Testosterone levels in isolated ovary (A) and brain (B) tissues in control (black bars) and drug treated (grey bars) animals. Note the increase of testosterone levels after drug treatment. Data represents the means ± SE (n = 5 in each group). *p < 0.05; **p < 0.01; ns not significant.

Follicle development and ovulation in control and PCOS zebrafish. (A,B) Histological sections of the ovary. In control fish (A), follicles at different stages could be identified. These include developing follicles (Stages I and II, black and green arrows), transitioning follicles (Stage III, red arrows), and mature follicles (Stages IV and V, red and black asterisks). In PCOS fish (B), more developing follicles but less mature follicles were observed. (C,D) Histological sections that show abnormal follicles in PCOS fish. These include zona pellucida invagination (black arrow), theca hypertrophy/granulosa cell invagination (blue arrow), basal membrane disintegration (red arrow), and basophilic granular accumulation (green arrow). Scale bar: 100 µm in panels (A) and (B), and 50 µm in panels (C) and (D). (E) Percentage of developing, transitioning, and mature follicles in control (black bars) and PCOS fish (grey bars). Note the increase of developing follicles and the decrease of mature follicles in PCOS animals. (F) Ovulation in control (black bar) and PCOS (grey bar) zebrafish. Note the decrease of ovulation in PCOS fish. Data represents the means ± SE (n = 8 in each group). *p < 0.05; **p < 0.01; ns not significant.

The expression of Tox3 (A) and Dennd1a (B) in isolated ovary and brain tissues from the control (black bars) and PCOS (grey bars) animals. The expression of Tox3 and Dennd1a in the control sample was normalized to 1, and their expression in PCOS fish was compared to the base value of 1. Note the decrease of Tox3 expression in PCOS samples. However, no significant changes in Dennd1a expression were detected. Data represents the means ± SE (n = 8 in each group). *p < 0.05; ns not significant.

Swimming patterns, sexuality scores, and mating rates of control and PCOS zebrafish. (A) Static positions of control (top panel, red circles) and PCOS fish (bottom panel, red circles) at 15-s intervals during a 30-min recording period in the morning. Bold dashed lines indicate the net. In the control setting, the female showed preferences to swim in areas close to the net. In the experimental setting, the PCOS fish swam in all areas, either near or far from the net. Blue circles show the static positions of the male fish at 15-s intervals. (B) Sexuality scores of control females (black bar) and PCOS fish (grey bar). The sexuality levels of control fish were normalized to 1, and the sexuality levels of PCOS fish were compared to the base value of 1. Note the decrease in sexuality levels in PCOS fish. (C) Mating rates of control (black bar) and PCOS fish (grey bar). Note the decrease of mating in PCOS fish. Data represents the Means ± SE (n = 12 in each group). **p < 0.01.

Swimming patterns, sexuality levels, and mating rates of PCOS zebrafish after 3 days (A), 14 days (B), 30 days (C), and 60 days (D) of rearing in different conditions. Left panels showed the static positions of the fish at 15-s intervals during the 30-min recording period. Bold dashed lines indicate the net. In all cases, the PCOS fish that had been previously kept alone displayed preferences to swim in areas close to the net. In contrast, the PCOS fish that had been previously kept with males swam randomly in all areas. Middle panels showed relative sexuality scores of PCOS fish that had been previously kept alone (black bars) or intermixed with males (grey bars). The scores of PCOS zebrafish kept alone were normalized to 1, and the scores of PCOS zebrafish that were kept with males were compared to the base value of 1. Note the decrease in sexuality levels in PCOS fish reared intermixed with males. Right panels showed the mating rate of PCOS zebrafish after they were kept alone (black bars) or intermixed with males (grey bars). After 3 or 14 days of rearing under different conditions, no statistical differences were detected in the mating rates. After 30 or 60 days of different rearing, the mating rate was increased in PCOS fish that had been previously reared alone as compared to PCOS fish that had been previously reared intermixed with males. Data represents the means ± SE (n = 8 in each group). *p < 0.05; **p < 0.01; ns not significant.