Proteomic insights into senescence of testicular peritubular cells from a nonhuman primate model
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF-X
ORGANISM(S): Callithrix Jacchus
TISSUE(S): Testis, Peritubular Myoid Cell
SUBMITTER: 
Jan Bernd Stöckl
LAB HEAD: Thomas Fröhlich
PROVIDER: PXD022165 | Pride | 2020-11-18
REPOSITORIES: Pride
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Project description:Smooth-muscle-like peritubular cells make up the wall of semniferous tubules of men. These human testicular peritubular cells (HTPC) have been shown to fulfill different roles. They transport sperm, secrete various factors like GDNF (glial cell line derived neurotrophic factor) and are immunologically active. They might contribute to spermatogonial stem cell niche altering and testicular ageing. Previous studies were limited regarding heterogeneity (due to lifestyle, age, medical history etc.) and accessibility of HTPCs. To circumvent this problem a cellular primate model should be established. The proteome of 6 MKTPCs of individual healthy and young (2 or 3 years) donors was assessed to investigate the suitability as a model.
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Project description:Aging of the human testis and associated cellular changes are difficult to assess, thus we used a translational non-human primate model to get insights into underlying biochemical processes. Using proteomics we analyzed testicular tissue of six young (age 2 to 3) and four old (age 10 to 12) C. jacchus individuals. Mass spectrometry identified 63124 peptides, which could be assigned to 5924 proteins. Among them we found proteins, which are specific for germ cells, Leydig cells, and somatic cells, indicating the relevance of the dataset. The quantitative analysis showed 31 differentially abundant proteins, of which 29 proteins were more abundant in old animals. An increased abundance of several anti-proliferative proteins, among them CDKN2A, indicate slowed cell proliferation in older testes. Immunohistochemistry localized CDKN2A in spermatogonia and spermatocytes, suggesting altered spermatogenesis. Additionally, an increased abundance of several extracellular proteins and small leucine rich repeat proteoglycans was observed, which may be related to impaired cell migration and fibrotic events. An increased abundance of proteins with inhibitory roles in smooth muscle cell contraction like CNN1, indicate functional alterations, specifically in peritubular cells and may mirror a reduced capacity of these cells, to contract in aged testes. In summary, these results document several age-associated changes in the testicular proteome of a non-human primate model and provides new insight into testicular aging.
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Project description:Peritubular cells of the human testis form a small compartment surrounding the seminiferous tubules. It contrast to rodents, it is composed of several cell layers and extracellular matrix. Peritubular cells are crucial for sperm transport and emerge as contributors to the spermatogonial stem cell niche, yet testicular peritubular cells are among the least known cell types of the human body. We employed single-cell RNA sequencing of cultured human testicular peritubular cells (HTPCs) isolated from testicular samples of donors with normal spermatogenesis and compared our data with recently published ex vivo data and, if available, with immunohistochemical data of the Human Protein Atlas. We observed a significant overlap between our results and genes expressed in vivo. Based on expression of a set of smooth muscle markers, HTPCs can be classified as smooth muscle cells. Small differences between in vivo/in vitro expressed genes may be due to plasticity/phenotypic switching. Plasticity was also shown upon addition of FCS to the culture medium. Based on transcriptome similarities four cellular states were observed. Characteristics of mesenchymal stromal cells and Leydig stem cells were noted, yet differentiation attempts into fat cells, bone cells or mature Leydig cells were not successful. Further analyses confirmed known stem cell niche-relevant factors (e.g. GDNF) and identified unknown functions e.g. the ability to produce retinoic acid. The results reveal that HTPCs upon isolation and culture are a highly adequate cellular model as they retain major characteristics. They therefore allow us to define the signature and functions of human testicular peritubular cells. The data may serve as a resource for future studies to better understand male (in)fertility.
