In vitro effects of endosulfan-based insecticides on mammalian sperm.

Endosulfan is an organochloride insecticide extensively used in several countries to protect crops from pests. As several studies indicate that endosulfan can affect human and animal development, the aim of this study was to analyse whether sperm parameters and the process of chromatin decondensation could be altered by endosulfan in mice sperm. Spermatozoa from cauda epididymis were obtained from mature male mice and incubated in the presence of two commercial formulations (CFs) of endosulfan (Master® and Zebra Ciagro®) or the active ingredient (AI) alone. A significant decrease in the percentage motility and viability of spermatozoa with respect to controls was found. In vitro decondensation was performed in the presence of glutathione and heparin. Spermatozoa incubated with the AI, endosulfan Master® and endosulfan Zebra Ciagro® showed an increase in chromatin decondensation. In addition, the TUNEL assay showed that DNA fragmentation was significantly higher when sperm were incubated with either one of the CFs when compared to the AI or controls. The ultrastructure analysis of sperm cells showed evident changes in the structure of the plasma and acrosome membranes of sperm incubated with endosulfan AI or the CFs. These results suggest that endosulfan can affect sperm integrity and in vitro chromatin decondensation as well as DNA fragmentation.

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.

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.

Changes in hematological parameters of Cichlasoma dimerus (Teleostei, Perciformes) exposed to sublethal concentrations of 4-tert-octylphenol.

Stress response involves various physiological changes, including alteration in hematological parameters closely related to the response of fish to the environment. 4-tert-octylphenol (OP) is one of the worldwide-used surfactants and can pollute the aquatic environment, both marine and freshwater. Previous studies have already shown estrogenic effects of this compound in various wildlife species, e.g., it can disrupt the reproductive system of males organisms including fish. The purpose of this study was to evaluate the effects caused by sublethal concentrations of OP on hematological parameters as biomarkers to assess environmental stress. Adult males and females of Cichlasoma dimerus were exposed to waterborne OP during 60 days under semistatic conditions. Experimental groups consisted of control (ethanol 0.005 %), OP 150, and OP 300 µg/L. OP caused hemodynamic stress in C. dimerus, which lead to normochromic and normocytic anemia, as well as erythrocytic pathologies such as a significant increase in erythroblasts and amitotic erythrocytes. All of the studied parameters can be used as biomarkers for the presence of xenobiotics in water.

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.

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.

Involvement of the gonadal germinal epithelium during sex reversal and seasonal testicular cycling in the protogynous swamp eel, Synbranchus marmoratus Bloch 1795 (Teleostei, Synbranchidae).

The swamp eel, Synbranchus marmoratus, is a protogynous, diandric species. During sex reversal, the ovarian germinal epithelium, which forms follicles containing an oocyte and encompassing follicle cells during the female portion of the life cycle, produces numerous invaginations, or acini, into the ovarian stroma. Within the acini, the gonia that formerly produced oocytes become spermatogonia, enter meiosis, and produce sperm. The acini are bounded by the basement membrane of the germinal epithelium. Epithelial cells of the female germinal epithelium, which formerly became follicle (granulosa) cells, now become Sertoli cells in the developing testis. Subsequently, lobules and testicular ducts form. The swamp eel testis has a lobular germinal compartment in both primary and secondary males, although the germinal compartment in testes of secondary males resides within the former ovarian lamellae. The germinal compartment, supported by a basement membrane, is composed of Sertoli and germ cells that give rise to sperm. Histological and immunohistochemical techniques were used to describe the five reproductive classes that were observed to occur during the annual reproductive cycle: regressed, early maturation, mid-maturation, late maturation, and regression. These classes are differentiated by the presence of continuous or discontinuous germinal epithelia and by the types of germ cells present. Synbranchus marmoratus has a permanent germinal epithelium. Differences between the germinal compartment of the testes of primary and secondary males were not observed.

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.

Regulation of metalloproteinases by nitric oxide in human trophoblast cells in culture.

The process of embryo implantation requires extensive remodelling of the endometrial extracellular matrix, a function largely performed by matrix-degrading metalloproteinases (MMPs). In the present study, we used trophoblast cells isolated from human term placentas to study the regulation of MMPs by nitric oxide (NO). Using a combination of zymography, Western blot and indirect immunofluorescence, we showed that MMP-2 and MMP-9 are increased during the conversion from low-motile cytotrophoblast cells to the highly motile and differentiated syncytiotrophoblast multinucleated cells. We also observed an increase in NO production and NO synthase (NOS) expression during this cellular differentiation process. In addition, we demonstrated a positive regulatory role of NO on the activity and protein expression of MMP-2 and MMP-9, because NO donors (NOC-18 and spermine-NONOate) or the NOS substrate (L-arginine) stimulate, whereas NOS inhibitors (N(G)-nitro-L-arginine methyl ester and N(G)-monomethyl-L-arginine) reduce the expression and gelatinolytic activity of MMP-2 and MMP-9 in isolated trophoblast cells. Taken together, these results suggest that, in differentiating trophoblasts, NO regulates the induction of matrix-degrading proteases required for invasion during embryo implantation.