Activin and follistatin interactions in the male reproductive tract: activin expression and morphological abnormalities in mice lacking follistatin 288.

Activin A is an important regulator of testicular and epididymal development and function, as well as inflammation and immunity. In the adult murine reproductive tract, activin A mRNA (Inhba) expression levels are highest in the caput epididymis and decrease progressively towards the distal vas deferens. The activin-binding protein, follistatin (FST), shows the opposite expression pattern, with exceptionally high levels of the Fst288 mRNA variant in the vas deferens. This unique pattern of expression suggests that activin A and follistatin, in particular FST288, play region-specific roles in regulating the epididymis and vas deferens. The cellular distribution of activin and follistatin and structural organization of the male reproductive tract was examined in wild-type and transgenic (TghFST315) mice lacking FST288. Compared to wild-type littermates, TghFST315 mice showed a 50% reduction in serum follistatin and a significant elevation of both activin A and B. Testicular, epididymal and seminal vesicle weights were reduced, but intra-testicular testosterone was normal. A decrease in the epididymal duct diameter in the corpus and thickening of the peritubular smooth muscle in the cauda, together with increased coiling of the proximal vas deferens, were observed in TghFST315 mice. No immune cell infiltrates were detected. Immunohistochemistry indicated that epithelial cells are the main source of activins and follistatin in the epididymis and vas deferens. Activin A, but not activin B, was also localized to sperm heads in the lumen of the epididymis and vas deferens. Expression of Inhba and another immunoregulatory gene, indoleamine-2,3-dioxygenase (Ido-1), was increased approximately twofold in the TghFST315 caput epididymis, but several other genes associated with immunoregulation, inflammation or fibrosis were unaffected. Our novel data indicate that disruption of follistatin expression has significant effects on the testis and epididymis, and suggest an association between activin A and indoleamine-2,3-dioxygenase in the caput epididymis, with implications for the epididymal immunoenvironment.

Epididymitis: ascending infection restricted by segmental boundaries.

STUDY QUESTION: Is the regionalization of epididymitis related to epididymal segmentation? SUMMARY ANSWER: We show for the first time that luminal ascent of bacteria is strictly gated by epididymal segment boundaries, involving ductal constriction adjacent to the infected area. WHAT IS KNOWN ALREADY: The epididymal duct is a continuous, unbranched tube, coiled into segments that are divided by connective tissue septa. Sonographic analysis indicates that swelling associated with epididymitis is predominant in the cauda region. Epididymal segmentation has never been investigated in the context of pathological alterations. STUDY DESIGN, SIZE, AND DURATION: We analyzed segment-specific changes in the epididymal duct in a mouse model and in men. In the mouse epididymitis model (3 days post-infection, injection of bacteria into the lumen of the vas deferens), two Escherichia coli strains were tested: a uropathogenic strain CFT073 (UPEC, n = 7) and a fecal non-pathogenic strain NPEC470 (NPEC, n = 5). Two control groups: phosphate-buffered saline, sham-treated animals (n = 4) and untreated mice (n = 8). In addition, segmentation was verified by ex vivo injection of dye into the interstitial spaces of untreated mouse epididymides. Histological findings were compared with specimens from epididymitis patients (n = 10, age range 14-78, median 60 years) who underwent surgical intervention; control: samples from patients without epididymitis (n = 16, age range 38-87, median 73 years). PARTICIPANTS/MATERIALS, SETTING, AND METHODS: We investigated the ascending infections by detailed histological analysis in correlation with local infection status in a mouse epididymitis model. As a proof of concept, rare patient material from two archives was analyzed: epididymides from patients who underwent surgical intervention for persisting epididymitis, and for control, histologically normal epididymides from men who underwent orchiectomy for therapy of prostatic carcinoma. MAIN RESULTS AND THE ROLE OF CHANCE: Luminal ascent of E. coli in mice was strictly gated by epididymal segment boundaries. In the mouse model, both strains of E. coli were detected exclusively in the distal cauda segment associated with damage of the epithelium and muscle layer. Ductal constriction occurred in the non-infected upstream segments of infected area, putatively blocking further luminal ascent of bacteria in UPEC-infected animals. Corresponding histological and morphological changes were found in epididymitis patients. The caput region was found to be unaffected in patients and the mouse model. LIMITATIONS, REASONS FOR CAUTION: Patient samples represented advanced cases of epididymitis that made surgical intervention necessary. WIDER IMPLICATIONS OF THE FINDINGS: Our data demonstrate the impact of epididymal segmentation, presumably a protective response mechanism against infectious invasion and bacterial ascent, during epididymitis and affirm the importance of rapid intervention. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from the State of Hessen (LOEWE-MIBIE) and the DFG (KFO 181). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: No clinical trial involved.

