Estrogenic and anti-androgenic effects of the herbicide tebuthiuron in male Nile tilapia (Oreochromis niloticus).

Tebuthiuron is a phenylurea herbicide widely used in agriculture that can reach the aquatic environments, possibly posing negative effects to the aquatic biota. Phenylurea herbicides, such as diuron, are known to cause estrogenic and anti-androgenic effects in fish, but no such effects were yet reported for tebuthiuron exposure. Thus, the aim of this study was to evaluate if tebuthiuron, at environmentally relevant concentrations (100 and 200ng/L) and after 25days of exposure have estrogenic and/or anti-androgenic effects on male of Nile tilapia (Oreochromis niloticus), through the evaluation of plasmatic testosterone (T) and estradiol (E2) levels, brain aromatase (CYP19) levels (western-blot), and by evaluating the histology of the testicles. When compared to the control group, plasmatic T levels decreased about 76% in the animals exposed to 200ng/L of tebuthiuron, while E2 levels increased about 94%, which could be related to a significant increase (77%) in CYP19A1 levels, an enzyme that catalyzes the conversion of androgens into estrogens. Histological analyses of the testicles also demonstrated that tebuthiuron at both tested concentrations caused a decrease in the diameter of the seminiferous tubules and in the diameter of the lumen. Therefore, the gonadosomatic index (GSI) was reduced by 36% % in the animals exposed 200ng/L to tebuthiuron. Indeed, the relative frequency of spermatocytes and spermatids increased respectively 73% (200ng/L) and 61% (100ng/L) in the tebuthiuron exposed animals, possibly due to the impairment of sperm release into the lumen, that was decreased 93% (200ng/L) in the treated animals compared to the control. These results confirm that tebuthiuron causes estrogenic and anti-androgenic effects in Nile tilapias at environmentally relevant concentrations.

Evolution of cadmium effects in the testis and sperm of the tropical fish Gymnotus carapo.

The present study investigated the testis and sperm morphology of the tropical fish Gymnotus carapo after exposure to increasing CdCl2 concentrations (5-40 µM) for 24 and 96 h. The treatments induced Cd accumulation in the testis and a decrease in the gonadosomatic index from a 10 µM. Cd induced alterations in testis since 24h; however the extension and severity of damages increased after 96 h in all tested concentrations. Marked variations in the cysts size, proliferation of the interstitial tissue, infiltration of inflammatory cells, necrosis, reduction of germ cells and sperm aggregation was observed in 96 h treated fishes. In this time, there was a complete absence of germ cells in the testis of fish treated with 40 µM. The ultrastructural analysis allowed for the visualization of the initial damages over germ cells, such as the presence of vacuoles in the cytoplasm of spermatogonia, spermatocytes, and spermatids. Exposed fish (20 µM for 24 and 96 h) had alterations in sperm number and morphology. These results are important for establishing a direct correlation between the Cd accumulation and incidence of damages and can help characterize the mechanism of Cd-induced pathogenesis in the male reproductive system.

Endocrine and testicular changes induced by olanzapine in adult Wistar rats.

Olanzapine is an atypical antipsychotic drug that has been increasingly used in acute treatment of, and therapeutic support for, schizophrenia, bipolar disorder and other psychoses. Considering that olanzapine acts on the dopaminergic receptor and this receptor is detected in germ cells, the present study aims to investigate the effects of treatment with different doses of olanzapine on spermatogenesis, plasma testosterone and weight of androgen-dependent organs in rats. Results showed reduced plasma testosterone levels, and reduced testis, epididymis and prostate weights. Histopathologic and histomorphometric analysis of spermatogenesis indicated testicular degeneration. Furthermore, germ cell desquamation, syncytial multinucleated cells, Sertoli cell vacuolization and presence of necrotic and apoptotic germ cells wwew observed. Olanzapine treatment in rats promoted endocrinological changes and lesions in the testis, leading to a disturbance in spermatogenesis.

A molecular evaluation of germ cell death induced by etoposide in pubertal rat testes.

Etoposide is widely used in the treatment of patients with testicular cancer. The mechanism underlying apoptosis induction in cancer cells has been studied in different cell types, but it is not known whether the same factors participate in viable germ cells undergoing programmed cell death. Since testicular cancer primarily affects young males, we used pubertal rats (21 days old) as a model to determine different apoptotic parameters after etoposide treatment in healthy testes. We found that one intratesticular injection of etoposide (1.2 microg/testis) induced a significant increase in spermatocytes undergoing apoptosis, along with activation of caspase-9, -8 and -3 after 24 h of treatment. Spermatocyte apoptosis was inhibited when a general caspase inhibitor was added along with etoposide. Etoposide induces a significant stabilization/activation of p53, resulting in an increase level of this protein. The mRNA of Bcl-2 antagonist of cell death (BAD), a pro-apoptotic gene and a transcriptional target of p53, was significantly increased after etoposide treatment. Thus, our results suggest a single injection of etoposide induces apoptosis in healthy pachytene spermatocytes mediated by p53 and caspase activation. These findings will assist the search for new therapies to prevent the deleterious effect of cancer drugs upon normal cells.

