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Characterization by histological and molecular tools of the reproductive cycle and endocrine disruption effects on a thicklip grey mullet (Chelon labrosus) population from the South Bay of Biscay
Abstracts / Comparative Biochemistry and Physiology, Part A 163 (2012) S32–S38
performed on collected larvae from the same individual mother fish from 5 of the sites. Analysis on the whole data set using within-region comparison shows that eelpouts from different polluted sites had different gene expression profiles for e.g. cytochrome p450's, possibly reflecting the different types of exposures and stress conditions in these sites. Results also indicate that genes involved in xenobiotic and lipid metabolic processes are found in females and DNA replication and tissue morphogenesis are pronounced in larvae. There are also genes and processes that have similar expression patterns for female and larvae which are also unique for specific sites. This is of great interest for studies of maternal transfer of e.g. specific pollutants. Preliminary data also indicate that females with a high reproductive success and females with low reproductive success have different gene expression profiles; thus these findings can be used in understanding mechanisms behind reproductive success of individual females and how some females can “protect” their brood. These results are part of an EU-financed project (www.science.balcofish.gu.se). doi:10.1016/j.cbpa.2012.05.095
3. Characterization by histological and molecular tools of the reproductive cycle and endocrine disruption effects on a thicklip grey mullet (Chelon labrosus) population from the South Bay of Biscay C. Bizarro (CBET Research Group, University of the Basque Country UPV/EHU, Basque Country, Spain); P. Aragón, A. Maquieira (IRMDT, UPV, Valencia, Spain); M.P. Cajaraville, M. Ortiz-Zarragoitia (CBET Research Group, UPV/EHU, Basque Country, Spain) Mugilids such as thicklip grey mullets Chelon labrosus are used worldwide as sentinel organisms of coastal pollution. Biomarkers of exposure and effect have been applied in mullets, together with contaminant bioaccumulation studies and endocrine disruption works. With the aim of characterizing the gametogenic cycle of mullets (C. labrosus) in the South Bay of Biscay, a monthly survey was completed from September 2010 to August 2011. After weighing and sizing, a piece of gonad tissue was collected for histological and histopathological studies and brain, liver and gonad portions for gene expression (TaqMan) assays. Expression levels of vitellogenin (vtg), estrogen receptor (er), retinoid-X-receptor (rxr) and aromatases cyp19a1 (ovarian form) and cyp19a2 (brain form) were assessed. Blood samples were also obtained for the quantification of vitellogenin plasma levels by specific ELISA. Gametogenesis started at the end of summer and proceeded during autumn. Mature gonads were observed at the end of autumn and spawning occurred during winter months. Postspawning gonads were identified during spring–early summer months. No significant differences were detected in gamete development between male and female mullets, but males reached gonad maturity slightly earlier than females. Gonad expression levels of cyp19a1 and er in females followed the same trend described for gametogenesis, as it was also the case for plasma vitellogen protein levels in females. No such trends were observed in the case of male mullets. rxr expression levels in male gonads decreased in parallel to gamete maturation. In females, rxr expression levels showed the opposite trend. Finally, endocrine disruption effects such as the occurrence of intersex gonads and high expression and plasma vitellogenin levels in immature and male mullets were detected. Work funded by: Basque Water Agency (URA 10/02, URA 11/03, URA 12/04); Basque Government (K-EGOKITZEN (IE09-245) and grant to consolidated research groups (GIC07/26-IT-393-07), UPV/EHU (UFI 11/37) and GVA Prometeo (2010/008). C.B. is recipient of a predoctoral fellowship from the UPV/EHU. doi:10.1016/j.cbpa.2012.05.096
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4. Bioavailable environmental antiandrogens and their potential effects on endpoints relevant to reproduction in fish A. Lange, M. Sebire (Biosciences, College of Life & Environmental Sciences, University of Exeter, UK); P. Rostkowski, J. Horwood (School of Life Sciences, University of Sussex, UK); S. Miyagawa, T. Mizutani, T. Iguchi (Division of Molecular Environmental Endocrinology, Department of Environmental Biology, National Institute for Basic Biology, Okazaki Institute for Integrative Biosciences, Japan); E.M. Hill (School of Life Sciences, University of Sussex, UK); C.R. Tyler (Biosciences, College of Life & Environmental Sciences, University of Exeter, UK) Chemicals that interact with the androgen receptor (AR; both agonists and antagonists) have been associated with adverse reproductive health effects in humans. Antiandrogens (AAs) are also increasingly recognised as potentially