Human Testicular Peritubular Cells Research Articles

Dexamethasone is a regulator of clock genes in testicular peritubular cells.

We recently found that peritubular cells of the human testis are a dominant site of expression of the glucocorticoid receptor (GR; encoded by NR3C1). Activation of GR by dexamethasone (Dex) strongly influences the phenotype of cultured human testicular peritubular cells (HTPCs), causing massive changes of their proteome and secretome. As glucocorticoids (GC) are also known to set the internal clock of peripheral organs by regulating clock genes, we tested such an influence of Dex in HTPCs. We performed cellular studies with HTPCs and immortalized nonhuman primate (Callithrix jacchus; Cj)-derived peritubular cells, organotypic incubations of testicular fragments of Cj, qPCR and proteomic, as well as immunohistochemical studies. Basal clock gene expression levels, when monitored by qPCR under standard culture conditions, showed alterations over 24 h, suggesting an endogenous circadian rhythm, especially for BMAL1. Dex (1 µM) when added to cells, caused a strong and significant increase of PER1, followed by elevations of BMAL1, and other clock genes. This action was observed as early as 4 h after the addition of Dex. Immunohistochemistry and data mining revealed GR in testicular peritubular cells and other somatic cells of Cj, in situ. We therefore performed organotypic incubations of testicular fragments of Cj (n = 3) and found that upon addition of Dex (1 µM), mRNA levels of BMAL1 and PER1 also increased in samples of two out of three animals after 6 h. Mass spectrometry did, however, not reveal significant alterations of the testicular proteome, possibly due to the short time point and/or the fact that the somatic GR-expressing cells represent only a small portion of the testis. In support for this assumption, Dex (1 µM; 6 h) significantly increased mRNA levels of BMAL1 and PER1 in Cj-derived immortalized testicular peritubular cells. The results indicate that an internal clock system likely exists in peritubular cells of the testis and that Dex, via testicular GR expressed by peritubular cells and other somatic cells, is a strong regulator of this system. In a physiological situation, GC thus may be important regulators of the testicular clock, while in a situation of prolonged stress or GC-medication, derangements in clock gene expression may result.

Identification of Catecholamine and Drug Target α2A-Adrenoceptor in Human Testis and Human Testicular Peritubular Cells.

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.

Prolonged exposure to dexamethasone alters the proteome and cellular phenotype of human testicular peritubular cells.

Human testicular peritubular cells (HTPCs) are smooth muscle cells, which in the testis form a small compartment surrounding the seminiferous tubules. Contractions of HTPCs are responsible for sperm transport, HTPCs contribute to spermatogenesis, have immunological roles and are a site of glucocorticoid receptor expression. Importantly, HTPCs maintain their characteristics in vitro, and thus can serve as an experimental window into the male gonad. Previously we reported consequences of 3-day treatment with Dexamethasone (Dex), a synthetic glucocorticoid and multi-purpose anti-inflammatory drug. However, as glucocorticoid therapies in man often last longer, we now studied consequences of a prolonged 7-day exposure to 1 µM Dex. Combining live cell imaging with quantative proteomics of samples taken from men, we confirmed our recent findings but more importantly, found numerous novel proteomic alterations induced by prolonged Dex treatment. The comparison of the 7-day treatment with the 3-day treatment dataset revealed that extracellular matrix- and focal adhesion-related proteins become more prominent after 7 days of treatment. In contrast, extended stimulation is, for example, associated with a decrease of proteins related to cholesterol and steroid metabolism. Our dataset, which describes phenotypic and proteomic alterations, is a valuable resource for further research projects investigating effects of Dex on human testicular cells.

The Molecular Signature of Human Testicular Peritubular Cells Revealed by Single-Cell Analysis.

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.

Profound Effects of Dexamethasone on the Immunological State, Synthesis and Secretion Capacity of Human Testicular Peritubular Cells

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.

Testicular adenosine acts as a pro-inflammatory molecule: role of testicular peritubular cells.

