Male-transmitted transgenerational effects of the herbicide linuron on DNA methylation profiles in Xenopus tropicalis brain and testis.

The herbicide linuron can cause endocrine disrupting effects in Xenopus tropicalis frogs, including offspring that were never exposed to the contaminant. The mechanisms by which these effects are transmitted across generations need to be further investigated. Here, we examined transgenerational alterations of brain and testis DNA methylation profiles paternally inherited from grandfathers developmentally exposed to an environmentally relevant concentration of linuron. Reduced representation bisulfite sequencing (RRBS) revealed numerous differentially methylated regions (DMRs) in brain (3060 DMRs) and testis (2551 DMRs) of the adult male F2 generation. Key genes in the brain involved in somatotropic (igfbp4) and thyrotropic signaling (dio1 and tg) were differentially methylated and correlated with phenotypical alterations in body size, weight, hind limb length and plasma glucose levels, indicating that these methylation changes could be potential mediators of the transgenerational effects of linuron. Testis DMRs were found in genes essential for spermatogenesis, meiosis and germ cell development (piwil1, spo11 and tdrd9) and their methylation levels were correlated with the number of germ cells nests per seminiferous tubule, an endpoint of disrupted spermatogenesis. DMRs were also identified in several genes central for the machinery that regulates the epigenetic landscape including DNA methylation (dnmt3a and mbd2) and histone acetylation (hdac8, ep300, elp3, kat5 and kat14), which may at least partly drive the linuron-induced transgenerational effects. The results from this genome-wide DNA methylation profiling contribute to better understanding of potential transgenerational epigenetic inheritance mechanisms in amphibians.

Impaired spermatogenesis and associated endocrine effects of azole fungicides in peripubertal Xenopus tropicalis.

Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3ß-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis.

Pubertal sexual development and endpoints for disrupted spermatogenesis in the model Xenopus tropicalis.

Peripubertal models to determine effects of anti-androgenic endocrine disrupting chemicals are needed. Using the toxicological model species Xenopus tropicalis, the aims of the study were to 1) provide data on sexual maturation and 2) characterise effects of short-term exposure to an anti-androgenic model substance. Juvenile (2.5 weeks post metamorphosis old) X. tropicalis were exposed to 0, 250, 500 or 1000 µg flutamide/L (nominal) for 2.5 weeks. Upon exposure termination, histology of gonads and Müllerian ducts was characterised in detail. New sperm stages were identified: pale and dark spermatogonial stem cells (SSCs). The testes of control males contained spermatozoa, indicating pubertal onset. The ovaries were immature, and composed of non-follicular and pre-vitellogenic follicular oocytes. The Müllerian ducts were more mature in females than males indicating development/regression in the females and males, respectively. In the 500 µg/L group, the number of dark SSCs per testis area was decreased and the number of secondary spermatogonia was increased. No treatment effects on ovaries or Müllerian ducts were detected. To conclude, our present data provide new knowledge on spermatogenesis, and pubertal onset in X. tropicalis. New endpoints for evaluating spermatogenesis are suggested to be added to existing assays used in endocrine and reproductive toxicology.

Proposal for regulatory risk mitigation measures for human pharmaceutical residues in the environment.

Environmental risks of human pharmaceutical products should be made transparent and mitigated as far as possible. We propose to apply a risk mitigation scheme to the marketing authorisation of human medicinal products which is pragmatic and tailored, and thus will not increase the burden to regulators and industry too much. This scheme takes into account increasing knowledge and accuracy of the environmental risk estimates, applying preliminary risk mitigation when risks are determined based on model estimates, and definitive, more strict and far-reaching risk mitigation when risks are based on actual measured environmental concentrations. Risk mitigation measures should be designed to be effective, proportional, easy to implement, and in line with current (other) legislation, as well as not being a burden to the patient/health care professionals. Furthermore, individual risk mitigation measures are proposed for products showing environmental risks, while general risk mitigation measures can be applied to all products to reduce the overall burden of pharmaceuticals in the environment. In order to effectively mitigate risk, linking marketing authorisation legislation to environmental legislation is essential.

Improving the regulatory environmental risk assessment of human pharmaceuticals: Required changes in the new legislation.

