Achim Trubiroha

Endocrine disruption in aquatic vertebrates.

Environmental compounds can interfere with endocrine systems of wildlife and humans. The main sink of such substances, called endocrine disrupters (ED), are surface waters. Thus, aquatic vertebrates, such as fish and amphibians, are most endangered. ED can adversely affect reproductive biology and the thyroid system. ED act by (anti)estrogenic and (anti)androgenic modes of action, resulting in abnormal sexual differentiation and impaired reproduction. These effects are mainly driven by direct interferences of ED with sex steroid receptors rather than indirectly by impacting synthesis and bioavailability of sex steroids, which in turn might affect the hypothalamic–pituitary–gonadal axis. Recent findings reveal that, in addition to the human‐produced waste of ED, natural sources, such as parasites and decomposition of leaves, also might act as ED, markedly affecting sexual differentiation and reproduction in fish and amphibians. Although the thyroid system has essential functions in both fish and amphibians, amphibian metamorphosis has been introduced as the most sensitive model to detect thyroidal ED; no suitable fish model exists. Whereas ED may act primarily on only one specific endocrine target, all endocrine systems will eventually be deregulated as they are intimately connected to each other. The recent ecotoxicological issue of pharmaceutically active compounds (PhACs) present in the aquatic environment indicates a high potential for further endocrine modes of action on aquatic vertebrates by ED derived from PhACs, such as glucocorticoids, progestins, and ß‐agonists.

The Challenge Presented by Progestins in Ecotoxicological Research: A Critical Review

Around 20 progestins (also called gestagens, progestogens, or progestagens) are used today in assisting a range of medical conditions from endometrial cancer to uterine bleeding and as an important component of oral contraception. These progestins can bind to a wide range of receptors including progestin, estrogen, androgen, glucocorticoid, and mineralocorticoid receptor, as well as sex hormone and corticosteroid binding globulins. It appears that only five of these (four synthetic and one natural) progestins have so far been studied in sewage effluent and surface waters. Analysis has reported values as either nondetects or low nanograms per liter in rivers. Seven of the progestins have been examined for their effects on aquatic vertebrates (fish and frogs). The greatest concern is associated with levonorgestrel, norethisterone, and gestodene and their ability to reduce egg production in fish at levels of 0.8-1.0 ng/L. The lack of environmental measurements, and some of the contradictions in existing values, however, hampers our ability to make a risk assessment. Only a few nanograms per liter of ethynodiol diacetate and desogestrel in water would be needed for fish to receive a human therapeutic dose for these progestins according to modeled bioconcentration factors. But for the other synthetic progestins levels would need to reach tens or hundreds of nanograms per liter to achieve a therapeutic dose. Nevertheless, the wide range of compounds, diverse receptor targets, and the effect on fish reproduction at sub-nanogram-per-liter levels should prompt further research. The ability to impair female reproduction at very low concentrations makes the progestins arguably the most important pharmaceutical group of concern after ethinylestradiol.

Influence of temperature on puberty and maturation of pikeperch, Sander lucioperca

Among external factors, temperature is known to exhibit a prominent role in reproduction of temperate fish species. Here, temperature related induction of puberty in pikeperch Sander lucioperca was investigated. For the first time the key factors of the pikeperch brain-pituitary-gonad axis, targeting the mRNA expression of the luteinising hormone (LH) and the follicle stimulating hormone (FSH), as well as the plasma sex steroids estradiol (E2), testosterone (T), 11-ketotestosteron (11-KT) and 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) were addressed in the experiment. Concomitant the maturational stages were described histologically. After 3 months, female pikeperch kept at 12°C revealed significant increases in the GSI and plasma E2 concentration and 90% of the females were mid-vitellogenic. After 5 months, females kept between 9 and 15°C exhibited significant up-regulation of E2 and GSI as well as comparable histological outcome. At 6 and 23°C in nearly all females stagnation of oogenesis was recorded. Congruently, T was increased at 12 and 15°C. Expression analysis revealed a significant up-regulation of LHβ and FSHβ mRNA in females from early-vitellogenesis, and from mid-spermatogenesis in males, correlated to elevated plasma concentrations of steroids (except for E2 in males). In conclusion, moderate temperatures (12-15°C for) for at least 3 months were required to proceed with first maturation in juvenile pikeperch. The most efficient effect was observed at 12°C, while high (23°C) or low (6°C) temperatures prevented gonadal maturation. So temperature was identified as a prime factor in the induction of puberty in pikeperch, as revealed by histological as well as endocrine parameters.

