Mechanisms For The Effects Of Environmental Estrogens On The Reproductive System In Zebrafish

Scientists and government have paid extentive attention to the effects of environmental endocrine disrupting chemicals (EDCs) on wildlife and human health. EDCs are several kinds of synthetic or natural chemicals which can interfere with endocrine and reproductive system of creatures. The most effective kind among EDCs is environmental estrogens, for they can imitate the function of natural estrogen in vivo and course reproduction defect or intersexuality.17α-ethinylestradiol (EE2), a component of contraceptions, flow into aquatic environment through sewage treatment work. Recent studies reported that concentration of EE2 in natural aquosystem was range from non-detectable to 42ng/L. In USA,5.7% of rivers contain EE2 and the concentrations may be higher than 5ng/L. The EE2 has long half-life and can be bioconcentrated, so the concentration of EE2 in fish can be hundreds of fold higher than that in the aquosystem.Thousands of studies have shown that EE2 has been able to induce the expression of female-specific vitellogenin (VTG) in male and junvenile fish, and disrupts sexual differentiation and normal reproduction.Regarding to the extensive existence and serious harm of environmental estrogens, with the characteristic of induced VTG expression, we cloned the promoter and transcriptional regulation region and generated a transgenic fishline named ere-vtg:egfp. EEs could be detected by ere-vtg:egfp in vivo. We identified the sensitivity and specificity of the transgenic fishline, and discuss its practical application.Previous studies have made a fully description on the morphology and disfunction of the reproductive system in zebrafish after EEs exposure. Our researches focus on the potential mechanisms involved in the effects of EEs on the reproductive system.EE2 binding with fish estrogen receptors, combine to the upstream estrogen-response element, activate the vtg peomoter, start the transcription and at last induce the vtg expression. To understand the function of estrogen receptors in the progress of EE2 induced VTG espression, antagonist and morphlino were used to wild type and ere-vtg:egfp transgenic zebrafish, after many times of exposure and statistics, the result showed that estrogen receptor 2b (ESR2b) is ensential for the synthesis of VTG induced by EE2.Previous studies reported EDCs exposure has been able to disrupt the migration of primordial germ cells (PGCs) in zebrafish. To investigate the effect of EE2 on PGCs migration in zebrafish, we label PGCs with gfp:nanos mRNA and expose the labeled fish to EE2. The esposure results showed that the PGCs migration was disrupted by high concentration of EE2 (1ug/L). This progress needs the participant of ESR2a.It is understanding that EDCs can induce heritable epigenetic modification, lsd1 is an important epigenetic gene coding a demethylase target to histone 3 lysine 4 (H3K4). In C. elegant, bimethylzation of H3K4 is a stable epigenetic memory, which can be changed by loss of lsdl gene. Cross generation, loss of lsd1 results in increased sterility. In mammal, the protein level of LSD 1 is higher in testes than that in somatic tissues, LSD1 take part in the progress of spermatogenesis. EEs reduce the quantity and quanlity of sperm, even induce sterility in zebrafish. Both EE2 exposure and lsdl expression level change can impact on the quantity and quality of zebrafish sperm, is there any relation or interaction between the two reproductive interferents? To investigate the function of lsd1 gene, we target lsdl gene with zinc finger nuclease (ZFN) and generate the lsdl gene knock-out fishline. lsdl-ZFN has been reconstructed and lsdlgene knock-out fishline has been searching.