scholarly journals Insights into replicative senescence of human testicular peritubular cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nina Schmid ◽  
Florian Flenkenthaler ◽  
Jan B. Stöckl ◽  
Kim-Gwendolyn Dietrich ◽  
Frank M. Köhn ◽  
...  
Keyword(s):  
Replicative Senescence ◽  
Ion Beam ◽  
Immune Surveillance ◽  
Human Testis ◽  
Mrna Levels ◽  
Dipeptidyl Peptidase 4 ◽  
Telomere Attrition ◽  
Age Related ◽  
Peritubular Cells

Abstract There is evidence for an age-related decline in male reproductive functions, yet how the human testis may age is not understood. Human testicular peritubular cells (HTPCs) transport sperm, contribute to the spermatogonial stem cell (SSC) niche and immune surveillance, and can be isolated and studied in vitro. Consequences of replicative senescence of HTPCs were evaluated to gain partial insights into human testicular aging. To this end, early and advanced HTPC passages, in which replicative senescence was indicated by increased cell size, altered nuclear morphology, enhanced β-galactosidase activity, telomere attrition and reduced mitochondrial DNA (mtDNA), were compared. These alterations are typical for senescent cells, in general. To examine HTPC-specific changes, focused ion beam scanning electron microscopy (FIB/SEM) tomography was employed, which revealed a reduced mitochondrial network and an increased lysosome population. The results coincide with the data of a parallel proteomic analysis and indicate deranged proteostasis. The mRNA levels of typical contractility markers and growth factors, important for the SSC niche, were not significantly altered. A secretome analysis identified, however, elevated levels of macrophage migration inhibitory factor (MIF) and dipeptidyl peptidase 4 (DPP4), which may play a role in spermatogenesis. Testicular DPP4 may further represent a possible drug target.

scholarly journals Human Aging DNA Methylation Signatures are Conserved but Accelerated in Cultured Fibroblasts

2019 ◽  
Author(s):  
Gabriel Sturm ◽  
Andres Cardenas ◽  
Marie-Abèle Bind ◽  
Steve Horvath ◽  
Shuang Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Replicative Senescence ◽  
Human Fibroblasts ◽  
High Temporal Resolution ◽  
Global Changes ◽  
Mrna Levels ◽  
Cultured Fibroblasts ◽  
Age Related ◽  
Epigenetic Aging

SummaryAging is associated with progressive and site-specific changes in DNA methylation (DNAm). These global changes are captured by DNAm clocks that accurately predict chronological age in humans but relatively little is known about how clocks perform in vitro. Here we culture primary human fibroblasts across the cellular lifespan (∼6 months) and use four different DNAm clocks to show that age-related DNAm signatures are conserved and accelerated in vitro. The Skin & Blood clock shows the best linear correlation with chronological time (r=0.90), including during replicative senescence. Although similar in nature, the rate of epigenetic aging is approximately 62x times faster in cultured cells than in the human body. Consistent with in vivo data, cells aged under hyperglycemic conditions exhibit an approximately three years elevation in baseline DNAm age. Moreover, candidate gene-based analyses further corroborate the conserved but accelerated biological aging process in cultured fibroblasts. Fibroblasts mirror the established DNAm topology of the age-related ELOVL2 gene in human blood and the rapid hypermethylation of its promoter cg16867657, which correlates with a linear decrease in ELOVL2 mRNA levels across the lifespan. Using generalized additive modeling on twelve timepoints across the lifespan, we also show how single CpGs exhibit loci-specific, linear and nonlinear trajectories that reach rates up to −47% (hypomethylation) to +23% (hypermethylation) per month. Together, these high temporal resolution global, gene-specific, and single CpG data highlight the conserved and accelerated nature of epigenetic aging in cultured fibroblasts, which may constitute a system to evaluate age-modifying interventions across the lifespan.Graphical Abstract

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

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2498
Author(s):  
Jan B. Stöckl ◽  
Nina Schmid ◽  
Florian Flenkenthaler ◽  
Charis Drummer ◽  
Rüdiger Behr ◽  
...  
Keyword(s):  
Nonhuman Primate ◽  
Human Testis ◽  
Nonhuman Primate Model ◽  
Primate Model ◽  
Morphological Alterations ◽  
Age Related ◽  
Cell Niche ◽  
Peritubular Cells