Uropathogenic Escherichia coli (UPEC) induced antimicrobial gene expression in the male reproductive tract of rat: evaluation of the potential of Defensin 21 to limit infection.

Escherichia coli (E. coli) is a common pathogen in epididymitis, which represents a prevalent entity in male reproductive tract infections (RTI). Although current treatment regimens using antibiotics are satisfactory, development of antimicrobial resistance by the pathogen represents a challenge in the management of RTI. Hence, identification of antimicrobial peptides as alternatives to antibiotics has gained importance. We demonstrate that in a rat epididymo-orchitis model induced with uropathogenic E. coli (UPEC) strain MTCC 729, the expression of defensins and defensin-like Spag11 genes are induced in the epididymis and testes. The induction of antimicrobial gene expression is paralleled by phosphorylation of the NF-kB subunit p65 and the inhibitor of NFkB (IkB-alpha), decreased levels of histone deacetylase 1 and increased methylation of Histone 3, indicating the role of classical Toll-like receptor mediated signaling and epigenetic regulation. Recombinant Defensin 21, when administered to UPEC-infected rats, substantially reduced the bacterial load in the epididymis and testis and proved to be more effective than gentamycin. The ability of Defensin 21 to limit RTI provides support that antibacterial proteins of the male reproductive tract may be used as potential alternatives to antibiotics in treatment of this disease.

H3K79 methylation directly precedes the histone-to-protamine transition in mammalian spermatids and is sensitive to bacterial infections.

In both mammalian and Drosophila spermatids, the completely histone-based chromatin structure is reorganized to a largely protamine-based structure. During this histone-to-protamine switch, transition proteins are expressed, for example TNP1 and TNP2 in mammals and Tpl94D in Drosophila. Recently, we demonstrated that in Drosophila spermatids, H3K79 methylation accompanies histone H4 hyperacetylation during chromatin reorganization. Preceding the histone-to-protamine transition, the H3K79 methyltransferase Grappa is expressed, and the predominant isoform bears a C-terminal extension. Here, we show that isoforms of the Grappa-equivalent protein in humans, rats and mice, that is DOT1L, have a C-terminal extension. In mice, the transcript of this isoform was enriched in the post-meiotic stages of spermatogenesis. In human and mice spermatids, di- and tri-methylated H3K79 temporally overlapped with hyperacetylated H4 and thus accompanied chromatin reorganization. In rat spermatids, trimethylated H3K79 directly preceded transition protein loading on chromatin. We analysed the impact of bacterial infections on spermatid chromatin using a uropathogenic Escherichia coli-elicited epididymo-orchitis rat model and showed that these infections caused aberrant spermatid chromatin. Bacterial infections led to premature emergence of trimethylated H3K79 and hyperacetylated H4. Trimethylated H3K79 and hyperacetylated H4 simultaneously occurred with transition protein TNP1, which was never observed in spermatids of mock-infected rats. Upon bacterial infection, only histone-based spermatid chromatin showed abnormalities, whereas protamine-compacted chromatin seemed to be unaffected. Our results indicated that H3K79 methylation is a histone modification conserved in Drosophila, mouse, rat and human spermatids and may be a prerequisite for proper chromatin reorganization.

Prostatitis and andrological implications.

