RESUMEN
Nutrients and environmental chemicals, including endocrine disruptors, have been incriminated in the current increase in male reproductive dysfunction, but the underlying mechanisms remain unknown. The gastrointestinal tract represents the largest surface area exposed to our environment and thereby plays a key role in connection with exposure of internal organs to exogenous factors. In this context the gut microbiome (all bacteria and their metabolites) have been shown to be important contributors to body physiology including metabolism, cognitive functions and immunity. Pivotal to male reproduction is a proper development of the testis, including the formation of the blood-testis barrier (BTB) that encapsulates and protects germ cells from stress induced environmental cues, e.g. pathogenic organisms and xenobiotics. Here we used specific pathogen free (SPF) mice and germ-free (GF) mice to explore whether gut microbiota and/or their metabolites can influence testis development and regulation of BTB. Lumen formation in the seminiferous tubules, which coincides with the development of the BTB was delayed in the testes of GF mice at 16 days postpartum. In addition, perfusion experiments (Evans blue) demonstrated increased BTB permeability in these same mice. Reduced expressions of occludin, ZO-2 and E-cadherin in GF testis suggested that the microbiota modulated BTB permeability by regulation of cell-cell adhesion. Interestingly, exposure of GF mice to Clostridium Tyrobutyricum (CBUT), which secrete high levels of butyrate, restored the integrity of the BTB and normalized the levels of cell adhesion proteins. Moreover, the GF mice exhibited lower serum levels of gonadotropins (LH and FSH) than the SPF group. In addition, the intratesticular content of testosterone was lower in GF compared to SPF or CBUT animals. Thus, the gut microbiome can modulate the permeability of the BTB and might play a role in the regulation of endocrine functions of the testis.
Asunto(s)
Microbiota , Testículo/crecimiento & desarrollo , Animales , Barrera Hematotesticular/crecimiento & desarrollo , Adhesión Celular , Fertilidad , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos , Permeabilidad , Organismos Libres de Patógenos EspecíficosRESUMEN
Gonadotropins and testosterone are important regulators of spermatogenesis, even though gonadotropin receptors and the androgen receptor are not expressed by germ cells. However, a functional role for estrogens in connection with male reproduction has been postulated on the basis of the phenotypes of mice lacking estrogen receptor (ER) and cytochrome P-450 aromatase. This has further support by findings of ER expression in the testis, including that of ERbeta in spermatogonia. 5alpha-Androstane-3beta, 17beta-diol (3betaAdiol), a metabolite of testosterone produced via the intermediate potent androgen 5alpha-dihydrotestosterone (DHT), has been reported to selectively bind ERbeta rather than EpsilonRalpha, but not androgen receptor. Here, we have characterized the influence of 17beta-estradiol (E), the major physiological estrogen, 3betaAdiol, and DHT on DNA synthesis in vitro by segments of the seminiferous epithelium at different stages of the seminiferous epithelial cycle in the rat. E and 3betaAdiol exerted similar stimulatory effects on premitotic DNA synthesis in stage I segments, whereas other stages tested (V, VIIa, and XIII-IX) remained unresponsive. In contrast, DHT had no effect on this process. 5-bromo-2'-deoxyuridine labeling of stage I segments revealed a 30-fold higher labeling index in the presence than in the absence of E, and the labeled cells were identified as spermatogonia. Moreover, high levels of 3betaAdiol were found in the testis of intact rats as well as in primary cultures of rat Leydig cells in response to human chorionic gonadotropin. We suggest that 3betaAdiol may serve as a growth factor for germ cells stimulating premitotic DNA synthesis in connection with spermatogenesis via an ERbeta-dependent pathway.
