (iTRAQ) coupled with liquid chromatography-tandem mass spectrometry. A total of 1477 proteins were identified in murine TIF. The abundance of 706 proteins showed a linear change trend with age, of which 360 and 346 proteins increased and decreased, respectively. In addition, 45 age-related DEPs were identified ( P < 0.05, with at least 1.2-fold up- or downregulation). Bioinformatic analyses revealed that these proteins were involved in “actin cytoskeleton organization,” “intrinsic apoptotic signaling pathway ,” and “regulation of protein transport.” Comparative analysis with relevant proteomes previously reported further revealed the characteristics of mouse TIF proteome. Moreover, two of the age-related DEPs (Fga and Qsox1) were also found to be age-related differentially expressed proteins in human blood plasma and senescence-related secretome of human peritubular myoid cells. Taken together, these findings may represent the foundation for a better understanding of the molecular function of TIF and testicular aging. • Testicular interstitial fluid (TIF) bathing the seminiferous tubules constitutes the main microenvironment of testis. • The TIF proteome of mice with 1477 proteins were first analyzed by iTRAQ-based method. • The TIF proteome dynamically altered with age, with 45 age-related proteins between the young, middle and old age groups. • Functional analysis interpreted the proteomic changes with age, such as “actin cytoskeleton organization”.