Testicular steroid sulfatase overexpression is associated with Leydig cell dysfunction in primary spermatogenic failure.

BACKGROUND: Decreased testosterone (T) to LH ratio and increased 17ß-estradiol (E2) serum concentrations represent a common finding among patients with severe spermatogenic failure, suggesting a concurrent Leydig cell steroidogenic dysfunction. Aromatase overexpression has been associated with increased serum and intratesticular E2 in these patients. However, it is unknown whether the sulfatase pathway contributes to the increased availability of active estrogens in patients with primary spermatogenic failure. OBJECTIVES: To assess estrogen sulfotransferase (SULT1E1) and steroid sulfatase (STS) mRNA abundance in testicular tissue of patients with Sertoli cell-only syndrome (SCOS) and normal tissues, its association with serum and intratesticular hormone levels, and to explore the mRNA and protein testicular localization of both enzymes. MATERIALS AND METHODS: Testicular tissues of 23 subjects with SCOS (cases) and 22 patients with obstructive azoospermia and normal spermatogenesis (controls) were obtained after biopsy. SULT1E1 and STS transcripts accumulation was quantified by RT-qPCR. For mRNA and protein localization, we performed RT-qPCR in Leydig cell clusters and seminiferous tubules isolated by laser-capture microdissection and immunofluorescence in testicular tissues. Serum and intratesticular hormones were measured by immunoradiometric assays. RESULTS: SULT1E1 mRNA accumulation was similar in both groups. The amount of STS mRNA was higher in cases (p = 0.007) and inversely correlated with T/LH ratio (r = -0.402; p = 0.02). Also, a near significant correlation was observed with intratesticular E2 (r = 0.329, p = 0.057), in agreement with higher intratesticular E2 in cases (p < 0.001). Strong STS immunoreaction was localized in the wall of small blood vessels but not in Leydig cells. Both SULT1E1 and STS mRNA abundance was similar in Leydig cell clusters and the tubular compartment, except for lower SUTL1E1 mRNA in the seminiferous tubules of SCOS patients (p = 0.001). CONCLUSIONS: Our results suggest that an unbalance of the STS/SULT1E1 pathway contributes to the testicular hyperestrogenic microenvironment in patients with primary spermatogenic failure and Leydig cell dysfunction.

Role of indoleamine 2,3-dioxygenase in testicular immune-privilege.

Male meiotic germ cell including the spermatozoa represent a great challenge to the immune system, as they appear long after the establishment of normal immune tolerance mechanisms. The capacity of the testes to tolerate autoantigenic germ cells as well as survival of allogeneic organ engrafted in the testicular interstitium have led to consider the testis an immunologically privileged site. Disruption of this immune privilege following trauma, tumor, or autoimmune orchitis often results in male infertility. Strong evidence indicates that indoleamine 2,3-dioxygenase (IDO) has been implicated in fetal and allograft tolerance, tumor immune resistance, and regulation of autoimmune diseases. IDO and tryptophan 2,3-dioxygenase (TDO) catalyze the same rate-limiting step of tryptophan metabolism along a common pathway, which leads to tryptophan starvation and generation of catabolites collectively known as kynurenines. However, the relevance of tryptophan metabolism in testis pathophysiology has not yet been explored. Here we assessed the in vivo role of IDO/TDO in experimental autoimmune orchitis (EAO), a model of autoimmune testicular inflammation and immunologically impaired spermatogenesis. EAO was induced in adult Wistar rats with testicular homogenate and adjuvants. Control (C) rats injected with saline and adjuvants and normal untreated rats (N) were also studied. mRNA expression of IDO decreased in whole testes and in isolated Sertoli cells during EAO. TDO and IDO localization and level of expression in the testis were analyzed by immunostaining and Western blot. TDO is expressed in granulomas from EAO rats, and similar protein levels were observed in N, C, and EAO groups. IDO was detected in mononuclear and endothelial cells and reduced IDO expression was detected in EAO group compared to N and C rats. This phenomenon was concomitant with a significant reduction of IDO activity in EAO testis measured by tryptophan and kynurenine concentrations (HPLC). Finally, in vivo inhibition of IDO with 1-methyl-tryptophan increased severity of the disease, demonstrating down regulation of IDO-based tolerance when testicular immune regulation was disrupted. We present evidence that an IDO-based mechanism is involved in testicular immune privilege.