Ultrastructural findings in murine seminiferous tubules as a consequence of subchronic vanadium pentoxide inhalation.

Vanadium (V) is a transition metal emitted to the atmosphere during the combustion of fossil fuels. Its current status as an atmospheric pollutant increases the need for information about the effects that this element might have on the reproductive health of exposed populations. The present study investigated changes in testicular ultrastructure following inhalation exposure of male mice to V (as vanadium pentoxide). Tissue V level was constant during the 12-week time period. We observed necrosis of spermatogonium, spermatocytes and Sertoli cells, as well as pseudo-nuclear inclusion and disruption of cellular junctions. Our findings stressed the importance of the hemato-testicular barrier in supporting the function of Sertoli cells and suggest as a possible target of V, tight junction proteins. Further analysis is needed in order to identify the role that reactive oxidative species (ROS) might have on these cellular junctions, and if a specific protein is the target of its toxic effects. The relevance of this report concerns the impact that metal air pollution could have on male fertility in dense cities with vehicular traffic problems.

Effect of di-(2-ethylhexyl) phthalate on N-cadherin and catenin protein expression in rat testis.

This study investigated the effect of DEHP exposure on N-cadherin and alpha-, beta- and p120-catenin immunoreactivities in the rat testis. DEHP was administered by daily gavage to 25-day-old male Sprague-Dawley rats at a dose of 2 g DEHP/5 ml corn oil/kg body weight for 2 days or 7 days. Control rats were treated with corn oil vehicle under the same conditions. Animals were killed at 24h after the last treatment. Another group of rats treated with DEHP or corn oil vehicle (control group) for 2 days were held for 30 days without treatment to observe recovery. Testes were analyzed for histopathology, TUNEL staining, immunofluorescence (IF) and Western blot analyses. Animals exposed to DEHP for 2 days or 7 days showed severe alterations of seminiferous tubules characterized by germ cell sloughing. Animals from the longer term recovery group treated with DEHP showed foci of delayed spermatogenesis. A linear and continuous pattern of N-cadherin was observed in the basal compartment of the seminiferous tubules. A similar pattern but with higher IF intensity was observed for N-cadherin in rats treated with DEHP for 2 days or 7 days, compared to control animals. The alpha-, beta- and p120-catenins were detected in the basal compartment of seminiferous tubules in similar localization and IF pattern for DEHP and control groups. A significant increase in testicular N-cadherin and alpha-catenin levels was detected by Western blot analysis in DEHP-exposed versus control rats. No variations in N-cadherin or catenin expression were detected in the recovery groups. These findings demonstrate that DEHP induces an up-regulation of N-cadherin and alpha-catenin expression and may perturb cell-cell adhesion phenomena in the seminiferous tubule.

Hexose transporters GLUT1 and GLUT3 are colocalized with hexokinase I in caveolae microdomains of rat spermatogenic cells.

Postmeiotic spermatogenic cells, but not meiotic spermatogenic cells respond differentially with glucose-induced changes in [Ca2+]i indicating a differential transport of glucose via facilitative hexose transporters (GLUTs) specifically distributed in the plasma membrane. Several studies have indicated that plasma membrane in mammalian cells is not homogeneously organized, but contains specific microdomains known as detergent-resistant membrane domains (DRMDs), lipid rafts or caveolae. The association of these domains and GLUTs isoforms has not been characterized in spermatogenic cells. We analyzed the expression and function of GLUT1 and GLUT3 in isolated spermatocytes and spermatids. The results showed that spermatogenic cells express both glucose transporters, with spermatids exhibiting a higher affinity glucose transport system. In addition, spermatogenic cells express caveolin-1, and glucose transporters colocalize with caveolin-1 in caveolin-enriched membrane fractions. Experiments in which the integrity of caveolae was disrupted by pretreatment with methyl-beta-cyclodextrin, indicated that the involvement of cholesterol-enriched plasma membrane microdomains were involved in the localization of GLUTs and uptake of 2-deoxyglucose. We also observed cofractionation of GLUT3 and caveolin-1 in low-buoyant density membranes together with their shift to higher densities after methyl-beta-cyclodextrin treatment. GLUT1 was found in all fractions isolated. Immunofluorescent studies indicated that caveolin-1, GLUT1, and hexokinase I colocalize in spermatocytes while caveolin-1, GLUT3, and hexokinase I colocalize in spermatids. These findings suggest the presence of hexose transporters in DRMDs, and further support a role for intact caveolae or cholesterol-enriched membrane microdomains in relation to glucose uptake and glucose phosphorylation. The results would also explain the different glucose-induced changes in [Ca2+]i in both cells.

Spermatogenesis and blood-testis barrier in rats after long-term Vitamin A deprivation.

