Effects Of Exposure To Nonylphenol Combined With Anti-estrogen Drugs On Spermatogenesis In Zebrafish (Danio Rerio)

Nonylphenol (NP) is a chemical material that is degraded from nonionic surfactant nonylphenol ethoxylate (NPEOs). NPEOs have been widely used in textile industries, pesticides, emulsifiers and so on. Although, the use of NPEOs have been restricted by the governments in some countries, there is still a considerable amount of production in most countries. A lot of NP has been discharged into water and the environment residual amount is still very high. NP is known as one of endocrine disrupting chemicals (EDCs), or estrogenic compounds. McClusky reported that there were serious damages in the reproductive system of rats exposed to NP, including changes in the synthesis of steroids, the damage of testis structural, reduction of the sperms and so on. The reduction of sperm density, damage of the structure of seminal vesicle, and condensation of sperms was found in fish exposed to high concentrations (greater than 250μg/L) of NP. It was proved that NP disturb the synthesis of sex steroid hormone, testicular development, and significantly increase the expression of ERα and CYP19A mRNA in adult male zebrafish. Present studies on the impacts of NP on fish testis development only focused on the endocrine-disrupting effects, as well as morphology. However, there is less specific datas on the impact in the process of spermatogenesis in the testis.Spermatogenesis in fish is a highly organized and coordinated process, including self-renewal of spermatogonial stem cells, spermatogonial stem cells mitosis, spermatocyte meiosis and sperm formation. The whole process is regulated by hormone. By analyzing the whole fish spermatogenesis, we can better evaluate the development of the reproductive system. Each stage gamete cells have its own specific gene expression biomarker. To research the relations of EDCs and its related gene expression, we can better reveal the interference effect of EDCs and provide a more complete basis for the interference of EDCs in male reproduction.In this study, we took adult male zebrafish as our research objects, and studied from:1)the molecular level, using real-time quantitative fluorescent PCR (q-PCR) to detect the expression of spermatogonia cell marker gene PIWIL 1, spermatogonia stem cell renewal factor GDNF and FGF2, spermatocytes marker genes SYCP3 and CYCLIN B3; 2) the biochemical level, using double-antibody sandwich enzyme-linked immune assay (ELISA) to detect levels of zebrafish testis 11-KT and E2; 3) the level of tissue morphology, using paraffin tissue sections to study the changes in zebrafish testis structure; 4) the level of development, we counted the number of germ cells at different stages, gonad index over the period of spermatogenesis. We studied the impacts of spermatogenesis and exposed adult male zebrafish to NP (125μg·L-1), TAM (300μg·L-1), LET (300μg·L-1), NP+ TAM, NP+LET. The exposure period was 21d, and sampled at 7d and 21 d respectively. Results are as follows:1. The expression of spermatogonia marker PIWIL 1, spermatogonial stem cell renewal factor GDNF, FGF2 and meiosis spermatocytes marker genes SYCP3 and CYCLIN B3 was induced by exposure 21d to NP. In addition to the expression of CYCLIN B3 mRNA of the NP+TAM group was significantly higher than the control, the rest of combined exposure were no significant differences compared to the control group.2. There were histology changes resulting from NP exposure:the spermatogonia (SG) increased slightly, the SG/SZ ratio increased significantly whereas spermatogenesis and sperm maturation was inhibited (compared to control).3. The level of male zebrafish estrogen E2 was increased while androgen 11-KT levels was down regulated after exposure to NP:Conclusion:Zebrafish testis GDNF, FGF2 and SYCP3 gene well reflected the male gametes mitosis and pre-meiosis and can act as effective molecular marker genes. These genes were upregulated after 21d exposed to 125μg·L-1 NP, promoting spermatogonial stem cell renewal and meiosis, but the formation of sperm was suppressed and sperm cell number was decreased significantly. The level of endogenous E2 was up-regulated by NP, while the level of androgen 11-KT was down-regulated and affected the process of spermatogenesis. In addition, anti-estrogen drugs TAM and LET have eased the interference effects caused by NP.