Immunolocalization of antioxidant enzymes in testis of rams submitted to long-term heat stress.

Long-term heat stress (HS) induced by testicular insulation generates oxidative stress (OS) on the testicular environment; consequently activating antioxidant enzymes such as superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx). The aim of this work was to immunolocalize antioxidant enzymes present in different cells within the seminiferous tubule when rams were submitted to HS. Rams were divided into control (n = 6) and treated group (n = 6), comprising rams subjected to testicular insulation for 240 h. After the testicular insulation period, rams were subjected to orchiectomy. Testicular fragments were submitted to immunohistochemistry for staining against SOD, GR and GPx enzymes. We observed immunolocalization of GPx in more cell types of the testis after HS and when compared with other enzymes. In conclusion, GPx is the main antioxidant enzyme identified in testicular cells in an attempt to maintain oxidative balance when HS occurs.

Biometria escroto-testicular de carneiros da raça Santa Inês e mestiços (Dorper + Santa Inês) criados sob sistema intensivo / Biometry scrotal-testicular of rams race Santa Inês and mestizos (Dorper + Santa Inês) crated under intensive system

The objective of this study was to evaluate comparatively the scrotum, scrotal circumference of thebreed rams Santa Ines and mestizos (Dorper + Santa Inês) created under intensive system. Used four rams eachrace healthy, aged between 18 and 24 months and scores ranging from 3 to 3.5. There were two collections ofscrotum-testis to determine the characteristics for each group, seven days apart. The data were submitted toanalysis of variance (ANOVA) for a randomized block design. The variables analyzed were submitted to Tukeytest at 5% probability, to compare the scrotum-testicular parameters between the races. Based on the resultsobtained, it is concluded that the scrotum-testicular parameters animal Santa Inês and mestizos (Dorper + SantaInês) created under confinement do not differ significantly (P <0.05).(AU)

Biometria escroto-testicular de carneiros da raça Santa Inês e mestiços (Dorper + Santa Inês) criados sob sistema intensivo / Biometry scrotal-testicular of rams race Santa Inês and mestizos (Dorper + Santa Inês) crated under intensive system

The objective of this study was to evaluate comparatively the scrotum, scrotal circumference of thebreed rams Santa Ines and mestizos (Dorper + Santa Inês) created under intensive system. Used four rams eachrace healthy, aged between 18 and 24 months and scores ranging from 3 to 3.5. There were two collections ofscrotum-testis to determine the characteristics for each group, seven days apart. The data were submitted toanalysis of variance (ANOVA) for a randomized block design. The variables analyzed were submitted to Tukeytest at 5% probability, to compare the scrotum-testicular parameters between the races. Based on the resultsobtained, it is concluded that the scrotum-testicular parameters animal Santa Inês and mestizos (Dorper + SantaInês) created under confinement do not differ significantly (P <0.05).

Mechanism of action of endosulfan as disruptor of gonadal steroidogenesis in the cichlid fish Cichlasoma dimerus.

