Proteome profile of the cerebellum from α7 nicotinic acetylcholine receptor deficient mice.

The alpha7 nicotinic acetylcholine receptor (α7 nAChR; CHRNA7) is expressed in the nervous system and in non-neuronal tissues. Within the central nervous system, it is involved in various cognitive and sensory processes such as learning, attention, and memory. It is also expressed in the cerebellum, where its roles are; however, not as well understood as in the other brain regions. To investigate the consequences of absence of CHRNA7 on the cerebellum proteome, we performed a quantitative nano-LC-MS/MS analysis of samples from CHRNA7 knockout (KO) mice and corresponding wild type (WT) controls. Liver, an organ which does not express this receptor, was analyzed, in comparison. While the liver proteome remained relatively unaltered (three proteins more abundant in KOs), 90 more and 20 less abundant proteins were detected in the cerebellum proteome of the KO mice. The gene ontology analysis of the differentially abundant proteins indicates that the absence of CHRNA7 leads to alterations in the glutamatergic system and myelin sheath in the cerebellum. In conclusion, our dataset provides new insights in the role of CHRNA7 in the cerebellum, which may serve as a basis for future in depth-investigations.

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.

Reduced oxygen concentrations regulate the phenotype and function of human granulosa cells in vitro and cause a diminished steroidogenic but increased inflammatory cellular reaction.

Oxygen (O2) concentrations have recently been discussed as important regulators of ovarian cells. Human IVF-derived granulosa cells (human GCs) can be maintained in vitro and are a widely used cellular model for the human ovary. Typically, GCs are cultured at atmospheric O2 levels (approximately around 20%), yet the O2 conditions in vivo, especially in the preovulatory follicle, are estimated to be much lower. Therefore, we comprehensively evaluated the consequences of atmospheric versus hypoxic (1% O2) conditions for 4 days on human GCs. We found lower cellular RNA and protein levels but unchanged cell numbers at 1% O2, indicating reduced transcriptional and/or translational activity. A proteomic analysis showed that 391 proteins were indeed decreased, yet 133 proteins were increased under hypoxic conditions. According to gene ontology (GO) enrichment analysis, pathways associated with metabolic processes, for example amino acid-catabolic-processes, mitochondrial protein biosynthesis, and steroid biosynthesis, were downregulated. Pathways associated with glycolysis, chemical homeostasis, cellular response to hypoxia, and actin filament bundle assembly were upregulated. In accordance with lower CYP11A1 (a cholesterol side-chain cleavage enzyme) levels, progesterone release was decreased. A proteome profiler, as well as IL-6 and IL-8 ELISA assays, revealed that hypoxia led to increased secretion of pro-inflammatory and angiogenic factors. Immunofluorescence studies showed nuclear localization of hypoxia-inducible factor 1α (HIF1α) in human GCs upon acute (2 h) exposure to 1% O2 but not in cells exposed to 1% O2 for 4 days. Hence, the role of HIF1α may be restricted to initiation of the hypoxic response in human GCs. The results provide a detailed picture of hypoxia-induced phenotypic changes in human GCs and reveal that chronically low O2 conditions inhibit the steroidogenic but promote the inflammatory phenotype of these cells.

TRPV2, a novel player in the human ovary and human granulosa cells.

The cation channel 'transient receptor potential vanilloid 2' (TRPV2) is activated by a broad spectrum of stimuli, including mechanical stretch, endogenous and exogenous chemical compounds, hormones, growth factors, reactive oxygen species, and cannabinoids. TRPV2 is known to be involved in inflammatory and immunological processes, which are also of relevance in the ovary. Yet, neither the presence nor possible roles of TRPV2 in the ovary have been investigated. Data mining indicated expression, for example, in granulosa cells (GCs) of the human ovary in situ, which was retained in cultured GCs derived from patients undergoing medical reproductive procedures. We performed immunohistochemistry of human and rhesus monkey ovarian sections and then cellular studies in cultured GCs, employing the preferential TRPV2 agonist cannabidiol (CBD). Immunohistochemistry showed TRPV2 staining in GCs of large antral follicles and corpus luteum but also in theca, endothelial, and stromal cells. TRPV2 transcript and protein levels increased upon administration of hCG or forskolin. Acutely, application of the agonist CBD elicited transient Ca2+ fluxes, which was followed by the production and secretion of several inflammatory factors, especially COX2, IL6, IL8, and PTX3, in a time- and dose-dependent manner. CBD interfered with progesterone synthesis and altered both the proteome and secretome, as revealed by a proteomic study. While studies are somewhat hampered by the lack of highly specific TRPV2 agonist or antagonists, the results pinpoint TRPV2 as a modulator of inflammation with possible roles in human ovarian (patho-)physiology. Finally, as TRPV2 is activated by cannabinoids, their possible ovarian actions should be further evaluated.

An ovarian phenotype of alpha 7 nicotinic receptor knockout mice.