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.

scholarly journals Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yan Gong ◽  
Jesse Li-Ling ◽  
Dongsheng Xiong ◽  
Jiajing Wei ◽  
Taiqing Zhong ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Clinical Trial Registry ◽  
Altered Expression ◽  
Vitro Fertilization ◽  
Age Related ◽  
Tertiary Teaching ◽  
Poor Ovarian Responders

Abstract Background Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes play important roles in folliculogenesis. Altered expression of the two have been found among patients with poor ovarian response (POR). In this prospective cohort study, we have determined the expression of the GDF9 and BMP15 genes in follicle fluid (FF) and granulosa cells (GCs) derived from poor ovarian responders grouped by age, and explored its correlation with the outcome of in vitro fertilization and embryo transfer (IVF-ET) treatment. Methods A total of 196 patients with POR were enrolled from a tertiary teaching hospital. The patients were diagnosed by the Bologna criteria and sub-divided into group A (< 35 year old), group B (35–40 year old), and group C (> 40 year old). A GnRH antagonist protocol was conducted for all patients, and FF and GCs were collected after oocyte retrieval. Expression of the GDF9 and BMP15 genes in the FF and GCs was determined with enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Results Compared with group C, groups A and B had significantly more two pronuclei (2PN) oocytes and transplantable embryos, in addition with higher rates of implantation and clinical pregnancy (P <  0.05). The expression level of GDF9 and BMP15 genes in the FF and GCs differed significantly among the three groups (P <  0.05), showing a trend of decline along with age. The ratio of GDF9/BMP15 mRNA levels were similar among the three groups (P > 0.05). The relative levels of GDF9 and BMP15 proteins in GCs have correlated with the relative mRNA levels in GCs and protein concentrations in FF (P <  0.05). Conclusions For poor ovarian responders, in particular those over 40, the expression of GDF9 and BMP15 is declined along with increased age and in accompany with poorer oocyte quality and IVF outcome, whilst the ratio of GDF9/BMP15 mRNA levels remained relatively constant. Trial registration Chinese Clinical Trial Registry Center (ChiCTR1800016107). Registered on 11 May 2018.

scholarly journals Effects of CREG1 on Age-Associated Metabolic Phenotypes and Renal Senescence in Mice

2021 ◽  
Vol 22 (3) ◽  
pp. 1276
Author(s):  
Michihiro Hashimoto ◽  
Ayumi Goto ◽  
Yuki Endo ◽  
Masataka Sugimoto ◽  
Jun Ueda ◽  
...  
Keyword(s):  
Renal Dysfunction ◽  
Body Weight Gain ◽  
Mrna Levels ◽  
Wild Type ◽  
Metabolic Phenotypes ◽  
Age Related ◽  
Secretory Phenotype ◽  
Filtering Function ◽  
Renal Senescence

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a secreted glycoprotein that accelerates p16-dependent cellular senescence in vitro. We recently reported the ability of CREG1 to stimulate brown adipogenesis using adipocyte P2-CREG1-transgenic (Tg) mice; however, little is known about the effect of CREG1 on aging-associated phenotypes. In this study, we investigated the effects of CREG1 on age-related obesity and renal dysfunction in Tg mice. Increased brown fat formation was detected in aged Tg mice, in which age-associated metabolic phenotypes such as body weight gain and increases in blood glucose were improved compared with those in wild-type (WT) mice. Blood CREG1 levels increased significantly in WT mice with age, whereas the age-related increase was suppressed, and its levels were reduced, in the livers and kidneys of Tg mice relative to those in WT mice at 25 months. Intriguingly, the mRNA levels of Ink4a, Arf, and senescence-associated secretory phenotype (SASP)-related genes and p38MAPK activity were significantly lowered in the aged kidneys of Tg mice, in which the morphological abnormalities of glomeruli as well as filtering function seen in WT kidneys were alleviated. These results suggest the involvement of CREG1 in kidney aging and its potential as a target for improving age-related renal dysfunction.

scholarly journals A translational cellular model for the study of peritubular cells of the testis

