Recovery of the Reproductive Capability Following Exposure to 4-tert-Octylphenol in the Neotropical Cichlid Fish Cichlasoma dimerus.

Here, we analyzed the recovery of the reproductive capability in male Cichlasoma dimerus after exposure to sublethal concentrations of 4-tert-octylphenol (OP), a demonstrated estrogenic chemical. Adult fish were exposed to 0, 30, 150 and 300 µg/L OP during 60 days and subsequently transferred to OP-free water for another 60 days. At 150 and 300 µg/L, absence of fertilized spawnings were recorded during the first 4 weeks following OP exposure, which could be explained by the impairment of testis architecture recorded at the highest OP concentrations. The restoration of the testicular organization began by day 14 in OP-free water, when the germinal epithelium re-started to proliferate. Testicular functionality was recovered by day 28, yielding fertilized eggs and viable F1 embryos. These results show that pathological features induced in the testes of C. dimerus by OP exposure are not permanent since fish recover their fertilization capacity after an adequate depuration period.

Ultrastructure of the testis in Synbranchus marmoratus (Teleostei, Synbranchidae): the germinal compartment.

Synbranchus marmoratus, is a protogynic diandric species in which two types of males, primary and secondary, are found. In both types, the germinal compartment in the testes is of the unrestricted lobular type, but in secondary (sex reversed females) males the lobules develop within the former ovarian lamellae. In the present study, the germinal compartment was examined in both types of males using light microscopy as well as scanning and transmission electron microscopy. Germinal compartment is limited by a basement membrane and contains Sertoli and germ cells. During maturation, processes of Sertoli cells form the borders of spermatocysts containing isogenic germ cells. Characteristically, type A and type B spermatogonia have a single nucleolus and grouped mitochondria associated with dense bodies or nuage. Type B spermatogonia, spermatocytes and spermatids are joined by cytoplasmatic bridges and are confined within spermatocysts. Secondary spermatocytes are difficult to find, indicating that this stage is of short duration. Biflagellated spermatozoa have a rounded head, no acrosome, and possess a midpiece consisting of two basal bodies, each of which produces a flagellum with a typical 9+2 microtubular composition. No associations occur between sperm and Sertoli cells. There were no differences between spermatogenesis in primary and secondary males in this protogynic, diandric fish.

Nonmonotonic response of vitellogenin and estrogen receptor α gene expression after octylphenol exposure of Cichlasoma dimerus (Perciformes, Cichlidae).

In oviparous vertebrates, vitellogenin (VTG) is mainly produced by the liver in response to estrogen (E2) and its synthesis is traditionally coupled to estrogen receptor alpha induction. Even though VTG is a female-specific protein, chemicals that mimic natural estrogens, known as xenoestrogens, can activate its expression in males causing endocrine disruption to wildlife and humans. Alkylphenols such as nonylphenol (NP) and octylphenol (OP) are industrial additives used in the manufacture of a wide variety of plastics and detergents, and can disrupt endocrine functions in exposed animals. For more than a decade, the freshwater cichlid fish Cichlasoma dimerus has been used for ecotoxicological studies in our laboratory. We recently found an up-regulation of VTG gene expression in livers of male fish exposed to OP, from a silent state to values similar to those of E2-induced fish. To better understand the underlying mechanisms behind the action of xenoestrogens, the aim of this study was to analyze the dose-response relationship of C. dimerus VTG and estrogen receptors (ERs) gene expression after waterborne exposure to 0.15, 1.5, 15, and 150µg/L OP for up to 1 month (0, 1, 3, 7, 14, 21, and 28 days). At the end of the experiment, histological features of exposed fish included active hepatocytes with basophilic cytoplasm and high eosinophilic content in their vascular system due to augmented expression of VTG. In testis, high preponderance of sperm was found in fish exposed to 150µg/L OP. A classic dose-response down-regulation of the expression of Na(+)/K(+)-ATPase, a "non-gender specific gene" used for comparison, was found with increasing OP concentrations. No VTG and very low levels of ERα were detected in control male livers, but an up-regulation of both genes was found in males exposed to 0.15 or 150µg/L OP. Moreover, VTG transcripts were significant as early as day 3 or day 1 of exposure to these OP concentrations, respectively. Nearly no response was detected in 1.5 and 15µg/L OP exposed-fish. Data was curve-fitted evidencing a nonmonotonic dose-response curve. Interestingly, ERß2 mRNA expression was augmented above baseline levels only when males were exposed to the lowest OP concentration. We speculate that genomic control of vitellogenesis is under control of multiple steroid receptors with different affinities for ligands. ERß isoform, only up-regulated with very low concentrations of ligand, would act as a sensors of OP (or E2) to induce ERα and VTG. With high OP concentrations, the expression of ERα isoform is promptly augmented, with the concomitant VTG transactivation.

Exposure to waterborne 4-tert-octylphenol induces vitellogenin synthesis and disrupts testis morphology in the South American freshwater fish Cichlasoma dimerus (Teleostei, Perciformes).

Exposure to environmental pollutants may disrupt endocrine functions and cause reproductive effects in human and wildlife populations. Various groups of chemicals have estrogen-like effects, including degradation products of alkylphenol polyethoxylates, such as 4-tert-octylphenol (OP). Laboratory studies have shown that exposure of male fish to xenoestrogens results in induction of circulating vitellogenin (Vtg), inhibition of testicular growth, testis abnormalities and formation of intersex gonads. In this study, the impact of the exposure to waterborne OP on reproductive aspects in the South American cichlid fish Cichlasoma dimerus was evaluated using qualitative changes in the levels of Vtg in plasma and surface mucus and histological alterations in the liver and gonads as endpoints. Adult males and females were exposed to OP via immersion during 60 days in aquaria under semi-static conditions, water changes being made every 84 h. Treatment groups were: control (ethanol 0.005%), OP 30, 150 and 300 microg/L. Using Western and Dot blot analysis, Vtg was detected in plasma and mucus of control and treated females and treated males, while no Vtg was observed in samples from control males. Morphological changes in the hepatocytes due to the accumulation of Vtg were observed in OP-exposed males. Impairment of testicular structure became apparent in males treated with the highest OP concentrations. The most salient pathological change was the alteration of lobular organization with increased testicular fibrosis and progressive disruption of spermatogenesis. No major changes were observed in ovarian architecture. Our results indicate that detection of Vtg in surface mucus may be a sensitive and non-invasive biomarker of the endocrine disrupting effects of environmental estrogens, resulting in a useful method for field monitoring.

Expression of pejerrey gonadotropin-releasing hormone in three orders of fish.

Molecular variants of GnRH were characterized by reverse-phase, high-performance liquid chromatography from brain extracts of fish in three different orders: Synbranchiformes (swamp eel [Synbranchus marmoratus]), Cyprinidontiformes (platyfish [Xiphophorus maculatus] and green swordtail [X. helleri]), and Atheriniformes (Patagonia pejerrey [Odontesthes hatchery]). Also, pituitary gland extracts from the pejerrey O. bonariensis (Atheriniformes) were characterized. Eluted fractions were tested in radioimmunoassays with antisera specific to GnRH, including both antisera that detected only one form of GnRH and those that detected several forms. The results show that brain extracts obtained from all species contained the same three molecular forms of GnRH, which were immunologically and chromatographically undistinguishable from chicken GnRH-II, pejerrey GnRH (pjGnRH), and salmon GnRH. This study supports the hypothesis that expression of these three forms is common in different fish orders and that pjGnRH is the main regulator of pituitary function in these fish.