Effect of aging on rat testicular interstitial compartment: characterization by single-cell-RNA-sequence analysis
Description
This dataset is a part of study addressing the difference in testicular cell transcriptomes between young and old BN rats. The study utilized single-cell RNA sequencing and immunofluorescence staining to characterize the testicular interstitial cells of young (3 month-old) and old (21 month-old) of BN rats. The findings revealed that the interstitial cells do not age in a synchronous manner and display remarkable heterogeneity. Specifically, Leydig cells (LCs), endothelial cells (ECs), and immune cells are predominantly affected. The LC population experiences a loss of heterogeneity and associated functions, such as xenobiotics metabolism and cytokine signaling, accompanied by an increase in cardiomyocyte-related functions. ECs exhibit a reduction in biological functions and an enhancement in immune response and leukocyte chemotaxis. Among the immune cell populations, dendritic cell number decreases, while that of lymphocytes rises, accompanied by a shift from innate to adaptive immunity. These alterations may lead to the formation of proinflammatory microenvironment and further accelerate overall testicular aging.
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ANIMALS USED: 4 young (3 month-old) and 4 old (21 month-old) BN male rats. METHOD DETAILS 1) Steps for interstitial cell preparation and scRNA-seq analysis can be found in a previously published article: Front Cell Dev Biol 10, 805249. DOI: 10.3389/fcell.2022.805249 2) Enrichment analysis of DEGs The total number of DEGs (if less than 100) or the top 100 (if more than 100) were used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. GO and KEGG terms with a corrected P-value less than 0.05 were considered significantly enriched by the cell types or ages. The top 5-10 terms for each enrichment were presented. GO and/or KEGG enrichments of the top 200 DEGs for each cell type were carried out by the online resource of OmicStudio Biotech Inc (Huangzhou, China; https://www.omicstudio.cn/tool). 3) Cell communication analysis by CellChat software Ligand-receptor pair analysis provides a unique opportunity to characterize cell-cell communications within an organ. To understand the interactions among various testicular cell types and how aging may affect them, we first identified DEGs across all cell groups within a given scRNA-seq dataset using the Wilcoxon rank sum test with a significance level of 0.05. Cell-cell communication (ligand-receptor pairing) analysis was conducted using the CellChat DB software (version 1.1.0) [23]. Only ligands and receptors with a p value < 0.05 in their expressions were retained for predicting cell-cell interactions. 4) Tissue preparation and immunofluorescence staining Young and old rat testes were initially fixed with 4% paraformaldehyde for 36 hours, dehydrated with sucrose (15% for 24 hours followed by 30% for 24 hours), and finally embedded in OCT at - 20°C. For some of staining paraffin-sections were first processed. Frozen or dewaxed paraffin sections (6 µm thick) were first washed with PBS three times and then incubated with the primary antibody overnight at 4°C, followed by the secondary antibody for 1 hour at room temperature in the dark. Before microscopic examination, a drop of cover-slide sealing solution containing DAPI was applied to visualize the cell nucleus. The manufacturers and dilutions of the antibodies can be found in supplementary Table S1. 5) Cell quantifications of the tissue sections Immunofluorescently labelled interstitial cells were counted within a square of 500μm × 500μm in size. Also, the total interstitial cells in the same area were counted by the number of nuclei. There were about 10-60 areas counted for each animal that came from at least 3 different sections for each cell type. The percentages of the cell type were calculated by dividing the number of positive cells for each marker protein by the total interstitial cell nuclei counted in the same area. Total of three animals were used for cell quantification.
Institutions
- Wenzhou Medical University Second Affiliated HospitalZhejiang, Wenzhou
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Funders
- Natural Science Foundation of Zhejiang ProvinceGrant ID: LZ23H040002
- Natural Science Foundation of Zhejiang Province, CNGrant ID: LQ23H040001