Extracellular ATP has been described to be involved in inflammatory cytokine production by human testicular peritubular cells (HTPCs). The ectonucleotidases ENTPD1 and NT5E degrade ATP and have been reported in rodent testicular peritubular cells. We hypothesized that if a similar situation exists in human testis, ATP metabolites may contribute to cytokine production. Indeed, ENTPD1 and NT5E were found in situ and in vitro in HTPCs. Malachite green assays confirmed enzyme activities in HTPCs. Pharmacological inhibition of ENTPD1 (by POM-1) significantly reduced pro-inflammatory cytokines evoked by ATP treatment, suggesting that metabolites of ATP, including adenosine, are likely involved. We focused on adenosine and detected three of the four known adenosine receptors in HTPCs. One, A2B, was also found in situ in peritubular cells of human testicular sections. The A2B agonist BAY60-6583 significantly elevated levels of IL6 and CXCL8, a result also obtained with adenosine and its analogue NECA. Results of siRNA-mediated A2B down-regulation support a role of this receptor. In mouse peritubular cells, in contrast to HTPCs, all four of the known adenosine receptors were detected; when challenged with adenosine, cytokine expression levels significantly increased. Organotypic short-term testis cultures yielded comparable results and indicate an overall pro-inflammatory action of adenosine in the mouse testis. If transferable to the in vivo situation, our results may implicate that interference with the generation of ATP metabolites or interference with adenosine receptors could reduce inflammatory events in the testis. These novel insights may provide new avenues for treatment of sterile inflammation in male subfertility and infertility.

Palmitic Acid Targets Human Testicular Peritubular Cells and Causes a Pro-Inflammatory Response

Palmitic acid (PA) is a major fatty acid, derived from diet and endogenous production, which is being linked to inflammation. While such actions of PA at the level of the testis remain difficult to examine, we reasoned that studies in human testicular cells may be instructive. Human testicular peritubular cells (HTPCs) can be isolated from men and cultured. They have contractile properties but also produce Interleukin 6 (IL6), express the inflammasome member NLRP3, and via glia cell line derived neurotrophic factor (GDNF), they contribute to the spermatogonial stem cell niche. We found that PA at 100 µM significantly increased the levels of IL6, while NLRP3 or the related Interleukin 1 beta (IL1beta) were not affected. The contractility marker calponin (CNN1) and the growth factor GDNF were likewise not affected. ELISA studies confirmed the stimulatory PA actions on IL6. Hence, PA derived from diet and/or endogenous sources may be able to foster a pro-inflammatory milieu in the testis. A possible link of these results to diet and high fat intake and obesity is indicated by the about 12-fold elevated testicular levels of IL6 in testes of obese rhesus monkeys (n = 3), fed with a Western Style diet. They had elevated 2–5-fold increased body fat and increased circulating triglyceride levels. Further consequences of PA and obesity for testicular functions remain to be evaluated.

A translational cellular model for the study of peritubular cells of the testis.

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.

The Glucocorticoid Receptor NR3C1 in Testicular Peritubular Cells is Developmentally Regulated and Linked to the Smooth Muscle-Like Cellular Phenotype.

Whether glucocorticoids (GC) can directly affect human testicular functions is not well understood. A predominant site of GC receptor (GR; NR3C1) expression in the adult testis are peritubular smooth muscle-like cells, which express smooth muscle actin (ACTA2), contract and thereby are involved in sperm transport. In contrast to the adult, neither GR nor ACTA2, or elastin (ELN) were detected in the peritubular compartment before puberty in non-human primate testes. In isolated human testicular peritubular cells (HTPCs), activation of GR by dexamethasone (Dex) caused the translocation of GR to the nucleus and stimulated expression of ACTA2 and ELN, without affecting the expression of collagens. Cytoskeletal ACTA2-rearrangements were observed and were associated with an increased ability to contract. Our results indicate post-pubertal testicular roles of GC in the maintenance of the contractile, smooth muscle-like phenotype of peritubular cells.

Insights into replicative senescence of human testicular peritubular cells

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.

Peritubular cells of the human testis: prostaglandin E2 and more.

