A game of two? Gene expression analysis of brain (cyp19a1b) and gonadal (cyp19a1a) aromatase in females of a Neotropical cichlid fish through the parental care period and removal of the offspring.

For many species parental behavior is essential for the survival of the offspring. While the ultimate causes of teleost parental behavior have been widely studied, comparatively little is known about its proximate causes. The aim of this study was to analyze the yet unexplored, potential dual role of brain and gonadal aromatases, the enzymes responsible for the conversion of androgens to estrogens in the brains and gonads of teleosts, respectively, on the different stages of the maternal care period of the biparental cichlid Cichlasoma dimerus, locally known as chanchita. By immunohistochemistry we analyzed the neural distribution of brain aromatase and observed it exclusively within the forebrain, including areas involved in the regulation of parental behavior. We next analyzed the gene expression of brain aromatase in the brain, and gonadal aromatase in the ovary, of female chanchitas through the parental care period. To further characterize the physiological environment associated to maternal care, we also evaluated sex steroid levels (17ß-estradiol, testosterone and 11-ketotestoterone) and ovarian follicle percentage. The onset of parental behavior specifically downregulated sex steroids synthesis and the rate of ovarian maturation, as denoted by a more than 10-fold decrease in steroid levels and delayed detection of mature follicles in females with offspring, compared to females which eggs were removed. Gene expression levels of both aromatases were independent of maternal care at the evaluated time points, even though they varied during the parental care period.

Endosulfan affects GnRH cells in sexually differentiated juveniles of the perciform Cichlasoma dimerus.

Endosulfan (ES) is an organochlorine pesticide widely used in agriculture despite its high toxicity towards non-target organisms such as fish. It has been demonstrated that ES can cause negative effects on aquatic animals, including disruption of hormonal systems. However, the alterations produced by this pesticide on the reproductive axis of fish prior to sexual maturity, as well as possible modes of action have hardly been studied. This study aimed at assessing the effect of waterborne exposure to the pesticide ES on the reproductive axis during sexual differentiation of juveniles of the South American freshwater cichlid fish Cichlasoma dimerus. No mortality was observed due to ES subchronic exposure (90 days post-fertilization). Exposure to ES did not affect body weight nor morphometric parameters, indicating that larvae nutritional state was not affected. Timing of sexual differentiation, gonadal morphology and sex ratio were likewise not altered by ES. However, ES acted as an endocrine disrupting chemical in this species as the morphometry of gonadotropin-releasing hormones (GnRH) producing cells was altered. Exposure to ES altered nuclear area, cell area and nucleus/cytoplasm ratio of GnRH II neurons, and cell and nuclear area and diameter of GnRH III neurons. Interestingly, in our previous study, exposure before sex differentiation (30 day exposure) caused no alteration to GnRH II and III, and did alter GnRH I and FSH cells. These alterations could lead to changes in circulating hormone levels, especially when fish are exposed for prolonged periods, ultimately impairing reproductive fitness. C. dimerus juveniles can be an interesting biological model to perform toxicological studies with the intent to assess early disruption endpoints in the reproductive axis during development.

Endocrine disruption in the sub Antarctic fish Patagonotothen tessellata (Perciformes, Notothenidae) from Beagle Channel associated to anthropogenic impact.

Situated in the sub-Antarctic region, Beagle Channel represents a unique marine ecosystem due to the connection between the Pacific and the Atlantic Oceans, and its proximity to the Antarctic Peninsula. Ushuaia city, the biggest settlement on the channel, exerts an increasing anthropogenic pressure by discharges of urban and industrial effluents. In the present work, we use Patagonotothen tessellata, one of the most abundant and widespread species in the channel, as a bioindicator species in order to evidence anthropic impact from Ushuaia Bay and surrounding areas. We first analyzed and characterized real time gene expression of androgen receptor, estrogen receptor and different forms of vitellogenin (VTG), under laboratory conditions. This was achieved by induction with estradiol of P. tessellata males. Then, the selected genes were used as biomarkers for an environmental biomonitoring study. Morphometric indices and circulating sex steroids (estradiol and testosterone) were also quantified in male fish collected from different sites. The qPCR analysis showed that vtgAb form is more inducible than vtgAa or vtgC forms after estrogen induction. The field survey revealed the up-regulation of vtgAb and the androgen receptor in fish from sites with higher anthropogenic influence. Sex steroids followed seasonal variations according to their reproductive cycle, with higher levels of estradiol and testosterone in winter and summer seasons. The use of biomarkers such as gene expression of VTG demonstrates that fish from Ushuaia Bay are likely to be exposed to endocrine disrupting compounds. To our knowledge, this research is the first attempt to assess the endocrine disruption associated to anthropic impact in a widespread fish of the Beagle Channel and contributes to a better understanding of the reproductive physiology of sub Antarctic ichthyofauna.

