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- Jansson, Erika
(författare)
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Ontogenetic Characterization of Müllerian Duct Development and Expression of anti-Müllerian hormone and Other Genes : New Endpoints in the Frog Xenopus tropicalis Test System
- 2015
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The amphibian model-species Xenopus tropicalis is used in reproductive toxicology studies. Lifecycle studies are requested but they are time consuming and susceptible to several of the risks attributed to long-term studies. Herein, the ontogenetic expression of genes associated to gonads and Müllerian ducts and the early Müllerian duct development were characterized as well as explored as suitable early juvenile endpoints after developmental levonorgestrel (LNG) exposure. The urogenital mRNA expression of the anti-Müllerian hormone (amh) was male-biased while the cytochrome P450 19a1 (cyp19a1) mRNA expression was female-biased from the Nieuwkoop and Faber developmental stage (NF) 50, to 4 weeks post-metamorphosis. amh expression in combination with cyp19a1 expression is proposed to constitute a robust marker of phenotypic sex during gonadal differentiation in amphibians. The expression of the amh receptor 2 (amhr2) was characterized for the first time in amphibians. amhr2 was expressed higher in putative females than in males from NF 56, suggesting a role in ovarian development. The mRNA levels of intracellular and membrane progesterone receptors (ipgr and mpgr beta) respectively increased and decreased as the tadpole developed into a juvenile frog, indicating different roles for these genes during development. Exposure to LNG modulated the mRNA expression of the amh and pgrs in a sex and developmental stage dependent manner, suggesting the involvement of these genes in the LNG mode of action. The Müllerian ducts were first observed at NF 64 in both sexes and grew into distinct tubules, larger in females than in males at 4 weeks post-metamorphosis. Therefore, 4 weeks post-metamorphosis is suggested as a suitable early juvenile stage for investigation into disruptions of female reproductive organ development. In conclusion, the protein Amh is suggested to be involved in testicular differentiation and to be a reliable testicular marker during gonadal differentiation, even after developmental LNG exposure, in amphibians.
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| 2. |
- Jansson, Erika, et al.
(författare)
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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
- 2016
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Ingår i: General and Comparative Endocrinology. - : Elsevier BV. - 0016-6480 .- 1095-6840. ; 229, s. 132-144
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
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| 4. |
- Orton, F., et al.
(författare)
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Exposure to an anti-androgenic herbicide negatively impacts reproductive physiology and fertility in Xenopustropicalis
- 2018
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Amphibians are threatened on a global scale and pollutants may be contributing to population declines, but how chemicals impact on their reproduction is poorly understood. We conducted a life cycle analysis to investigate the impacts of early life exposure to two anti-androgens (exposure until completion of metamorphosis;stage 66): flutamide, (50 µg/L)/linuron (9 and 45 µg/L)) on sexual development and breeding competence in Xenopus tropicalis. Our analyses included: mRNA levels of dmrt1, cyp17, amh, cyp19, foxl2 and ar (tadpoles/metamorphs), gonadal histomorphology (metamorphs/adults), mRNA levels of ar/gr (adult male brain/gonad/forelimb), testosterone/corticosterone levels (adult males), secondary sexual characteristics (forelimb width/nuptial pad: adult males) and breeding competence (amplexus/fertility: adult males). Compared to controls, feminised sex ratios and increased number of spermatogonia (adults) were observed after exposure to flutamide and the lower linuron concentration. Exposure to the lower linuron concentration also resulted in demasculinisation of secondary sexual characteristics and reduced male fertility. Flutamide exposure resulted in masculinisation of the nuptial pad and elevated mRNA levels of dmrt1, cyp17, amh and foxl2 in brains (metamorphs). Testosterone levels were higher in all treatment groups, however, overall few effects were observed in response to the higher linuron concentration. Our findings advance understanding of reproductive biology of X. tropicalis and illustrate negative effects of linuron on reproductive processes at a concentration measured in freshwater environments.
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| 5. |
- Säfholm, Moa, et al.
(författare)
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Mixture effects of levonorgestrel and ethinylestradiol : Estrogenic biomarkers and hormone receptor mRNA expression during sexual programming
- 2015
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Ingår i: Aquatic Toxicology. - : Elsevier BV. - 0166-445X .- 1879-1514. ; 161, s. 146-153
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Tidskriftsartikel (refereegranskat)abstract
- 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 (0.1 nM), or EE2 (0.1 nM) singly, or in combination with LNG (0.01, 0.1, 1.0 nM), 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 (or) receptors were quantified using quantitative PCR. All EE2-exposed groups showed female-biased sex ratios and increased vtgbeta1 mRNA levels compared with the controls. Compared with the EE2-alone group (positive control) there were no significant alterations in vtg betal 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 or 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 or 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. (C) 2015 Elsevier B.V. All rights reserved.
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| 6. |
- Säfholm, Moa, et al.
(författare)
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Molecular and histological endpoints for developmental reproductive toxicity in Xenopus tropicalis : Levonorgestrel perturbs anti-Mullerian hormone and progesterone receptor expression
- 2016
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Ingår i: Comparative Biochemistry and Physiology - Part C. - : Elsevier BV. - 1532-0456 .- 1878-1659. ; 181, s. 9-18
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Tidskriftsartikel (refereegranskat)abstract
- 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-Mullerian 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 Mullerian 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 Mullerian duct development were found, implying that the exposure window was not critical with regard to these endpoints. In juveniles, folliculogenesis had initiated and the Mullerian 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.
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