Several layers of slender, smooth muscle-like, peritubular cells and extracellular matrix (ECM) form the peritubular compartment of the human testis. Peritubular cells are the least explored testicular cells. Human testicular peritubular cells (HTPCs) can be isolated from small testicular fragments of patients and studied in vitro. We have used this cellular model, in combination with human testicular samples, to examine how peritubular cells may contribute to male (in)fertility. Human testicular peritubular cells(HTPCs) retain contractile abilities in vitro and secrete many proteins. Among them are factors, which serve intra-testicular roles, for example, glial cell line-derived neurotrophic factor (GDNF), thought to be important for the renewal of spermatogonial stem cells (SSCs). Studies in mutant mice indicated that peritubular cell-derived GDNF is crucial for lifelong spermatogenesis. Thus, peritubular cells are a functional part of the SSC niche. Peritubular cells of mice and men express androgen receptors (AR). In mouse peritubular cells, androgens enhanced GDNF production, but not in HTPCs. Rather, AR activation increased the levels of AR and smooth muscle proteins and thereby enhanced the smooth muscle-like phenotype. Following the lead of a proteomic analysis, which identified the key prostaglandin (PG)-synthesizing enzyme (PTGS1=COX1), we found that HTPCs secrete PGE2 . COX1, and PGE2 receptors (EP1, 2, and 4) were identified in peritubular cells in situ, supporting in vivo relevance. In HTPCs, activation of EP1/4 increased GDNF and a smooth muscle protein. Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID), which blocks PG synthesis. Added to HTPCs it reduced PGE2 and GDNF production and lowered smooth muscle protein levels. If applicable to the in vivo situation, the results suggest that ibuprofen and possibly other NSAIDs may impair important peritubular cell functions and consequently testicular functions. The few examples highlighted, together with others not mentioned here, indicate that HTPCs provide an experimental window into the human testis.

Human testicular peritubular cells, mast cells and testicular inflammation.

In man, the wall of seminiferous tubules forms a testicular compartment, which contains several layers of smooth muscle-like, "myoid", peritubular cells and extracellular matrix. Its architecture and its cellular composition change in male infertility associated with impaired spermatogenesis. Increased deposits of extracellular matrix, changes in the smooth muscle-like phenotype of peritubular cells and accumulation of immune cells, especially mast cells, are among the striking alterations. Taken together, the changes indicate that inflammatory events take place in particular within this compartment. This short review summarises recent studies, which pinpoint possible mechanisms of the interplay between peritubular cells and mast cells, which may contribute to sterile inflammation and impairments of testicular function. These insights are based mainly on cellular studies, for which we used isolated human testicular peritubular cells (HTPCs), and on the examination of human testicular sections. Recent data on immunological properties of peritubular cells, unexpected roles of the extracellular matrix factor, biglycan, which is secreted by peritubular cells and functions of mast cell products (chymase, tryptase and ATP) are presented. We believe that the results may foster a better understanding of peritubular cells, their roles in the human testis and specifically their involvement in infertility.

Ca2+ Signaling and IL-8 Secretion in Human Testicular Peritubular Cells Involve the Cation Channel TRPV2.

Peritubular cells are part of the wall of seminiferous tubules in the human testis and their contractile abilities are important for sperm transport. In addition, they have immunological roles. A proteomic analysis of isolated human testicular peritubular cells (HTPCs) revealed expression of the transient receptor potential channel subfamily V member 2 (TRPV2). This cation channel is linked to mechano-sensation and to immunological processes and inflammation in other organs. We verified expression of TRPV2 in peritubular cells in human sections by immunohistochemistry. It was also found in other testicular cells, including Sertoli cells and interstitial cells. In cultured HTPCs, application of cannabidiol (CBD), a known TRPV2 agonist, acutely induced a transient increase in intracellular Ca2+ levels. These Ca2+ transients could be blocked both by ruthenium red, an unspecific Ca2+ channel blocker, and tranilast (TRA), an antagonist of TRPV2, and were also abolished when extracellular Ca2+ was removed. Taken together this indicates functional TRPV2 channels in peritubular cells. When applied for 24 to 48 h, CBD induced expression of proinflammatory factors. In particular, mRNA and secreted protein levels of the proinflammatory chemokine interleukin-8 (IL-8/CXCL8) were elevated. Via its known roles as a major mediator of the inflammatory response and as an angiogenic factor, this chemokine may play a role in testicular physiology and pathology.

NLRP3 in somatic non-immune cells of rodent and primate testes.

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. NLRP3 and associated genes (PYCARD, CASP1, IL1B) 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 (AROM+) develop testicular inflammation and impaired spermatogenesis during aging, and the present data show that this is associated with strikingly elevated Nlrp3 expression in the testes compared to WT controls. Interference by aromatase inhibitor treatment significantly reduced increased Nlrp3 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.

