Diethylstilbestrol, flutamide and their combination impaired the spermatogenesis of male adult zebrafish through disrupting HPG axis, meiosis and apoptosis

Both diethylstilbestrol (DES, an environmental estrogen) and flutamide (FLU, an anti-androgen) are found to impair spermatogenesis by disrupting hypothalamic-pituitary-gonadal (HPG) axis and altering androgen levels through different mechanisms/modes of action in fish with poorly understood underlying mechanisms. Furthermore, it is not known whether and how a combined exposure of DES and FLU has a stronger effect than the compounds alone. In this study, male zebrafish adults were exposed to DES, FLU and their combination (DES+FLU) for 30days, and their effects on histological structure and sperm count in testis, androgen level in plasma, as well as the mRNA levels of genes involved in HPG axis, meiotic regulation and apoptosis were analyzed. After exposure, DES and FLU disrupted spermatogenesis in zebrafish, and their combination resulted in even more severe impairment, indicating the inhibitory roles of these chemicals on spermatogenesis and their additive effects on zebrafish. The different regulation of vtg1 expression in the liver in response to DES and FLU further confirmed the different modes of action of these drugs. Gene expression and plasma steroid level analyses demonstrated the suppressed mRNA levels of the key genes (such as gnrh3, fshβ and lhβ in brain and dmrt1, sf1, cyp17a1 and cyp11b2 in testis) in HPG axis and decreased 11-ketotestosterone (11-KT) levels in plasma. The declined level of 11-KT was thus supposed to be closely related to the down-regulation of cyp26a1 (encoding the catabolic enzyme of retinoic acid) and suppression of genes involved in meiotic regulation (nanos1, dmc1 and sycp3). In fish exposed to DES and DES+FLU, enhanced apoptosis (elevated bax/bcl-2 expression ratio) was also observed. The suppression of meiotic regulation in response to all the exposures and enhanced apoptosis in response to DES were thus supposed to result in the spermatogenic impairment in zebrafish. The present study greatly extends our understanding on the mechanisms underlying of reproductive toxicity of environment estrogens and anti-androgens in fish.