Jane Ebsen Morthorst

Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations

Feminization of fish caused by certain estrogenic compounds e.g. 17 alpha-ethinylestradiol (EE2) has been shown to be partly reversible. So far it has not been studied if this applies for androgenic compounds too. The androgenic steroid trenbolone acetate (TbA) is used as growth promoter in beef cattle in the United States, South America, and Australia. TbA metabolites are stable in animal waste and have been detected in surface waters associated with feedlot areas and studies on both fish and mammals have demonstrated a strong androgenic effect of those metabolites. Zebrafish (Danio rerio) were exposed to environmentally relevant concentrations of the TbA metabolite 17beta-trenbolone from 0 to 60 days post-hatch (dph) and either sacrificed at 60 dph, transferred to clean water for 170 days or kept in exposure for 170 days. At 60 dph gonadal histology and vitellogenin analyses revealed all-male populations in groups exposed to 15.5 and 26.2 ng/L, and at 9.2 ng/L a skewed sex ratio towards males was observed. After the depuration period no sign of reversibility was observed. Environmentally relevant concentrations of 17beta-trenbolone cause a strong and irreversible masculinization of zebrafish and that raises concern about the effects of androgenic discharges in the aquatic environment. In addition this study also aids in understanding of the so far unknown sex determination process in zebrafish.

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.

Laser capture microdissection of gonads from juvenile zebrafish

BackgroundInvestigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation.MethodsThe laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads.ResultsThe juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling.ConclusionThe study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species.

Evaluation of yolk protein levels as estrogenic biomarker in bivalves; Comparison of the alkali labile phosphate method (ALP) and a species specific immunoassay (ELISA)

Altered concentration of the vertebrate yolk protein precursor vitellogenin is a recognized biomarker for endocrine disruption in fish, and within recent years yolk protein alteration has also been associated with endocrine disruption in bivalves. Species-specific, direct and sensitive methods for quantification of vitellogenin in fish have been available for years whereas bivalve yolk protein levels have been estimated indirectly by alkali-labile phosphate (ALP) liberated from high molecular weight proteins because the sequence and biochemical structure of most bivalve yolk proteins are unknown. By applying a species-specific enzyme-linked immunosorbent assay (ELISA) for accurate determination of yolk protein level the impact of 17β-estradiol (57, 164 and 512 ng/L) on the freshwater bivalve Unio tumidus was investigated and compared with ALP estimations. Seven weeks of exposure during the pre-spawning and spawning period had no consistent effect on yolk protein concentration in hemolymph, and ALP levels in hemolymph also remained unchanged in both males and females. Further, basal male and female ALP levels were indistinguishable whereas the ELISA demonstrated that yolk protein levels of females exceeded male levels at the time of sampling, although male basal levels were high compared to fish. Altogether it is shown that individual ALP levels do not reflect yolk protein levels and hence hemolymph ALP levels cannot serve as biomarker for estrogenic exposure during the pre-spawning and spawning period in U. tumidus. The necessity of sensitive and validated biomarkers for reliable interpretation of data and the utility of ALP and yolk protein levels as biomarkers in bivalves are discussed.

17β-estradiol causes abnormal development in embryos of the viviparous eelpout

Elevated frequencies of malformations among the offspring of Baltic eelpout (Zoarces viviparus) have been observed in aquatic environments receiving high anthropogenic input suggesting that manmade chemicals could be the causative agent. However, causal links between exposure to chemicals and abnormal development have never been confirmed in laboratory experiments. The purpose of this study was to investigate if exposure to 17β-estradiol (E2) causes abnormal development in larvae of the viviparous eelpout. Wild female eelpout were collected immediately after fertilization and exposed to E2 concentrations ranging from 5.7 to 133 ng L(-1) for 6 weeks in a flow through test system. The experiment shows that E2 concentrations of 53.6 and 133 ng L(-1) cause severe abnormal development among eelpout embryos. Reduced amount of ovarian fluid and increased weight of the ovarian sac indicate disturbance of ovarian function. Female plasma concentrations of E2 and vitellogenin increase in a monotonic concentration-response relationship with significant induction in the low concentration range. Our findings support the plausibility that the abnormal development among eelpout embryos encountered in monitoring programs may actually be caused by exposure to chemicals in the environment.

Expression of prostaglandin synthases (pgds and pges) during zebrafish gonadal differentiation.

