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Single-cell atlas of rhesus monkey testis reveals aging- and season-dependent regulation of spermatogenesis and microenvironment homeostasis [RNA-Seq]

ABSTRACT: Spermatogenesis is a highly orchestrated process that depends on precise coordination between germ cells and the testicular microenvironment. However, how aging and seasonal shifts jointly regulate this process in nonhuman primates remains largely unexplored. Here, we generated a comprehensive single-cell transcriptomic atlas of the rhesus monkey testis across different ages and reproductive seasons. Our analyses reveal that Sertoli cells actively regulate the blood–testis barrier (BTB) by dynamically remodeling tight and adherens junctions, thereby mediating seasonal fluctuations in meiotic germ cells. We further identified a previously uncharacterized SSC subpopulation (U3) enriched in the non-breeding season, defined by PDGF pathway activation. By integrating this pathway into a three-dimensional collagen–PDGF scaffold, we successfully established an in vitro rhesus SSC culture system with enhanced clonogenicity and long-term propagation. In parallel, testicular immunosenescence emerged as a key determinant of age-related reproductive decline. We provide the first evidence that double-negative T cells (DNTs) markedly accumulate in aged testes, while pro-inflammatory macrophages (Mφ_SPAG16, M1-like) aberrantly expand during the non-breeding season, together disrupting immune homeostasis and niche support. Collectively, this study delineates the age- and season-dependent regulation of spermatogenesis in rhesus monkeys by integrating Sertoli–BTB dynamics, SSC niche signaling, and immune remodeling, thereby providing new mechanistic insights into testicular aging and reproductive homeostasis. experimental design：Following the manufacturer's protocol, cells were isolated from rhesus monkey testicular tissue for single-cell RNA sequencing, with results validated via immunocytochemistry and qPCR.

ORGANISM(S): Macaca mulatta

PROVIDER: GSE306702 | GEO | 2026/08/05

REPOSITORIES: GEO

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Project description:Embryonic stem cells (ESCs) may be able to cure or alleviate the symptoms of various degenerative diseases. However, unresolved issues regarding apoptosis, maintaining function and tumor formation mean a prudent approach should be taken towards advancing ESCs into human clinical trials. The rhesus monkey provides the ideal model organism for developing strategies to prevent immune rejection and test the feasibility, safety and efficacy of ESC-based medical treatments. Transcriptional profiling of rhesus ESCs provides a foundation for future pre-clinical ESC research using non-human primates as the model organism. In this research we use microarray, immunocytochemistry, real-time and standard RT-PCR to characterize and transcriptionally profile rhesus monkey embryonic stem cells. We identify 367 rhesus monkey stemness genes, we demonstrate the high level (>85%) of conservation of rhesus monkey stemness gene expression across five different rhesus monkey embryonic stem cell lines, we demonstrate that rhesus monkey ESC lines maintain a pluripotent undifferentiated state over a wide range of Pou5f1 (Oct-4) expression levels and we compare rhesus monkey, human and murine stemness genes to identify the key mammalian stemness genes. The supplementary tables list the genes that have been upregulated in each undifferentiated rhesus monkey embryonic stem cell line (GSM99998, GSM99999,GSM100000, GSM100001, GSM100002, GSM99965, GSM99966) in comparison analysis with the pooled differentiated embryonic stem cells (GSM99840). Supplemental Table 1 contains the comparison analysis for all 52,865 probe sets on the rhesus monkey gene chip, Supplemental Table 2 contains the rhesus monkey genes that were significantly upregulated (FC>3) in the ORMES-6 biological replicates, Supplemental Table 3 contains the rhesus monkey genes that were significantly upregulated (FC>3) in the pooled differentiated EBs and Supplemental Tables 4-8 represent genes that were significantly upregulated in ORMES 6A, 7, 9, 10 and 13 respectively. Supplemental Table 9 contains the RT-PCR primers used in this project. Keywords: Rhesus monkey embryonic stem cell microarray

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Single-cell atlas of rhesus monkey testis reveals aging- and season-dependent regulation of spermatogenesis and microenvironment homeostasis [RNA-Seq]

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