The organochlorine pesticide endosulfan (ES) is used in several countries as a wide spectrum insecticide on crops with high commercial value. Due to its high toxicity to non-target animals, its persistence in the environment and its ability to act as an endocrine disrupting compound in fish, ES use is currently banned or restricted in many other countries. Previous studies on the cichlid fish Cichlasoma dimerus have shown that waterborne exposure to ES can lead to both decreased pituitary FSH content and histological alterations of testes. As gonadotropin-stimulated sex steroids release from gonads was inhibited by ES in vitro, the aim of the present study was to elucidate possible mechanisms of disruption of ES on gonadal steroidogenesis in C. dimerus, as well as compare the action of the active ingredient (AI) with that of currently used commercial formulations (CF). Testis and ovary fragments were incubated with ES (AI or CF) and/or steroidogenesis activators or precursors. Testosterone and estradiol levels were measured in the incubation media. By itself, ES did not affect hormone levels. Co-incubation with LH and the adenylate cyclase activator forskolin caused a decrease of the stimulated sex steroids release. When co-incubated with precursors dehydroandrostenedione and 17αhydroxyprogesterone, ES did not affect the increase caused by their addition alone. No differences were observed between the AI and CFs, suggesting that the effect on steroidogenesis disruption is mainly caused by the AI. Results indicate that action of ES takes place downstream of LH-receptor activation and upstream of the studied steroidogenic enzymes.

Comparison of antioxidant effects of kolaviron and vitamin C interventions on testicular structures following nevirapine administration in Sprague-Dawley rats / Comparación de los efectos antioxidantes en la intervención con kolaviron y vitamina C sobre las estructuras testiculares después de la administración de nevirapina en ratas Sprague-Dawley

Testicular toxicity has been implicated in highly active anti-retroviral therapy (HAART) treatment. Hence there is need to identify an effective antioxidant product that can alleviate testicular necrosis due to HAART administration. Forty eight adult male Sprague-Dawley rats were used in this study. The animals were divided into eight (8) groups: A-H (n= 6). Group A animals received normal saline as the control; Group B was given Nevirapine (Nv); Group C was given Kolaviron (Kv); Group D was given vitamin C; Group E was given Nv and Kv; Group F was given Nv and Vitamin C; Group G was given Nv for 56 d and Kv for 28 d serving as a withdrawal group; Group H was given corn oil. Nv, Kv and Vit. C were given at 1.54, 200 and 250 (mg·kg)/bw respectively while all administrations were through oral gavage. The body weights were taken every other day. Thereafter, they were anaesthetized with halothane. The testes were excised, weighed, fixed in Bouin's fluid and stained with H&E while the epididymes removed for semen fluid analyses. The results showed a significant (P<0.05) decrease in sperm motility in group E (Nevirapine + kolaviron) when compared with group F (Nevirapine + Vitamin C) while Sperm count was not significantly different (P>0.05) across the groups. The testicular histoarchitectural studies revealed indistinct spermatogonia, necrotic interstititial endocrine cells in the altered interstitial space, fragmented spermatids, atrophy of mature spermatocytes, degenerated germ cells, obliterated seminiferous tubules lumen, undifferentiated spermatogonia and cellular debris in the somniferous tubules lumen of nevirapine administered group but normal across the other groups. In the testis, there were no significant reduction in SOD, Catalase and GPx activities but a significant decrease in GST activity (P<0.001) when group E was compared with group F. In conclusion, vitamin C presents a better remediation in nevirapine induced spermiotoxicity compared to kolaviron in Sprague-Dawley rats.