Insights into the role of androgen receptor in human testicular peritubular cells

Contractile smooth muscle-like peritubular cells build the wall of seminiferous tubules in men. They are crucial for sperm transport and complement the functions of Sertoli cells by secreting factors, including glial cell line-derived neurotrophic factor. Previous studies revealed that they also secrete the chemokine C-X-C motif chemokine ligand 12 (CXCL12), which has known roles in spermatogenesis. Peritubular cells express the androgen receptor (AR), which is retained in isolated human testicular peritubular cells. We aimed to explore AR-regulated functions in human testicular peritubular cells. Bearing in mind that infertile men often have high aromatase activity, which may lower intratesticular androgen concentrations, an animal model for male infertility was studied. These mice display an age-dependent loss in spermatogenesis due to high aromatase activity. Human testicular peritubular cells were exposed to dihydrotestosterone or the antiandrogen flutamide. We studied AR, smooth muscle cell markers, glial cell line-derived neurotrophic factor and 15 secreted factors previously identified, including CXCL12. We used qPCR, Western blotting, ELISA or selected reaction monitoring (SRM). In the animal model for male infertility, we employed qPCR and immunohistochemistry. Dihydrotestosterone increased AR and flutamide prevented these actions. The smooth muscle cell markers calponin and smooth muscle actin were likewise increased, while cell size or cellular proliferation was not changed. Dihydrotestosterone did not increase glial cell line-derived neurotrophic factor or CXCL12 secretion but increased levels of serine proteinase inhibitor (SERPIN) E1. The animal model for male infertility with high aromatase activity showed reduced numbers of AR-immunoreactive testicular peritubular cells, suggesting that altered androgen and/or oestrogen levels could influence AR-mediated responses in peritubular cells. Androgens act on human testicular peritubular cells to enhance AR levels, their contractile phenotype and to modulate the secretion of some secreted factors. This study suggests that some aspects of human peritubular cell functions are regulated by androgens.

Characterization of a non-human primate model for the study of testicular peritubular cells-comparison with human testicular peritubular cells.

Are monkey testicular peritubular cells (MKTPCs) from the common marmoset monkey (Callithrix jacchus) a suitable translational model for the study of human testicular peritubular cells (HTPCs)? MKTPCs can be isolated and propagated in vitro, retain characteristic markers for testicular peritubular cells and their proteome strongly (correlation coefficient of 0.78) overlaps with the proteome of HTPCs. Smooth-muscle-like peritubular cells form the wall of seminiferous tubules, transport sperm, are immunologically active, secrete a plethora of factors and may contribute to the spermatogonial stem cell niche. Mechanistic studies are hampered by heterogeneity of human samples. We established a culture method for MKTPCs and characterized these cells from six young adult animals (2-3 years). To examine whether they qualify as a translational model we also examined HTPCs from seven men and compared the proteomes of both groups. We used explant cultures to obtain MKTPCs, which express smooth muscle markers (calponin (CNN1), smooth muscle actin (ACTA2)), lack FSH-receptors (FSHR) and LH-receptors (LHCGR), but possess androgen receptors (AR). MKTPCs can be passaged at least up to eight times, without discernable phenotypic changes. Mass-spectrometry-based analyses of the MKTPC and HTPC proteomes were performed. We established a method for isolation and cultivation of MKTPCs, and provide a comprehensive analysis of their protein repertoire. The results let us conclude that MKTPCs are suitable as a non-human primate model to study peritubular cell functions. List of identified proteins in MKTPCs by liquid chromatography-tandem mass spectrometry is accessible at the ProteomeXchange (identifier PXD009394). This is an in vitro cellular non-human primate model used to provide a window into the role of these cells in the human testis. Previous studies with HTPCs from patients revealed a degree of heterogeneity, possibly due to age, lifestyle and medical history of the individual human donors. We anticipate that the new translational model, derived from young healthy non-human primates, may allow us to circumvent these issues and may lead to a better understanding of the role of peritubular cells. This work was supported by grants from the Deutsche Forschungsgemeinschaft (MA 1080/27-1; AR 362/9-1; BE 2296/8-1). The authors declare no competing financial interests.