AIM: The prostatitis syndrome is a frequent disease affecting men in their reproductive age. The prostatitis syndrome is classified according to the National Institutes of Health (NIH) definition. Andrological implications of the prostatitis syndrome might encompass fertility issues, sexual dysfunctions and endocrinological alterations and influences. METHODS: A medline query using the terms prostatitis AND andrological implication, fertility, sexual dysfunction or endocrinology was performed. RESULTS: Acute bacterial prostatitis and andrological implications have not been adequately addressed. Patients with chronic bacterial prostatitis and chronic pelvic pain syndrome have been investigated evaluating sperm parameters. Some studies showed impaired sperm parameters. In chronic bacterial prostatitis, half of the patients reveal significant bacteriospermia with still debatable deleterious effects on sperm quality. Few interventional studies have addressed fertility issues in those patients. Anti-inflammatory treatment perhaps could have a positive impact on sperm parameters. Sexual dysfunction can be described by different components such as erectile, ejaculatory, orgasmic and sexual desire dysfunctions. Sexual dysfunction in chronic prostatitis adds to the number of positive symptom phenotypes and correlates therefore with increasing symptom scores in patients with chronic prostatitis syndromes. However, prospective interventional studies on the role of sexual dysfunctions are missing. Hormones have been found to modulate the inflammatory response via different receptors, particularly via estrogen receptor alpha. This evidence, however, is mainly limited to pre-clinical studies currently. CONCLUSION: Andrological implications are heterogenous and frequently described in patients with chronic prostatitis syndrome. Nonetheless, andrological factors have not been routinely addressed as primary variables in the different studies, which makes further research necessary.

Argon reduces neurohistopathological damage and preserves functional recovery after cardiac arrest in rats.

BACKGROUND: Xenon has profound neuroprotective effects after neurological injury and is currently undergoing phase 2 clinical trials in cardiac arrest patients. However, xenon is very costly, which might preclude its widespread use. We hypothesized argon, which is more available, might also protect central nervous tissues and allow better functional recovery in a rodent model of global cerebral ischaemia. METHODS: Fourteen male Sprague-Dawley rats were subjected to 7 min of cardiac arrest and 3 min of cardiopulmonary resuscitation (CPR). One hour after successful CPR, animals were randomized to either ventilation with 70% argon in oxygen (n = 7) for 1 h or 70% nitrogen (controls, n=7). A neurological deficit score (NDS) was calculated daily for the following 7 days, then the animals were killed and the brains harvested for histopathological analyses. RESULTS: All animals survived. Control rats had severe neurological dysfunction, while argon-treated animals showed significant improvements in the NDS at all time points. This was paralleled by a significant reduction in the neuronal damage index in the neocortex and the hippocampal CA 3/4 region. CONCLUSIONS: Our study demonstrates that a single 1 h application of 70% argon significantly reduced histopathological damage of the neocortex and hippocampus, associated with a marked improvement in functional neurological recovery.

Overexpression of Lis1 in different stages of spermatogenesis does not result in an aberrant phenotype.

Previous studies showed that in the mouse mutant Lis1(GT/GT) gene trap integration in intron 2 of Lis1 gene leads to male infertility in homozygous Lis1(GT/GT) mice. We further analyzed this line and could confirm the suggested downregulation of a testis-specific Lis1 transcript in mutant animals in a quantitative manner. Moreover, we analyzed the gene trap mutation on different genetic backgrounds in incipient congenic animals and could exclude a genetic background effect. To gain further insights into the role and requirement of LIS1 in spermatogenesis, 3 transgenic lines were generated, that overexpress Lis1 under control of the testis-specific promoters hEF-1α, which is exclusively active in spermatogonial cells, PGK2, which is active in pachytene spermatocytes and following stages of spermatogenesis, and Tnp2 which is active in round spermatids and following stages of spermatogenesis, respectively. All 3 transgenic lines remained fertile and testis sections displayed no abnormalities. To overcome the infertility of Lis1(GT/GT) males, these transgenic Lis1-overexpressing animals were mated with Lis1(GT/GT) mice to generate 'rescued' Lis1(GT/GT)/Lis1(Tpos) males. 'Rescued' animals from all transgenic lines remained infertile, thus overexpression of Lis1 in different stages of spermatogenesis could not rescue the infertility phenotype of homozygous gene trap males.

