NLRP3 is part of the NLRP3 inflammasome and a global sensor of cellular damage. It was recently discovered in rodent Sertoli cells. We investigated NLRP3 in mouse, human and non-human primate ...(marmoset and rhesus macaque) testes, employing immunohistochemistry. Sertoli cells of all species expressed NLRP3, and the expression preceded puberty. In addition, peritubular cells of the adult human testes expressed NLRP3.
and associated genes (
,
,
) were also found in isolated human testicular peritubular cells and the mouse Sertoli cell line TM4. Male infertility due to impairments of spermatogenesis may be related to sterile inflammatory events. We observed that the expression of NLRP3 was altered in the testes of patients suffering from mixed atrophy syndrome, in which tubules with impairments of spermatogenesis showed prominent NLRP3 staining. In order to explore a possible role of NLRP3 in male infertility, associated with sterile testicular inflammation, we studied a mouse model of male infertility. These human aromatase-expressing transgenic mice (
) develop testicular inflammation and impaired spermatogenesis during aging, and the present data show that this is associated with strikingly elevated
expression in the testes compared to WT controls. Interference by aromatase inhibitor treatment significantly reduced increased
levels. Thus, throughout species NLRP3 is expressed by somatic cells of the testis, which are involved in testicular immune surveillance. We conclude that NLRP3 may be a novel player in testicular immune regulation.
The cation channel TRPV2 is known to be expressed by murine macrophages and is crucially involved in their functionality. Macrophages are frequent cells of the mouse testis, an immune-privileged and ...steroid-producing organ. TRPV2 expression by testicular macrophages and possible changes associated with age or inflammation have not been investigated yet. Therefore, we studied testes of young adult and old wild-type (WT) and AROM
mice, i.e., transgenic mice overexpressing aromatase. In these animals, inflammatory changes are described in the testis, involving active macrophages, which increase with age. This is associated with impaired spermatogenesis and therefore AROM
mice are a model for male infertility associated with sterile inflammation. In WT animals, testicular TRPV2 expression was mapped to interstitial CD206
and peritubular MHC II
macrophages, with higher levels in CD206
cells. Expression levels of TRPV2 and most macrophage markers did not increase significantly in old mice, with the exception of CD206. As the number of TRPV2
testicular macrophages was relatively small, their possible involvement in testicular functions and in aging in WT mice remains to be further studied. In AROM
testis, TRPV2 was readily detected and levels increased significantly with age, together with macrophage markers and TNF-α. TRPV2 co-localized with F4/80 in macrophages and further studies showed that TRPV2 is mainly expressed by unusual CD206
MHC II
macrophages, arising in the testis of these animals. Rescue experiments (aromatase inhibitor treatment and crossing with ERαKO mice) restored the testicular phenotype and also abolished the elevated expression of TRPV2, macrophage and inflammation markers. This suggests that TRPV2
macrophages of the testis are part of an inflammatory cascade initiated by an altered sex hormone balance in AROM
mice. The changes in testis are distinct from the described alterations in other organs of AROM
, such as prostate and spleen. When we monitored TRPV2 levels in another immune-privileged organ, namely the brain, we found that levels of TRPV2 were not elevated in AROM
and remained stable during aging. In the adrenal, which similar to the testis produces steroids, we found slight, albeit not significant increases in TRPV2 in both AROM
and WT mice, which were associated with age. Thus, the changes in the testis are specific for this organ.
Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors Schmid, Nina; Dietrich, Kim-Gwendolyn; Forne, Ignasi ...
International journal of molecular sciences,
02/2021, Letnik:
22, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Sirtuins (SIRTs) are NAD
-dependent deacetylases that regulate proliferation and cell death. In the human ovary, granulosa cells express sirtuin 1 (SIRT1), which has also been detected in human ...tumors derived from granulosa cells, i.e., granulosa cell tumors (GCTs), and in KGN cells. KGN cells are an established cellular model for the majority of GCTs and were used to explore the role of SIRT1. The SIRT1 activator SRT2104 increased cell proliferation. By contrast, the inhibitor EX527 reduced cell numbers, without inducing apoptosis. These results were supported by the outcome of siRNA-mediated silencing studies. A tissue microarray containing 92 GCTs revealed nuclear and/or cytoplasmic SIRT1 staining in the majority of the samples, and also, SIRT2-7 were detected in most samples. The expression of SIRT1-7 was not correlated with the survival of the patients; however, SIRT3 and SIRT7 expression was significantly correlated with the proliferation marker Ki-67, implying roles in tumor cell proliferation. SIRT3 was identified by a proteomic analysis as the most abundant SIRT in KGN. The results of the siRNA-silencing experiments indicate involvement of SIRT3 in proliferation. Thus, several SIRTs are expressed by GCTs, and SIRT1 and SIRT3 are involved in the growth regulation of KGN. If transferable to GCTs, these SIRTs may represent novel drug targets.