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Project description:The functions of human testicular peritubular cells (HTPCs), which form a small compartment located between the seminiferous epithelium and the interstitials areas of the testis, go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid receptor (GR/NR3C1) by these cells indicates that they are a target of glucocorticoids (GCs), yet detailed insights into consequences of GC actions are unknown. To address this point, we studied cultured HTPCs, which serve as a window into the human testis. We used a cytokine profiler assay to screen for changes in secreted cytokines upon dexamethasone (Dex) treatment and employed qPCR and ELISA studies to verify the results. Dex, used in a therapeutic concentration, decreased proinflammatory factors, including IL6, IL8, MCP1 and CXCL1 within 24 - 48 h. An siRNA-mediated knockdown of GR reduced these actions. Exposure to Dex resulted in reduced GR levels, implying that exposure to Dex could also reduce anti-inflammatory actions of Dex. To evaluate the influence of Dex on HTPCs further, we performed a proteomic analysis of cellular components and secreted proteins. Strong responses were detectable after 24 h (31 significantly altered cellular proteins) and more pronounced ones after 72 h (30 less abundant and 42 more abundant cellular proteins). Dex also altered the composition of the secretome (33 proteins decreased, 13 increased) after 72 h. Among the regulated proteins were ECM- and basement membrane components (e.g. FBLN2, COL1A2, COL3A1), as well as PTX3 and StAR. These results reveal that Dex via GR exerts profound actions in HTPCs. If transferrable to the human testis, changes specifically in ECM and the immunological state of the testis may result in men upon treatment with Dex for medical reasons. -Secretome Data Subset-
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Project description:The functions of human testicular peritubular cells (HTPCs), which form a small compartment located between the seminiferous epithelium and the interstitials areas of the testis, go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid receptor (GR/NR3C1) by these cells indicates that they are a target of glucocorticoids (GCs), yet detailed insights into consequences of GC actions are unknown. To address this point, we studied cultured HTPCs, which serve as a window into the human testis. We used a cytokine profiler assay to screen for changes in secreted cytokines upon dexamethasone (Dex) treatment and employed qPCR and ELISA studies to verify the results. Dex, used in a therapeutic concentration, decreased proinflammatory factors, including IL6, IL8, MCP1 and CXCL1 within 24 - 48 h. An siRNA-mediated knockdown of GR reduced these actions. Exposure to Dex resulted in reduced GR levels, implying that exposure to Dex could also reduce anti-inflammatory actions of Dex. To evaluate the influence of Dex on HTPCs further, we performed a proteomic analysis of cellular components and secreted proteins. Strong responses were detectable after 24 h (31 significantly altered cellular proteins) and more pronounced ones after 72 h (30 less abundant and 42 more abundant cellular proteins). Dex also altered the composition of the secretome (33 proteins decreased, 13 increased) after 72 h. Among the regulated proteins were ECM- and basement membrane components (e.g. FBLN2, COL1A2, COL3A1), as well as PTX3 and StAR. These results reveal that Dex via GR exerts profound actions in HTPCs. If transferrable to the human testis, changes specifically in ECM and the immunological state of the testis may result in men upon treatment with Dex for medical reasons. -Proteome Data Subset 24 h-
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Project description:The functions of human testicular peritubular cells (HTPCs), which form a small compartment located between the seminiferous epithelium and the interstitials areas of the testis, go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid receptor (GR/NR3C1) by these cells indicates that they are a target of glucocorticoids (GCs), yet detailed insights into consequences of GC actions are unknown. To address this point, we studied cultured HTPCs, which serve as a window into the human testis. We used a cytokine profiler assay to screen for changes in secreted cytokines upon dexamethasone (Dex) treatment and employed qPCR and ELISA studies to verify the results. Dex, used in a therapeutic concentration, decreased proinflammatory factors, including IL6, IL8, MCP1 and CXCL1 within 24 - 48 h. An siRNA-mediated knockdown of GR reduced these actions. Exposure to Dex resulted in reduced GR levels, implying that exposure to Dex could also reduce anti-inflammatory actions of Dex. To evaluate the influence of Dex on HTPCs further, we performed a proteomic analysis of cellular components and secreted proteins. Strong responses were detectable after 24 h (31 significantly altered cellular proteins) and more pronounced ones after 72 h (30 less abundant and 42 more abundant cellular proteins). Dex also altered the composition of the secretome (33 proteins decreased, 13 increased) after 72 h. Among the regulated proteins were ECM- and basement membrane components (e.g. FBLN2, COL1A2, COL3A1), as well as PTX3 and StAR. These results reveal that Dex via GR exerts profound actions in HTPCs. If transferrable to the human testis, changes specifically in ECM and the immunological state of the testis may result in men upon treatment with Dex for medical reasons. -Proteome Data Subset 72 h-
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Project description:Transcriptional profiling of human control and Néstor-Guillermo Progeria Syndrome (NGPS) mesenchymal stem cells (MSCs). Somatic cell reprogramming involves rejuvenation of adult cells and relies on the ability to erase age-associated molecular marks. Accordingly, reprogramming efficiency declines with ageing, and age-associated features such as genetic instability, cell senescence or telomere shortening negatively affect this process. However, the regulatory mechanisms that constitute age-associated barriers for cell reprogramming remain largely unknown. Here, by using cells from patients with premature ageing, we demonstrate that NF-κB activation is a critical barrier for the generation of induced pluripotent stem cells (iPSCs) in ageing. We show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs generation of iPSCs by eliciting reprogramming repressors DOT1L and YY1, reinforcing cell senescence signals and down-regulating pluripotency genes. We also show that genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo Progeria Syndrome (NGPS) and Hutchinson-Gilford Progeria Syndrome (HGPS) patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo ameliorates the accelerated ageing phenotype and extends lifespan in a progeroid animal model. Collectively, our results provide evidence for a novel role of NF-κB in the control of cell fate transitions and reinforce the interest of studying age-associated molecular impairments to implement cell reprogramming methodologies, and to identify new targets of rejuvenation strategies.
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