In brief: Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is expressed by various murine ovarian cells. Morphological and molecular investigations, including a proteomic study of adult Chrna7 knockout (KO) mouse ovaries, reveal the roles of these receptors in the local regulation of the ovary. Abstract: Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is involved in cellular functions ranging from synaptic transmission in neurons to regulation of inflammation, cell growth and metabolism to cell death in other cells. Our qPCR results and other studies indicated that nAChRa7 is expressed in the adult mouse ovary, while in situ hybridization and single-cell sequencing data suggested this expression may be shared by several ovarian cells, including fibroblast-like and steroidogenic stroma cells, macrophages and oocytes of small follicles. To explore a possible involvement of nAChRa7 in ovarian functions, we evaluated ovarian morphology of Chrna7-null mutant adult mice (KO) and wildtype mice (WT; 3 months, metestrus) by performing immunohistochemistry, qPCR studies, measurements of serum progesterone and proteomic analyses. The evaluation of serial sections indicated fewer primordial follicles but similar numbers of primary, secondary and tertiary follicles, as well as corpora lutea in KO and WT mice. Atresia was unchanged. Serum progesterone and mRNA levels of proliferation and most apoptosis markers were not changed, yet two typical macrophage markers were elevated. Furthermore, the proteomes of KO ovaries were significantly altered with 96 proteins increased and 32 decreased in abundance in KOs compared to WTs. Among the elevated proteins were markers for stroma cells. Hence, the lack of nAChRa7 causes changes in small follicle counts and alterations of the ovarian stroma cells. The ovarian phenotype of Chrna7 mutant mice links this channel protein to the local regulation of ovarian cells, including stroma cells.

Evidence of a role for cAMP in mitochondrial regulation in ovarian granulosa cells.

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.

The role of resveratrol, Sirtuin1 and RXRα as prognostic markers in ovarian cancer.

OBJECTIVE: Ovarian cancer is the most lethal gynecologic cancer. Resveratrol (RSV) is known to alter metabolism in cancer. It affects the nuclear retinoid-X-receptor (RXR), which implies a modulating effect of RXR to gynaecologic cancers. Furthermore, RSV targets Sirtuin1 (Sirt1), a histone deacetylase. STUDY DESIGN: 123 tissue samples of patients with serous or mucinous ovarian cancer were examined for expression of Sirt1 and RXR. Ovarian cell lines were treated with RSV and consequences on viability and apoptosis were evaluated. The influence of RSV to Sirt1 and RXR expression was analyzed by western blotting RESULTS: A correlation of nuclear Sirt1 and RXRα expression could be detected (p = 0.006). Co-expression of nuclear RXRα and cytoplasmic (p = 0.026) or nuclear (p = 0.041) Sirt1 was associated with significantly increased overall survival in advanced tumour stages. Viability was decreased in all cell lines after stimulation with resveratrol, while cell apoptosis was increased. RSV treatment led to significant lower Sirt1 expression in A2780 cells (p = 0.025) and significant increased RXR expression in cisA2780 cells (p = 0.012) CONCLUSION: In order to use RSV as medical target, studies could be developed to improve the understanding of drug resistance mechanisms and consequently improve treatment outcome.

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.

A rapid and robust method for the cryopreservation of human granulosa cells.

Human primary granulosa cells (GCs) derived from women undergoing oocyte retrieval can be cultured and used as a cellular model for the study of human ovarian function. In vitro, they change rapidly, initially resembling cells of the preovulatory follicle and then cells of the corpus luteum. They are derived from individual patients, whose different medical history, lifestyle and age lead to heterogeneity. Thus, cells can rarely be ideally matched for cellular experiments or, if available, only in small quantities. We reasoned that cryopreservation of human GCs may be helpful to improve this situation. Previous studies indicated the feasibility of such an approach, but low survival of human GCs was reported, and effects on human GC functionality were only partially evaluated. We tested a slow freezing protocol (employing FCS and DMSO) for human GCs upon isolation from follicular fluid. We compared cryopreserved and subsequently thawed cells with fresh, non-cryopreserved cells from the same patients. About 80% of human GCs survived freezing/thawing. No differences were found in cell morphology, survival rate in culture, or transcript levels of mitochondrial (COX4, OPA1, TOMM20), steroidogenic (CYP11A1, CYP19A1) or cell-cell contact genes (GJA1) between the two groups in cells cultured for 1-5 days. A proteomic analysis revealed no statistically significant change in the abundance of a total of 5962 proteins. The two groups produced comparable basal levels of progesterone and responded similarly to hCG with elevation of progesterone. Taken together, our results show this to be a rapid and readily available method for the cryopreservation of human GCs. We anticipate that it will allow future large-scale experiments and may thereby improve cellular studies with human ovarian cells.

Signal peptide peptidase-like 2c impairs vesicular transport and cleaves SNARE proteins.

