The effects of 17α‐methyltestosterone on gonadal histology and gene expression along hypothalamic‐pituitary‐gonadal axis, germ cells, sex determination, and hypothalamus‐pituitary‐thyroid axis in zebrafish (Danio rerio)

Abstract As a synthetic androgen, 17α‐methyltestosterone (MT) is widely used in aquaculture to induce sex reversal and may pose a potential risk to aquatic organisms. This ecological risk has attracted the attention of many scholars, but it is not comprehensive enough. Thus, the adverse effects of MT on zebrafish ( Danio rerio ) were comprehensively evaluated from gonadal histology, as well as the mRNA expression levels of 47 genes related to hypothalamic‐pituitary‐gonadal (HPG) axis, germ cell differentiation, sex determination, and hypothalamus‐pituitary‐thyroid (HPT) axis. Adult zebrafish with a female/male ratio of 5:7 were exposed to a solvent control (0.001% dimethyl sulfoxide) and three measured concentrations of MT (5, 51 and 583 ng/L) for 50 days. The results showed that MT had no significant histological effects on the ovaries of females, but the frequency of late‐mature oocytes (LMO) showed a downward trend, indicating that MT could induce ovarian suppression to a certain extent. The transcriptional expression of activating transcription factor 4b1 ( atf4b1 ), activating transcription factor 4b2 ( atf4b2 ), calcium/calmodulin‐dependent protein kinase II delta 1 ( camk2d1 ), calcium/calmodulin‐dependent protein kinase II delta 2 ( camk2d2 ) and calcium/calmodulin‐dependent protein kinase II inhibitor 2 ( camk2n2 ) genes in the brain of females increased significantly at all treatment groups of MT, and the mRNA expression of forkhead box L2a ( foxl2 ) and ovarian cytochrome P450 aromatase ( cyp19a1a ) genes in the ovaries were down‐regulated by 5 and 583 ng/L group, which would translate into inhibition of oocyte development. As compared to females, MT had relatively little effects on the reproductive system of males, and only the transcriptional alterations of synaptonemal complex protein 3 ( sycp3 ) and 17‐alpha‐hydroxylase/17,20‐lyase ( cyp17 ) genes were observed in the testes, not enough to affect testicular histology. In addition, MT at all treatments strongly increased corticotropin‐releasing hormone ( crh ) transcript in the brain of females, as well as deiodinase 2 ( dio2 ) transcript in the brain of males. The paired box protein 8 ( pax8 ) gene was significantly decreased at 51 or 583 ng/L of MT in both female and male brains. The above results suggest that MT can pose potential adverse effects on the reproductive and thyroid endocrine system of fish.