Endocrine and reproductive endpoints of Notothenia rossii and N. coriiceps: A baseline study for ecotoxicological monitoring in Antarctic waters.

Pollution threats Antarctica and scientists blame xenobiotics and anthropic activities. Yet little is known about their effect on Antarctic ichthyofauna. Accordingly, we investigated the endocrine system of male fish Notothenia rossii and N. coriiceps (Perciformes, Nototheniidae) collected during Austral summer. For N. rossii, hormone levels of estradiol, testosterone, and 11-ketotestosterone were higher in fish collected during March than those of January; whereas for N. coriiceps estradiol and androgens levels were higher and lower in March, respectively. Histological analysis of the testes showed an unrestricted lobular type with no pathological alterations. However, detection of vitellogenin-like in plasma and skin mucus were seen in 75% of N. coriiceps males and 7% of N. rossii males. This is the first report of mucus vitellogenin-like detection as a non-invasive biomarker of endocrine disruption in notothenioid males and settles a baseline for future studies of reproductive biology and endocrine disruption in Antarctic fishes.

The SSRI fluoxetine exhibits mild effects on the reproductive axis in the cichlid fish Cichlasoma dimerus (Teleostei, Cichliformes).

Among the wide variety of pharmaceuticals released into the environment, Fluoxetine (FLX), a selective serotonin reuptake inhibitor, is one of the most prescribed for the treatment of major depression. It inhibits serotonin (5-HT) reuptake at the presinaptic membrane, increasing serotonergic activity. In vertebrates, including fish, the serotonergic system is closely related to the Hypothalamic Pituitary Gonadal (HPG) axis which regulates reproduction. As FLX can act as an endocrine disrupting compound (EDC) by affecting several reproductive parameters in fish, the aim of this study was to provide an integral assessment of the potential effect of FLX on the reproductive axis of the Neotropical freshwater fish Cichlasoma dimerus. Adult fish were intraperitoneally injected with 2 µg g-1 FLX or saline every third day for 15 days. No significant differences were found on serotonergic turnover (5-HIAA/5-HT ratio). Pituitary ßLH content in FLX injected females was significantly higher than control females; no significant differences were seen for ßFSH content. Sex steroids remained unaltered, both in males and females fish, after FLX treatment. No plasma vitellogenin was induced in treated males. Some alterations were seen in testes of FLX injected males, such as the presence of foam cells and an acidophilic PAS positive, Alcian-Blue negative secretion in the lobular lumen. Although there is no clear consensus about the effect of this drug on reproductive physiology, these results indicate that FLX is acting as a mild EDC in adults of C. dimerus.

Toxicity of hexachlorobenzene and its transference from microalgae (Chlorella kessleri) to crabs (Chasmagnathus granulatus).

The aim of this work was to study the transference of hexachlorobenzene from a green alga (Chlorella kessleri) to an estuary crab (Chasmagnathus granulatus), and to analyze the toxic effects that the xenobiotic has on the latter. The effect of hexachlorobenzene uptake was evaluated measuring oxidative stress, Uroporphyrinogen decarboxylase activity and morphometric parameter alteration, and also performing a histological analysis of crab hepatopancreas. Results demonstrated that hexachlorobenzene enters the alga, is accumulated in it, and then transferred into the crab, causing a decrease in Uroporphyrinogen decarboxylase activity in both organisms. The high malondialdehyde levels detected in crab hepatopancreas after the toxic treatment suggested the existence of hexachlorobenzene-induced lipid peroxidation. Antioxidant defenses such as superoxide dismutase activity and reduced glutathione content fell below normal values on the fourth week of treatment. At the same time, the hepatosomatic index, used as a morphometric parameter, reduced 20% with respect to the control. The histological analysis revealed epithelium disorganization in hepatopancreas tubules, confirming the existence of structural damage caused by hexachlorobenzene.