Transgenic zebrafish illuminate the dynamics of thyroid morphogenesis and its relationship to cardiovascular development.

Among the various organs derived from foregut endoderm, the thyroid gland is unique in that major morphogenic events such as budding from foregut endoderm, descent into subpharyngeal mesenchyme and growth expansion occur in close proximity to cardiovascular tissues. To date, research on thyroid organogenesis was missing one vital tool-a transgenic model that allows to track the dynamic changes in thyroid size, shape and location relative to adjacent cardiovascular tissues in live embryos. In this study, we generated a novel transgenic zebrafish line, tg(tg:mCherry), in which robust and thyroid-specific expression of a membrane version of mCherry enables live imaging of thyroid development in embryos from budding stage throughout formation of functional thyroid follicles. By using various double transgenic models in which EGFP expression additionally labels cardiovascular structures, a high coordination was revealed between thyroid organogenesis and cardiovascular development. Early thyroid development was found to proceed in intimate contact with the distal ventricular myocardium and live imaging confirmed that thyroid budding from the pharyngeal floor is tightly coordinated with the descent of the heart. Four-dimensional imaging of live embryos by selective plane illumination microscopy and 3D-reconstruction of confocal images of stained embryos yielded novel insights into the role of specific pharyngeal vessels, such as the hypobranchial artery (HA), in guiding late thyroid expansion along the pharyngeal midline. An important role of the HA was corroborated by the detailed examination of thyroid development in various zebrafish models showing defective cardiovascular development. In combination, our results from live imaging as well es from 3D-reconstruction of thyroid development in tg(tg:mCherry) embryos provided a first dynamic view of late thyroid organogenesis in zebrafish-a critical resource for the design of future studies addressing the molecular mechanisms of these thyroid-vasculature interactions.

Naturally-induced endocrine disruption by the parasite Ligula intestinalis (Cestoda) in roach (Rutilus rutilus).

Fish represent the most frequently used vertebrate class for the investigation of endocrine disruption (ED) in wildlife. However, field studies are complicated by exposure scenarios involving a variety of anthropogenic and natural influences interfering with the endocrine system. One natural aspect rarely considered in ecotoxicological studies is how parasites modulate host physiology. Therefore, investigations were carried out to characterise the impacts of the parasitic tapeworm Ligula intestinalis on plasma sex steroid levels and expression of key genes associated with the reproduction in roach (Rutilus rutilus), a sentinel species for wildlife ED research. Parasitisation by L. intestinalis suppressed gonadal development in both genders of roach and analysis of plasma sex steroids revealed substantially lower levels of 17beta-oestradiol (E2) and 11-ketotestosterone (11-KT) in infected females as well as E2, 11-KT, and testosterone in infected males. Consistently, in both, infected females and males, expression of the oestrogen dependent genes such as vitellogenin and brain-type aromatase in liver and brain was reduced. Furthermore, parasitisation differentially modulated mRNA expression of the oestrogen and androgen receptors in brain and liver. Most prominently, liver expression of oestrogen receptor 1 was reduced in infected females but not in males, whereas expression of oestrogen receptor 2a was up-regulated in both genders. Further, insulin-like growth factor 1 mRNA in the liver was increased in infected females but not in males. Despite severe impacts on plasma sex steroids and pituitary gonadotropin expression, brain mRNA levels of gonadotropin-releasing hormone (GnRH) precursors encoding GnRH2 and GnRH3 were not affected by L. intestinalis-infection. In summary, the present results provide basic knowledge of the endocrine system in L. intestinalis-infected roach and clearly demonstrate that parasites can cause ED in fish.

