Andrea Lister

Inhibition of egg production in zebrafish by fluoxetine and municipal effluents: A mechanistic evaluation

This study explored the impact of nominal concentrations of ethinylestradiol (EE(2); 10ng/L), fluoxetine (FLU; 0.32, 3.2, 32microg/L), and 1-50% treated municipal effluent on reproduction and liver function in sexually mature female zebrafish over a 7-day period. Compared with the control groups, FLU (32microg/L) and 50% effluent treatment significantly reduced the average eggs spawned by approximately 4.5 and 2 fold, respectively. FLU also decreased ovarian levels of 17beta-estradiol (E(2)) without affecting the gonadosomatic indices of the fish. The expression of ovarian genes involved in the production of prostaglandins, steroid biosynthesis, and gonadotropin receptors were quantified to determine a potential mechanism underlying the reduced egg production in FLU and effluent exposed fish. Quantitative real-time PCR analysis determined that ovarian aromatase, follicle stimulating hormone receptor (FSHr), and luteinizing hormone receptor (LHr) gene expression were significantly reduced by FLU suggesting that disruptions to the synthesis of ovarian steroids and the actions of gonadotropins may underlie the negative influence of FLU on ovarian E(2) and spawning levels. Potential mechanisms involved in the modest effects of the effluent on reproduction remain unknown, but the elevated levels of total ammonia and nitrite in the 50% effluent treatment groups compared with the other treatments should not be discounted. Liver expression of CYP3A65 was significantly induced by all treatments of effluent, while EE(2) caused a reduction in the expression levels of CYP1A1, PXR, and CYP3A65. The results of the present study suggest that SSRI may disrupt reproductive functioning at concentrations greater than those found in receiving environments; yet, more research is warranted into to the possible effects of low levels of synthetic estrogens on liver function in exposed fish, particularly the PXR-CYP3A pathway.

An investigation into the role of prostaglandins in zebrafish oocyte maturation and ovulation

This study explored the potential for ovarian-derived prostaglandins (PGs) to be involved in the regulation of oocyte maturation and ovulation in zebrafish. It was demonstrated that cultured vitellogenic follicles have the capacity to produce prostaglandin E(2) (PGE(2)) and PGF(2alpha) in response to arachidonic acid (AA) in a concentration-dependent manner, and that AA stimulates the in vitro production of 17beta-estradiol (E(2)). The production of AA-stimulated PGF(2alpha) was significantly reduced by treatment with the non-selective cyclooxygenase (COX) inhibitor, indomethacin (INDO). Treatment of full-grown follicles with AA did not induce oocyte maturation as assessed by germinal vesicle breakdown, but INDO significantly decreased the rate of spontaneous maturation. Using Real-Time PCR, it was shown that follicles of different developmental size classes (primary growth and pre-vitellogenic, early-vitellogenic, and mid- to full-grown vitellogenic) express enzymes that release (cytosolic phospholipase A(2) (cPLA(2)); phospholipase Cgamma1) or metabolize (COX-1, COX-2, and prostaglandin synthase-2) AA to PG metabolites. The expression of cPLA(2) was found to be significantly greater in full-grown follicles compared to follicles of the pre- and early-vitellogenic stages. In vivo studies demonstrated that breeding groups of zebrafish exposed to 100 microg/L INDO exhibited reduced spawning rates and clutch sizes compared with control and 1 microg/L INDO exposed fish. In other studies, it was shown that naturally spawning groups of females exhibit increased ovarian levels of PGF(2alpha), E(2), and 17alpha,20beta-dihydroxy-4-pregnen-3-one (a maturation-inducing hormone in zebrafish) near the time of ovulation compared with non-breeding females. Collectively, these experiments indicate that the AA pathway in zebrafish ovaries is involved in the regulation of oocyte maturation and ovulation and a non-selective inhibitor of COX disrupts these processes.

Reproductive and stress hormone levels in goldfish (Carassius auratus) exposed to oil sands process-affected water