Endocrine disruptive potential of endosulfan on the reproductive axis of Cichlasoma dimerus (Perciformes, Cichlidae).

Endosulfan (ES), a persistent organochlorine pesticide, is widely used despite its toxicity to non-target animals. Upon reaching water bodies, ES can cause negative effects on aquatic animals, including disruption of hormonal systems. However, the action of ES on fish reproductive axis has been hardly studied thus far. The aim of the present work was to assess the endocrine disruptive potential of endosulfan on the pituitary gonadotropins levels and on the testes function due to ES in the South American freshwater fish Cichlasoma dimerus, using in vitro and in vivo approaches. In vitro experiments showed that ES inhibited the LH-stimulated steroidogenesis in gonads; no change was observed in gonadotropins release from pituitaries in culture. Laboratory waterborne ES (0.1, 0.3 and 1 µg/L) exposure for two months caused decrease in ßFSH pituitary content and γGT activity in the testes (Sertoli cell function marker). Testicular histology revealed pathologies such as scarce intermediate stages of spermatogenesis, release of immature germ cells into the lobular lumen, presence of foam cells and interstitial fibrosis. As FSH and FSH-mediated steroidogenesis regulate spermatogenesis and Sertoli cell function, the effect of ES on FSH could be responsible for the morphological alterations observed in testes. In vitro, ES disrupted steroidogenesis in gonads, therefore similar effects in vivo cannot be ruled out. Based on this evidence, ES exhibits an endocrine disruptive action on the reproductive axis of C. dimerus, causing disruption at the pituitary and/or at the gonad level. These effects could acquire ecological significance under prolonged exposure to the pesticide in nature.

Time-course recovery of estrogen-responsive genes of a cichlid fish exposed to waterborne octylphenol.

The aim of this study was to describe the time-course of estrogen-induced gene expression, corresponding plasma protein detection and histological alterations after cessation of octylphenol (OP) exposure of Cichlasoma dimerus, to test differential responses of biomarkers suitable for environmental monitoring. Male fish were exposed to a nominal concentration of 150 µg/L OP for 28 days, and later transferred to OP-free water aquaria for 1, 3, 7, 14, 21 or 28 days. Blood and mucus samples were obtained in order to analyze vitellogenin (VTG) and zona pellucida (ZP) proteins by Western blot; liver samples were used for gene expression and to assess tissue damage and further recovery of all the analyzed endpoints. Partial sequences of C. dimerus VTG and Na⁺/K⁺-ATPase were obtained. Comparison with VTGs of several teleosts supports that the partial sequence obtained for C. dimerus belongs to VTGAb type. ZP and VTG expression was highly up-regulated by OP. Immunoreactive (ir-) bands of 62, 52 and 50 kDa for ZP and 140, 103, 75 and 64 kDa for VTG, were detected after 28 days of OP exposure in plasma and mucus samples. After transfer of treated fish to clean water, ZP ir-bands in plasma disappeared rapidly (day 3), while VTG ir-bands decreased gradually; no ir-bands were detected on day 28 of recovery. Similarly, ZPB transcripts abruptly returned to background levels (day 3), earlier than for ZPC (day 7) or VTG (day 14). Liver from OP treated fish showed tissue disarrangement, eccentric and euchromatic hepatocytes nuclei and intense perinuclear basophilia. After the recovery period, these changes were still evident though less pronounced, accounting for irreversibility of tissue damage or the requirement for a longer period of depuration. The present results confirm that for biochemical and molecular biomarkers, such as induction of female proteins in male fish exposed to OP, complete recovery is achieved after adequate time of depuration (28 days). Male ZPB expression reflects a recent exposure to estrogenic contaminants, while VTG may reveal past exposures. The combination of biomarkers with different temporal responses such as C. dimerus ZP and VTG provides a more comprehensive interpretation of pollution status.

