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.

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.

Secretome analysis of testicular peritubular cells: a window into the human testicular microenvironment and the spermatogonial stem cell niche in man.

Spermatogonial stem cells (SSCs) are vital for lifelong spermatogenesis in man. In their niches, a special growth factor milieu and structural support by surrounding cells are thought to ensure their maintenance. In man, the cells of the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), are considered to contribute to this microenvironment and the overall testicular microenvironment via secreted proteins. Therefore, the secretome of cultured HTPCs from five individual men was analyzed by LC-MS/MS. Quantification and comparison to the proteome of HTPC lysates revealed 263 out of 660 identified secretome proteins to be at least 5-fold enriched in the culture media. To obtain additional evidence for secretion, signal peptide and gene ontology (GO) enrichment analyses were applied. The latter revealed--besides extracellular matrix (ECM) components--a significant over-representation of chemokines and growth factors acting in signaling pathways that appear critical for SSC maintenance. Immunohistochemistry, performed with human testicular sections, depicted expression of selected proteins in vivo. The significant enrichment of proteins related to cell adhesion and migration may indicate their involvement in SSC regulation. Our data strongly support the hypothesis of a crucial role of HTPCs in the composition of SSC niches in man.

Tunica albuginea underlap--a new modification of the Nesbit procedure: description of the technique and preliminary results.

INTRODUCTION: We describe a new surgical technique for the treatment of penile curvature that combines features of the Nesbit procedure with features of tunical plication. U-shaped flaps of tunica albuginea are freed from the corpus cavernosum. The flaps are brought under the remaining tunica albuginea and are fixated with single absorbable sutures. As the defects of the tunica are sealed tightly and with high tensile strength by double layers of tunica albuginea, correction of the abnormal curvature is achieved. AIM: To present our experience with a new surgical technique for the treatment of penile curvature. METHODS: Between 2008 and 2011, 50 patients underwent the underlap technique because of Peyronie's disease (37) or congenital penile deviation (13) in a single center. MAIN OUTCOME MEASURES: Preoperative and postoperative evaluation included the Erection Hardness Score (EHS) and the Symptom Score for Induratio penis plastica (IPP-SSC), a symptom score for penile deviation that was based on a consensus of regional andrologists. Clinical data concerning the early postoperative outcome were analyzed retrospectively using standardized items. RESULTS: Mean age ± standard deviation was 59.7 ± 8.4 years for patients with Peyronie's disease and 34.1 ± 7.8 years for patients with congenital penile deviation. The mean follow-up period was 27 months. The major complication rate was 4%, overall satisfaction 86%. Intraoperative correction of the curvature was achieved in 100%, significant relapse occurred in 6%. The mean difference of preoperative and postoperative IPP-SSC was 8.1 (95% confidence interval [CI] 7.24 to 8.96). The mean difference of preoperative and postoperative EHC was -0.03 (95% CI -0.16 to 0.09). CONCLUSIONS: Preliminary results obtained with the underlap technique showed promising outcome with minimal morbidity. The new technique might have three main advantages: more flexible intraoperative correctability of the curvature, tighter sealing of the tunical defects, and greater tensile strength of the plications.

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 ß-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.

Exploring human testicular peritubular cells: identification of secretory products and regulation by tumor necrosis factor-alpha.

Testicular peritubular cells are myofibroblastic cells, which represent the major cellular components of the wall of the seminiferous tubules. In men their phenotypic characteristics, including possible secretory activity and regulation, are not well known, in neither normal nor pathologically altered testes. Especially in testes of men with impaired spermatogenesis, the cytoarchitecture of the tubular wall is frequently remodeled and presents fibrotic thickening, increased innervation, and infiltration by macrophages and mast cells. The latter are two sources of TNF-alpha. The purpose of our study was to explore human testicular peritubular cells and mechanisms of their regulation. To this end we primarily studied cultured human testicular peritubular cells (HTPCs), isolated from adult human testes. Having established that HTPCs express TNF-alpha receptors 1 and 2 and respond to recombinant human TNF-alpha by a rapid phosphorylation of ERK1/2, we used complementary approaches, including gene array/RT-PCR studies, Western blotting/immunocytochemistry, and ELISA techniques to study phenotypic characteristics of HTPCs and actions of TNFalpha. We found that HTPCs express the nerve growth factor gene and TNF-alpha-stimulated mRNA levels and secretion of nerve growth factor in a dose- and time-dependent manner. Similarly, monocyte chemoattractant protein-1 was identified as a product of HTPCs, which was regulated by TNF-alpha in a concentration- and time-dependent manner. TNF-alpha furthermore strongly enhanced expression and/or synthesis of other inflammatory molecules, namely IL-6 and cyclooxygenase-2. Active cyclooxygenase-2 is indicated by increased prostaglandin D2 levels. In addition, intercellular adhesion molecule-1, which was not detected at protein level in the absence of TNF-alpha, was induced upon TNF-alpha stimulation. In conclusion, these results provide novel insights into the nature of human peritubular cells, which are able to secrete potent signaling molecules and are regulated by TNF-alpha. These results also hint to an as-yet-unknown role of peritubular cells in normal human testis and involvement in the pathomechanisms associated with impaired spermatogenesis in men.

ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation.

Peritubular myoid cells, which form the walls of seminiferous tubules in the testis, are functionally unexplored. While they transport sperm and contribute to the spermatogonial stem cell niche, specifically their emerging role in the immune surveillance of the testis and in male infertility remains to be studied. Recently, cytokine production and activation of Toll-like receptors (TLRs) were uncovered in cultured peritubular cells. We now show that human peritubular cells express purinergic receptors P2RX4 and P2RX7, which are functionally linked to TLRs, with P2RX4 being the prevalent ATP-gated ion channel. Subsequent ATP treatment of cultured peritubular cells resulted in up-regulated (pro-)inflammatory cytokine expression and secretion, while characteristic peritubular proteins, that is smooth muscle cell markers and extracellular matrix molecules, decreased. These findings indicate that extracellular ATP may act as danger molecule on peritubular cells, able to promote inflammatory responses in the testicular environment.

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, ß1 and ß2. 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-1ß 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 ß2-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.

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.

Isolation and cultivation of human testicular peritubular cells: a new model for the investigation of fibrotic processes in the human testis and male infertility.

CONTEXT: Fibrotic remodeling, especially of the tubule wall, in testes of infertile men is common, but reasons or consequences of these striking changes are not known. Based on cell culture and ex vivo studies, we previously suggested that mast cells via their products tryptase and histamine are involved in the development of fibrosis. However, studies in a relevant human testicular model are required to further test this hypothesis and the mechanisms of testicular fibrosis in general. OBJECTIVE: The objective of the study was the isolation, culture, and characterization of adult human testicular peritubular cells. PATIENTS AND INTERVENTIONS: Peritubular cells were obtained from biopsies of men suffering from obstructive azoospermia (n = 8) and varicocele (n = 2) but displaying normal spermatogenesis. RESULTS: Explant cultures were obtained from all biopsies. Immunostaining of the cultured cells and corresponding paraffin-embedded tissues with antibodies against markers of fibroblasts (CD90/Thy-1) and smooth muscle cells (alpha-smooth muscle actin) clearly proved their origin from the peritubular region. These cells displayed morphological features of myofibroblasts, and gene array analyses as well as immunohistochemistry revealed the predominant expression of extracellular matrix genes and genes coding for basement membrane components. The cultured cells retain receptors for the major mast cell products histamine and tryptase. The addition of histamine (100 microm) and the tryptase agonist peptide SLIGKV (10 microm) resulted in a transient increase in intracellular calcium levels, confirming the functionality of the receptors. CONCLUSIONS: We conclude that human peritubular cells are a novel model for the investigation of paracrine, including mast cell initiated, interactions in the human testis, which will allow the study of fibrotic processes underlying male idiopathic infertility.

Current status of vasectomy reversal.

Vasectomy reversal is the most common microsurgical intervention for the treatment of male infertility. Originally introduced in 1977, microsurgical vasectomy reversal has become highly sophisticated and is a minimally invasive, highly efficient and cost-effective treatment option for men with a desire to have children after vasectomy. It can be an effective physiological method of restoring fertility in more than 90% of vasectomized men. Although assisted reproductive technology (ART) is an alternative to vasectomy reversal, it is normally associated with higher costs without offering higher cumulative chances of a pregnancy. Recovery of physiological male fertility can take up to 2 years after vasectomy reversal, especially if reversal is performed >10 years after vasectomy, owing to impaired epididymal function. Under these circumstances, ART can be used to bridge the time until recovery of natural fertility. Although the basic principles of microsurgical vasovasostomy have been established since the late 1970s, there have since been numerous technical innovations to improve the delicate operation and promising new technical modifications, particularly for vasoepididymostomy, have been described. Robotic vasectomy reversal is an emerging field in specialized urologic centers, but whether the high quality of conventional microsurgical vasectomy reversal can be matched by robotic platforms is yet to be seen.