La toxicidad testicular ha sido implicada en la terapia antirretroviral altamente activa (TARAA). Por lo tanto existe la necesidad de identificar un producto antioxidante eficaz que pueda aliviar la necrosis testicular en la administración de la TARAA. Cuarenta y ocho ratas macho Sprague-Dawley adultas fueron utilizadas. Los animales se dividieron en ocho (8) grupos: AH (n= 6). Grupo A, animales recibieron solución salina normal como el control; Grupo B, recibió Nevirapina (Nv); Grupo C, recibió Kolaviron (Kv); Grupo D, recibió vitamina C; Grupo E, recibió Nv y Kv; Grupo F, recibió Nv y vitamina C; Grupo G, recibió Nv durante 56 d y Kv por 28 d como un grupo de retirada; Grupo H, recibió aceite de maíz. Nv, Kv y Vit. C se administraron en dosis de 1, 54, 200 y 250 (mg · kg) de peso corporal respectivamente; todas las administraciones fueron por sonda oral. Los pesos corporales se tomaron cada dos días. A partir de ese momento los animales fueron anestesiados con halotano. Los testículos fueron extirpados, pesados y fijados en solución de Bouin y teñidos con H&E, mientras que el epidídimo se retiró para analizar el semen. Los resultados mostraron un descenso (p<0,05) en la motilidad de los espermatozoides en el grupo E (Nevirapina + Kolaviron) en comparación con el grupo F (Nevirapina + vitamina C), mientras que el recuento espermático no mostró diferencias significativas (P>0,05) entre los grupos. El estudio de la histoarquitectura testicular reveló espermatogonias indiferenciadas, con células intersticiales necróticas en el espacio intersticial y espermátidas fragmentadas. Además, en el grupo que recibió Nevirapina mostró espermatocitos maduros atrofiados, degeneración de células germinales, lumen de los túbulos seminíferos obliterados, espermatogonias indiferenciadas y restos celulares en el lumen de los tubulos seminíferos. En el resto de los grupos los resultados fueron normales. En el testículo hubo una reducción significativa en las actividades de la superóxido dismutasa, catalasa y glutatión peroxidasa, pero una disminución significativa en la actividad glutatión S-transferasa (P <0,001) al comparar los grupo E y F.

Effects of Sodium Arsenite and Arsenate in Testicular Histomorphometry and Antioxidants Enzymes Activities in Rats.

The main source of environmental arsenic exposure in most countries of the world is drinking water in which inorganic forms of arsenic predominate. The present study was aimed to test the impact of two different compounds of inorganic arsenic in histomorphometric and enzymatic parameters in the testes by oral exposition. Adult Wistar male rats were exposed to sodium arsenite and arsenate in drinking water, testing for each chemical form the concentrations of 0.01 and 10 mg/L per 56 days. The animals intoxicated with arsenic, mainly sodium arsenite, showed reduction in the percentage of seminiferous epithelium and in proportion and volume of Leydig cells. Moreover, there was an increase in the percentage of tunica propria, lumen, lymphatic space, blood vessels, and macrophages. The activity of superoxide dismutase (SOD) did not change among the groups. However, the activity of catalase (CAT) decreased in animals exposed to both arsenic compounds. In addition, the higher concentration of arsenic, mainly as sodium arsenite, caused vacuolization in the seminiferous epithelium. The body and testes weight as well as testosterone concentration remained unchanged among the groups. In conclusion, exposition to arsenic, mainly as sodium arsenite, caused alteration in histomorphometric parameters and antioxidant defense system in the testes.

Simultaneous Administration of Dexamethasone and Vitamin E Reversed Experimental Varicocele-induced Impact in testicular tissue in Rats; Correlation with Hsp70-2 Chaperone Expression.

PURPOSE: This study aimed to investigate the protective effects of isolated and co-administration of vitamin E (VitE) and dexamethasone (DEX) on varicocele (VCL)-induced damages in testicular tissue. MATERIALS AND METHODS: Wistar rats were divided into five groups (n=6), including; control-sham, non-treated VCL-induced, VitE-treated VCL-induced (VitE, 150 mg/kg, orally), DEX-administrated VCL-induced (DEX, 0.125 mg/kg, i.p.), VitE+DEX-received VCL-induced animals. The antioxidant status analyses, histopathological examinations, hormonal assay and tissue levels of alkaline phosphatase (ALP) were analyzed. The germinal epithelium RNA damage and Leydig cells steroidogenesis were analyzed. Moreover, the Hsp70-2 protein expression was examined based on immunohistochemical and western blot analyses. The sperm parameters, DNA integrity and chromatin condensation were investigated. RESULTS: VitE and DEX in simultaneous form of administration significantly (P<0.05) down-regulated the tissue ALP level and attenuated the VCL-decreased GSH-px, SOD and TAC levels and remarkably (P<0.05) down-regulated the testicular malondialdehyde (MDA) and nitric oxide (NO) contents. The VCL-induced histopathological alterations significantly (P<0.05) improved in VitE and DEX-administrated animals. The VitE and DEX co-administration reduced the VCL-increased RNA damage and elevated the Leydig cells steroidogenic activity. The Hsp70-2 protein level completely (P<0.05) increased in VitE and DEX alone-and-simultaneous-administrated animals. Finally, the VitE and DEX could significantly (P<0.05) improve the VCL-decreased semen quality and improved the sperm DNA integrity and chromatin condensation. CONCLUSION: Our data suggest that Vit E by up-regulating the antioxidant status and DEX by reducing inflammation-dependent oxidative and nitrosative stresses could improve the VCL-reduced Hsp70-2 chaperone expression and ultimately protected the testicular endocrine activities and promoted the spermatogenesis process.