Asunto(s)
Androstano-3,17-diol/farmacología , Células Epiteliales/fisiología , Receptor beta de Estrógeno/fisiología , Túbulos Seminíferos/fisiología , Espermatogonias/fisiología , Animales , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Dihidrotestosterona/farmacología , Células Epiteliales/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Túbulos Seminíferos/citología , Túbulos Seminíferos/efectos de los fármacos , Espermatogonias/efectos de los fármacosRESUMEN
BACKGROUND: The high mobility group box chromosomal protein 1 (HMGB1) was originally shown to be a nuclear DNA-binding protein that activates transcription and promotes differentiation. More recently, there have been reports that HMGB1 may also function as a pro-inflammatory and antibacterial factor. The aim of this study was to investigate the testicular expression and antibacterial functions of HMGB1 to elucidate a possible role of HMGB1 in the testicular barrier defence against infections. METHODS AND RESULTS: RT-PCR and in situ hybridization revealed high-level testicular expression of HMGB1 mRNA and localization of this expression to the Sertoli cells and germ cells of the human and rat testis. In addition, immunohistochemical examination demonstrated the presence of the corresponding protein in Sertoli cells and spermatogonia in human and rat testes. Western blotting detected abundant amounts of the HMGB1 protein in the interstitial and intratubular fluids of the intact adult rat testis. Finally, the HMGB1 protein purified from both human and rat testis by reversed-phase high-performance liquid chromatography (HPLC) exerted antibacterial activity towards Bacillus megaterium in an inhibition zone assay. CONCLUSION: HMGB1 is expressed by Sertoli cells and germ cells in the mammalian testis. In addition, purified testicular HMGB1 shows antibacterial activity, indicating that this protein may function as a paracrine host defence factor in the testis.
Asunto(s)
Antibacterianos , Proteína HMGB1/genética , Proteínas del Grupo de Alta Movilidad/genética , Proteínas Represoras/genética , Testículo/fisiología , Adulto , Animales , Bacillus megaterium/efectos de los fármacos , Cartilla de ADN , Proteína HMGB1/farmacología , Proteínas del Grupo de Alta Movilidad/farmacología , Humanos , Hibridación in Situ , Masculino , ARN Mensajero/genética , Ratas , Proteínas Represoras/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Sertoli/fisiología , Espermatozoides/fisiologíaRESUMEN
Humanin (HN) is a 24 amino acids peptide with potent neuro-survival properties that protects against damage associated with Alzheimer's disease. In the present report, we have demonstrated by immunohistochemical analysis and Western blotting the pattern of expression of rat humanin (HNr) in the testis of 10- to 60-day-old rats. The Leydig cells of 10- and 40- day-old rats expressed this peptide at high levels; and in the testis of 60-day-old rats the expression of HNr expanded to include Leydig, endothelial, peritubular and germ cells. As monitored by Western blotting, HNr was released into the medium of cultures of Leydig cells isolated from 10-, 40-, and 60-days-old rats. HNr stimulated the incorporation of [(3)H]TdR into DNA of Leydig cells from 10-days-old rats, in a manner that indicated promotion of cell survival rather than an increase in the rate of cell multiplication. This peptide also enhanced steroidogenesis by cultured Leydig cells from 10- to 40-day-old rats both alone and synergistically with IGF-I. The expression of HNr in cultured Leydig cells increased in response to GH and IGF-I. In summary, we demonstrated here that HNr was expressed at all stages of maturation in the rat testis. This peptide promoted the survival of Leydig cells in culture and interacted with IGF-I to stimulate DNA synthesis and steroidogenesis. We propose that HNr is a novel testicular anti-apoptotic factor.
