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Acetaminophen (ACE; paracetamol) is one of the most widely used nonsteroidal anti-inflammatory drugs worldwide and is often found in aquatic systems, where it can act on nontarget species and impair fish reproduction. This study aimed to investigate the effects of chronic exposure to environmentally relevant ACE concentrations (0.5, 5 and 50 µg/L) on multiple reproductive parameters in zebrafish (Danio rerio). Gametogenesis was analyzed using histology, morphometry, cell proliferation, and apoptosis. This study also evaluated sex steroids, and prostaglandin E

Estrone (E1) is a common environmental contaminant found in rivers and streams due to the farming of animals, such as swine and cattle. Our study evaluated the effects of chronic E1 exposure at environmentally relevant concentrations on spermatogenesis and the semen quality of zebrafish (Danio rerio). We exposed the fish to E1 at concentrations of 20, 200, and 2000 ng/L diluted in 0.001% ethanol (v/v) for 49 days. There were two control groups: one was exposed to water only and the other to ethanol at the same concentration used in the E1 groups. Following exposure, we analyzed the proportion of testicular cell types and other components (%), rate of cell proliferation and death, and sex steroid concentrations. Furthermore, we analyzed the expression of insulin-like growth factor 1 (IGF1), IGF2, IGF1 receptor (IGF1R), and inducible nitric oxide synthase and assessed the semen quality. E1 exposure increased spermatogonia, spermatids, Sertoli cells, Leydig cells, and the proportion of inflammatory infiltrate but decreased the spermatozoa amount. These changes were reflected by reductions in the gonadosomatic index and levels of 11-ketotestosterone in the testes. On the other hand, E1 exposure increased testicular estradiol, IGF1R expression, and nitric oxide production. After an evaluation using a computer-assisted sperm analysis (CASA) system, we observed reduced progressive motility, curvilinear velocity, and beat cross frequency of 20 and 2000 ng/L E1 groups. Our findings support that E1 causes deleterious effects on the testicular function and semen quality of D. rerio even at environmental concentrations. Thus, E1 concentrations should be monitored in surface waters for the purposes of fish conservation.

Although herbicides have been developed to act on the physiological processes of plants, they are responsible for causing deleterious effects on animals. These chemical compounds are widely used throughout the world, but especially in countries that export agricultural products such as Central and South America, their use has increased in recent years. Aquatic environments are natural reservoirs of herbicides, which after being applied on crops, run off through the soil reaching rivers, lakes, and oceans. Fish are among the many organisms affected by the contamination of aquatic environments caused by herbicides. These animals play an important ecological role and are a major source of food for humans. However, few studies address the effects of herbicides on fish in this region. Thus, in the present review we discuss the morphophysiological and molecular consequences of herbicide exposure in Neotropical fish systems as well as how the environmental and land use characteristics in this region can influence the toxicity of these pollutants. A toxicity pathway framework was developed summarizing the mechanisms by which herbicides act and endpoints that need to be further investigated.

Atrazine (ATZ) is a herbicide that is frequently present in surface waters and may result in damage to the health of various organisms, including humans. However, most scientific literature reports injuries caused by ATZ at high concentrations, which are not found in the environment. Therefore, the scope of this study was to investigate the impacts of realistic concentrations of ATZ found in surface waters (1, 2, 5, 10, 15 and 20 μg/L) using the bioindicators Allium cepa, Daphnia magna and zebrafish (Danio rerio). ATZ elicited a genotoxic effect in A. cepa, manifested by the induction of chromosomal aberrations, and a mutagenic effect with increased incidence of micronuclei formation, promotion of cell death and reduction in nuclear size revealed by flow cytometry analysis. D. magna exposed to 10, 15 and 20 μg/L of ATZ showed significant reduction in body size after 21 days, delayed first-brood release, decreased egg production and total offspring, as well as swimming behavioral changes. ATZ exposure promoted physiological and developmental alterations in zebrafish embryos, including an increased spontaneous movement rate, which led to premature hatching at all concentrations investigated. Increase in total body length, decrease of the yolk sac area, pericardial edema and higher heart rate were also detected in ATZ-treated zebrafish. In summary, environmentally relevant concentrations of ATZ can induce substantial alterations in the three bioindicators investigated. This study evidences the deleterious effects of ATZ on three aquatic bioindicators employing established and current techniques, and may contribute to elucidate the risks caused by this widely used herbicide even at low concentrations and short-to-medium-term exposure.