One of the flagship actions of the Pharmaceutical Strategy for Europe is to address environmental challenges associated with pharmaceutical use. This includes strengthening the Environmental Risk Assessment (ERA) at marketing authorisation (MA) of pharmaceuticals, and revision of the pharmaceutical legislation where needed. The overall aim of an ERA should be to enable comprehensive and effective identification and management of environmental risks of pharmaceuticals without affecting the availability of pharmaceuticals to patients. As experts in the evaluation of ERAs of human medicinal products submitted by pharmaceutical industries (Applicants), we have summarized the current status of the ERA and suggest legislative changes to improve environmental protection without affecting availability. Six regulatory goals were defined and discussed, including possible ways forward: 1) mandatory ERAs in accordance to the EMA guideline at the time of the MA, 2) enforcement of risk mitigation measures including re-evaluation of the ERA, 3) facilitated exchange of environmental data between pharmaceutical and environmental legislations, 4) substance-based assessments, 5) transparency of data, and 6) a catching-up procedure for active pharmaceutical ingredients that lack an ERA. These legislative proposals can be considered as prerequisites for a harmonised assessment and effective management of environmental risks and hazards of human pharmaceuticals.

Experiences of physical activity and exercise among women with obstructive pulmonary disease.

BACKGROUND: With more women being diagnosed with obstructive pulmonary disease, it is important to know how women experience non-pharmacological rehabilitation including different types of physical activity and exercise. OBJECTIVE: This study aimed to explore how women with obstructive pulmonary disease experienced participating in pulmonary rehabilitation including yoga or strength- and endurance training to promote physical activity. A second aim included exploring experiences of physical activity and exercise through life. METHODS: Fifteen women with asthma or chronic obstructive pulmonary disease were interviewed about their experiences of participating in an exercise intervention and about their experiences of physical activity and exercise in their lives. The transcribed interviews were analyzed using qualitative content analysis. RESULTS: An overall theme, "Wishing to succeed in attending physical activity and exercise," emerged. Three categories were identified: 1) strategies to overcome insecurity; 2) a life situation which enables and hinders; and 3) an inner drive and focus on myself. CONCLUSIONS: The women's wishes to be physically activity and exercise involved hindering and enabling factors. Specifically, their gender roles as women were described as a hinder. This suggests a need to include a gender perspective when promoting physical activity and exercise to women with obstructive pulmonary disease.

Pesticide-induced multigenerational effects on amphibian reproduction and metabolism.

Underlying drivers of species extinctions need to be better understood for effective conservation of biodiversity. Nearly half of all amphibian species are at risk of extinction, and pollution may be a significant threat as seasonal high-level agrochemical use overlaps with critical windows of larval development. The potential of environmental chemicals to reduce the fitness of future generations may have profound ecological and evolutionary implications. This study characterized effects of male developmental exposure to environmentally relevant concentrations of the anti-androgenic pesticide linuron over two generations of offspring in Xenopus tropicalis frogs. The adult male offspring of pesticide-exposed fathers (F1) showed reduced body size, decreased fertility, and signs of endocrine system disruption. Impacts were further propagated to the grand-offspring (F2), providing evidence of transgenerational effects in amphibians. The adult F2 males demonstrated increased weight and fat body palmitoleic-to-palmitic acid ratio, and decreased plasma glucose levels. The study provides important cross-species evidence of paternal epigenetic inheritance and pollutant-induced transgenerational toxicity, supporting a causal and complex role of environmental contamination in the ongoing species extinctions, particularly of amphibians.

Developmental reproductive toxicity and endocrine activity of propiconazole in the Xenopus tropicalis model.

Environmental pollutants and especially endocrine disrupting chemicals (EDCs) are implicated as one of the drivers of the amphibian declines. To advance the understanding of the risks of EDCs to amphibians, methods to determine endocrine-linked adverse effects are needed. The aims were to 1) develop a partial life-cycle assay with the model frog Xenopus tropicalis to determine endocrine perturbation and adverse developmental effects, and 2) determine effects of propiconazole in this assay. Propiconazole is a pesticide with multiple endocrine modes of action in vitro. Its potential endocrine activity and adverse effects in amphibians remain to be elucidated. Tadpoles were exposed to 0, 33 and 384 µg propiconazole/L during critical developmental windows until completed metamorphosis. At metamorphosis, a sub-sample of animals was analysed for endpoints for disruption of estrogen/androgen (sex ratio, brain aromatase activity) and thyroid pathways (time to metamorphosis). The remaining individuals were kept unexposed for 2 months post-metamorphosis to analyze effects on sexual development including gonadal and Müllerian duct maturity and gametogenesis. At metamorphosis, brain aromatase activity was significantly increased in the high-dose group compared to control. In both propiconazole groups, an increased proportion of individuals reached metamorphosis faster than the mean time for controls, suggesting a stimulatory effect on the thyroid system. At 2 months post-metamorphosis, testis size, sperm and Müllerian duct maturity were reduced in the low-dose males, and the liver somatic index in males was increased in both propiconazole groups, compared with controls. In conclusion, our results show that propiconazole exposure caused endocrine perturbations and subsequent hepatic and reproductive effects evident at puberty, indicating persistent disruption of metabolism and male reproductive function. Our findings advance the development of methodology to determine endocrine and adverse effects of EDCs. Moreover, they increase the understanding of endocrine perturbations and consequent risk of adverse effects of azoles in amphibians.