Peritubular cells of the human testis form a small compartment surrounding the seminiferous tubules. They are crucial for sperm transport, and they emerge as contributors to the spermatogonial stem ...cell niche. They are among the least known cell types of the human body. We employed single-cell RNA sequencing of cultured human testicular peritubular cells (HTPCs), which had been isolated from testicular samples of donors with normal spermatogenesis. The significant overlap between our results and recently published ex vivo data indicates that HTPCs are a highly adequate cellular model to define and study these cells. Thus, based on the expression of several markers, HTPCs can be classified as testicular smooth muscle cells. Small differences between the in vivo/in vitro expressed genes may be due to cellular plasticity. Plasticity was also shown upon addition of FCS to the culture medium. Based on transcriptome similarities, four cellular states were identified. Further analyses confirmed the presence of known stem cell niche-relevant factors (e.g., GDNF) and identified unknown functions, e.g., the ability to produce retinoic acid. Therefore, HTPCs allow us to define the signature(s) and delineate the functions of human testicular peritubular cells. The data may also serve as a resource for future studies to better understand male (in)fertility.
Background: Clonidine has been used in clinical medicine, e.g., to treat high blood pressure and other conditions. Animal studies have linked its use to impairments of male reproductive functions, ...and although only a few reports exist for the human species, such actions may exist in man as well. The underlying reasons and, specifically, possible actions of clonidine at the level of the testis are not known. Introduction: Clonidine is an agonist at the α2A-adrenoceptor (ADRA2A), which, as data bank mining indicated, is expressed by several cells of the human testis. The human testis and most of its cells are, however, not readily accessible to experimental testing. Cells from the peritubular wall compartment (human testicular peritubular cells; HTPCs) are the exception. Methods and Results: As shown by immunohistochemical/immunocytochemical and PCR techniques these cells express ADRA2A and retain expression upon isolation and culture. When tested over a concentration range (1–1000 µM) and 24 h, clonidine did not visibly affect HTPC morphology but significantly stimulated IL6 mRNA levels in a concentration-dependent manner. ELISA measurements of cell culture supernatants confirmed a stimulatory action of clonidine (10 µM) on secreted IL6. When examined in collagen gel contraction assays of HTPCs, clonidine (10 µM) exerted a slight relaxing action, while a proteomic study revealed that clonidine (10 µM) did not significantly change cellular protein abundance of HTPCs after 24 h (data available via ProteomeXchange with identifier PXD052220). Conclusion: Thus, ADRA2A-bearing cells in the human testis are targets for catecholamines and drugs such as clonidine. The results of this HTPCs-focused study only show the tip of the iceberg. It is likely that catecholamines/catecholaminergic drugs have the potential to interfere with human testicular functions.
The functions of human testicular peritubular cells (HTPCs), forming a small compartment located between the seminiferous epithelium and the interstitial areas of the testis, are not fully known but ...go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid receptor (GR) indicates that they may be regulated by glucocorticoids (GCs). Herein, we studied the consequences of the GC dexamethasone (Dex) in cultured HTPCs, which serves as a unique window into the human testis. We examined changes in cytokines, mainly by qPCR and ELISA. A holistic mass-spectrometry-based proteome analysis of cellular and secreted proteins was also performed. Dex, used in a therapeutic concentration, decreased the transcript level of proinflammatory cytokines, e.g., IL6, IL8 and MCP1. An siRNA-mediated knockdown of GR reduced the actions on IL6. Changes in IL6 were confirmed by ELISA measurements. Of note, Dex also lowered GR levels. The proteomic results revealed strong responses after 24 h (31 significantly altered cellular proteins) and more pronounced ones after 72 h of Dex exposure (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 extracellular matrix (ECM) and basement membrane components (e.g., FBLN2, COL1A2 and COL3A1), as well as PTX3 and StAR. These results pinpoint novel, profound effects of Dex in HTPCs. If transferrable to the human testis, changes specifically in ECM and the immunological state of the testis may occur in men upon treatment with Dex for medical reasons.