Members of the GxGD-type intramembrane aspartyl proteases have emerged as key players not only in fundamental cellular processes such as B-cell development or protein glycosylation, but also in development of pathologies, such as Alzheimer's disease or hepatitis virus infections. However, one member of this protease family, signal peptide peptidase-like 2c (SPPL2c), remains orphan and its capability of proteolysis as well as its physiological function is still enigmatic. Here, we demonstrate that SPPL2c is catalytically active and identify a variety of SPPL2c candidate substrates using proteomics. The majority of the SPPL2c candidate substrates cluster to the biological process of vesicular trafficking. Analysis of selected SNARE proteins reveals proteolytic processing by SPPL2c that impairs vesicular transport and causes retention of cargo proteins in the endoplasmic reticulum. As a consequence, the integrity of subcellular compartments, in particular the Golgi, is disturbed. Together with a strikingly high physiological SPPL2c expression in testis, our data suggest involvement of SPPL2c in acrosome formation during spermatogenesis.

The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban.

Signal peptide peptidase (SPP) and the four homologous SPP-like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II-oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non-expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail-anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c-/- mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2)-regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c-/- mice.

Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors.

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.

Exploring the Ion Channel TRPV2 and Testicular Macrophages in Mouse Testis.

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.

Sirtuin1 expression and survival in endometrial and clear-cell uterine cancer.

Several risk factors like obesity and hyperlipidemia were described for endometrial cancer. Here, the nuclear NAD-dependent histone-deacetylase Sirtuin1 (SIRT1) seems to be important. SIRT1 is also involved in cell regulatory mechanisms and can serve as tumor promotor or suppressor. Its role in tumor biology is not clear yet. In this study, we evaluated and correlated the SIRT1 expression with patients' tumor characteristics in endometrioid and clear-cell cancer of the uterus. 65 paraffin-embedded samples of patients with endometrial and clear-cell cancer of the uterus were immunohistochemically stained and SIRT1 expression was evaluated by immunoreactive score. The results were correlated to clinical and pathological tumor characteristics as well as to the expression of ARID1A and ß-Catenin. The staining was significantly more intensive in uterine endometrioid carcinoma compared to uterine clear-cell carcinoma (p = 0.007). The expression of SIRT1 correlated significantly with the membranous expression of ß-Catenin (p = 0.028) and ARID1A (p = 0.021). Patients with positive Sirtuin1 expression had a significantly better progression-free survival (p = 0.042), the overall survival showed a trend towards a better prognosis (p = 0.070). SIRT1 expression seems to be associated with improved progression-free survival in uterine cancer (endometrioid and clear-cell) and is correlated to the tumor suppressors ß-Catenin and ARID1A. Further studies are necessary to elucidate the role of SIRT1 in uterine and ovarian cancer and its potential as a therapeutic target.

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.

Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.

The pituitary gonadotrophins and testosterone are the main hormonal regulators of spermatogenesis, but estradiol is also known to play a role in the process. The hormonal responses in the testis are partially mediated by somatic Sertoli cells that provide nutritional and physical support for differentiating male germ cells. Hydroxysteroid (17ß) dehydrogenase 1 (HSD17B1) is a steroidogenic enzyme that especially catalyzes the conversion of low potent 17keto-steroids to highly potent 17ß-hydroxysteroids. In this study, we show that Hsd17b1 is highly expressed in Sertoli cells of fetal and newborn mice, and HSD17B1 knockout males present with disrupted spermatogenesis with major defects, particularly in the head shape of elongating spermatids. The cell-cell junctions between Sertoli cells and germ cells were disrupted in the HSD17B1 knockout mice. This resulted in complications in the orientation of elongating spermatids in the seminiferous epithelium, reduced sperm production, and morphologically abnormal spermatozoa. We also showed that the Sertoli cell-expressed HSD17B1 participates in testicular steroid synthesis, evidenced by a compensatory up-regulation of HSD17B3 in Leydig cells. These results revealed a novel role for HSD17B1 in the control of spermatogenesis and male fertility, and that Sertoli cells significantly contribute to steroid synthesis in the testis.-Hakkarainen, J., Zhang, F.-P., Jokela, H., Mayerhofer, A., Behr, R., Cisneros-Montalvo, S., Nurmio, M., Toppari, J., Ohlsson, C., Kotaja, N., Sipilä, P., Poutanen, M. Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.

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.

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.

The Rapamycin-Sensitive Complex of Mammalian Target of Rapamycin Is Essential to Maintain Male Fertility.

The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin and its analogs are being increasingly used in solid-organ transplantation. A commonly reported side effect is male subfertility to infertility, yet the precise mechanisms of mTOR interference with male fertility remain obscure. With the use of a conditional mouse genetic approach we demonstrate that deficiency of mTORC1 in the epithelial derivatives of the Wolffian duct is sufficient to cause male infertility. Analysis of spermatozoa from Raptor fl/fl*KspCre mice revealed an overall decreased motility pattern. Both epididymis and seminal vesicles displayed extensive organ regression with increasing age. Histologic and ultrastructural analyses demonstrated increased amounts of destroyed and absorbed spermatozoa in different segments of the epididymis. Mechanistically, genetic and pharmacologic mTORC1 inhibition was associated with an impaired cellular metabolism and a disturbed protein secretion of epididymal epithelial cells. Collectively, our data highlight the role of mTORC1 to preserve the function of the epididymis, ductus deferens, and the seminal vesicles. We thus reveal unexpected new insights into the frequently observed mTORC1 inhibitor side effect of male infertility in transplant recipients.