Athabasca oil sands mining in northern Alberta produces process-affected waters that are characterized by the presence of naphthenic acids, polycyclic aromatic hydrocarbons, and high salinity. The purpose of this study was to examine the impact of these process-affected waters on reproductive and stress related endpoints in mature goldfish, Carassius auratus. In two separate studies, testosterone and 17beta-estradiol levels in the plasma were significantly reduced in both male and female goldfish caged for 19 days in process-affected waters relative to controls. This effect was most pronounced in goldfish caged at a site containing mature fine tailing and tailings pond water (P5). Ovarian and testicular tissues from fish in the caging studies were incubated in vitro to evaluate potential differences in basal steroid production levels and responsiveness to human chorionic gonadotropin (hCG). Basal levels of testosterone were reduced significantly in males and females from P5 compared with the control pond (P1) demonstrating that the gonads from exposed fish had a diminished steroidogenic capacity. Gonadal tissues of fish from all ponds responded similarly to hCG suggesting that the steroid biosynthetic pathway remained functionally intact. Plasma cortisol levels were significantly higher in male goldfish caged in a pond containing mature fine tailings and capped with uncontaminated water (P3) and in P5 compared with P1. Collectively, these studies suggest that waste products of oil sands mining have the potential to disrupt the normal endocrine functioning in exposed fish through alterations to both reproductive and glucocorticoid hormone biosynthesis. In additional laboratory studies, exposure of goldfish to a naphthenic acid extract for 7 days failed to replicate the effects of processes-affected waters on plasma steroid levels and the causative agent(s) responsible for the effects on steroid biosynthesis remains to be identified.

A toxicity identification evaluation approach to studying estrogenic substances in hog manure and agricultural runoff

Spreading liquid manure on agricultural fields is a routine way of disposing of animal manure and optimizing the use of nutrients for crops. Limited studies suggest that these wastes may contain a variety of endocrine-disrupting compounds (EDCs) that may be released into aquatic environments through runoff. The purpose of this study was to apply a toxicity identification and evaluation approach to isolate and identify estrogenic compounds in hog manure. A recombinant yeast estrogen screen bioassay was used to detect estrogenicity of high-performance liquid chromatography--separated hog manure fractions. Further analytical analyses of the fractions and comparison to authentic standards resulted in the identification of the endogenous estrogens 17 beta-estradiol (E2) and estrone, and the phytoestrogen metabolite, equol. High levels of equol (6.9-16.6 ppm) were found to be present in manure that was stored for several months. The endocrine-disrupting potential of equol was characterized further by using fish hormone estrogen receptor (ER), sex hormone binding protein (SSBP), and goldfish androgen receptor (AR) radioligand binding assays. Equol was found to be approximately 1,000- and 200-fold less potent that E2 in competing for binding sites of the SSBP and ER, respectively. Equols potency was 2,200-fold less than testosterone for the AR. Additional studies confirmed the presence of compounds with estrogenic activity in tile drain water after application of hog manure to an agriculture field. In this case, the contribution of equol to the total estrogenicity of the tile drain water was minimal relative to that of natural estrogens. Overall, this study indicates that further work is warranted to assess the impact that EDCs that originate from agricultural runoff may have on the ecology or physiology of exposed biota.

Regulation of prostaglandin synthesis in ovaries of sexually‐mature zebrafish (Danio rerio)

This study investigates the regulation of prostaglandin (PG) synthesis in the ovaries of sexually‐mature zebrafish (Danio rerio). We examined the ovarian expression of genes within the arachidonic acid (AA) pathway, and the ovarian levels of 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17α,20β‐P), 17β‐estradiol (E2), and PGF2α in spawning and nonspawning fish during the ovulatory cycle. Real‐time RT‐PCR analysis revealed that the expression levels of cytosolic phospholipase A2 (cpla2) and cyclooxygenases (COX)‐2 (ptgs2) in ovarian fragments and in isolated full‐grown follicles of spawning fish were highest at 6:00 when ovulation was expected to occur. In nonspawning fish, cpla2 expression levels declined over time while ptgs2 expression displayed the same temporal pattern as in spawning fish. Elevated levels of 17α,20β‐P in the spawning fish occurred at 3:30, but there were no changes in the nonspawning fish. In other studies conducted to investigate the hormonal regulation of AA pathway genes, fish exposed via the water for 24 or 96 hr to 17α,20β‐P or E2 exhibited reduced ovarian expression levels of COX‐1 (ptgs1) and PG E synthase‐2 (ptgsl), and E2 reduced the expression of cpla2. Injection of human chorionic gonadotropin (hCG) (100 IU) led to increased expression levels of cpla2 and ptgs2 at 2 and 18 hr post‐treatment, but consistently reduced ptgs1 and ptgsl expression. In these fish, ovarian levels of 17α,20β‐P were elevated at all time points and PGF2α levels in the hCG‐treated group were significantly higher than the control fish at 18 hr. Collectively, these in vivo results suggest that gonadotropins and steroids are involved in the regulation of the AA pathway in ovarian follicles of zebrafish. Mol. Reprod. Dev. 76: 1064–1075, 2009.