The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E2 synthase (pges) and especially the lipocalin-type prostaglandin D2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found in other species. In mice and chicken, the lipocalin-type Pgds is specifically expressed in pre-Sertoli cells just after Sry and Sox9 and is involved in masculinisation of the developing testis. Furthermore, Pges are implicated in female reproduction including follicular development and ovulation. In this study, a sexually dimorphic expression of pgds was found in gonads of adult zebrafish with expression in testis but not in ovaries. To determine whether the sex-specific expression pattern of pgds was present in gonads of juvenile zebrafish and therefore could be an early marker of sex in zebrafish, we microdissected gonads from four randomly selected individual zebrafish for every second day in the period 2-20 days post hatch (dph) and 0-1 dph. The temporal expression of pgds and pges was investigated in the microdissected gonads, however, no differential expression that could indicate sex-specific difference between individual juvenile zebrafish was observed.

Vitellogenin concentrations in feral Danish brown trout have decreased: An effect of improved sewage treatment in rural areas?

Feminization of male and juvenile fish because of exposure to estrogens or estrogenic chemicals in effluents from central wastewater treatment plants (WWTPs) is a worldwide issue of concern. Intersex and induction of the female yolk protein, vitellogenin, in male and juvenile fish are robust biomarkers for estrogenic exposure, and feminized fish have been observed downstream of WWTP outlets in many countries. Danish central WWTPs reduce effluent estrogenicity effectively by advanced sewage treatment, and feminizations have not been observed downstream of central WWTP outlets. However, between 2000 and 2004, investigations of Danish streams not receiving sewage from central WWTPs revealed a high variation in vitellogenin concentrations of male juvenile brown trout (Salmo trutta); some individuals had high concentrations, probably as a result of estrogenic point sources, and the plasma concentration was >50 ng mL-1 in 79% of the juvenile males. The streams were reinvestigated in 2010 to 2016, and the average male level had decreased to a hitherto unseen baseline level; in 2010 only 0.7% (one individual) of the males had a vitellogenin concentration >50 ng mL-1 , which could indicate that the estrogenicity of the streams decreased after 2004. We examined possible estrogenic sources in streams unaffected by central WWTP effluents, and found that the reduced vitellogenin levels are most likely explained by a national effort to improve on-site wastewater treatment in scattered houses not connected to central WWTPs. Environ Toxicol Chem 2018;37:839-845.

Two common mild analgesics have no effect on general endocrine mediated endpoints in zebrafish ( Danio rerio )

Mild analgesics such as acetylsalicylic acid (ASA) and acetaminophen (APAP) exert their pain-relieving effect in humans by inhibition of prostaglandin synthesis. Prostaglandins play key roles in developmental and reproductive processes in vertebrates, and in recent years, it has been suggested that weak analgesics might also act as endocrine disrupters. In a set of experiments we investigated if ASA and APAP affect well-established endocrine endpoints in zebrafish (Danio rerio), which is a commonly used model organism in the investigation of endocrine disrupting chemicals. Zebrafish were exposed to APAP (0.22, 2.3, and 30mgL-1) or ASA (0.2, 0.5, 1.7, and 8.2mgL-1) from hatch to sexual maturity in a test design resembling the OECD Fish Sexual Development Test. No effects on sex ratio and vitellogenin levels were observed. Adult zebrafish were exposed to high concentrations (mgL-1) of ASA or APAP for eight or 14days. ASA reduced the levels of prostaglandin E2, but had no effect on the concentration of 11-ketotestosterone and vitellogenin. Overall, ASA decrease prostaglandin E2 concentrations, but well-established endpoints for endocrine disruption in zebrafish are generally not affected by aquatic exposure neither during development nor adulthood. According to the WHO/IPCS definition of an endocrine disrupter, the present results do not define APAP and ASA as endocrine disrupters.

A field study of hemolymph yolk protein levels in a bivalve (Unio tumidus) and future considerations for bivalve yolk protein as endocrine biomarker

Induction of yolk protein in male fish is a recognized biomarker for estrogenic exposure because the estrogen-dependent induction mechanism is well investigated and there is a clear difference in yolk protein levels of unexposed males and females. Attempts have been made to use induction of bivalve yolk protein as biomarker for estrogenic exposure. However, several biomarker validation criteria have not yet been investigated e.g. an in-depth understanding of the induction mechanism and background variability is needed and reliable detection assays are yet to be developed. To obtain background knowledge about yolk protein levels freshwater bivalves (Unio tumidus) were collected in an uncontaminated Danish lake over the course of a year (33 collection dates). The hemolymph yolk protein concentration of 569 individuals was determined by a species specific enzyme-linked immunosorbent assay (ELISA) and male and female gonadal development cycles were established. The average yolk protein levels of males and females collected at each sampling date overlapped in some periods; the male and female range was 66,946 - 169,692 ng/mL and 88,731 - 681,667 ng/mL, respectively. Because male and female hemolymph yolk protein levels overlap, great care should be taken if yolk protein induction in bivalve hemolymph is considered as endocrine biomarker.