It has been established that experimental avitaminosis A in rats results in a 'Sertoli cell-only situation' after about 10 weeks, and that replacing the vitamin immediately reinitiates spermatogenesis. The present study deals with testicular recovery after prolonged deprivation (up to 19 weeks). The Sertoli cell-only situation reached under this condition was thought to be refractory to Vitamin A replacement. However, spermatogenesis did reinitiate about 11 weeks after vitamin restoration, although in an atypical manner. The blood-testis barrier, normally assembled when spermatocytes reaches the zygotene stage, remained under this condition permeable to the lanthanum intercellular tracer. Concomitantly, primary spermatocytes normally found in the adluminal compartment isolated by the barrier (zygotene onward) became massively apoptotic. All the tubules containing early spermatocytes (preleptotene or leptotene cells), normally found in the basal compartment, exhibited normal features with no signs of degeneration. Based on these results, a possible relationship between blood-testis barrier assembly and spermatocyte differentiation is proposed.

The goitrogen 6-n-propyl-2-thiouracil (PTU) given during testis development increases Sertoli and germ cell numbers per cyst in fish: the tilapia (Oreochromis niloticus) model.

The main objectives of the present study were to investigate the effects of 6-n-propyl-2-thiouracil (PTU) on Sertoli cell proliferation, germ cell number, and testis size in Nile tilapias (Oreochromis niloticus). In this regard, young fish (approximately 1 g BW and approximately 3.5 cm total in length) were treated for a period of 40 d with different concentrations (100 and 150 ppm) of PTU. The animals were killed and analyzed on d 1, 30, 40, 98, and 208 after the beginning of the treatment. On d 30 and 40 the spermatogenic process was delayed in fish treated with PTU compared with the control group. Also at these periods, treated tilapia had decreased (P < 0.05) body weight and total length. On d 98 body weight and total length had recovered in PTU-treated fish and were similar (P > 0.05) to those of the controls. However, testis weight and gonadosomatic index (testis mass/body weight) were approximately 100% higher (P < 0.05) in treated tilapia. Similarly, the area occupied by seminiferous tubules, the number of Sertoli cells and germ cells per cyst, and the number of Leydig cells per testis were significantly (P < 0.05) greater in treated fish. Nevertheless, nuclear volume and individual Leydig cell volume were significantly lower (P < 0.05) in tilapia receiving PTU treatment. Compared with controls, at 208 d all parameters analyzed presented the same trend as that observed at 98 d. In general, at 98 d the different PTU concentrations used during the treatment period induced similar effects. However, at 208 d the mean values observed for several parameters were significantly higher (P < 0.05) in fish exposed to 150 ppm. Probably due to the higher density of Sertoli cells per cyst in treated tilapia, these cells presented a smaller (P < 0.05) nucleolus and a trend to decrease its support capacity (efficiency). However, the meiotic index (germ cell loss during the two meiotic divisions) was similar (P > 0.05) in the three groups of fish investigated. Remarkably, the results found in tilapia were similar to those found for rats treated with PTU. This suggests strongly that the mechanisms of control of Sertoli cell and Leydig cell proliferation seem to be preserved during vertebrate evolution.

Synaptonemal complex damage in mouse spermatocytes exposed to tequila and brandy.

We evaluated the capacity of tequila and brandy to induce synaptonemal complex (SC) breaks in mouse spermatocytes. The alcoholic beverages were 20% diluted in distilled water and administered daily by oral intubation for 21 days (1, 2, and 3 g/kg, six animals per group). A positive control group was administered cyclophosphamide (CP) (20 mg/kg), and another group of mice was treated with distilled water. The results indicated the following: (i) tequila induced a statistically significant increase in SC damage with 2 g/kg and 3 g/kg, and brandy was genotoxic only with 3 g/kg, (ii) CP induced almost a duplication of the total number of breaks produced by tequila or brandy; (iii) the average weight increase in the animals was 3.3 g, but the group treated with the highest dose of tequila showed a slight weight decrease; (iv) the weight of the testes did not show any significant difference among groups of animals; and (v) there were no significant differences between groups with respect to the frequency of pachytenes (mean number: 195 in 1000 cells).

Effects of long-term zearalenone administration on spermatogenesis and serum luteinizing hormone, follicle-stimulating hormone, and prolactin values in male rats.

Body and testis weights, serum luteinizing hormone, follicle-stimulating hormone, and prolactin values and volume fractions of Sertoli cells, spermatogonia, early and late primary spermatocytes, and round and long spermatids were evaluated in 70-day-old male rates treated orally with 20 mg of zearalenone/kg of body weight daily for 5 weeks. Significant (P < 0.05) increase in serum prolactin concentration was consistently observed during the 5 weeks of treatment with zearalenone. Significant changes were not observed in any of the other variables evaluated.

Cytogenetic effects of malathion insecticide on somatic and germ cells of mice.

Male mice dermally exposed to single or multiple treatment (5 days/2 weeks) showed that the ability of malathion to induce chromosome aberrations in somatic (bone marrow) and germ cells (primary spermatocytes) was related to the type of treatment and dose used. Statistically significant increases of chromosome aberrations in bone marrow cells occurred after single treatment (500 and 2000 mg/kg body wt) when chromatid gaps were included and after multiple treatment (250 and 500 mg/kg) when they were excluded. No dose-response relationships were observed for either treatment. In germ cells, malathion induced a significant increase of univalents in both types of treatment but structural chromosome aberrations were induced only by multiple treatment. Malathion induced a significant decrease of the mitotic indices in the bone marrow.