contributing to endocrine disruption in wildlife. Modelling studies on roach (R. rutilus) in UK rivers have shown associations between feminised responses and exposure to environmental antiandrogens as well as oestrogens. Evidence from laboratory-based in vivo studies has further shown that AAs cause a range of adverse effects in fish including ovarian atresia, intersexuality, reduced sperm count, alterations in secondary sex characteristics and disruption of androgen-dependent reproductive behaviours in males. Using a bioassay-directed fractionation process we identified an array of AAs in bile of fish exposed to sewage effluent. Screening of pure standards of the identified structures in two in vitro bioassays confirmed their interaction with the human AR. The compounds include biocides (e.g. triclosan) and resin acids. We subsequently investigated the antiandrogenicity of four identified AAs and their possible involvement in the feminisation of roach through a series of in vivo exposures. These experiments examined the ability of AAs to suppress induction of the androgen-dependent biomarker spiggin in the three-spined stickleback (G. aculeatus) and whether they induce gonadal feminisation in roach exposed during early life (covering of gonadal differentiation). As yet we have found no significant effects of AAs alone or as mixture on these endpoints. Interestingly, however, a mixture of three AAs combined with the oestrogen ethinyloestradiol (nominal 3 ng/L) resulted in an enhanced feminising effect in roach (formation of an ovarian cavity in males). We are now employing fish AR transactivation assays to provide a more relevant in vitro bioassay system for informing in vivo studies in fish. Funding: NERC, Defra (UK) and MoE (J). doi:10.1016/j.cbpa.2012.05.097
5. Expressional changes by environmental concentrations of progestins and anti-progestin RU486 in zebrafish (Danio rerio) embryos S. Zucchi (University of Applied Sciences Northwestern Switzerland, Switzerland); S. Castiglioni (Department of Environmental Health Sciences, Mario Negri Institute for Pharmacological Research, Italy); K. Fent (Department of Environmental Sciences, Swiss Federal Institute of Technology, Switzerland)
Progesterone (P4) and synthetic analogs (called gestagenes, progestogens or progestins) find application in medicine (such as hormone therapy) and oral contraceptives. Synthetic progestins include a broad range of steroids with progesterone-like activity, derived from different parent structures. They ensure the prevention of fertilization by inhibiting ovulation. In comparison to androgens and estrogens, very little attention is given to the environmental fate and effects of P4 and
performed on collected larvae from the same individual mother fish from 5 of the sites. Analysis on the whole data set using within-region comparison shows that eelpouts from different polluted sites had different gene expression profiles for e.g. cytochrome p450's, possibly reflecting the different types of exposures and stress conditions in these sites. Results also indicate that genes involved in xenobiotic and lipid metabolic processes are found in females and DNA replication and tissue morphogenesis are pronounced in larvae. There are also genes and processes that have similar expression patterns for female and larvae which are also unique for specific sites. This is of great interest for studies of maternal transfer of e.g. specific pollutants. Preliminary data also indicate that females with a high reproductive success and females with low reproductive success have different gene expression profiles; thus these findings can be used in understanding mechanisms behind reproductive success of individual females and how some females can “protect” their brood. These results are part of an EU-financed project (www.science.balcofish.gu.se). doi:10.1016/j.cbpa.2012.05.095
3. Characterization by histological and molecular tools of the reproductive cycle and endocrine disruption effects on a thicklip grey mullet (Chelon labrosus) population from the South Bay of Biscay C. Bizarro (CBET Research Group, University of the Basque Country UPV/EHU, Basque Country, Spain); P. Aragón, A. Maquieira (IRMDT, UPV, Valencia, Spain); M.P. Cajaraville, M. Ortiz-Zarragoitia (CBET Research Group, UPV/EHU, Basque Country, Spain) Mugilids such as thicklip grey mullets Chelon labrosus are used worldwide as sentinel organisms of coastal pollution. Biomarkers of exposure and effect have been applied in mullets, together with contaminant bioaccumulation studies and endocrine disruption works. With the aim of characterizing the gametogenic cycle of mullets (C. labrosus) in the South Bay of Biscay, a monthly survey was completed from September 2010 to August 2011. After weighing and sizing, a piece of gonad tissue was collected for histological and histopathological studies and brain, liver and gonad portions for gene expression (TaqMan) assays. Expression levels of vitellogenin (vtg), estrogen receptor (er), retinoid-X-receptor (rxr) and aromatases cyp19a1 (ovarian