| Occasionally,environmental endocrine disruptors(EEDs)produced by industry,agriculture and daily life with a higher concentration,are accidentally discharged into waters through wastewater effluent.Due to their high strong estrogenic effect,these EEDs,though the discharge period is limited within several days,can cause severe pathologic response(or even lethal)to aquatic animals(including fish).17?-ethynylestradiol(EE2),a common xenoestrogen,exerts strong estrogenic effects to aquatic animals.Our preliminary study showed that,exposure of EE2 with a high concentration(100 ng/L)for six days induced severe toxicological adverse effects on zebrafish,while the underlying mechanisms are not clear.In this study,six-day exposure to EE2 caused decreased ovary weight and gonadosomatic index(GSI),as well as several types of ovary injuries to zebrafish adult females.These adverse effects included atretic oocytes,rupture of follicular layer,and cluster of early follicles and the decreased.When paired with unexposed male,both the fecundity of exposed female and the number of survived larva were significantly reduced,and no egg was observed in EE2-high.Meanwhile,the mRNA levels of er? and vtg1 in liver were dramatically elevated,indicating an enhanced estrogenic effect of EE2 on zebrafish.However,the plasma levels of estradiol(E2)and(DHP)declined significantly,suggesting an arrested steroidogensis.Further studies revealed that,EE2 down-regulated the m RNA levels of genes involved in estrogen production(cyp19a1b)and gonadotrophin-releasing hormone gnrh-III in brain.EE2-high also significantly decreased the mRNA level of gnrhr-II.Besides,the mRNA levels of the fsh?(follicle stimulating hormone subunit ?)and lh?(luteinizing hormone subunit?)were also down-regulated.Furthermore,the mRNA levels of fshr and lhr in the ovary decreased as well.The suppressed expressions of these genes were then supposed to be responsible for the down-regulation of cyp17a1,cyp19a1 a,20?-hsd,and cyp17a2 which are responsible for the biosynthesis of E2 and DHP in ovary.On the other hand,after EE2 exposure,the m RNA levels of nanos1(the biomarker for primordial germ cells and gonocytes),aldh1a2(RA synthase)andcyp26a1(RA catalyse)were all up-regulated.Most importantly,the ratio of aldh1a2/cyp26a1(instructor for meiotic initiation)was also enhanced,indicating an elevated meiotic initiation.Meanwhile,the expressions of sycp3 and dmc1(both are biomarkers for meiosis)were also up-regulated,suggesting an enhanced meiosis or increased number of germ cells in meiotic phase.The altered expressions of genes in germ cell development agreed well with the cluster of early follicles and disordered reproduction,indicating a disrupted oogenesis in zebrafish.Except for disrupted oogenesis,other adverse effects(such as edema of the body,dermahemia,angiectasis)were also observed.Further study revealed the increased blood volume and plasma volume in EE2 treated fish,as well as decreased blood cell concentration,indicating an imbalanced osmoregulation.Histological results further confirmed pathological alterations in gill of the treated fish,such as swelling of the epithelial cells,disrupted arrangement of the bronchial filaments and expanded capillary.In kidney,EE2 exposure caused swelling,degeneration and necrosis in epithelial cells of the renal tubule,as well as multiple hemorrhage of the peritubular capillaries.These impairments indicated the injury of imbalanced osmoregulation in on these tissues.In fish,aquapoins(AQPs)play essential roles in water reabsorption in the kidney,regulating the circulating blood volume.In this study,EE2 exposure significantly up-regulated the mRNA levels of aqp1?1 and aqp4,suggesting enhanced water reabsorption.These data agreed well with the increased blood volume and decreased blood cell concentrations.Therefore,it was highly suggested that,the altered expressions of aqp1?1 and aqp4 might be one of the reason responsible for the imbalanced osmoregulation.Argininevasopressin(AVP),also known as antidiuretic hormone,is also important on water reabsorption.However,the mRNA level of avp was significantly suppressed.We supposed that,the down-regulation of avp might be responsible for the feedback action of the increased circulating blood volume induced by elevated expressions of aqps.Besides,adrenocortical hormones(glucocorticoid and mineralocorticoid),particularly the mineralocorticoid,plays pivotal role in osmoregulation of vertebrates.Corticotropin releasing hormone(CRH)released from hypothalamus,acts on pituitary,regulating the synthesis of adrenocorticotropic hormone(ACTH),through CRHR.ACTH then acts on adrenal gland(head kidney in fish),triggering the synthesis of adrenocortical hormones.In this study,the mRNA levels of crh,crhr and acth were all significantly suppressed in EE2 treated fish,indicating thepossible inhibited synthesis of adrenocortical hormones.Different from terrestrial tetrapods,mineralocorticoid is absent in teleost,instead,glucocorticoid(such as cortisol)play critical role in osmoregulation.In this study,the plasma level of cortisol was dramatically declined.Accordingly,the activity of Na+-K+-ATPase and the mRNA level of atp1a1 a.1,atp1a1 a.4 in gill and kidney were all suppressed,suggesting an enhanced osmotic pressure in epithelial cells in these tissues.The altered expressions of these genes agreed well with the histological observation which showed swelling of the epithelial cells in gill and kidney,also suggesting an imbalanced osmoregulation.In summary,our data suggested the subacute toxicity of EE2 on reproduction and osmoregulation in zebrafish adult females.The present study greatly extended our understanding on the mechanisms underlying of reproductive and osmotic toxicities of environment estrogens on fish. |
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