Reproduction ◽  
2020 ◽  
Vol 160 (2) ◽  
pp. 259-268 ◽  
Author(s):  
Nina Schmid ◽  
Annika Missel ◽  
Stoyan Petkov ◽  
Jan B Stöckl ◽  
Florian Flenkenthaler ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Replicative Senescence ◽  
Smooth Muscle Actin ◽  
Proteome Analysis ◽  
Inflammatory Factors ◽  
Seminiferous Tubules ◽  
Cellular Model ◽  
Mechanistic Studies ◽  
Peritubular Cells

Testicular peritubular cells (TPCs) are smooth muscle-like cells, which form a compartment surrounding the seminiferous tubules. Previous studies employing isolated human testicular peritubular cells (HTPCs) indicated that their roles in the testis go beyond sperm transport and include paracrine and immunological contributions. Peritubular cells from a non-human primate (MKTPCs), the common marmoset monkey, Callithrix jacchus, share a high degree of homology with HTPCs. However, like their human counterparts these cells age in vitro and replicative senescence limits in-depth functional or mechanistic studies. Therefore, a stable cellular model was established. MKTPCs of a young adult animal were immortalized by piggyBac transposition of human telomerase (hTERT), that is, without the expression of viral oncogenes. Immortalized MKTPCs (iMKTPCs) grew without discernable changes for more than 50 passages. An initial characterization revealed typical genes expressed by peritubular cells (androgen receptor (AR), smooth-muscle actin (ACTA2), calponin (CNN1)). A proteome analysis of the primary MKTPCs and the derived immortalized cell line confirmed that the cells almost completely retained their phenotype. To test whether they respond in a similar way as HTPCs, iMKTPCs were challenged with forskolin (FSK) and ATP. As HTPCs, they showed increased expression level of the StAR protein (StAR) after FSK stimulation, indicating steroidogenic capacity. ATP increased the expression of pro-inflammatory factors (e.g. IL1B; CCL7), as it is the case in HTPCs. Finally, we confirmed that iMKTPCs can efficiently be transfected. Therefore, they represent a highly relevant translational model, which allows mechanistic studies for further exploration of the roles of testicular peritubular cells.

scholarly journals Aldosterone up-regulates voltage-gated potassium currents and NKCC1 protein membrane fractions

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Parveen Bazard ◽  
Bo Ding ◽  
Harish K. Chittam ◽  
Xiaoxia Zhu ◽  
Thomas A. Parks ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mineralocorticoid Receptor ◽  
Therapeutic Potential ◽  
Potassium Currents ◽  
Chloride Ions ◽  
Mrna Levels ◽  
Mineralocorticoid Receptor Antagonist ◽  
Voltage Gated ◽  
Age Related

Abstract Na+–K+–2Cl− Cotransporter (NKCC1) is a protein that aids in the active transport of sodium, potassium, and chloride ions across cell membranes. It has been shown that long-term systemic treatment with aldosterone (ALD) can enhance NKCC1 protein expression and activity in the aging cochlea resulting in improved hearing. In the present work, we used a cell line with confirmed NKCC1 expression to demonstrate that in vitro application of ALD increased outward voltage-gated potassium currents significantly, and simultaneously upregulated whole lysate and membrane portion NKCC1 protein expression. These ALD-induced changes were blocked by applying the mineralocorticoid receptor antagonist eplerenone. However, application of the NKCC1 inhibitor bumetanide or the potassium channel antagonist Tetraethyl ammonium had no effect. In addition, NKKC1 mRNA levels remained stable, indicating that ALD modulates NKCC1 protein expression via the activation of mineralocorticoid receptors and post-transcriptional modifications. Further, in vitro electrophysiology experiments, with ALD in the presence of NKCC1, K+ channel and mineralocorticoid receptor inhibitors, revealed interactions between NKCC1 and outward K+ channels, mediated by a mineralocorticoid receptor-ALD complex. These results provide evidence of the therapeutic potential of ALD for the prevention/treatment of inner ear disorders such as age-related hearing loss.

scholarly journals Human and rat testis express two mRNA species encoding variants of NRD convertase, a metalloendopeptidase of the insulinase family

1997 ◽  
Vol 327 (3) ◽  
pp. 773-779 ◽  
Author(s):  
Véronique HOSPITAL ◽  
Annik PRAT ◽  
Catherine JOULIE ◽  
Dorra CHÉRIF ◽  
Robert DAY ◽  
...  
Keyword(s):  
Significant Degree ◽  
Human Testis ◽  
Mrna Levels ◽  
Gene Encoding ◽  
Peptide Substrates ◽  
Rat Testis ◽  
Processing Enzymes ◽  
Nucleotide Insertion ◽  
Testis Cdna