Expression of gonadotropin subunits in roach (Rutilus rutilus, Cyprinidae) infected with plerocercoids of the tapeworm Ligula intestinalis (Cestoda).

Plerocercoids of the tapeworm Ligula intestinalis (Cestoda: Bothriocephalidea) have been reported to inhibit gametogenesis of their intermediate fish hosts. However, mechanistic studies are rare and the proximate cues leading to impaired reproduction still remain unknown. In the present study we investigated the effects of infection by L. intestinalis on reproductive parameters of roach (Rutilus rutilus, Cyprinidae), a common fish host of this parasite. Field studies on roach demonstrated that in both genders infection prevented gonad development. As revealed by quantitative PCR, infection was accompanied by essentially lower pituitary expression of follicle-stimulating hormone beta-subunit (FSHbeta) and luteinizing hormone beta-subunit (LHbeta) mRNA compared with uninfected roach, providing clear evidence for gonadotropin-insufficiency as the cause of arrested gametogenesis. Under controlled laboratory conditions infected roach showed lower mRNA levels of FSHbeta but not of LHbeta, despite histology revealing similar gonad stages as in uninfected conspecifics. These findings indicate the involvement of FSH rather than LH in mediating effects of infection early during gonad development in roach. Moreover, the impact of L. intestinalis on reproductive parameters of roach appeared to be independent of the parasite burden. Together, these data provide valuable information on the role of FSH and LH as mediators of parasite-induced sterilization in a vertebrate and implicate the selective inhibition of host reproduction by L. intestinalis as a natural source of endocrine disruption in fish.

The progestin levonorgestrel disrupts gonadotropin expression and sex steroid levels in pubertal roach (Rutilus rutilus).

The aim of the present study was to investigate the effects of the synthetic progestin levonorgestrel (LNG) on the reproductive endocrine system of a teleost fish, the roach (Rutilus rutilus). Pubertal roach were exposed for 28 days in a flow-through system to four concentrations of LNG (3, 31, 312, and 3124 ng/l). Both males and females treated with 3124 ng/l LNG exhibited the upregulated levels of vitellogenin and oestrogen receptor 1 mRNA in the liver. At the same concentration, LNG caused a significant upregulation of the mRNA expression of the gene encoding luteinising hormone β-subunit (lhβ) and the suppression of the mRNA expression of the gene encoding follicle-stimulating hormone β-subunit (fshβ) in the pituitary of both male and female roach. A lower LNG concentration (312 ng/l) suppressed mRNA expression of fshβ in males only. Females treated with 3124 ng/l LNG exhibited significantly lower plasma 11-ketotestosterone (11-KT) and oestradiol (E2) concentrations, whereas their testosterone (T) level was higher compared with the control. Females exposed to 312 ng/l LNG presented significantly lower plasma E2 concentrations. Males exposed to ≥31 ng/l LNG exhibited significantly reduced 11-KT levels. As determined through a histological analysis, the ovaries of females were not affected by LNG exposure, whereas the testes of males exposed to 31 and 312 ng/l LNG exhibited a significantly higher percentage of spermatogonia B compared with the control. The results of the present study demonstrate that LNG disrupts the reproductive system of pubertal roach by affecting the pituitary gonadotropin expression and the sex steroid levels. This disruption was determined to occur in males after exposure to an environmentally relevant concentration (31 ng/l). Moreover, the highest tested concentration of LNG (3124 ng/l) exerted an oestrogenic effect on fish of both sexes.

The Synthetic Gestagen Levonorgestrel Disrupts Sexual Development in Xenopus laevis by Affecting Gene Expression of Pituitary Gonadotropins and Gonadal Steroidogenic Enzymes