Asunto(s)
Apoptosis/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Intersticiales del Testículo/fisiología , Espermatogénesis , Testículo/citología , Testículo/metabolismo , Animales , Técnicas de Cultivo de Célula , Muerte Celular/fisiología , Células Cultivadas , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/química , Células Intersticiales del Testículo/citología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein important for viral binding to target cells. Using RT-PCR, Western analysis, GST pull-down assay and indirect immunofluorescence, it was shown that CAR is expressed in male germ cells from mice, rats, and humans. CAR was detected in round spermatids in the testis as well as in purified, mature spermatozoa. The two membrane-bound isoforms of CAR occupied different subcellular sites in the acrosomal region of the spermatozoa. CAR was exposed on the surface of acrosome-reacted, but not acrosome-intact cells. Two CAR-binding proteins belonging to the ligand-of-numb protein-X (LNX) family also occupied distinct regions in spermatozoa. Finally, co-immunoprecipitation experiments demonstrated an interaction between CAR and JAM-C, a protein required for spermatid differentiation. Together, these findings imply a function for CAR in male fertility. The results also suggest that CAR in spermatozoa is inaccessible to adenovirus-based gene therapy vectors, and that the risk of germ line infection therefore is low.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Moléculas de Adhesión Celular/fisiología , Regulación de la Expresión Génica , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Virales/metabolismo , Testículo/citología , Acrosoma/metabolismo , Animales , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Epidídimo/química , Epidídimo/fisiología , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Receptores Virales/genética , Receptores Virales/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Túbulos Seminíferos/química , Túbulos Seminíferos/citología , Túbulos Seminíferos/fisiología , Espermatogénesis/genética , Espermatogénesis/fisiología , Espermatozoides/química , Espermatozoides/citología , Espermatozoides/fisiología , Testículo/química , Testículo/fisiología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Although host-defence mechanisms, designed to preserve the integrity of the developing germ cells are operative in the testis, the components of this protective system have yet to be characterised in detail. Here, we report that the cytokine interleukin-18 (IL-18) is expressed in the rat testis and may contribute to these defences. Thus, analysis by RT-PCR and Western blotting revealed pronounced testicular expression of pro-IL-18 from postnatal day 5 and onwards. Expression of both IL-18 mRNA and protein was found to be localised to meiotic and post-meiotic germ cells as evaluated by in situ hybridisation and immunohistochemistry, respectively. The mRNA species coding for the IL-18 receptor and IL-1beta converting enzyme, which activates pro-IL-18, were also shown to be expressed by the seminiferous tubules. Recombinant IL-18 was seen to stimulate spermatogonial DNA synthesis in cultures of staged segments of rat seminiferous tubules, without influencing germ cell apoptosis. These results suggest that IL-18 may have host-protective and growth-promoting functions in the testis, but further investigations need to be done to confirm this.
Asunto(s)
Interleucina-18/metabolismo , Espermatogénesis/inmunología , Espermatozoides/crecimiento & desarrollo , Testículo/metabolismo , Animales , Apoptosis , Replicación del ADN/efectos de los fármacos , Interleucina-18/genética , Interleucina-18/farmacología , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Masculino , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Espermatozoides/química , Testículo/citología , Testosterona/biosíntesisRESUMEN
Helicobacter pylori infection is associated with a variety of outcomes ranging from seemingly asymptomatic coexistence to peptic ulcer disease and gastric cancer. The cag pathogenicity island (PAI) contains genes associated with a more aggressive phenotype and has been suggested to be a determinant of severe disease outcome. The cagA gene has served as a marker for the cag PAI. However, the presence of this single gene does not necessarily indicate the presence of a complete set of cag PAI genes. We have analyzed the composition of the cag PAI in 66 clinical isolates obtained from patients with duodenal ulcer, gastric cancer, and nonulcer dyspepsia. Hybridization of DNA to microarrays containing all the genes of the cag PAI showed that 76 and 9% of the strains contained all or none of the cag PAI genes, respectively. Partial deletions of the cag PAI were found in 10 isolates (15%), of which 3 were cagA negative. The ability to induce interleukin-8 (IL-8) production in AGS cells was correlated to the presence of a complete cag PAI. Strains carrying only parts of the island induced IL-8 at levels significantly lower than those induced by cag PAI-positive isolates. The presence of an intact cag PAI correlates with development of more severe pathology, and such strains were found more frequently in patients with severe gastroduodenal disease (odds ratio, 5.13; 95% confidence interval, 1.5 to 17.4). Partial deletions of the cag PAI appear to be sufficient to render the organism less pathogenic.