A laboratory investigation into features of morphology and physiology for their potential to predict reproductive success in male frogs.

Amphibian populations are declining globally, however, the contribution of reduced reproduction to declines is unknown. We investigated associations between morphological (weight/snout-vent length, nuptial pad colour/size, forelimb width/size) and physiological (nuptial pad/testis histomorphology, plasma hormones, gene expression) features with reproductive success in males as measured by amplexus success and fertility rate (% eggs fertilised) in laboratory maintained Silurana/Xenopus tropicalis. We explored the robustness of these features to predict amplexus success/fertility rate by investigating these associations within a sub-set of frogs exposed to anti-androgens (flutamide (50 µg/L)/linuron (9 or 45 µg/L)). In unexposed males, nuptial pad features (size/colour/number of hooks/androgen receptor mRNA) were positively associated with amplexus success, but not with fertility rate. In exposed males, many of the associations with amplexus success differed from untreated animals (they were either reversed or absent). In the exposed males forelimb width/nuptial pad morphology were also associated with fertility rate. However, a more darkly coloured nuptial pad was positively associated with amplexus success across all groups and was indicative of androgen status. Our findings demonstrate the central role for nuptial pad morphology in reproductive success in S. tropicalis, however, the lack of concordance between unexposed/exposed frogs complicates understanding of the utility of features of nuptial pad morphology as biomarkers in wild populations. In conclusion, our work has indicated that nuptial pad and forelimb morphology have potential for development as biomarkers of reproductive health in wild anurans, however, further research is needed to establish this.

The Xenopus tropicalis Model for Studies of Developmental and Reproductive Toxicity.

The reproductive cycle encompasses processes such as sex organ differentiation and development in the early life stages and maturation of the gametes in the adult organism. During the early life stages, critical developmental programming of the endocrine and reproductive systems occurs, and exposure to chemicals during these critical developmental windows can result in impaired reproductive function later in life. It is therefore important to evaluate long-term consequences of early life stage exposure to endocrine-disrupting chemicals. The African clawed frog Xenopus tropicalis has several characteristics that facilitate studies of developmental and reproductive toxicity. Here I present a X. tropicalis life cycle test protocol including study design, exposure regimes, and endpoints for chemical disruption of sex differentiation, gonadal and Müllerian duct development, the thyroxin-regulated metamorphosis, estrogen synthesis (activity of the CYP19 aromatase enzyme), spermatogenesis, oogenesis, puberty and fertility.

Scientific Opinion on the state of the science on pesticide risk assessment for amphibians and reptiles.

Following a request from EFSA, the Panel on Plant Protection Products and their Residues developed an opinion on the science to support the potential development of a risk assessment scheme of plant protection products for amphibians and reptiles. The coverage of the risk to amphibians and reptiles by current risk assessments for other vertebrate groups was investigated. Available test methods and exposure models were reviewed with regard to their applicability to amphibians and reptiles. Proposals were made for specific protection goals aiming to protect important ecosystem services and taking into consideration the regulatory framework and existing protection goals for other vertebrates. Uncertainties, knowledge gaps and research needs were highlighted.

Sex-dependent expression of anti-Müllerian hormone (amh) and amh receptor 2 during sex organ differentiation and characterization of the Müllerian duct development in Xenopus tropicalis.