Prostaglandin E2 (PGE2) is a testicular peritubular cell-derived factor involved in human testicular homeostasis

In man, blockage of prostaglandin (PG)-production e.g. by non-steroidal anti-inflammatory drug (NSAIDs) may have negative testicular side effects, implying beneficial actions of PGs in the testis. We examined human testicular samples and isolated human testicular peritubular cells (HTPCs) to explore sites of PG-synthesis and targets. HTPCs express cyclooxygenase 1 (COX1) and secrete PGE2. Receptors (EP1, 2, 4) were specifically identified in peritubular cells. In HTPCs PGE2 significantly increased mRNA levels of the contractility protein calponin, but did not induce contractions. PGE2, as well as EP1 and EP4 receptor agonists, significantly increased glia cell line derived neurotrophic factor (GDNF) mRNA and/or protein levels. Importantly, the NSAID ibuprofen reduced PGE2 and this action also lowered SMA and calponin mRNA levels and levels of secreted GDNF protein. The results reveal an unknown PGE2 system in the human testis, in involving peritubular cells, which may be prone to interference by NSAIDs.

Alpha 1 adrenergic receptor-mediated inflammatory responses in human testicular peritubular cells.

Stress activates the sympathetic nervous system and is linked to impaired fertility in man. We hypothesized that catecholamines by acting on testicular cells have a role in these events, possibly by fostering an inflammatory environment. The cells of the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), express adrenergic receptors (ADRs) α1B, α1D, β1 and β2. A selective α1-ADR agonist, phenylephrine, increased intracellular Ca2+-levels in cultured HTPCs and induced COX-2, IL-6 and MCP-1 mRNA expression without affecting IL-1β mRNA. These changes were paralleled by a significant increase in the secretion of IL-6 and MCP-1. Epinephrine was also effective, but salbutamol, a selective β2-ADR agonist was not. Our results suggest that stress-associated elevation of catecholamines may be able to promote inflammatory events by targeting peritubular cells in the human testis. Blockage of α1-ADRs may therefore be a novel way to interfere with stress-related impairment of male reproductive functions.

Sterile inflammation as a factor in human male infertility: Involvement of Toll like receptor 2, biglycan and peritubular cells.

Changes in the wall of seminiferous tubules in men with impaired spermatogenesis imply sterile inflammation of the testis. We tested the hypothesis that the cells forming the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), orchestrate inflammatory events and that Toll like receptors (TLRs) and danger signals from the extracellular matrix (ECM) of this wall are involved. In cultured HTPCs we detected TLRs, including TLR2. A TLR-2 ligand (PAM) augmented interleukin 6 (IL-6), monocyte chemo-attractant protein-1 (MCP-1) and pentraxin 3 (PTX3) in HTPCs. The ECM-derived proteoglycan biglycan (BGN) is secreted by HTPCs and may be a TLR2-ligand at HTPCs. In support, recombinant human BGN increased PTX3, MCP-1 and IL-6 in HTPCs. Variable endogenous BGN levels in HTPCs derived from different men and differences in BGN levels in the tubular wall in infertile men were observed. In testes of a systemic mouse model for male infertility, testicular sterile inflammation and elevated estradiol (E2) levels, BGN was also elevated. Hence we studied the role of E2 in HTPCs and observed that E2 elevated the levels of BGN. The anti-estrogen ICI 182,780 blocked this action. We conclude that TLR2 and BGN contribute to sterile inflammation and infertility in man.

Human testicular peritubular cells secrete pigment epithelium-derived factor (PEDF), which may be responsible for the avascularity of the seminiferous tubules.

Male fertility depends on spermatogenesis, which takes place in the seminiferous tubules of the testis. This compartment is devoid of blood vessels, which are however found in the wall of the seminiferous tubules. Our proteomic study using cultured human testicular peritubular cells (HTPCs) i.e. the cells, which form this wall, revealed that they constitutively secrete pigment epithelium-derived factor, PEDF, which is known to exert anti-angiogenic actions. Immunohistochemistry supports its presence in vivo, in the human tubular wall. Co-culture studies and analysis of cell migration patterns showed that human endothelial cells (HUVECs) are repulsed by HTPCs. The factor involved is likely PEDF, as a PEDF-antiserum blocked the repulsing action. Thus testicular peritubular cells, via PEDF, may prevent vascularization of human seminiferous tubules. Dihydrotestosterone (DHT) increased PEDF (qPCR) in HTPCs, however PEDF expression in the testis of a non-human primate occurs before puberty. Thus PEDF could be involved in the establishment of the avascular nature of seminiferous tubules and after puberty androgens may further reinforce this feature. Testicular microvessels and blood flow are known to contribute to the spermatogonial stem cell niche. Hence HTPCs via control of testicular microvessels may contribute to the regulation of spermatogonial stem cells, as well.