PMC

Embryo at developmental stage 19 was exposed to 0.5-μL 95% ethanol/g E.W. (top panel), 0.5-μg E₂/g E.W. (E₂) (middle panel), or 5.0 μg/g E.W. of PPT (bottom panel) at MPT. PPT induced enlarged cloaca, pelvic bone, and Müllerian duct. Circle, arrow, and arrowhead indicate cloaca, pelvic bone, and Müllerian duct, respectively.

Embryo at developmental stage 19 was exposed to 0.5 μL/g E.W. of 95% ethanol, various doses of E₂, PPT, or WAY 200070 at FPT or MPT. Existences of Müllerian duct were observed and counted, and data were shown as percentage of Müllerian ductal presence or absence (B). An exposure to E₂ and PPT increased the existence of Müllerian duct at MPT, whereas WAY 200070 exposure did not alter it. Numbers on the graph indicate number of animals. Asterisks indicate significant difference in χ² test (P < .05).

Embryo at developmental stage 19 was exposed to 0.5 μL/g E.W. of 95% ethanol, various doses of E₂, PPT, or WAY 200070 at FPT or MPT. Gonadal characteristics were observed and counted, and data were shown as percentage of ovarian or testicular characteristics (B) and score of lacunae (C). An exposure to E₂ and PPT induced the ovarian development at MPT, whereas WAY 200070 exposure and control induced the testicular development with seminiferous tubules at MPT. PPT exposure at developmental stage 19 induced higher score of lacunae development. Numbers on the graph indicate number of animals. Asterisks indicate significant difference in χ² test or (P < .05).

Embryo at developmental stage 19 was exposed to 0.5 μL/g E.W. of 95% ethanol, various doses of E₂, PPT, or WAY 200070 at FPT or MPT. Gonadal tissue was speared out from GAM complex at developmental stage 27, and their mRNA abundances were quantified by Q-PCR. Male pattern was defined with lower abundance of CYP19 mRNA and higher abundance of AMH, whereas female pattern was defined with high CYP19 and low AMH mRNA abundances. An exposure to E₂ and PPT induced the female pattern of mRNA abundance at MPT, whereas WAY 200070 exposure and control induced the male pattern of mRNA abundance at MPT. Numbers on the graph indicate number of animals. Asterisks indicate significant difference in χ² test (P < .05).

Embryo at developmental stage 19 was exposed to 0.5 μL/g E.W. of 95% ethanol, various doses of E₂, PPT, or WAY 200070 at FPT or MPT. Gonadal tissue was speared out from GAM complex at developmental stage 27, and their mRNA abundances were quantified by Q-PCR. The embryos from Lake Apopka were also analyzed at stage 27 after incubation at FPT without exogenous exposure. CYP19 (aromatase), DAX1, FST, FOXL2, GDF9, and AMH were quantified. Asterisks indicate significant difference as compared with vehicle controls (EtOH) at FPT (P < .05). Based on the quantified mRNA abundances, the 2-way hierarchical clustering was conducted with GENE-E software (B) and classified the individual embryos into 2 major groups.

HEK293 cells were transiently transfected with the 4 estrogen-responsive elements containing vector together and an alligator ESR expression vector (A and B) or were transfected with pG5luc vector containing 5 GAL4 binding sites and LBD of each ESR in pBIND vector, including the GAL4 DNA-binding domain (C and D). Cells were incubated with serial concentrations of E₂, PPT, DPN, FERb 033, or WAY 200070 at 10⁻¹⁴M–10⁻⁵M. Data were represented as fold inductions of luciferase signals as compared with vehicle (DMSO) controls. PPT and WAY 200070 showed the highest specificity on ESR1 and ESR2, respectively. Each column represents the mean of triplicate determinations, and vertical bars represent the mean ± 1 SEM.