Simultaneous Administration of Dexamethasone and Vitamin E Reversed Experimental Varicocele-induced Impact in testicular tissue in Rats; Correlation with Hsp70-2 Chaperone Expression

ABSTRACTPurpose:This study aimed to investigate the protective effects of isolated and co-administration of vitamin E (VitE) and dexamethasone (DEX) on varicocele (VCL)-induced damages in testicular tissue.Materials and Methods:Wistar rats were divided into five groups (n=6), including; control-sham, non-treated VCL-induced, VitE-treated VCL-induced (VitE, 150 mg/kg, orally), DEX-administrated VCL-induced (DEX, 0.125 mg/kg, i.p.), VitE+DEX-received VCL-induced animals. The antioxidant status analyses, histopathological examinations, hormonal assay and tissue levels of alkaline phosphatase (ALP) were analyzed. The germinal epithelium RNA damage and Leydig cells steroidogenesis were analyzed. Moreover, the Hsp70-2 protein expression was examined based on immunohistochemical and western blot analyses. The sperm parameters, DNA integrity and chromatin condensation were investigated.Results:VitE and DEX in simultaneous form of administration significantly (P<0.05) down-regulated the tissue ALP level and attenuated the VCL-decreased GSH-px, SOD and TAC levels and remarkably (P<0.05) down-regulated the testicular malondialdehyde (MDA) and nitric oxide (NO) contents. The VCL-induced histopathological alterations significantly (P<0.05) improved in VitE and DEX-administrated animals. The VitE and DEX co-administration reduced the VCL-increased RNA damage and elevated the Leydig cells steroidogenic activity. The Hsp70-2 protein level completely (P<0.05) increased in VitE and DEX alone–and-simultaneous-administrated animals. Finally, the VitE and DEX could significantly (P<0.05) improve the VCL-decreased semen quality and improved the sperm DNA integrity and chromatin condensation.Conclusion:Our data suggest that Vit E by up-regulating the antioxidant status and DEX by reducing inflammation-dependent oxidative and nitrosative stresses could improve the VCL-reduced Hsp70-2 chaperone expression and ultimately protected the testicular endocrine activities and promoted the spermatogenesis process.

Impairment of male reproductive function after sleep deprivation.

OBJECTIVE: To evaluate the influence of sleep loss on sexual behavior, hormone levels, sperm parameters, and testis-specific gene expression in male rats. DESIGN: Experimental research. SETTING: Animal laboratory. ANIMAL(S): Male adult Wistar-Hannover rats. INTERVENTION(S): Sexually experienced rats were subjected to paradoxic sleep deprivation (PSD) for 96 hours or sleep restriction (SR) for 21 days or kept in their home cage as control (CTRL). MAIN OUTCOME MEASURE(S): Sexual behavior, hormone levels, sperm parameters and expression of stress and nitric oxide-related genes were evaluated. RESULT(S): PSD significantly decreased sexual behavior compared with the CTRL group, whereas SR had no effect. The PSD group had significantly lower testosterone levels than the CTRL group. Both PSD and SR groups had lower sperm viabilities than the CTRL group. The decrease in the number of live sperm compared with the CTRL group was larger in the PSD group than in the SR group. Regarding testicular gene expression, both PSD and SR led to an increase of iNOS and hydroxysteroid 11ß-dehydrogenase 1 expressions compared with the CTRL group. These changes were more pronounced in the PSD group. A significant increase in endothelial nitric oxide synthase expression was observed in the PSD groups compared with the CTRL group. No changes were observed in dimethylarginine dimethylaminohydrolase 1 and casein kinase 2ß-polypeptide expressions. CONCLUSION(S): Sleep loss can promote marked changes in the male reproductive system of rats, particularly affecting spermatic function in part by interfering in the testicular nitric oxide pathway.