Amphibian gonadal differentiation involves the action of sex steroids. Recent research indicates that the anti-Müllerian hormone (AMH) is involved in testicular development in some lower vertebrate species. For amphibians there is a lack of data on ontogenetic expression of the AMH receptor AMHR2/amhr2 and of progesterone receptors (PGRS/pgrs). Here we expand the knowledge on amphibian sex differentiation by characterizing ontogenetic mRNA levels of amh, amhr2, intracellular and membrane pgrs (ipgr and mpgr beta) and cytochrome P450 19a1 (cyp19a1) (ovarian marker) in the urogenital complex of the model species Xenopus (Silurana) tropicalis. Furthermore, we characterized the ontogenetic development of the Müllerian ducts (precursors of the female reproductive tract) histologically. The developmental period investigated spanned from beginning of gonadal differentiation, Nieuwkoop and Faber (NF) stage 51, to 4weeks post-metamorphosis. The Müllerian ducts were first observed at NF 64 in both sexes. Male-enhanced amh mRNA levels from NF 53/54 to 6days post-metamorphosis and female-enhanced cyp19a1 levels from NF 53 to 4weeks post-metamorphosis were noted. The sexually dimorphic mRNA level profile was more distinct for amh than for cyp19a1. The pgrs mRNA levels increased over the studied period and showed no sex differences. At later developmental stages, the amhr2 mRNA level was increased in putative females compared with males. Our findings suggest that AMH has a role in gonadal differentiation in X. tropicalis. We propose relative gonadal amh mRNA level as a testicular marker during early gonadal development in amphibians.

Molecular and histological endpoints for developmental reproductive toxicity in Xenopus tropicalis: Levonorgestrel perturbs anti-Müllerian hormone and progesterone receptor expression.

There is an increasing concern regarding the risks associated with developmental exposure to endocrine disrupting chemicals and the consequences for reproductive capability. The present study aimed to refine the Xenopus (Silurana) tropicalis test system for developmental reproductive toxicity by characterising molecular and histological features of sexual development, and to explore effects of exposure to the progestagen levonorgestrel (LNG). Larvae were exposed to LNG (0, 3, 30, 300 ng/L) over the first three weeks of development, encompassing the beginning of gonadal differentiation. mRNA levels of amh (anti-Müllerian hormone), amhr2 (amh receptor 2), ipgr (intracellular progesterone receptor), mpgr beta (membrane progesterone receptor beta), and cyp19a1 (cytochrome p450 19a1) were quantified in larvae and juveniles (4 weeks post-metamorphosis). Relative cyp19a1 and amh expression was used as a molecular marker for phenotypic sex of larvae. Gonadal and Müllerian duct development were characterised histologically in juveniles. Compared to controls, LNG exposure increased the expression of amh and ipgr in male larvae. In juveniles, mpgr beta expression was increased in both sexes and amhr2 expression was decreased in males, implying persistent effects of developmental progestagen exposure on amh and pgr expression signalling. No effects of LNG on the gonadal or Müllerian duct development were found, implying that the exposure window was not critical with regard to these endpoints. In juveniles, folliculogenesis had initiated and the Müllerian ducts were larger in females than in males. This new knowledge on sexual development in X. tropicalis is useful in the development of early life-stage endpoints for developmental reproductive toxicity.

Improving environmental risk assessment of human pharmaceuticals.

This paper presents 10 recommendations for improving the European Medicines Agency's guidance for environmental risk assessment of human pharmaceutical products. The recommendations are based on up-to-date, available science in combination with experiences from other chemical frameworks such as the REACH-legislation for industrial chemicals. The recommendations concern: expanding the scope of the current guideline; requirements to assess the risk for development of antibiotic resistance; jointly performed assessments; refinement of the test proposal; mixture toxicity assessments on active pharmaceutical ingredients with similar modes of action; use of all available ecotoxicity studies; mandatory reviews; increased transparency; inclusion of emission data from production; and a risk management option. We believe that implementation of our recommendations would strengthen the protection of the environment and be beneficial to society. Legislation and guidance documents need to be updated at regular intervals in order to incorporate new knowledge from the scientific community. This is particularly important for regulatory documents concerning pharmaceuticals in the environment since this is a research field that has been growing substantially in the last decades.

Mixture effects of levonorgestrel and ethinylestradiol: estrogenic biomarkers and hormone receptor mRNA expression during sexual programming.

