Bisphenol A (BPA), a very important raw material in the plastics industry, is an endocrine‐disrupting chemical in teleost fish. Although BPA induces testis‐ova and sex reversal in teleost fish species, the molecular mechanism remains unclear. We evaluated the effects of BPA (measured concentrations: 45, 92, 326, 1030 and 3406 μg/L) on Japanese medaka (Oryzias latipes) using OECD TG234 (2011, Fish Sexual Development Test, OECD Guidelines for the Testing of Chemicals, Section 2). BPA at 1030 and 3406 μg/L induced testis‐ova and sex reversal with female‐type secondary sexual characteristics in XY males at 30 and 60 days posthatching (dph). Then we examined the BPA effect on the expression of sex differentiation genes related to the testis‐ova and sex reversal in XY medaka. BPA exposure (1030 and 3406 μg/L) suppressed gsdf mRNA expression and increased cyp19a1a mRNA expression in XY individuals at stage 38 and 30 dph, although foxl2 mRNA expression showed no change. Interestingly, the concentration of BPA that suppressed gsdf mRNA expression at the larval stage was consistent with that needed to induce testis‐ova and sex reversal. These results suggest that the gsdf gene at the embryonic stage can be used as a useful biomarker for predicting the impact of estrogenic endocrine‐disrupting chemicals on sexual differentiation in Japanese medaka.
The present study attempted to examine whether clonal cell lines of the oral epithelium can differentiate into ameloblasts and regenerate tooth when combined with dental germ mesenchyme. Clonal cell lines with a distinct morphology were established from the oral epithelium of p53-deficient fetal mice at embryonic day 18 (E18). The strain of mouse is shown to be a useful source for establishing clonal and immortalized cell lines from various tissues and at various stages of development. Tooth morphogenesis is almost completed and the oral epithelium is segregated from the dental epithelium at E18. In RT-PCR analysis of cell lines, mucosal epithelial markers (cytokeratin 14) were detected, but ameloblast markers such as amelogenin and ameloblastin were not detected when cells were cultured on plastic dish. They formed stratified epithelia and expressed a specific differentiation marker (CK13) in the upper layer when cultured on feeder layer or on collagen gel for 1–3 wk, demonstrating that they are of oral mucosa origin. Next, bioengineered tooth germs were prepared with cell lines and fetal molar mesenchymal tissues and implanted under kidney capsule for 2–3 wk. Five among six cell lines regenerated calcified structures as seen in natural tooth. Our results indicate that some oral epithelial cells at E18 possess the capability to differentiate into ameloblasts. Furthermore, cell lines established in the present study are useful models to study processes in tooth organogenesis and tooth regeneration.
Chemicals with androgenic or estrogenic activity induce the sex reversal and/or intersex condition in various teleost fish species. Previously, we reported that exposure to 17α‐methyltestosterone, bisphenol A, or 4‐nonylphenol induces changes in expression of the gonadal soma‐derived factor (gsdf) gene accompanied by disruption of gonadal differentiation in Japanese medaka (Oryzias latipes). These findings suggest that gsdf expression might be a useful biomarker for predicting the potential effect of chemicals on gonadal differentiation. We examined the gsdf expression in Japanese medaka exposed to chemicals with estrogenic or androgenic activity. Exposure to the androgenic steroid 17β‐trenbolone at 0.5–22.1 μg/L induced the development of ovotestis (presence of ovarian tissue with testicular tissue) and female‐to‐male sex reversal in XX embryos, and exposure at 6.32 and 22.1 μg/L significantly increased gsdf expression in XX embryos compared with controls at developmental stage 38 (1 day before hatching). In the present study, no statistically significant difference in gsdf mRNA expression was observed after exposure to 17β‐estradiol, 17α‐ethinylestradiol, and 4‐t‐octylphenol, which have estrogenic activity. In addition, antiandrogenic chemicals or chemicals without endocrine‐disrupting activity did not induce changes in gsdf expression in XX or XY embryos. Thus, an increase in gsdf expression after androgen exposure was observed in XX embryos. Together, these findings indicate that gsdf expression might be useful for predicting the adverse effect of chemicals on gonadal differentiation. Environ Toxicol Chem 2022;41:1875–1884. © 2022 SETAC
We compared the influence of thyroid hormone-disrupting chemicals (heptafluorobutanoic acid, PFBA and tris[1, phosphate, TDCPP) and thyroid hormone (3,3 0 ,5-triiodo-L-thyronine, T3) on swim bladder inflation and thyroid hormone-related gene expression in Japanese medaka and zebrafish. The swim bladder of most larvae had inflated at 4 h post hatching (hph) in Japanese medaka and at 48 hph in zebrafish in controls. In both fish species, the swim bladder inflation was inhibited in larvae exposed to PFBA (lowest observed effect concentration [LOEC] in medaka: 40 mg/L; in zebrafish: 80 mg/L), TDCPP (LOEC in medaka: 1 mg/L; in zebrafish: 0.5 mg/L), and T3 (no inhibition in Japanese medaka; LOEC in zebrafish: 7.5 μg/L). We also examined the influence of PFBA, TDCPP, and T3 on the expression of thyroid stimulating hormone subunit beta (tshβ) or thyroid hormone receptor alpha (trα) and beta (trβ). No changes were observed in the expression of genes after PFBA and TDCPP exposure; however, T3 exposure upregulated trα and trβ expression in both fish species. When the results were compared between Japanese medaka and zebrafish, swim bladder inflation in both species was found to be inhibited by exposure to thyroid hormone-disrupting chemicals. Our results show that inhibition of the swim bladder inflation at 4 hph in Japanese medaka and 48 hph in zebrafish is a potential indicator of thyroid hormone-disturbing activity of chemicals.
The branched isomer mixture 4‐nonylphenol (4‐NP) has been used worldwide as a surfactant, and can have endocrine‐disrupting effects on aquatic organisms. For instance, 4‐NP induces the formation of testis‐ova (i.e., testicular and ovarian tissue in the same gonad) or male to female sex reversal of various teleost fishes. Recently, our group revealed that altered gsdf gene expression is associated with disruption of gonadal differentiation in Japanese medaka (Oryzias latipes) embryos exposed to methyltestosterone or bisphenol A, suggesting that gsdf might be useful as a biomarker for predicting the impact of endocrine‐disrupting chemicals (EDCs) on gonadal differentiation. Here, we used 4‐NP to examine further whether gsdf expression at the embryo stage is useful for predicting EDC impact on gonadal sex differentiation. When fertilized medaka eggs were exposed to 32 or 100 μg/L 4‐NP, testis‐ova in genetic males and sex reversal from genetic male to phenotypic female were observed. At stage 38 (just before hatching), 4‐NP exposure at 1‐100 μg/L did not affect gsdf expression in XX embryos compared with the nontreated control; however, in XY embryos, the gsdf expression in the 100 μg/L‐exposed group was significantly lower than that in the controls. The 4‐NP concentration at which gsdf expression was suppressed was equal to that at which testis‐ova and sex reversal were induced. These results indicate that expression of the gsdf gene at the embryonic stage in medaka is a useful biomarker for predicting the impact of EDCs on sexual differentiation.
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