Method for in vivo ovary measurement in Casper zebrafish. The calibrated line tool in ImageJ was used to measure the diameter (height and width cross hairs) of larger, vitellogenic ovarian follicles with opaque yolk (indicated by O with arrowheads). Panel A: Left and right ovaries of a moderately gravid female Casper zebrafish. Data represent mean ± SD of larger follicle size class and N = number of the larger follicles, bar equals 1 mm. Panel B: Left and right ovaries of a less gravid female Casper zebrafish. Data represent mean ± SD of larger follicle size class and N = number of the larger follicles, bar equals 1 mm.

Effects of diethylstilbestrol (DES) on breeding tubercles of adult male Casper zebrafish. Panels A – C show pectoral fins of same control treated male. Panel A: control male at 17 WPF. Panel B: control male at 20 WPF. Panel C: control male at 22 WPF. Panels A – C show no reduction in breeding tubercles over time. Panels D – F show pectoral fins of same 0.01 μg/L DES treated male. Panel D: DES male at 17 WPF, the day of treatment, before 0.01 μg/L DES was applied. Panel E: DES male at 20 WPF after DES treatment for 21 days. Panel F: DES male at 22 WPF, 14 days after DES withdrawal. Panels D – F show a modest reduction in the height and pointedness of the breeding tubercles after treatment. Panels G – I show pectoral fins of same male treated with 0.1 μg/L DES. Panel G: DES male at 17 WPF, the day of treatment, before 0.1 μg/L DES was applied. Panel H: DES male at 20 WPF after 21 days of DES treatment. Panel I: DES male at 23 WPF, 21 days after DES withdrawal. Images show a clear reduction in breeding tubercles after treatment and reformation with DES withdrawal. Images were taken at 32× magnification; all scale bars represent 400 μm, except inset which are 64× and scale bar represents 200 μm.

Effects of diethylstilbestrol (DES) on testes and vent of Casper male zebrafish. Panels A and B: Image of left and right testes, respectively, and vent of a sexually mature male (17 WPF) before DES treatment. Black arrowheads point to opaque lower area of testes connecting to vent (white arrows). Panels C and D: Image of left and right testes, respectively, and vent of sexually mature male (21 WPF) after 4 weeks of 0.1 μg/L DES treatment. Testes are notably more transparent without obvious connection to vent. Images show reduction in testes opacity and increase in vent size after treatment. Images were taken at 12× magnification. Scale bar represents 1 mm.

Representative male vent changes with diethylstilbestrol (DES) treatment and withdrawal. Adult male Casper zebrafish before treatment (A), after 21 days of 0.1 μg/L DES (B) and after 14 days of DES withdrawal (C). The vent (white arrows) has been outlined in black with the imageJ line tool for determining area in each image. The scale bar represents 1 mm.

Effects of diethylstilbestrol (DES) on female spawning: ovarian morphology. Panels A and B: Left and right sides of female C62 before DES treatment at 25.5 WPF (O with black arrows indicate large follicles, black outlines with white arrows denote vents). Panels C and D: Left and right sides of female C62 after 28 days of 1 μg/L DES exposure, 29.5 WPF, note the atretic ovaries and increased vent size (black outline and white arrow). All DES females in the study exhibited regression of the ovary. Images were taken at 8× magnification. Scale bar represents 1 mm.