In the present study, Xenopus laevis tadpoles were chronically exposed to four concentrations of the synthetic gestagen Levonorgestrel (LNG; 10(-11), 10(-10), 10(-9), and 10(-8)M) starting at Nieuwkoop and Faber (NF) stage 48 until completion of metamorphosis. At NF 58 and 66, brain-pituitary and gonad samples were taken for gene expression analyses of gonadotropins and gonadal steroidogenic enzymes. Exposure to 10(-9) and 10(-8)M LNG until NF 58 repressed messenger RNA (mRNA) expression of luteinizing hormone (LH) β in both genders. This decrease was persistent after further treatment until NF 66 in the 10(-8)M LNG treatment. Expression of follicle-stimulating hormone (FSH) β was affected sex-specifically. No effect was present in NF 58 females, whereas LNG at 10(-9) and 10(-8)M significantly increased FSHβ mRNA levels in males. In NF 66 females, 10(-9)M LNG treatment increased FSHβ gene expression, whereas a decrease was observed in NF 66 males exposed to 10(-8)M LNG. In gonads, expression of steroid-5-alpha-reductase was affected sex-specifically with increased mRNA levels in females but repressed levels in males. Gene expression of further gonadal steroidogenic factors was decreased by 10(-8)M LNG in both genders at NF 66. Assessment of gonad gross morphology and histology revealed poorly developed testes in the 10(-8)M LNG treatment. Our results reveal considerable effects of chronic LNG exposure on sexual development of amphibians. The persistent inhibition of LHβ expression concomitant with decreased mRNA levels of gonadal steroidogenic enzymes is suggested to result in the disruption of reproduction in adult amphibians.

Stage-dependent differences in RNA composition and content affect the outcome of expression profiling in roach (Rutilus rutilus) ovary.

The influence of changing composition and content of RNA on the results of expression profiling was studied in the group-synchronous ovaries of roach (Rutilus rutilus) over the course of their maturation. The highest yield of total RNA was detected in the primary growth and early cortical alveolus stages. The total RNA yield gradually decreased through the late cortical alveolus and late vitellogenic stages. In the primary growth and early cortical alveolus stages, total RNA was characterized by a low percentage of 18S and 28S rRNA and a high percentage of smaller-sized RNAs (tRNA, 5S and 5.8S rRNA), whereas 18S and 28S rRNA had increased by the late cortical alveolus stage and dominated by the late vitellogenic stage. The ratio of mRNA to total RNA was highest at the primary growth stage but decreased significantly in later ovarian stages. When total RNA was used for reverse transcription (RT), the shift in the mRNA/total RNA ratio influenced the results of qPCR expression profiling of several commonly used reference genes (ribosomal protein L8, elongation factor-1α, RNA polymerase-subunit B5, and β2-microglobulin) and of two target genes, gonad-type aromatase (cyp19a1a) and follistatin (fst). We conclude that the expression of target genes should be related to the mRNA pool using the same input of either mRNA to RT or cDNA to qPCR. Furthermore, gene expression was related to tissue-specific RNA yield per body mass (RNA yield x ovary mass x body mass⁻¹) thereby reflecting the massive increase in the size and cellular composition of the ovary during the reproductive cycle.

Inhibition of gametogenesis by the cestode Ligula intestinalis in roach (Rutilus rutilus) is attenuated under laboratory conditions.

Reproductive parameters of Ligula intestinalis-infected roach (Rutilus rutilus) which were held under long-tem laboratory conditions with unlimited food supply were investigated. Although uninfected and infected roach showed no difference in condition factor and both groups deposited perivisceral fat, the gonadosomatic-index was significantly lower in infected female and male roach. Quantitative histological analysis revealed that gonad development was retarded upon parasitization in both genders. In contrast to the phenotype described in the field, infected females were able to recruit follicles into secondary growth, but a high percentage of secondary growth follicles underwent atresia. In both genders, the histological data corresponded well with reduced expression of pituitary gonadotropins and lowered plasma concentrations of sex steroids, as revealed by real-time RT-PCR and ELISA, respectively. Furthermore, a reduction of vitellogenin mRNA and modulated expression of sex steroid receptors in the liver was demonstrated. Like in the field, there was a significant adverse impact of L. intestinalis on host reproductive physiology which could not be related to parasite burden. Our results show, for the first time, that maintenance under laboratory conditions can not abolish the deleterious effect of L. intestinalis on gametogenesis in roach, and indicate a specific inhibition of host reproduction by endocrine disruption.