Long-term exposure of xenoestrogens with environmental relevant concentrations disrupted spermatogenesis of zebrafish through altering sex hormone balance, stimulating germ cell proliferation, meiosis and enhancing apoptosis

Environmental estrogens are capable of interfering with the spermatogenesis and fertility of fish. However in natural waters, these chemicals are more likely to occur as a combination rather than a single stressor. Whether and how the mixture of xenoestrogens with environmental relevant concentrations may affect fish spermatogenesis remains largely unknown. In this study, male zebrafish adults were administered to 17alpha-ethinylestradiol (EE2) and a mixture of xenoestrogens (Mix (E2, EE2, DES, 4-t-OP, 4-NP and BPA)), with the estrogenic potency equivalent to EE2. After a 60-day exposures, elevated mRNA levels of vitellogenin 1 (vtg1) and estrogen receptor 1 (esr1) in the liver of fish in both treated groups were observed. Moreover, the plasma level of E2 declined significantly in the Mix group and the ratio of 11-KT/E2 was significantly elevated in both treated groups. Consistently, the mRNA level of P450 side-chain cleavage (scc) in the EE2 group and ovarian type aromatase (cyp19a1a) in the Mix group was significantly suppressed. In addition, decreased gonadosomatic index and sperm count in the fish of Mix group were present. Furthermore, increased number of the proliferating germ cells (such as spermatogonia and spermatocytes) was observed in the fish of both groups, suggesting a stimulated germ cell proliferation and meiosis. Accordingly, both exposures significantly up-regulated the mRNA levels of genes in mitosis (cyclinb1) and meiosis (cyp26a1 in EE2 group, aldh1a2, cyp26a1, sycp3 and spo11 in Mix). In addition, decreased number of spermatozoa and increased number of TUNEL-positive signals were present in the testis of fish in the Mix group, indicating an enhanced apoptosis. Further analyses demonstrated the significant elevated expressions of tnfrsf1a and the ratio of tnfrsf1a/tnfrsf1b in the Mix group, suggesting an elevated apoptosis in the testis of fish in the Mix group via extrinsic pathway. The present study greatly extends our understanding of the underlying mechanisms of the reproductive toxicity of xenoestrogens on fish.