The cholinergic system in rat testis is of non-neuronal origin.

The cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7; also known as ChT1), vesicular ACh transporter (SLC18A3; also known as VAChT), organic cation transporters (SLC22s; also known as OCTs), the nicotinic ACh receptors (CHRN; also known as nAChR) and muscarinic ACh receptors. The cholinergic system is not restricted to neurons but plays an important role in the structure and function of non-neuronal tissues such as epithelia and the immune system. Using molecular and immunohistochemical techniques, we show in this study that non-neuronal cells in the parenchyma of rat testis express mRNAs for Chat, Slc18a3, Slc5a7 and Slc22a2 as well as for the CHRN subunits in locations completely lacking any form of innervation, as demonstrated by the absence of protein gene product 9.5 labelling. We found differentially expressed mRNAs for eight α and three ß subunits of CHRN in testis. Expression of the α7-subunit of CHRN was widespread in spermatogonia, spermatocytes within seminiferous tubules as well as within Sertoli cells. Spermatogonia and spermatocytes also expressed the α4-subunit of CHRN. The presence of ACh in testicular parenchyma (TP), capsule and isolated germ cells could be demonstrated by HPLC. Taken together, our results reveal the presence of a non-neuronal cholinergic system in rat TP suggesting a potentially important role for non-neuronal ACh and its receptors in germ cell differentiation.

Non-Hodgkin's lymphoma in adolescents: experiences in 378 adolescent NHL patients treated according to pediatric NHL-BFM protocols.

Age-related differences in the distribution, biology and treatment response of non-Hodgkin's lymphoma (NHL) in adolescents remain to be elucidated. The current analyses present clinical parameters and outcomes of adolescents treated in pediatric NHL-BFM trials. Patients were stratified by histological subtype: lymphoblastic lymphoma (LBL); mature B-NHL, including Burkitt's lymphoma/leukemia (BL/B-AL), diffuse B-cell lymphoma (DLBCL-CB) and mediastinal B-cell lymphoma (PMLBL); and anaplastic large cell lymphoma (ALCL). Between October 1986 and December 2007, 2915 patients were registered, including 378 (13%) adolescents (15-18 years) with BL/B-AL (n=101), ALCL (n=74), DLBCL-CB (n=55), T-LBL (n=45), PMLBL (n=24), pB-LBL (n=13) and rare or not-specified NHL subtypes (n=66). The 5-year event-free survival (EFS) was 79±2% for adolescents compared with 85±1% for patients aged <15 years (P=0.014). EFS was 83±7% for adolescents with T-LBL, 82±4% with BL/B-AL, 85±5% with DLBCL-CB, 57±10% with PMLBL and 70±6% with ALCL. According to sex, the 5-year EFS in females versus males, respectively, was 70±5 versus 83±2% overall (P=0.004), 57±17 versus 92±6% (P=0.0036) for T-LBL patients and 71±9 versus 97±3% (P=0.0067) for DLBCL-CB patients. Adolescents with NHL treated according to pediatric NHL-BFM protocols had an EFS of 79±2%, which is marginally inferior to that of children. In adolescents with T-LBL and DLBCL-CB, female sex was associated with a worse prognosis.

Characterization of dendritic cells in testicular draining lymph nodes in a rat model of experimental autoimmune orchitis.

