Proteomic Insights into Senescence of Testicular Peritubular Cells from a Nonhuman Primate Model

Abstract

Age-related changes in the human testis may include morphological alterations, disturbed steroidogenesis, and impaired spermatogenesis. However, the specific impact of cell age remains poorly understood and difficult to assess. Testicular peritubular cells fulfill essential functions, including sperm transport, contributions to the spermatogonial stem cell niche, and paracrine interactions within the testis. To study their role in age-associated decline of testicular functions, we performed comprehensive proteome and secretome analyses of repeatedly passaged peritubular cells from Callithrix jacchus. This nonhuman primate model better reflects the human testicular biology than rodents and further gives access to young donors unavailable from humans. Among 5095 identified proteins, 583 were differentially abundant between samples with low and high passage numbers. The alterations indicate a reduced ability of senescent peritubular cells to contract and secrete proteins, as well as disturbances in nuclear factor (NF)-κB signaling and a reduced capacity to handle reactive oxygen species. Since this in vitro model may not exactly mirror all molecular aspects of in vivo aging, we investigated the proteomes and secretomes of testicular peritubular cells from young and old donors. Even though the age-related alterations at the protein level were less pronounced, we found evidence for impaired protein secretion, altered NF-κB signaling, and reduced contractility of these in vivo aged peritubular cells.

Keywords: aging; cellular model; marmoset monkey; nonhuman primate; proteome; secretome; senescence; testis.

Figure A1

Volcano plot analysis of proteomes (a) and secretomes (b) of MKTPCs from older and younger individual donors. Proteins above the significance cutoff curve (s = 0.1; FDR < 0.05) are considered significant.

Figure 1

(a) Cell size measurement of low-passage-number vs high-passage-number testicular peritubular cells from the common marmoset (lp-MKTPCs vs. hp-MKTPCs) from the same donor animal. A significant increase in cellular diameter of hp-MKTPCs was shown. Columns indicate the mean; bars indicate the standard deviation; for statistics, a paired t-test was used. (b) Light micrograph of senescence-associated β-galactosidase staining of lp-MKTPCS and hp-MKTPCs. ** p-value < 0.005

Figure 2

Principal component analysis (PCA) of cellular proteomes (a) and secretomes (b) of MKTPCs derived from lp-MKTPCs and hp-MKTPCs; each square represents an individual donor. Heat map and unsupervised hierarchical clustering of cellular proteomes (c) and secretome (d).

Figure 3

Volcano plots of hp-MKTPC vs. lp-MKTPC proteomes (a) and secretomes (b). For statistics, a paired Welch t-test was used. Results were corrected for multiple testing with a false discovery rate (FDR) of 0.05. Each colored dot represents a differentially abundant protein fulfilling the significance criteria.

Figure 4

Enriched Database for Annotation, Visualization, and Integrated Discovery (DAVID) annotation clusters from proteins more abundant (a) and less abundant (b) in hp-MKTPCs. The number in brackets behind the term indicates the number of proteins in one cluster. Results with an enrichment score >1.3 were considered significant. Categories used for the DAVID tool were as follows: Gene Ontology (GO) biological process, GO cellular component, GO molecular function, and reactome. NADH: nicotinamide-adenine dinucleotide, reduced, GDP: guanosine diphosphate PAK: p21 activated kinase

Figure 5

(a) Cells size measurement of MKTPCs from young vs. old monkeys revealed significantly increased cell size of MKTPCs from older monkeys. (b) Proportion of β-galactosidase-positive MKTPCs from young vs. old monkeys. (c) Light micrograph of senescence-associated β-galactosidase staining of MKTPCs from young (3 years) and old (11 years) monkey in passage 2. (d) Maximal passage numbers before offset of cell division. Columns indicate the mean; bars indicate the standard deviation. For statistical analysis, unpaired t-tests were used (a,b,d). * p-value < 0.05; **** p-value < 0.00005

Figure 6

Principal component analysis of cell proteomes (a) and secretomes (b) of MKTPCs derived from young and older individual donors.

Figure 7

The 20 most significant gene set hits of MKTPC proteomes from older vs. young individual donors. Significant Gene Set Enrichment Analysis (GSEA) results (FDR q-value < 0.05) were summarized by reducing redundancy with REVIGO. Only gene sets from the GO biological process category were utilized. Gene sets enriched in the MKTPC proteome of old donors (a) and enriched gene sets in the MKTPC proteome of young donors (b) are displayed.