Cadmium exposure inhibits MMP2 and MMP9 activities in the prostate and testis.

Matrix metalloproteinases (MMPs) are zinc (Zn(2+)) and calcium (Ca(2+)) dependant endopeptidases, capable of degradation of numerous components of the extracellular matrix. Cadmium (Cd(2+)) is a well known environmental contaminant which could impair the activity of MMPs. In this sense, this study was conducted to evaluate if Cd(2+) intake inhibits these endopeptidases activities at the rat prostate and testicles and if it directly inhibits the activity of MMP2 and MMP9 at gelatinolytic assays when present in the incubation buffer. To investigate this hypothesis, Wistar rats (5 weeks old), were given tap water (untreated, n = 9), or 15 ppm CdCl2 diluted in drinking water, during 10 weeks (n = 9) and 20 weeks (n = 9). The animals were euthanized and their ventral prostate, dorsal prostate, and testicles were removed. These tissue samples were processed for protein extraction and subjected to gelatin zymography evaluation. Additionally, we performed an experiment of gelatin zymography in which 5 µM or 2 mM cadmium chloride (CdCl2) was directly dissolved at the incubation buffer, using the prostatic tissue samples from untreated animals that exhibited the highest MMP2 and MMP9 activities in the previous experiment. We have found that CdCl2 intake in the drinking water led to the inhibition of 35% and 30% of MMP2 and MMP9 (p < 0.05) at the ventral prostate and testis, respectively, in Cd(2+) treated animals when compared to controls. Moreover, the activities of the referred enzymes were 80% and 100% inhibited by 5 µM and 2 mM of CdCl2, respectively, even in the presence of 10 mM of CaCl2 within the incubation buffer solution. These important findings demonstrate that environmental cadmium contamination may deregulate the natural balance in the extracellular matrix turnover, through MMPs downregulation, which could contribute to the toxic effects observed in prostatic and testicular tissue after its exposure.

Identification and characterization of a novel splice variant of the PLCζ1 gene in bull testis tissues.

Phospholipase C zeta 1 (PLCζ1), which transcribes a key protein, has an important function in oocyte activation and embryo development because PLCζ1 can trigger a series of intracellular Ca2+ oscillations in mammals. In this study, a novel splice variant in the testis tissues of adult and fetal Chinese Holstein bulls was characterized by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing analysis. The novel splice variant PLCζ1-sv1 was derived from the PLCζ1 complete transcript (PLCζ1-complete) by alternative splicing; the alternative splicing pattern exhibited alternative 5'-splice sites. The full-length transcript, PLCζ1-complete, is the main transcript found in fetal and adult cow testis tissue. Quantitative real-time PCR (qPCR) analysis demonstrated that the expression levels of the PLCζ1-complete transcript were significantly higher than those of the PLCζ1-sv1 splice variant in bovine testis tissues. PLCζ1 protein sequencing analysis showed that the amino acids at positions 453 to 457 were deleted in PLCζ1-sv1, thereby terminating transcription prematurely. In summary, this study provided information to elucidate the structure and function of the bovine PLCζ1 gene.

Levels of the retinoic acid synthesizing enzyme aldehyde dehydrogenase-1A2 are lower in testicular tissue from men with infertility.