Discharge of municipal wastewater promotes the entry of diverse oestrogenic compounds into the water bodies. This complex mixture of substances interferes in the steroidogenic pathway, being able to promote severe reproductive impairment in freshwater fish populations. The purpose of the present study was to evaluate the effects of oestrogenic endocrine disruptors (EDCs) mixture on gonadal sex steroids (testosterone, T; 11-ketotestosterone, 11-KT; 17β-oestradiol, E2; 17-hydroxyprogesterone, 17-OHP) in the peak of the reproductive season of Astyanax rivularis, correlating the results obtained with the proportion of germ cells and gonadal histopathology. Three sampling sites were chosen to conduct the study, one reference site (S1), without contamination by municipal wastewater and two sites (S2 and S3) receiving discharge of municipal wastewater. Males of A. rivularis presented higher concentrations of E2, lower androgens (T and 11-KT) in gonads when compared to males from site S1. Concentrations of 17-OHP did not present significant difference among sites. In sites S2 and S3, the proportion of early spermatocytes, spermatids and Leydig cells increased while spermatozoa decreased compared to fish from S1. The following gonadal histopathologies were detected in the male fishes: intersex gonads (28% in S3) and testicular degeneration with germinal epithelium exhibiting agglutinated germ cells masses and empty cysts (57% in S2 and 71% in S3). In females, concentrations of T, E2 and 17-OHP did not present significant difference among the sites, however higher 11-KT concentrations were detected in females from sites S2 and S3. A lower proportion of perinucleolar follicles and a higher incidence of vitellogenic follicles, besides, aged oocytes and the presence of eosinophilic proteinaceous fluid in the interstitial compartment were also found in females from impacted sites. These results indicate that the urbanization and consequent release of municipal wastewater containing oestrogenic compounds in the headwater creeks are altering the levels of sex hormones and gametogenesis of A. rivularis. Further studies should be performed to determine whether oestrogenic endocrine disrupters are disrupting the reproduction of A. rivularis.

Bisphenol A (BPA) is an organic synthetic compound used in the plastic industry with endocrine disrupting activity. Although it is frequently found in surface waters, few studies have investigated its impact on fish gametogenesis, particularly when associated with natural stressors. In this regard, the present study evaluated BPA toxicity on spermatogenesis in the lambari Astyanax bimaculatus under controlled conditions and its interactive effects with water temperature. Adult specimens were exposed in duplicate to 40 μg/L and 400 μg/L BPA at 23 °C and 28 °C for 21 days; the control group did not receive BPA. Testicular samples were collected and analyzed using different cellular and molecular techniques. The results showed a significant reduction in the gonadosomatic index in the BPA-treated groups at both temperatures. A decrease in the testicular levels of 11-ketotestosterone was observed in the 400 μg/L BPA group at 23 °C, 17β-estradiol increased significantly in the treated groups at 28 °C, and vitellogenin showed no difference between the treatments. The morphometric analysis of spermatogenesis revealed a significant increase in the proportion of spermatogonia, spermatocytes, and Sertoli cells in the treated groups, with a higher proportion at 23 °C than at 28 °C. Otherwise, the proportion of spermatozoa was significantly lower in the BPA-treated groups, with a greater reduction at 23 °C. In addition, BPA also stimulated spermatogonial proliferation in the treated groups, but apoptosis was significantly increased in spermatids at 23 °C. Testis-ova, cell degeneration, and chromatin alterations in spermatids and Sertoli cells were observed in the germinal epithelium of the BPA-treated groups. The integrated biomarker response (IBR) index revealed that the analyzed endpoints are suitable for assessing estrogenic contamination. Taken together, our results indicate that the interactive effects of BPA and temperature contribute to the impairment of spermatogenesis in A. bimaculatus with more severe effects observed on sperm production at 23 °C than at 28 °C.

In the last decades, oestrogenic compounds have often been reported in environmentally relevant concentrations in aquatic environments around the world. Most laboratory studies of oestrogens try to understand the effects of a single contaminant, but in natural environments, the effects may be quite different due to interactions with other compounds. The present study aimed to compare the action of oestrone (E1) and bisphenol-A (BPA), acting singularly and in combination, on the spermatogenesis of Astyanax bimaculatus. After exposure to 100 ng/L of E1, BPA and a mixture of the two for 15 days, our results showed that E1 and the E1 + BPA mixture significantly altered the number of spermatogenic cells. BPA presented high cytotoxicity when compared to other treatments. Analysis of the two oestrogenic compounds suggests that the E1 + BPA mixture has no additive or synergistic effects. Together, the results of the present study indicate that endocrine-disrupting chemicals (EDCs) analysed alone may behave differently than when administered with other substances.

