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.

Radiation-induced late effects in two affected individuals of the Lilo radiation accident.

Radiation exposure leads to a risk for long-term deterministic and stochastic late effects. Two individuals exposed to protracted photon radiation in the radiological accident at the Lilo Military site in Georgia in 1997 received follow-up treatment and resection of several chronic radiation ulcers in the Bundeswehr Hospital Ulm, Germany, in 2003. Multi-parameter analysis revealed that spermatogenetic arrest and serum hormone levels in both patients had recovered compared to the status in 1997. However, we observed a persistence of altered T-cell ratios, increased ICAM1 and beta1-integrin expression, and aberrant bone marrow cells and lymphocytes with significantly increased translocations 6 years after the accident. This investigation thus identified altered end points still detectable years after the accident that suggest persistent genomic damage as well as epigenetic effects in these individuals, which may be associated with an elevated risk for the development of further late effects. Our observations further suggest the development of a chronic radiation syndrome and indicate follow-up parameters in radiation victims.

Ionizing radiation induces degranulation of human mast cells and release of tryptase.

PURPOSE: Skin fibrosis is a hallmark of ionizing radiation-induced tissue injury and we hypothesized that mast cells via their products (especially tryptase) are involved in this event. We therefore investigated whether: (i) irradiation with 5 Gray (Gy) is able to induce the release of the typical mast cell mediator tryptase from human mast cells (HMC-1) in vitro, (ii) this effect can be influenced by application of clinically relevant mast cell blockers, and (iii) irradiation leads to mast cell degranulation in ex vivo skin culture models. MATERIALS AND METHODS: The human mast cell line (HMC)-1, as well as ex vivo skin tissue served as experimental models. Fluorescence activated cell sorting (FACS), Enzyme linked immunosorbent assays (ELISA), mast cell degranulation assays and immunohistochemistry were applied. RESULTS: Ionizing radiation induces a time-dependent, statistically significant increase in the release of tryptase by HMC-1 cultured in vitro. Mast cell degranulation and secretion of tryptase was partially, but not significantly, inhibited by pre-incubation with the histamine-1 receptor (H1) blocker cetirizine. Mast cell degranulation was also clearly evident after irradiation using an ex vivo skin culture model of mastocytoma tissue. CONCLUSIONS: We propose that ionizing radiation leads to a degranulation of dermal mast cells, an event which is accompanied by the release of tryptase.

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.

Number, distribution pattern, and identification of macrophages in the testes of infertile men.

OBJECTIVE: To investigate the number, location, and secretory products of macrophages in human testes showing normal and abnormal spermatogenesis. DESIGN: Evaluation of testicular biopsies with the use of immunohistochemistry, laser capture microdissection, and reverse transcriptase polymerase chain reaction. SETTING: University research and clinical institutes. PATIENT(S): Infertile men with germ cell arrest (n = 10), Sertoli cell only (n = 8), or mixed atrophy (n = 7) syndromes, and with cases of idiopathic infertility showing normal spermatogenesis (n = 8). INTERVENTION(S): Diagnostic testicular biopsy was performed on participants. MAIN OUTCOME MEASURE(S): We recorded the location, number, distribution, and cytokine expression of human testicular macrophages. RESULT(S): CD68-positive macrophages were found in the testes of all groups analyzed. These macrophages expressed the genes for interleukin 1 and tumor necrosis factor-alpha, and were located in the interstitium, tubular wall, and tubular lumen. In Sertoli cell only and germ cell arrest syndromes, the overall macrophage number was increased over twofold. In all pathologic states, there was a significant shift of these cells from the interstitium to the tubules. CONCLUSION(S): Our study suggests that increased numbers of CD68-positive macrophages directly (via phagocytosis) or indirectly (via paracrine actions exerted through their secretory products) are involved in the regulation of steroidogenesis, Sertoli cell activity, germ cell survival, and, in consequence, in the pathogenesis or maintenance of infertility states in the human testes.

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.

Mast cells in human testicular biopsies from patients with mixed atrophy: increased numbers, heterogeneity, and expression of cyclooxygenase 2 and prostaglandin D2 synthase.

OBJECTIVE: To determine intratesticular abundance and distribution of tryptase-positive mast cells (MCs) and to examine the expression of key enzymes of prostaglandin (PG) synthesis, cyclooxygenase 2 (COX2), and PGD2 synthase in the testes of men with mixed atrophy (MA) syndrome and in normal samples. DESIGN: Retrospective study. SETTING: Academic research institute and andrology practice. PATIENT(S): Nineteen men. INTERVENTION(S): Testicular biopsies. MAIN OUTCOME MEASURE(S): Immunohistochemistry and evaluation of COX2 and tryptase-positive MCs, laser microdissection of immunoreactive cells followed by reverse transcriptase polymerase chain reaction for COX2 and PGDS-H mRNA, and transmission electron microscopy. RESULT(S): In line with previous studies, few tryptase-positive MCs, but no COX2-positive cells, were observed in testes with normal spermatogenesis. In MA samples, the number of tryptase-positive MCs was significantly increased and the cells accumulated in the walls of the seminiferous tubules. In 11 of 13 MA samples, COX2 protein was detected. In 2 cases, Leydig cells were positive; however, in all 11 of 13 cases, COX2 was localized to MCs, coexpressing tryptase. The proportion of MCs coexpressing COX2 varied from 4% to 35%. Laser microdissection of tryptase/COX2-positive MCs followed by reverse transcriptase polymerase chain reaction revealed PGDS-H mRNA. Transmission electron microscopy identified typical MCs with abundant granules and another subtype with only a few granules, implying that MCs may differentiate in the testes. CONCLUSION(S): In patients with MA, testicular MC numbers and phenotypes change with respect to the ability to express COX2 and synthesize PGs. MCs and PGs have emerged as players in spermatogenic dysfunction.