The maturation state of dendritic cells (DC) is regarded as a control point for the induction of peripheral tolerance or autoimmunity. Experimental autoimmune orchitis (EAO) serves as a model to investigate inflammatory-based testicular impairment, which ranks as a significant cause of male infertility. This work aimed to determine whether DC enrichment occurs organotypically in testicular draining lymph nodes (TLN) compared with LN draining the site of immunization (ILN) and thus contributes to the pathogenesis of autoimmune orchitis. In this regard, we quantified and characterized the DC from TLN and ILN in rats with EAO. Flow cytometric analysis showed a significant increase in the percentage of DC (OX62+) only in TLN from EAO rats compared with normal (N) and adjuvant control (C) groups. The number of DC from ILN and TLN expressing CD80, CD86 and major histocompatibility complex (MHC) II was comparable among N, C and experimental (E) groups at 30 and 50 days after the first immunization. However, TLN DC from EAO rats (50 days) showed an increase in mean fluorescence intensity for MHC II compared with N, C and E groups (30 days). The mRNA expression level of IL-10 and IL-12p35 was significantly upregulated in enriched DC fraction from TLN in EAO rats with no significant changes observed in ILN DC. The expression of IL-23p19 mRNA remained unchanged. Functional data, using proliferation assays showed that EAO-DC from TLN, but not from ILN, significantly enhanced the proliferation of naïve T cells compared with C-DC. In summary, our data suggest that the DC in TLN from orchitis rats are mature, present antigens to T cells and stimulate an autoimmune response against testicular antigens, thus causing immunological infertility.

[Orchitis and male infertility]. / Orchitis und Infertilität.

Infections and inflammations of the genital tract are considered the most frequent causes of reduced male fertility, but conclusive epidemiological data are not available. In view of the exposure of germ cells to pathogenic components as well as the cells and mediators involved in the inflammatory processes, irreversible damage to spermatogenesis and corresponding decline of ejaculate quality are to be expected, particularly in cases of chronic orchitis. While the consequences of orchitis and epididymo-orchitis that exhibit clinical symptoms due to systemic or local infections are well known, including testicular atrophy and complete loss of fertility, those cases of inflammatory reactions of the testicles that manifest an asymptomatic or subclinical course, or are not even due to an infection, have received little attention until now. However, systematic histopathological analyses have shown a high prevalence of asymptomatic inflammatory reactions in testicular biopsies from infertile men. The mostly focal lymphocytic infiltrates correlate with the degree of damage to spermatogenesis and corresponding clinical and endocrinological parameters of testicular function. Noninvasive diagnostic techniques are not yet available so that chronic asymptomatic inflammations of the testicles as the primary cause or cofactor of male fertility disorders are underestimated. Except for administration of pathogen-specific antibiotics, treatment recommendations are to a large extent still lacking.

Efecto de la orquiepidádimitis por Escherichia coli uropatogénica sobre el epitelio germinal y la producción de testosterona / Effects of orchiepididimitis due to uropathogenic Escherichia coli on germinal epithelium and testosterone secretion

Introducción: Una de las complicaciones de la orquiepidédimitis (OE) aguda al largo plazo es la infertilidad secundaria a la atrofia testicular y el deterioro de la espermatogénesis. Los mecanismos fisiopatológicas exactos a través de los cuales la infección deteriora la espermatogénesis aún no están claros. Objetivo: Evaluar el impacto de la OE por Escherichia coli uropatógena (ECUP) sobre parámetros específicos de la espermatogénesis y esteroidogénesis. Materiales y método: El estudio incluyó 40 ratas Wistar macho. Post anestesia general se realizó una incisión escrotal media con posterior exposición de ambos epidídimos y conductos deferentes. Mediante la punción con una aguja 27G en el conducto deferente a un centímetro del epidídimo se inyectaron 50 ul de suspensión ECUP (10x6 UFC/ml) (grupo infectados) o suero fisiológico (grupo control). Las ratas fueron divididas en 4 grupos de 10 animales cada uno: el primer grupo infectado (n= 10) y control (n= 10) fue sacrificado a los 7 días y el segundo grupo infectado (n= 10) y control (n= 10) fue sacrificado a los 30 días post inoculación. Resultados: Respecto de los controles los animales infectados tuvieron menor (p< 0,05): concentración espermática (promedio +/- ds: 7 días = 100,2 +/- 56,8 vs 249,8 +/- 111,6; 30 días= 20,7 +/- 38,3 vs 75,3 +/- 98,7 millones/grm tejido en cola epidídimo); y mayor (p< 0,05): (i) degeneración del epitelio germinal e infiltración celular inflamatoria a los 30 días, y (ii) número de células espermatogénicas apoptóticas (índice apoptótico)detectadas por prueba de TUNEL a los 7 y a los 30 días. El ensayo inmunohistoquímico antivimentina reveló que las células apoptóticas dentro de los túbulos seminíferos fueron casi exclusivamente células germinales y no de Sertoli. No se observaron diferencias significativas en los niveles plasmáticos de testosterona entre el grupo infectado y control a los siete y treinta días de ser inyectados. Conclusiones: La OE aguda provocada...