No relationship between biopsy sites near the main testicular vessels or rete testis and successful sperm retrieval using conventional or microdissection biopsies in 220 non-obstructive azoospermic men.

In 220 consecutive patients with non-obstructive azoospermia, sperm retrieval was attempted by a combination of conventional and microdissection testicular sperm extraction (TESE). For sperm retrieval, 2-3 conventional biopsies were performed followed by a microdissection TESE in cases of negative conventional biopsies. During the surgery, the vasculature of the testis was assessed using the operative microscope, and the location of positive biopsies was registered in relation to the blood supply. The overall sperm retrieval rate was 58.2%. From the initial conventional biopsies, sperm could be retrieved in 46.8% of the patients. With microdissection TESE, sperm could be retrieved from an additional 11.4% of the patients. The further use of microdissection TESE improved the sperm retrieval rate significantly (P=0.017). No significant accumulation of positive biopsies was found towards the rete testis or the main testicular vessels.

Penile corporoplasty using tunica albuginea free graft from proximal corpus cavernosum: a new technique for treatment of penile curvature in Peyronie's disease.

OBJECTIVES: In Peyronie's disease, any kind of plication technique for correcting penile deformities is associated with penile shortening in addition to the disease-related shrinkage. To minimize penile shortening we describe a new technique of penile corporoplasty using a free graft from the tunica albuginea. PATIENTS AND METHODS: From October 2001 to February 2003 we treated 18 patients with the new technique. All patients had stable Peyronie's disease with relevant curvature and sufficient erectile rigidity without any signs of inflammatory disease. Penile corporoplasty was performed by incision of the plaques to produce straightening. The resulting gap was covered with a free graft of tunica albuginea removed from the crural segment of the corpora cavernosa. RESULTS: In a preliminary follow-up of 16 patients, 12 penises were straight and 4 had a residual curvature less than 20 degrees. Two patients needed sildenafil for sufficient penile rigidity. Fourteen of 16 patients were satisfactory with the result of penile straightening. No severe perioperative complication was noted. CONCLUSION: The technique of penile straightening using a free tunica albuginea graft is effective and avoids additional shortening of the penis. As the results are preliminary, the study is continued to gain experience with a larger number of patients.

Sperm retrieval procedures and intracytoplasmatic spermatozoa injection with epididymal and testicular sperms.

INTRODUCTION: Male infertility caused by azoospermia due to non-reconstructable obstruction or non-obstructive azoospermia can be treated by microsurgical epididymal aspiration (MESA) or testicular sperm extraction (TESE) followed by an intracytoplasmatic spermatozoa injection (ICSI). MATERIAL AND METHODS: From 9/93 to 6/01, we carried out 1,025 ICSI procedures with aspirated epididymal or testicular sperms in 684 cases. 163 ICSI cycles were performed with epididymal sperms and 862 ICSI cycles with testicular sperms or spermatids. The TESE was carried out by open biopsy, frequently in a multilocular technique. The aspirated spermatozoas were used after cryopreservation (frozen) or immediately after aspiration (fresh). RESULTS: 538 patients had obstructive azoospermia or ejaculation failure. In 487 cases the underlying cause of azoospermia was an impaired spermatogenesis, following maldescensus testis, chemotherapy, radiotherapy, or caused by Sertoli-cell-only syndrome, a genetic disorder or an unknown etiology. The transfer rates, pregnancy rates and birth rates per ICSI cycle showed no statistically significant differences between testicular and epididymal sperms in the cases of seminal obstruction (28% average birth rates in both cases). However, highly significant was the difference in birth rates with regard to the underlying cause of infertility. In contrast, in treating non-obstructive azoospermia we observed a birth rate of 19% per cycle. In all patient groups the birth rate with fresh spermatozoas did not differ from those with cryopreserved spermatozoa. 40% of patients after multilocular TESE showed clinical signs of testicular lesion. CONCLUSION: The underlying cause of azoospermia is the most important factor for the outcome of ICSI using epididymal and testicular sperms. In cases of non-obstructive azoospermia, the pregnancy rate is low compared with the results in cases of obstructive azoospermia. There is no difference between fresh and cryopreserved sperms. TESE with ICSI is the most efficient treatment of azoospermia caused by hypergonadotropic hypogonadism. The morbidity of the TESE procedure is highly relevant and must be considered if this technique is indicated.