OBJECTIVE: To determine whether decreased testicular levels of enzymes necessary for retinoic acid biosynthesis were associated with male infertility, as retinoic acid is known to be necessary for spermatogenesis. DESIGN: Observational analysis of testicular tissue samples, sperm indices, and serum hormone concentrations. SETTING: Two infertility centers in Chile. PATIENT(S): 32 infertile men and 11 control men. INTERVENTION(S): Measurement of the three enzymes necessary for retinoic acid biosynthesis, aldehyde dehydrogenase (ALDH) 1A1, 1A2, and 1A3, in testicular tissue by a novel liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) peptide assay. MAIN OUTCOME MEASURE(S): ALDH isozyme levels compared by type of infertility and correlated with testicular germ cell numbers, sperm parameters, and serum and intratesticular hormone concentrations. RESULT(S): Men with infertility had statistically significantly reduced levels of ALDH1A2 but not ALDH1A1 or ALDH1A3 in their testicular tissue compared with men with normal spermatogenesis. The ALDH1A2 protein levels were strongly correlated with the number of germ cells found via testicular biopsy. CONCLUSION(S): These findings suggest that ALDH1A2 is the enzyme involved in retinoic acid biosynthesis in human germ cells. Further study of the relationship between intratesticular ALDH1A2 and male infertility is warranted to determine whether men with infertility have a reduced ability to synthesize retinoic acid within their germ cells that could impair spermatogenesis.

Serotonin in testes of bat Myotis velifer during annual reproductive cycle: expression, localization, and content variations.

The mechanism of reproduction in mammals is very complex and in some cases is quite particular. For example in some bat species, the male presents a reproductive mechanism characterized by an annual testicular cycle that goes from recrudescence to regression (spermatogenesis to inactivity period, respectively). After recrudescence, the spermatozoa arrive at epididymis and wait to be expelled at the time of ejaculation during the mating period, which occurs some months later. Because serotonin (5-HT) has gained reproductive importance in the last years, the aim of the present study was to analyze the expression of this indolamine and both tryptophan hydroxylase and monoamine oxidase isoform A-enzymes involved in its metabolism-in Myotis velifer testes, a seasonal reproductive bat species that shows temporal asynchrony in its sexual cycle, across the principal periods of their reproductive cycle. By using both Falck-Hillarp histochemistry and immunofluorescence techniques, we found serotonin in vesicles of Leydig cells and probably Sertoli cells too; interestingly, both intracellular localization and concentration was variable across the different stages of the reproductive cycle, being lower during spermatogenesis phase and increasing during the mating phase. These results suggest that 5-HT is present in bat testes and it could play an important role in testicular function during their reproductive cycle.

Muscle glycogen synthase isoform is responsible for testicular glycogen synthesis: glycogen overproduction induces apoptosis in male germ cells.

Glycogen is the main source of glucose for many biological events. However, this molecule may have other functions, including those that have deleterious effects on cells. The rate-limiting enzyme in glycogen synthesis is glycogen synthase (GS). It is encoded by two genes, GYS1, expressed in muscle (muscle glycogen synthase, MGS) and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase, LGS). Expression of GS and its activity have been widely studied in many tissues. To date, it is not clear which GS isoform is responsible for glycogen synthesis and the role of glycogen in testis. Using RT-PCR, Western blot and immunofluorescence, we have detected expression of MGS but not LGS in mice testis during development. We have also evaluated GS activity and glycogen storage at different days after birth and we show that both GS activity and levels of glycogen are higher during the first days of development. Using RT-PCR, we have also shown that malin and laforin are expressed in testis, key enzymes for regulation of GS activity. These proteins form an active complex that regulates MGS by poly-ubiquitination in both Sertoli cell and male germ cell lines. In addition, PTG overexpression in male germ cell line triggered apoptosis by caspase3 activation, proposing a proapoptotic role of glycogen in testis. These findings suggest that GS activity and glycogen synthesis in testis could be regulated and a disruption of this process may be responsible for the apoptosis and degeneration of seminiferous tubules and possible cause of infertility.

γ-Oryzanol protects against acute cadmium-induced oxidative damage in mice testes.