Synthetic progesterone (progestins) and estrogens are widely used pharmaceuticals. Given that their simultaneous unintentional exposure occurs in wildlife and also in human infants, data on mixture effects of combined exposures to these hormones during development is needed. Using the Xenopus (Silurana) tropicalis test system we investigated mixture effects of levonorgestrel (LNG) and ethinylestradiol (EE2) on hormone sensitive endpoints. After larval exposure to LNG (0.1nM), or EE2 (0.1nM) singly, or in combination with LNG (0.01, 0.1, 1.0nM), the gonadal sex ratio was determined histologically and hepatic mRNA levels of genes encoding vitellogenin (vtg beta1) and the estrogen (esr1, esr2), progesterone (ipgr) and androgen (ar) receptors were quantified using quantitative PCR. All EE2-exposed groups showed female-biased sex ratios and increased vtg beta1 mRNA levels compared with the controls. Compared with the EE2-alone group (positive control) there were no significant alterations in vtg beta1 levels or in sex ratios in the co-exposure groups. Exposure to LNG-alone caused an increase in ar mRNA levels in females, but not in males, compared to the controls and the co-exposed groups, indicating that co-exposure to EE2 counteracted the LNG-induced ar levels. No treatment related impacts on the mRNA expression of esr1, esr2, and ipgr in female tadpoles were found, suggesting that these endpoints are insensitive to long-term exposure to estrogen or progestin. Due to the EE2-induced female-biased sex ratios, the mRNA expression data for the low number of males in the EE2-exposed groups were not statistically analyzed. In conclusion, our results suggest that induced vtg expression is a robust biomarker for estrogenic activity in exposure scenarios involving both estrogens and progestins. Developmental exposure to LNG caused an induction of hepatic ar mRNA expression that was antagonized by combined exposure to EE2 and LNG. To our knowledge this is the first study to report effects of combined exposures to EE2 and LNG during the period of sexual programming.

Risks of hormonally active pharmaceuticals to amphibians: a growing concern regarding progestagens.

Most amphibians breed in water, including the terrestrial species, and may therefore be exposed to water-borne pharmaceuticals during critical phases of the reproductive cycle, i.e. sex differentiation and gamete maturation. The objectives of this paper were to (i) review available literature regarding adverse effects of hormonally active pharmaceuticals on amphibians, with special reference to environmentally relevant exposure levels and (ii) expand the knowledge on toxicity of progestagens in amphibians by determining effects of norethindrone (NET) and progesterone (P) exposure to 0, 1, 10 or 100 ng l(-1) (nominal) on oogenesis in the test species Xenopus tropicalis. Very little information was found on toxicity of environmentally relevant concentrations of pharmaceuticals on amphibians. Research has shown that environmental concentrations (1.8 ng l(-1)) of the pharmaceutical oestrogen ethinylestradiol (EE2) cause developmental reproductive toxicity involving impaired spermatogenesis in frogs. Recently, it was found that the progestagen levonorgestrel (LNG) inhibited oogenesis in frogs by interrupting the formation of vitellogenic oocytes at an environmentally relevant concentration (1.3 ng l(-1)). Results from the present study revealed that 1 ng NET l(-1) and 10 ng P l(-1) caused reduced proportions of vitellogenic oocytes and increased proportions of previtellogenic oocytes compared with the controls, thereby indicating inhibited vitellogenesis. Hence, the available literature shows that the oestrogen EE2 and the progestagens LNG, NET and P impair reproductive functions in amphibians at environmentally relevant exposure concentrations. The progestagens are of particular concern given their prevalence, the range of compounds and that several of them (LNG, NET and P) share the same target (oogenesis) at environmental exposure concentrations, indicating a risk for adverse effects on fertility in exposed wild amphibians.

Developmental exposure to fluoxetine modulates the serotonin system in hypothalamus.

The selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLU, Prozac®) is commonly prescribed for depression in pregnant women. This results in SSRI exposure of the developing fetus. However, there are knowledge gaps regarding the impact of SSRI exposure during development. Given the role of serotonin in brain development and its cross-talk with sex hormone function, we investigated effects of developmental exposure to pharmacologically relevant concentrations of FLU (3 and 30 nM (measured)) on brain neurotransmitter levels, gonadal differentiation, aromatase activity in brain and gonads, and the thyroid system, using the Xenopus tropicalis model. Tadpoles were chronically exposed (8 weeks) until metamorphosis. At metamorphosis brains were cryosectioned and levels of serotonin, dopamine, norepinephrine, and their metabolites 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid, and homovanillic acid were measured in discrete regions (telencephalon, hypothalamus and the reticular formation) of the cryosections using high-performance liquid chromatography. Exposure to 30 nM FLU increased the concentration of 5-hydroxyindoleacetic acid in hypothalamus compared with controls. FLU exposure did not affect survival, time to metamorphosis, thyroid histology, gonadal sex differentiation, or aromatase activity implying that the effect on the serotonergic neurotransmitter system in the hypothalamus region was specific. The FLU concentration that impacted the serotonin system is lower than the concentration measured in umbilical cord serum, suggesting that the serotonin system of the developing brain is highly sensitive to in utero exposure to FLU. To our knowledge this is the first study showing effects of developmental FLU exposure on brain neurochemistry. Given that SSRIs are present in the aquatic environment the current results warrant further investigation into the neurobehavioral effects of SSRIs in aquatic wildlife.