Introduction: In the long term, one of the complications of epidydimo-orchitis (EO) is infertility due to testicular atrophy. The exact pathophisiologic mechanisms for impairment of spermatogenesis are not clear Objective: To evaluate the impact of infection by uropathogenic Escherichia coli (UPEC) on spermatogenesis and stereoidogenesis. Materials and method: The study included 40 Wistar male rats. Under general anesthesia a mid scrotal incision was performed. Both epidydimis and vas deferens were exposed. Using a 27G needle, 50 ul of either UPEC suspension (10x6 CFU/ml) or saline were injected. Rats were divided into 4 groups: the first study group (n =10) and controls ( n=10) were sacrificed at 7 days; the second study group (n =10) and controls (n =10) were sacrificed at 30 days post inoculation.Results: Compared to controls, infected animals had lower sperm concentration (p <0.05). The average +/- SD at 7 days was 100.2 +/- 56.8 vs 249.8 +/- 111.6 millions/gram of tissue from the tail of the epidydimis. At 30 days the results were 20.7 +/- 38.3 vs 75.3 +/- 98.7. The study group had more degeneration of germinal epithelium and inflammation at 30 days. The apoptotic index studied by TUNEL test at 7 and 30 days was higher. Immunohistochemistry with anti vimentin antibodies revealed that the apoptotic cells within seminifirous tubules were almost exclusively germ cells and not Sertoli cells. No significant differences on testosterone plasma levels between the study and control groups were found at 7 or 30 days post injection. Conclusions: Acute EO caused by retrograde inoculation of UPEC in rats generates a significant decrease in sperm concentration. This effect is secondary between others to an impairment of sperm activity at the seminiferous tubules generated by an increased apoptotic index of the sperm cells. The effect of EO on testosterone production seems irrelevant in the clinical setting.

PHF5A represents a bridge protein between splicing proteins and ATP-dependent helicases and is differentially expressed during mouse spermatogenesis.

PHF5A is a highly conserved protein from yeast to man, and based on studies in yeast, it was suggested that the homologous protein RDS3P in S. cerevisiae takes part in the organization of U2 snRNP particles. By using the yeast two-hybrid assay we could demonstrate that PHF5A interacted both with ATP-dependent helicases EP400 and DDX1 and with arginine-serine (RS)-rich domains of splicing factors U2AF1 and SFRS5 in mouse. Furthermore, domain interaction studies revealed that PHF5A interaction with EP400 and DDX1 is restricted to the N-terminal part of PHF5A, whereas the C-terminal region of PHF5A was found to be responsible for the association with U2AF1 and SFRS5. By using the yeast three-hybrid assay, we could further show that both EP400 and DDX1 interacted only indirectly with U2AF1 and SFRS5 proteins via the bridge protein PHF5A. The subcellular localization of a PHF5A-GFP fusion protein was predominantly observed in the nucleus and, in addition, PHF5A co-localized with both U2AF1 and SFRS5 proteins in nuclear speckles of NIH3T3 cells. Moreover, expression analyses demonstrated that PHF5A and U2AF1 gene expression coincided in spermatocytes during murine spermatogenesis and interaction between these proteins was also detectable in the spermatocyte-specific cell line GC-4spc by using in vivo co-immunoprecipitation studies. Taken together, our results indicate that PHF5A resembles a protein which interacts with splicing factors U2AF1 and SFRS5 and helicases EP400 and DDX1 and functions as a bridge protein between these proteins.