Endocrine Toxicants and Reproductive Success in Fish

There is compelling evidence on a global scale for compromised growth and reproduction, altered development, and abnormal behaviour in feral fish that can be correlated or in some cases causally linked with exposure to endocrine disrupting chemicals (EDCs). Attributing cause and effect relationships for EDCs is a specific challenge for studies with feral fish as many factors including food availability, disease, competition and loss of habitat also affect reproduction and development. Even in cases where there are physiological responses of fish exposed to EDCs (e.g., changes in reproductive hormone titres, vitellogenin levels), the utility of these measures in extrapolating to whole animal reproductive or developmental outcomes is often limited. Although fish differ from other vertebrates in certain aspects of their endocrinology, there is little evidence that fish are more sensitive to the effects of EDCs. Therefore, to address why endocrine disruption seems so widespread in fish, it is necessary to consider aspects of fish physiology and their environment that may increase their exposure to EDCs. Dependence on aquatic respiration, strategies for iono-osmotic regulation, and maternal transfer of contaminants to eggs creates additional avenues by which fish are exposed to EDCs. This paper provides an overview of responses observed in feral fish populations that have been attributed to EDCs and illustrates many of the factors that need consideration in evaluating the risks posed by these chemicals.

Ibuprofen reduces zebrafish PGE2 levels but steroid hormone levels and reproductive parameters are not affected

Prostaglandins are important regulators of reproductive function in fish. Analgesics like aspirin and ibuprofen are prostaglandin inhibitors and have been detected in freshwater systems at ng/L-μg/L levels. We investigated whether ibuprofen would affect prostaglandin and sex steroid hormone levels in adult zebrafish (Danio rerio) and if expression levels of genes involved in steroidogenesis and prostaglandin synthesis were affected. Zebrafish were exposed to moderate concentrations of ibuprofen (21, 201 or 506 μg/L) for 7 days in a semi-static test system. Ibuprofen concentrations were close to nominal levels and decreased by a maximum of 12-13% over 24 h. Prostaglandin E(2) (PGE(2)) levels in whole body homogenates of males and ovaries of females decreased in a monotonic dose-response relationship whereas male 11-ketotestosterone levels and ovarian 17β-estradiol levels remained unchanged. Ibuprofen did not have an influence on vitellogenin levels, female gonadosomatic index or cumulative egg production and no dose-response relationship in ovarian and testicular expression levels of the investigated genes was observed. This study shows that ibuprofen reduces PGE(2) levels in male and female zebrafish but has no consistent effects on other investigated reproductive parameters.

The Inhibitory Control of Oocyte Maturation in the Zebrafish (Danio rerio): The Role of the G Protein-Coupled Estrogen Receptor and Epidermal Growth Factor

The development and maturation of oocytes in fish have been studied extensively [1, 2]. The complex and coordinated actions of pituitary gonadotropins, ovarian-derived growth factors, and sex steroids occur in concert to ensure the ovulation and spawning of hundreds of viable oocytes from fish ovaries. It is well established that steroid hormones, notably estrogens and progestins, are involved with oocyte growth and resumption of meiosis, respectively. The ovarian follicle produces large amounts of 17b-estradiol (E2) to support the synthesis of vitellogenin by the liver; this protein is sequestered by the growing oocytes. During this period of growth, the oocytes are arrested at the first meiotic prophase by high intracellular levels of cAMP. Intraoocyte levels of cAMP decline when gonadotropin (luteinizing hormone) levels surge during the periovulatory period, leading to the increased synthesis of a maturation-inducing steroid (MIS; e.g., 17a,20bdihydroxy-4-pregnen-3-one and related progestins) that induces final maturation of the oocyte via nongenomic actions involving a membrane progesterone receptor [3]. The number of studies investigating the factors and biochemical pathways involved in the inhibition of the resumption of meiosis in fish has increased during recent years. The Thomas research group has contributed substantially to our understanding of the inhibitory role that E2 plays in controlling the onset of oocyte maturation, including the receptor through which it acts and the signal transduction pathways that are responsive downstream of receptor activation [4–7]. The following sections highlight these advancements and specifically address the recent contributions made in the manuscript by Peyton and Thomas published in this issue of Biology of Reproduction [7].

Mummichog (Fundulus heteroclitus) continue to successfully produce eggs after exposure to high levels of 17α-ethinylestradiol.

17α-Ethinylestradiol (EE2) is a potent estrogen used in birth-control pills. Previous laboratory and field studies have shown negative impacts in a variety of fish species after exposure to low levels of EE2, most notably a nearly complete shutdown of egg production. The present study demonstrates that mummichog (Fundulus heteroclitus), a small-bodied estuarine species, is able to continue to produce eggs after exposure for 28 d to 100 ng of EE2/L. No effect of EE2 on egg production was observed, whereas a >35-fold increase in vitellogenin (vtg 1) gene expression in males was found. The lack of response in egg production in fish exposed to high levels of EE2 warrants further investigations on species-specific responses to estrogens and endocrine disruptors in general.