Early expression of zona pellucida proteins under octylphenol exposure in Cichlasoma dimerus (Perciformes, Cichlidae).

An increasing number of widely used industrial and agricultural chemicals are being found to cause endocrine disruption. In fishes, xenoestrogens can induce female proteins, and in some cases, the development of testis-ova, demonstrating feminization of males. In this study we analyzed the effect of an acute exposure of adult male Cichlasoma dimerus fish to estradiol (E(2)) and octylphenol (OP). E(2) and OP were injected at 10 and 50 µg/g body weight doses, respectively. After a single OP dose, liver was processed for RNA extraction at 1, 3, 12, 24, and 72 h. PCR was performed using cDNA and primers for egg coat or zona pellucida proteins (ZP). Genes encoding ZPB and ZPC isoforms were sequenced. E(2)-induced fish were sacrificed at 72 h. Using multiple OP or E(2) injections, blood and surface mucus were sampled on days 0, 3, 6, 9, and 13. On day 13 fish were sacrificed for liver and testis dissection. Histological examination of E(2) and OP-treated fish livers showed cellular disarray and intense cytoplasmatic basophilia within hepatocytes, probably due to increased mRNA synthesis, as well as hypertrophied euchromatic nuclei, and conspicuous nucleoli, indicative of augmented cell activity. An abnormal amount of sperm and immature germ cells within the testis lumen were seen in treated fish, suggesting reproductive impairment. Both plasma and mucus revealed the presence of ZP (and vitellogenin) at day 3 and thereafter with E(2) treatment, using Western and Dot blot techniques; OP effects were delayed in time. These results validate the analysis of mucus by Dot blot as an easy and rapid technique to address endocrine disruption caused by OP. Quantitative gene expression showed induction of liver ZPB and ZPC upon OP injection; muscle, brain, and intestine did not express any ZP. Both ZPs were induced at 1h post injection, but only ZPB expression was statistically significant. At 12h, both ZPs increased significantly, reaching the same levels of E(2)-challenged males after 72 h. Therefore, OP mimicked the action of E(2) with a prompt and strong xenoestrogenic effect, evidenced by the early response through mRNA and protein expression of ZP and the concomitant histological liver and testis alterations.

Dopaminergic regulation of ion transport in gills of the euryhaline semiterrestrial crab Chasmagnathus granulatus: interaction between D1- and D2-like receptors.

The effects of dopamine (DA) and dopaminergic agonists and antagonists on ion transport were studied in isolated perfused gills of the crab Chasmagnathus granulatus. DA applied under steady state conditions (perfusion with hemolymph-like saline) produced a transient increase of the transepithelial potential difference (V(te)) from 2.2+/-0.2 to 4.8+/-0.3 mV, describing an initial cAMP-dependent stimulating phase followed by an inhibitory phase. Spiperone and domperidone (antagonists of D2-like DA receptors in vertebrates) completely blocked the response to DA, while the D1-like antagonist SCH23390 blocked only the inhibitory phase. Theophylline (phosphodiesterase inhibitor) and okadaic acid (protein phosphatases PP1 and PP2A inhibitor) were also able to block the inhibitory phase, suggesting that it depends on adenylyl cyclase inhibition and on protein phosphatases. When the gills were perfused with hypo-osmotic solution, or with the adenylyl cyclase activator forskolin, V(te) was increased several-fold. DA applied under these stimulated conditions partially reversed the V(te) increase by 54% and 25%, respectively. Similarly, the D1-like agonist, fenoldopam, produced a 33% reduction in the stimulated V(te). We propose that, in C. granulatus gills, DA stimulates adenylyl cyclase and therefore ion transport through D1-like receptors linked to a Gs protein, although they respond to antagonists that interact with D2-like receptors in vertebrates. The inhibitory phase seems to be mediated by D2-like receptors linked to a Gi/o protein, which inhibits adenylyl cyclase, although these receptors can be activated or blocked by agonists or antagonists that interact with D1-like receptors in vertebrates and insects.