Under-maturation of oogenesis was shown in topiramate-treated female fish. Developmental status of oocyte in maternal fish was determined by Hematoxylin and eosin (H & E) staining. (A) Generally, the ovary was filled with mature oocytes in control fish in a high percentage; (B–F) however, a decreased percentage of mature oocytes was detected in different topiramate-treated female fish. Identification of the maturation stage was based on morphologies (I, primary growth stage; II, early cortical alveolus stage; II *, mid-cortical alveolus stage III, vitellogesis stage; IV, mature oocyte); (G) statistical analysis of oogenesis stages. Columns represent the number of cells at the four stages as percentages of the total counted (averages ± SD; *** p < 0.005; control n = 8; topiramate-treated n = 7).

Embryonic epiboly deficiency in topiramate-treated offspring. (A) Embryonic development observation from control and topiramate-treated offspring. Morphology at 8 hpf in the control group showed normal epiboly progression. Embryos from topiramate-treated female fish were abnormally expressed during epiboly progression from 5.3 hpf; (B) embryonic development observation from control and topiramate-injected embryos at 5.3 and 8 hpf. Scale bar: 200 μm.

Cartilage development of offspring was impaired through maternal transmission of topiramate. (A) Ventral and lateral views of cartilage structure were detected by Alcian blue staining at 4 dpf larvae. Compared to the control group, there was a lack or shortage of cranial and pharyngeal development in topiramate-treated offspring; (B) embryonic number and abnormal rate were calculated from 14 female zebrafish; (C) length and width from individual maternal fish were measured and statistically analyzed. (averages ± SD; * p < 0.5; ** p < 0.01; *** p < 0.005; cb, ceratobranchial; ch, ceratohyal; ep, ethmoid plate; hs, hyosymplectic; M, Meckel’s cartilage; pq, palatoquadrate; Scale bar: 200 μm).

Bone development of offspring was impaired through maternal transmission of topiramate. (A–F) Ossification status of cranium and vertebral regions was detected by Alizarin red staining of zebrafish at 10 dpf; (G) Vertebrae numbers from offspring of control or topiramate-treated female zebrafish (♀25) (averages ± SD; *** p < 0.005; control n = 15; topiramate-treated n = 82). (ch, ceratohyal; cl, cleithrum; m, Meckel’s cartilage; Scale bar: 100 μm).

Craniofacial cartilage abnormalities upon diverse topiramte-treated offspring.