A prostaglandin D2 system in the human testis.

As shown recently, cyclooxygenase 2 (COX2), the inducible key enzyme for the prostaglandin (PG) biosynthetic pathway, is abundantly present in interstitial cells of testes of men suffering from different forms of impaired spermatogenesis and sub- or infertility, but it is absent in human testes with normal spermatogenesis. Although the spectrum of the downstream products of COX2 action in testis, namely PGs, and their effects are not known, our results show that Prostaglandin D2 (PGD2) likely plays a role. We describe (a) PGD2 synthetases, as well as receptors for PGD2 (DP) in testicular interstitial cells of men suffering from spermatogenic damage and infertility, and report that (b) PGD2 is produced by and can affect Leydig cells of an animal model, which expresses testicular COX2 and DP.

Evidence for a histaminergic system in the human testis.

The complete lack of information about mast cells as a source of histamine and potential target cells for histamine in human testes prompted us to investigate these issues in testes of fertile and infertile patients using a combination of laser microdissection, reverse transcription-polymerase chain reaction (RT-PCR), and immunohistochemistry. We show for the first time the expression of the rate-limiting enzyme in histamine synthesis-histidine decarboxylase-by human testicular mast cells and the expression of the histamine (H) receptors 1 (H1) and 2 (H2) by germinal, interstitial, and peritubular cells in the testes of fertile and infertile patients.

Proliferative action of mast-cell tryptase is mediated by PAR2, COX2, prostaglandins, and PPARgamma : Possible relevance to human fibrotic disorders.

Mast-cell products can stimulate fibroblast proliferation, implying that these cells are key players in fibrosis. One mast-cell product, the serine protease tryptase, is known to activate protease-activated receptor 2 (PAR2) and cause proliferation of fibroblasts. We found that recombinant tryptase, human mast-cell (HMC-1) supernatant, which contains tryptase, and the PAR2-activating peptide SLIGKV exert fibroproliferative actions in human fibroblasts. Here we report insights into this action, which after activation of PAR2 leads to increased expression of cyclooxygenase 2 (COX2), a key enzyme in the biosynthesis of prostaglandins, and consequently to enhanced prostaglandin synthesis. Subsequent cell proliferation is mediated by the prostaglandin 15-deoxy-Delta(12,14)-prostaglandin J(2), which acts via the nuclear peroxisome proliferator-activated receptor gamma (PPARgamma). Fibroblast proliferation induced by tryptase and PAR2 agonist peptide can be blocked by antagonists of COX2 and PPARgamma, implying that the proliferative effect of tryptase is PAR2-initiated but depends on COX2, 15-deoxy-Delta(12,14)-prostaglandin J(2), and PPARgamma. This previously uncharacterized pathway could be of relevance for human fibrotic diseases. For instance, increased numbers of activated mast cells are correlated with fibrosis in testes of infertile men. In these cases all components of the signaling pathway of tryptase were detected as well as expression of COX2. Therefore, our study describes as-yet-unknown interactions between mast cells and fibroblasts, which could be relevant for human fibrotic diseases.

Evidence for a GABAergic system in rodent and human testis: local GABA production and GABA receptors.

The major neurotransmitter of the central nervous system, gamma-aminobutyric acid (GABA), exerts its actions through GABA(A), GABA(B) and GABA(C) receptors. GABA and GABA receptors are, however, also present in several non-neural tissues, including the endocrine organs pituitary, pancreas and testis. In the case of the rat testis, GABA appears to be linked to the regulation of steroid synthesis by Leydig cells via GABA(A) receptors, but neither testicular sources of GABA, nor the precise nature of testicular GABA receptors are fully known. We examined these points in rat, mouse, hamster and human testicular samples. RT-PCR followed by sequencing showed that the GABA-synthesizing enzymes glutamate decarboxylase (GAD) 65 and/or GAD67, as well as the vesicular GABA transporter vesicular inhibitory amino acid transporter (VIAAT/VGAT) are expressed. Testicular GAD in the rat was shown to be functionally active by using a GAD assay, and Western blot analysis confirmed the presence of GAD65 and GAD67. Interstitial cells, most of which are Leydig cells according to their location and morphological characteristics, showed positive immunoreaction for GAD and VIAAT/VGAT proteins. In addition, several GABA(A) receptor subunits (alpha1-3, beta1-3, gamma1-3), as well as GABA(B) receptor subunits R1 and R2, were detected by RT-PCR. Western blot analysis confirmed the results for GABA(A) receptor subunits beta2/3 in the rat, and immunohistochemistry identified interstitial Leydig cells to possess immunoreactive GABA(A) receptor subunits beta2/3 and alpha1. The presence of GABA(A) receptor subunit alpha1 mRNA in interstitial cells of the rat testis was further shown after laser microdissection followed by RT-PCR analysis. In summary, these results describe molecular details of the components of an intratesticular GABAergic system expressed in the endocrine compartment of rodent and human testes. While the physiological significance of this peripheral neuroendocrine system conserved throughout species remains to be elucidated, its mere presence in humans suggests the possibility that clinically used drugs might be able to interfere with testicular function.