An amphibian model for studies of developmental reproductive toxicity.

The developmental programming of the reproductive system is vulnerable to chemical exposure. It is therefore important to evaluate long-term consequences of early life-stage exposure to endocrine disrupting chemicals. The African clawed frog Xenopus tropicalis has several characteristics which facilitates studies of developmental reproductive toxicity. Here, I present a X. tropicalis test protocol, including study design, exposure regime, and endpoints for chemical disruption of sex differentiation, reproductive organ development, the thyroxin-regulated metamorphosis, oestrogen synthesis (activity of the CYP19 aromatase enzyme), and fertility.

Disrupted oogenesis in the frog Xenopus tropicalis after exposure to environmental progestin concentrations.

Levonorgestrel is a synthetic progesterone commonly used in pharmaceuticals (e.g., in contraceptives). It is found in sewage treatment plant effluents at concentrations up to 30 ng/L and was recently shown to pose a threat to egg laying in fish. Information on the susceptibility of adult amphibians to progestin toxicity is lacking. The present study aimed to 1) characterize progestogenic effects on the full cycle of oogenesis (egg development) in frogs and 2) determine female amphibians' susceptibility to reproductive impacts from progestogenic compounds in the environment. Sexually mature female Xenopus tropicalis were exposed to levonorgestrel via the surrounding water for 7 days (0, 51, or 307 ng/L) or 28 days (0, 1.3, 18, 160, or 1240 ng/L). Their ovaries were analyzed histologically with respect to frequencies of immature (in early meiotic prophase I), previtellogenic, vitellogenic, mature, and atretic oocytes. The 28-day exposure caused reduced proportions of oocytes at immature, vitellogenic, and mature stages, and increased proportions of previtellogenic oocytes compared with the control. The lowest tested concentration, 1.3 ng/L, increased the proportions of previtellogenic oocytes and reduced the proportions of vitellogenic oocytes, indicating inhibited vitellogenesis. The present study shows that progestin concentrations found in the aquatic environment impaired oogenesis in adult frogs. Our results indicate that progestogenic effects on oocyte development include interrupted germ cell progression into meiosis and inhibited vitellogenesis. Considering the crucial role of oogenesis in female fertility, our results indicate that progestogenic pollutants may pose a threat to reproduction in wild amphibian populations.

Early life progestin exposure causes arrested oocyte development, oviductal agenesis and sterility in adult Xenopus tropicalis frogs.

Levonorgestrel (LNG) is a commonly used pharmaceutical progestin found in the environment. Information on the long-term toxicity of progestins following early life exposure is scant. We investigated the effects of developmental LNG exposure on sex differentiation, reproductive organ development and fertility in the model frog Xenopus tropicalis. Tadpoles were exposed to 0, 0.06 or 0.5nM LNG via the water from hatching until metamorphosis. At metamorphosis effects on gonadal differentiation were evaluated using a subsample of frogs. Remaining animals were held unexposed for nine months, at which time reproductive organ structure, function and fertility were determined. LNG exposure severely impaired oviduct and ovary development and fertility. All adult females in the 0.5nM group (n=10) completely lacked oviducts. They also displayed a significantly larger fraction of immature oocytes, arrested in meiotic prophase, than control females. Upon mating with unexposed males, only one of 11 LNG-exposed females laid eggs, whereas all control females did. No effects on testicular development, sperm count or male fertility were observed. At metamorphosis, no effects on sex ratio or gonadal histology were evident. The effects on ovarian and oviductal development were detected at adult age but not at metamorphosis, emphasising the importance of investigating the long-term consequences of developmental exposure. This is the first developmental reproductive toxicity study of a progestin in an aquatic vertebrate. Considering that several progestins are present in contaminated surface waters, further investigation into the sensitivity of frogs to progestins is warranted to understand the risk such compounds may pose to wild frog populations.