Chronic orchitis: a neglected cause of male infertility?

Infection and inflammation of the male reproductive tract are accepted as important aetiological factors of infertility. With regard to their impact on male reproductive function, orchitis and epididymo-orchitis due to local or systemic infection as well as noninfectious aetiological factors are of particular concern. There is clinical and pathological evidence that chronic inflammatory conditions of the testes can disrupt spermatogenesis and irreversibly alter both sperm number and quality. In the majority of patients, however, diagnosis is hampered by an asymptomatic course of the disease and unspecific clinical signs. Hence, respective epidemiological data are scarce. On the other hand, systematic histopathological work-up of testicular biopsies from infertile men indicates a high prevalence of inflammatory reactions. A characteristic pattern of inflammatory lesions with focal or multifocal, predominantly peritubular lymphocyte infiltration and concomitant damage of seminiferous tubules is seen in chronic orchitis of various origins. This supports the concept that induction of testicular inflammation is associated with a T-cell-mediated autoimmune response, i.e. disruption of the immune privilege. Moreover, despite the patchy distribution of the lesions, testicular volume and score counts for spermatogenesis may be significantly reduced. In conclusion, asymptomatic inflammatory reactions in the testis should not be neglected as an underlying cause or co-factor of male infertility. However, definitive diagnosis of chronic asymptomatic orchitis still requires testicular biopsy and guidelines for the therapeutic management are not yet available.

Asthenoteratozoospermia in mice lacking testis expressed gene 18 (Tex18).

Testis expressed gene 18 (Tex18) is a small gene with one exon of 240 bp, which is specifically expressed in male germ cells. The gene encodes for a protein of 80 amino acids with unknown domain. To investigate the function of (Tex18) gene, we generated mice with targeted disruption of the (Tex18) gene by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fertile, while they are subfertile on the 129/Sv background, although mating is normal. We showed that Tex18(-/-) males are subfertile because of abnormal sperm morphology and reduced motility, which is called asthenoteratozoospermia, suggesting that (Tex18) affects sperm characteristics. Maturation of spermatids is unsynchronized and partially impaired in seminiferous tubules of Tex18(-/-) mice. Electron microscopical examination demonstrated abnormal structures of sperm head. In vivo experiments with sperm of Tex18(-/-) 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility, as determined by the computer-assisted semen analysis system, is significantly affected, compared to wild-type spermatozoa. Generation of transgenic mice containing Tex18-EGFP fusion construct revealed a high transcriptional activity of (Tex18) during spermiogenesis, a process with morphological changes of haploid germ cells and development to mature spermatozoa. These results indicate that (Tex18) is expressed predominantly during spermatid differentiation and subfertility of the male Tex18(-/-) mice on the 129/Sv background is due to the differentiation arrest, abnormal sperm morphology and reduced sperm motility.

Inhibition of tumor growth and metastasis in vitro and in vivo by targeting macrophage migration inhibitory factor in human neuroblastoma.

Macrophage migration inhibitory factor (MIF) has been defined as a novel oncogene. Our previous results have shown that MIF may contribute to the progression of neuroblastoma by (a) inducing N-Myc expression and (b) upregulating the expression of angiogenic factors. The aim of this study was to test whether tumor growth could be inhibited by reduction of endogenous MIF expression in neuroblastoma and clarify the molecular mechanisms underlying MIF reduction on the control of neuroblastoma growth. We established human neuroblastoma cell lines stably expressing antisense MIF (AS-MIF) cDNA. These stable transfectants were characterized by cell proliferation, gene expression profile, tumorigenicity and metastasis in vitro and in vivo. Decreased MIF expression was observed after transfection with AS-MIF in neuroblastoma cells and downregulation of MIF expression significantly correlated with decreased expression of N-Myc, Ras, c-Met and TrkB at protein level. Affymetrix microarray analysis revealed that expression of IL-8 and c-met was inhibited and neuroblastoma-favorable genes such as EPHB6 and BLU were upregulated in MIF reduced cells. Neuroblastoma cell growth exhibited a nearly 80% reduction in AS-MIF transfectants in vitro. Furthermore, mice in which tumors formed after subcutaneous injection of AS-MIF transfectants showed a 90% reduction in tumor growth compared to control. Metastasis in mice was also suppressed dramatically. Our data demonstrate that targeting MIF expression is a promising therapeutic strategy in human neuroblastoma therapy, and also identifies the MIF target genes for further study.