Cell proliferation and apoptosis regulate germ cells stock and sperm production, eliminate anomalous gametes, and are essential parameters to consider in fish farming. Herein, spermatogenic activity as well as germ cell proliferation and apoptosis were assessed in Leporinus taeniatus, a seasonal breeding species from the Sao Francisco River basin, Brazil. Testes of 24 adult fishes from a farming station were sampled between December and July and processed for light and transmission electron microscopy and immunohistochemistry for PCNA and TUNEL assay. The gonadosomatic index and seminiferous tubule diameters presented higher values during the breeding season (December/January and February/March), and then significantly reduced during the regression and resting stages (April/May and June/July). Phagocytosis of spermatozoa by Sertoli cells was evident during gonadal regression, but a significant number (up to 30%) remained at the tubular lumen during the resting stage. A higher PCNA/TUNEL ratio occurred in the breeding period, leading to an elevated proportion (%) of spermatogonia (GA and GB) in resting. Moreover, a higher TUNEL/PCNA ratio indicates the contribution of apoptosis to the reduction of germ cells during testicular regression. Together, these results indicate a shift in the balance between cell proliferation and apoptosis that contributes to the regulation of the spermatogenic cycle and germ cells pool of L. taeniatus kept in captivity.

Understanding the influence of different temperature conditions on the spermatogenesis is important for improvement of the fish aquaculture. This study evaluated the influence of low temperature on structural and quantitative dynamics of the spermatogenesis in Oreochromis niloticus. Adult males were cultivated with room temperature water (20.28-22.46°C) and testes were collected for histological, ultrastructural and morphometric analyses. This species has unrestricted lobular testis with cystic spermatogenesis and type I spermiogenesis that results in a single anacrosomal aquasperm. Seminiferous lobules and spermatogenic cells had a radial arrangement toward the spermatic duct. Superior and central portions of testes had a greater lobular area than the inferior portion in all samplings. Spermatogonia (9.3%) were distributed in the inferior portion of testes, spermatocytes (25.3%) and spermatids (34.4%) in the central portion, while spermatozoids (39.4%) and secretory cells (4.6%) in the superior portion. Throughout the study, correlation between water temperature and lobular area characteristics was significant only in the inferior portion of testes (r(2)=0.95), although the lobular area in the other testicular portions increased when the water temperature increased by 2°C. Correlation between the water temperature and spermatogenic cells was significant for undifferentiated spermatogonia (r(2)=0.54) and number of spermatids (r(2)=0.67). It is concluded that low cultivation temperatures may positively influence the generation of primary spermatogonia in the inferior periphery of O. niloticus testes. In addition, males maintain reservoirs of germ cells at low temperatures and the radial zonation of spermatogenesis has an important role in the renewal and production of germ cells.

Food restriction is part of the life cycle of many fish species; however, nutritional deficiency may negatively influence gametogenesis and gonadal maturation. The aim of this study was to evaluate the effects of food restriction on the spermatogenesis of Nile tilapia. For this, adult males were submitted to starvation and refeeding cycles (alternating periods of starvation and feeding) for 7, 14, 21, and 28 days. After 7 days of starvation, glycaemic and lipid levels were significantly reduced, followed by reduction of plasma testosterone (T) and 11-ketotestosterone (11-KT). In addition, reduced proliferation of spermatogonia and increased apoptosis of spermatocytes, spermatids, and spermatozoa was observed in starvation groups. In the refeeding groups, the sex steroids and the proportion of germ cells had no significant alterations compared to the control group, except for spermatozoa. In this sense, the present study suggests that starvation after 7 days progressively reduces T and 11-TK, resulting in damage to the production of spermatogenic cells, while refeeding may delay spermatogenesis but does not lead to testicular impairment.

Continental waters salinisation is a global threat that has grown because of climate change and human activities, but little is known about how and what biological tracts are affected. The aim of this study was to investigate the influence of different water salinities on the expression of HSP70, PCNA and caspase-3 during spermatogenesis of Nile tilapia. Adult males were submitted to four salinity treatments: (S0) fresh water, (S7) 7 g L−1, (S14) 14 g L−1, and (S21) 21 g L−1 for 1, 4, and 9 days. All specimens were in spermatogenic activity and the highest values of the gonadosomatic index (GSI) occurred in the S0 and S7. In the morphometric analysis, spermatocytes were the most frequent germ cell detected in all treatments (>50%) and spermatids achieved about 20% of the testicular proportion, with few variations among treatments. Spermatozoa were significantly reduced only in S14 compared to S7. Leydig cells were significantly increased in S14 when compared to S7 but plasma concentrations of 11-KT showed no significant difference among treatments. ELISA assay showed higher testicular expression of HSP70 at 1 day in all groups, followed by a significant decrease at days 4 and 9 in S14 and S21. The expression of PCNA was significantly lower while the activity of caspase-3 was higher in S14 and S21 when compared to S0 and S7. These results indicate that higher salinities in S14 and S21 interfere with the relationship between testicular HSP70, PCNA, and caspase-3, but with few effects over spermatogenesis dynamics of Nile tilapia.