Rat testis NRD convertase (EC 3.4.24.61) is a Zn2+-dependent endopeptidase that cleaves, in vitro, peptide substrates at the N-terminus of Arg residues in dibasic sites. This putative processing enzyme of the insulinase family of metallopeptidases exhibits a significant degree of similarity to insulinase and two yeast processing enzymes, Axl1 and Ste23. We report the cloning of two human testis cDNA species encoding isoforms of NRD convertase, hNRD1 and hNRD2. Whereas the hNRD1 transcript (3.7 kb) is equivalent to the previously characterized rat cDNA (rNRD1), hNRD2 and rNRD2 are 3.9 kb novel forms containing a nucleotide insertion encoding a 68-residue segment. This motif, which is inserted N-terminal of the Zn2+-binding site, HXXEH, is contained within the most conserved region among the insulinase family members. Analysis of the deduced primary sequences revealed 92% identity between rat and human orthologues. The human gene encoding NRD convertase was localized to chromosome 1p32.1-p32.2. Whereas NRD convertase is mostly expressed in testis and in 24 cell lines, low mRNA levels were detected in most of the 27 other tissues tested.

scholarly journals Autophagy Upregulation by the TFEB Inducer Trehalose Protects against Oxidative Damage and Cell Death Associated with NRF2 Inhibition in Human RPE Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-18
Author(s):  
Samuel Abokyi ◽  
Sze wan Shan ◽  
Chi-ho To ◽  
Henry Ho-lung Chan ◽  
Dennis Yan-yin Tse
Keyword(s):  
Oxidative Stress ◽  
Oxidative Damage ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Mrna Levels ◽  
Neuroprotective Effects ◽  
Rpe Cells ◽  
Age Related ◽  
Protein Expressions

Trehalose is a natural dietary molecule that has shown antiaging and neuroprotective effects in several animal models of neurodegenerative diseases. The role of trehalose in the management of age-related macular degeneration (AMD) is yet to be investigated and whether trehalose could be a remedy for the treatment of diseases linked to oxidative stress and NRF2 dysregulation. Here, we showed that incubation of human retinal pigment epithelial (RPE) cells with trehalose enhanced the mRNA and protein expressions of TFEB, autophagy genes ATG5 and ATG7, as well as protein expressions of macroautophagy markers, LC3B and p62/SQTM1, and the chaperone-mediated autophagy (CMA) receptor LAMP2. Cathepsin D, a hydrolytic lysosomal enzyme, was also increased by trehalose, indicating higher proteolytic activity. Moreover, trehalose upregulated autophagy flux evident by an increase in the endogenous LC3B level, and accumulation of GFP-LC3B puncta and free GFP fragments in GFP-LC3 ̶ expressing cells in the presence of chloroquine. In addition, the mRNA levels of key molecular targets implicated in RPE damage and AMD, such as vascular endothelial growth factor- (VEGF-) A and heat shock protein 27 (HSP27), were downregulated, whereas NRF2 was upregulated by trehalose. Subsequently, we mimicked in vitro AMD conditions using hydroquinone (HQ) as the oxidative insult on RPE cells and evaluated the cytoprotective effect of trehalose compared to vehicle treatment. HQ depleted NRF2, increased oxidative stress, and reduced the viability of cells, while trehalose pretreatment protected against HQ-induced toxicity. The cytoprotection by trehalose was dependent on autophagy but not NRF2 activation, since autophagy inhibition by shRNA knockdown of ATG5 led to a loss of the protective effect. The results support the transcriptional upregulation of TFEB and autophagy by trehalose and its protection against HQ-induced oxidative damage in RPE cells. Further investigation is, therefore, warranted into the therapeutic value of trehalose in alleviating AMD and retinal diseases associated with impaired NRF2 antioxidant defense.

scholarly journals Age-related Beta-synuclein Alters the p53/Mdm2 Pathway and Induces the Apoptosis of Brain Microvascular Endothelial Cells In Vitro