Aging of human testis and associated cellular changes is difficult to assess. Therefore, we used a translational, non-human primate model to get insights into underlying cellular and biochemical ...processes. Using proteomics and immunohistochemistry, we analyzed testicular tissue of young (age 2 to 3) and old (age 10 to 12) common marmosets (Callithrix jacchus). Using a mass spectrometry-based proteomics approach, we identified 63,124 peptides, which could be assigned to 5924 proteins. Among them, we found proteins specific for germ cells and somatic cells, such as Leydig and Sertoli cells. Quantitative analysis showed 31 differentially abundant proteins, of which 29 proteins were more abundant in older animals. An increased abundance of anti-proliferative proteins, among them CDKN2A, indicate reduced cell proliferation in old testes. Additionally, an increased abundance of several small leucine rich repeat proteoglycans and other extracellular matrix proteins was observed, which may be related to impaired cell migration and fibrotic events. Furthermore, an increased abundance of proteins with inhibitory roles in smooth muscle cell contraction like CNN1 indicates functional alterations in testicular peritubular cells and may mirror a reduced capacity of these cells to contract in old testes.
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
. 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.
Aging men display reduced reproductive health; however, testis aging is poorly understood at the molecular and genomic levels. Here, we utilized single-cell RNA-seq to profile over 44,000 cells from ...both young and older men and examined age-related changes in germline development and in the testicular somatic cells. Age-related changes in spermatogonial stem cells appeared modest, whereas age-related dysregulation of spermatogenesis and somatic cells ranged from moderate to severe. Altered pathways included signaling and inflammation in multiple cell types, metabolic signaling in Sertoli cells, hedgehog signaling and testosterone production in Leydig cells, cell death and growth in testicular peritubular cells, and possible developmental regression in both Leydig and peritubular cells. Remarkably, the extent of dysregulation correlated with body mass index in older but not in younger men. Collectively, we reveal candidate molecular mechanisms underlying the complex testicular changes conferred by aging and their possible exacerbation by concurrent chronic conditions such as obesity.
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•A transcriptional single-cell atlas of testes from older men•Age-related changes in SSCs are modest•Age-related dysregulation of spermatogenesis and somatic cells are pronounced•Dysregulation correlated with BMI in older but not younger men
Guo, Cairns, and colleagues revealed aging-related alternations of human germ cells and testicular somatic cells through single-cell transcriptomic profiling, providing candidate molecular mechanisms underlying the complex testicular changes conferred by aging and their possible exacerbation by concurrent chronic conditions such as obesity.
In the ovary, proliferation and differentiation of granulosa cells (GCs) drive follicular growth. Our immunohistochemical study in a non-human primate, the Rhesus monkey, showed that the ...mitochondrial activity marker protein cytochrome c oxidase subunit 4 (COX4) increases in GCs in parallel to follicle size, and furthermore, its intracellular localization changes. This suggested that there is mitochondrial biogenesis and trafficking, and implicates the actions of gonadotropins, which regulate follicular growth and ovulation. Human KGN cells, i.e. granulosa tumour cells, were therefore used to study these possibilities. To robustly elevate cAMP, and thereby mimic the actions of gonadotropins, we used forskolin (FSK). FSK increased the cell size and the amount of mitochondrial DNA of KGN cells within 24 h. As revealed by MitoTracker™ experiments and ultrastructural 3D reconstruction, FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced the network formation. A proteomic analysis indicated that FSK elevated the levels of regulators of the cytoskeleton, among others (data available via ProteomeXchange with identifier PXD032160). The steroidogenic enzyme CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), located in mitochondria, was more than 3-fold increased by FSK, implying that the cAMP/PKA-associated structural changes occur in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, the observations show increases in mitochondria and suggest intracellular trafficking of mitochondria in GCs during follicular growth, and indicate that they may partially be under the control of gonadotropins and cAMP. In line with this, increased cAMP in KGN cells profoundly affected mitochondrial dynamics in a PKA-dependent manner and implicated cytoskeletal changes.
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