form) and cyp19a2 (brain form) were assessed. Blood samples were also obtained for the quantification of vitellogenin plasma levels by specific ELISA. Gametogenesis started at the end of summer and proceeded during autumn. Mature gonads were observed at the end of autumn and spawning occurred during winter months. Postspawning gonads were identified during spring–early summer months. No significant differences were detected in gamete development between male and female mullets, but males reached gonad maturity slightly earlier than females. Gonad expression levels of cyp19a1 and er in females followed the same trend described for gametogenesis, as it was also the case for plasma vitellogen protein levels in females. No such trends were observed in the case of male mullets. rxr expression levels in male gonads decreased in parallel to gamete maturation. In females, rxr expression levels showed the opposite trend. Finally, endocrine disruption effects such as the occurrence of intersex gonads and high expression and plasma vitellogenin levels in immature and male mullets were detected. Work funded by: Basque Water Agency (URA 10/02, URA 11/03, URA 12/04); Basque Government (K-EGOKITZEN (IE09-245) and grant to consolidated research groups (GIC07/26-IT-393-07), UPV/EHU (UFI 11/37) and GVA Prometeo (2010/008). C.B. is recipient of a predoctoral fellowship from the UPV/EHU. doi:10.1016/j.cbpa.2012.05.096
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4. Bioavailable environmental antiandrogens and their potential effects on endpoints relevant to reproduction in fish A. Lange, M. Sebire (Biosciences, College of Life & Environmental Sciences, University of Exeter, UK); P. Rostkowski, J. Horwood (School of Life Sciences, University of Sussex, UK); S. Miyagawa, T. Mizutani, T. Iguchi (Division of Molecular Environmental Endocrinology, Department of Environmental Biology, National Institute for Basic Biology, Okazaki Institute for Integrative Biosciences, Japan); E.M. Hill (School of Life Sciences, University of Sussex, UK); C.R. Tyler (Biosciences, College of Life & Environmental Sciences, University of Exeter, UK) Chemicals that interact with the androgen receptor (AR; both agonists and antagonists) have been associated with adverse reproductive health effects in humans. Antiandrogens (AAs) are also increasingly recognised as potentially contributing to endocrine disruption in wildlife. Modelling studies on roach (R. rutilus) in UK rivers have shown associations between feminised responses and exposure to environmental antiandrogens as well as oestrogens. Evidence from laboratory-based in vivo studies has further shown that AAs cause a range of adverse effects in fish including ovarian atresia, intersexuality, reduced sperm count, alterations in secondary sex characteristics and disruption of androgen-dependent reproductive behaviours in males. Using a bioassay-directed fractionation process we identified an array of AAs in bile of fish exposed to sewage effluent. Screening of pure standards of the identified structures in two in vitro bioassays confirmed their interaction with the human AR. The compounds include biocides (e.g. triclosan) and resin acids. We subsequently investigated the antiandrogenicity of four identified AAs and their possible involvement in the feminisation of roach through a series of in vivo exposures. These experiments examined the ability of AAs to suppress induction of the androgen-dependent biomarker spiggin in the three-spined stickleback (G. aculeatus) and whether they induce gonadal feminisation in roach exposed during early life (covering of gonadal differentiation). As yet we have found no significant effects of AAs alone or as mixture on these endpoints. Interestingly, however, a mixture of three AAs combined with the oestrogen ethinyloestradiol (nominal 3 ng/L) resulted in an enhanced feminising effect in roach (formation of an ovarian cavity in males). We are now employing fish AR transactivation assays to provide a more relevant in vitro bioassay system for informing in vivo studies in fish. Funding: NERC, Defra (UK) and MoE (J). doi:10.1016/j.cbpa.2012.05.097
5. Expressional changes by environmental concentrations of progestins and anti-progestin RU486 in zebrafish (Danio rerio) embryos S. Zucchi (University of Applied Sciences Northwestern Switzerland, Switzerland); S. Castiglioni (Department of Environmental Health Sciences, Mario Negri Institute for Pharmacological Research, Italy); K. Fent (Department of Environmental Sciences, Swiss Federal Institute of Technology, Switzerland)
Progesterone (P4) and synthetic analogs (called gestagenes, progestogens or progestins) find application in medicine (such as hormone therapy) and oral contraceptives. Synthetic progestins include a broad range of steroids with progesterone-like activity, derived from different parent structures. They ensure the prevention of fertilization by inhibiting ovulation. In comparison to androgens and estrogens, very little attention is given to the environmental fate and effects of P4 and