2018 ◽  
Vol 27 (5) ◽  
pp. 796-813 ◽  
Author(s):  
Katrin Brockhaus ◽  
Michael R. R. Böhm ◽  
Harutyun Melkonyan ◽  
Solon Thanos
Keyword(s):  
Endothelial Cells ◽  
Neurodegenerative Diseases ◽  
Microvascular Endothelial Cells ◽  
Mrna Levels ◽  
Brain Microvascular Endothelial Cells ◽  
Age Related ◽  
Microvascular Endothelial ◽  
Dose Dependent

Increased β-synuclein (Sncb) expression has been described in the aging visual system. Sncb functions as the physiological antagonist of α-synuclein (Snca), which is involved in the development of neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases. However, the exact function of Sncb remains unknown. The aim of this study was to elucidate the age-dependent role of Sncb in brain microvascular endothelial cells (BMECs). BMECs were isolated from the cortices of 5- to 9-d-old Sprague-Dawley rats and were cultured with different concentrations of recombinant Sncb (rSncb) up to 72 h resembling to some degree age-related as well as pathophysiological conditions. Viability, apoptosis, expression levels of Snca, and the members of phospholipase D2 (Pld2)/ p53/ Mouse double minute 2 homolog (Mdm2)/p19(Arf) pathway, response in RAC-alpha serine/threonine-protein kinase (Akt), and stress-mediating factors such as heme oxygenase (decycling) 1 (Hmox) and Nicotinamide adenine dinucleotide phosphate oxygenase 4 (Nox4) were examined. rSncb-induced effects were confirmed through Sncb small interfering RNA (siRNA) knockdown in BMECs. We demonstrated that the viability decreases, while the rate of apoptosis underly dose-dependent alterations. For example, apoptosis increases in BMECs following the treatment with higher dosed rSncb. Furthermore, we observed a decrease in Snca immunostaining and messenger RNA (mRNA) levels following the exposure to higher rScnb concentrations. Akt was shown to be downregulated and pAkt upregulated by this treatment, which was accompanied by a dose-independent increase in p19(Arf) levels and enhanced intracellular Mdm2 translocation in contrast to a dose-dependent p53 activation. Moreover, Pld2 activity was shown to be induced in rSncb-treated BMECs. The expression of Hmox and Nox4 after Sncb treatment was altered on BEMCs. The obtained results demonstrate dose-dependent effects of Sncb on BMECs in vitro. For example, the p53-mediated and Akt-independent apoptosis together with the stress-mediated response of BMECs related to exposure of higher SNCB concentrations may reflect the increase in Sncb with duration of culture as well as its impact on cell decay. Further studies, expanding on the role of Sncb, may help understand its role in the neurodegenerative diseases.

Differential response of cardiac fibroblasts from young adult and senescent rats to ANG II

2003 ◽  
Vol 284 (4) ◽  
pp. H1454-H1459 ◽  
Author(s):  
K. Shivakumar ◽  
David E. Dostal ◽  
Kenneth Boheler ◽  
Kenneth M. Baker ◽  
Edward G. Lakatta
Keyword(s):  
Young Adult ◽  
Transforming Growth Factor ◽  
Cardiac Fibroblasts ◽  
Receptor Subtype ◽  
Mrna Levels ◽  
Ang Ii ◽  
Adult Rats ◽  
Collagen Production ◽  
Age Related

The intracardiac ANG II-forming pathway is activated in the senescent myocardium, raising the possibility of enhanced ANG II effects on cardiac fibroblasts. This study established an in vitro model of cultured cardiac fibroblasts from aged rats to examine if the response of these cells to ANG II is modified in the aged heart. Levels of mRNA encoding renin, angiotensinogen, and the AT1 receptor subtype in cardiac fibroblasts from young adult and senescent rats were quantified by RT-PCR, net collagen production by a hydroxyproline-based assay, and transforming growth factor (TGF)-β levels using a commercial kit. In cardiac fibroblasts from young adult rats, ANG II significantly enhanced AT1mRNA levels, net collagen production, and TGF-β production. In fibroblasts from the aged myocardium, ANG II downregulated AT1 mRNA expression, had a less pronounced effect on net collagen production, and had no effect on TGF-β production. Such age-related modification of the response of cardiac fibroblasts to ANG II may counteract the effects of augmented intracardiac ANG II production in the senescent heart, limiting fibrogenesis.

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