Cadmium is a non-essential heavy metal that is present at low levels mainly in food and water and also in cigar smoke. The present study evaluated the testicular damage caused by acute cadmium exposure and verified the protective role of γ-oryzanol (ORY). Mice were administrated with a single dose of 2.5mg/kg of CdCl2, and then treated with ORY (50mM in canola oil, 5mL/kg). Testes were removed after 24h and tested for lipid peroxidation (TBARS), protein carbonylation, DNA breakage, ascorbic acid, cadmium and non-proteic thiols contents, and for the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and δ-aminolevulic acid dehydratase (δ-ALA-D). Cadmium presented a significant alteration in all parameters, except GPx and CAT activities. Therapy reduced in a slight degree cadmium concentration in testes (around 23%). ORY restored SOD and GST activities as well as TBARS production to the control levels. Furthermore, ORY partially recovered δ-ALA-D activity inhibited by cadmium. This study provides the first evidence on the therapeutic properties of ORY in protecting against cadmium-induced testicular toxicity.

Blueberry extracts protect testis from hypobaric hypoxia induced oxidative stress in rats.

Exposure to hypobaric hypoxia causes oxidative damage to male rat reproductive function. The aim of this study was to evaluate the protective effect of a blueberry extract (BB-4) in testis of rats exposed to hypobaric hypoxia. Morphometric analysis, cellular DNA fragmentation, glutathione reductase (GR), and superoxide dismutase (SOD) activities were evaluated. Our results showed that supplementation of BB-4 reduced lipid peroxidation, decreased apoptosis, and increased GR and SOD activities in rat testis under hypobaric hypoxia conditions (P < 0.05). Therefore, this study demonstrates that blueberry extract significantly reduced the harmful effects of oxidative stress caused by hypobaric hypoxia in rat testis by affecting glutathione reductase and superoxide dismutase activities.

Up regulation of nitric oxide synthase-nitric oxide system in the testis of rats undergoing autoimmune orchitis.

BACKGROUND: Male reproductive tract infection and inflammation are important aetiological factors of infertility. Experimental Autoimmune Orchitis (EAO) is a model of chronic inflammation useful to study mechanisms of inflammatory reactions leading to testicular impairment. EAO is characterised by interstitial cell infiltrate of lymphomonocytes, producers of pro-inflammatory cytokines involved in germ cell apoptosis. Nitric oxide (NO), a free radical promoting immune cell activation and apoptosis, is synthesised by conversion of l-arginine to l-citrulline catalysed by NO synthase (NOS). The NOS isoforms are: constitutively endothelial (e) and neuronal (n) NOS and inducible (i) NOS. OBJECTIVES: Although the NO-NOS system was found to be up-regulated by pro-inflammatory mediators in immune and non immune testicular cells, data on its regulation in chronic inflammatory states is lacking. METHODS AND RESULTS: EAO was induced in rats by active immunisation with spermatic antigens and adjuvants; control (C) rats were injected with adjuvants. Untreated normal (N) rats were also studied. We demonstrated that iNOS, eNOS and nNOS was mainly expressed by interstitial cells in N and C rats and that in EAO NOS was up-regulated and also expressed by tubular cells. Constitutive and inducible NOS content (Western blot) as well as NO production and activity increased in the testis of rats with EAO. iNOS content and activity were selectively up-regulated in the testis of rats with orchitis. Flow cytometric analysis of NOS isoforms in testicular macrophages (M) showed that the percentage of ED1(+)ED2(-) and ED1(+)ED2(+) M subsets, expressing constitutive and iNOS isoforms was significantly higher in EAO, but no change in the percentage of ED1(-)ED2(+) resident M was observed compared to C rats. M from EAO rats also released more NO than C and N rats. CONCLUSIONS: In testis of rats with EAO, NO-NOS system was up-regulated and both testicular M and cells from seminiferous tubules contributed to NO increase. NO over production in orchitis was generated mainly by increased iNOS content and activity.