Development of testicular inflammation in the rat involves activation of proteinase-activated receptor-2.

Mast cells are involved in early events crucial to inflammation and autoimmune disease. Recently, proteinase-activated receptor-2 (PAR(2)), a G-protein coupled receptor important to injury responses, was shown to be activated by mast cell tryptase. To investigate whether mast cells and PAR(2) are involved in the development and/or aggravation of testicular inflammation, we studied acute and chronic inflammatory models in the rat. In normal testes, PAR(2) was detected immunohistochemically in macrophages, in peritubular cells (PTCs) and in spermatid acrosomes. In experimentally induced autoimmune orchitis (EAO), PAR(2) was strongly upregulated in macrophages and peritubular-like cells, forming concentric layers around granulomas. Mast cells increased 10-fold in number, were more widely distributed throughout the interstitial tissue, and were partially degranulated. Isolated PTCs expressed functional PAR(2), responded to PAR(2) activation by phosphorylating extracellular signal-regulated kinases 1/2 (ERK1/2) and activating protein kinase c, and increased intracellular Ca(2+) concentrations as well as monocyte chemoattractant protein-1 (MCP-1), transforming growth factor beta(2) (TGFbeta(2)), and cyclooxygenase-2 (COX-2) mRNA expression. Expression of these inflammatory mediators, together with iNOS, also increased significantly in testes 50 days after EAO. In vivo, expression of cytokines and inflammatory mediators was upregulated after injection of recombinant tryptase (MCP-1, TGFbeta(2), and COX-2) and a specific PAR(2) peptide agonist (MCP-1, TGFbeta(2)) in the testis after 5 h. These results suggest that PAR(2) activation elicited on PTCs by mast cell tryptase contributes to acute testicular inflammation and that this pathogenetic mechanism may also play a role in autoimmune orchitis.

[Radiation-induced cardiac disease]. / Strahlenschaden des Herzens.

Radiation-induced effects may damage various cardiac structures chronically and cause heart valve dysfunction as well as occlusive lesions of coronary and other arteries exposed to radiation. A 72-year-old woman with a history of radiation treatment after breast cancer was admitted 25 years later with symptoms of tachycardia and acute dyspnea. We found valvular thickening, medium to severe valvular dysfunction and high grade occlusive coronary artery disease in proximal portions. The left subclavian artery also was affected. Surgical treatment was required immediately. Long-term follow-up cardiac evaluation even in asymptomatic patients is mandatory to uncover cardiac injuries by radiation. To lower the risk and maximize the benefit, early intervention by valvular replacement and myocardial revascularization is indicated. Restrictive myopathy and chronic pericarditis increase risk and have to be clarified. Diagnosis in these radiation exposed patients can be made by typical findings. Echocardiography is of eminent relevancy.

Generation and characterization of a transgenic mouse with a functional human TSPY.

To generate an animal model that is suitable for the analysis of regulation and expression of human testis-specific protein, Y-encoded TSPY, a transgenic mouse line, TgTSPY9, harboring a complete structural human TSPY gene was generated. Fluorescence in situ hybridization and Southern analyses show that approximately 50 copies of the human TSPY transgene are integrated at a single chromosomal site that maps to the distal long arm of the Y chromosome. The transgene is correctly transcribed and spliced according to the human pattern and is mainly expressed in testicular tissue, with spermatogonia and early primary spermatocytes (leptotene and zygotene) as expressing germ cells. TSPY transgenic mice are phenotypically normal, and spermatogenesis is neither impaired nor enhanced by the human transgene. The present study shows that a human TSPY gene integrated into the mouse genome follows the human expression pattern although murine tspy had lost its function in rodent evolution millions of years ago.