Studies On The Reproductive Toxicity Of Microcystin-LR In Female Zebrafish And Its Underlying Mechanisms

Cyanobacterial blooms and their secondary metabolites,microcystins(MCs),are not only toxic to aquatic organisms,but also to humans.There are more than 200 variants of MCs,of which microcystins-LR(MC-LR)is the most studied and most toxic variant.Studies have shown that MC-LR is reproductively toxic to female fish and is mainly manifested by disordered hormone levels in female fish.In addition,related research also shows that MC-LR affects the maturation and apoptosis of oocytes.However,the mechanism behind MC-LR interference in oocyte maturation and apoptosis remains largely unknown.In the present study,the reproductive toxicity of MC-LR to fish was studied from three levels of parent,oocyte and offspring,and from two aspects of oocyte quality and quantity,so as to provide experimental basis for evaluating the toxic effect of MC-LR.In the present study,adult female zebrafish were exposed to MC-LR(0,1,5,20 ?g/L)for 30 d.After exposure,fertilized eggs from treated females and healthy males were collected and cultured in water without MCLR.The effects of MC-LR on the maturation of zebrafish oocytes were studied by detecting morphology of ovary,rate of oocyte growth and rupture,and expression of oocyte maturation related proteins.On the other hand,the expression of endoplasmic reticulum stress(ERS)related genes and proteins in ovary and progeny were detected to explore the effects of MC-LR,including ERS and apoptosis of oocytes and toxic effects on progenies through maternal transmission.The main results are as follows:1.Histomorphological observations showed pathological damage in the ovary after MC-LR exposure,which was mainly characterized by enlarging intercellular spaces,detachment of follicular cells from oocytes,and vacuolation of parenchymal tissues.The 20 ?g/L MC-LR treatment caused a remarkable increase in the rate of the zebrafish oocytes germinal vesicle breakdown(GVBD)and a significant decrease in the levels of cyclic adenosine monophosphate(c AMP)and vitellogenin(VTG).In addition,the phosphorylation levels of the extracellular signal-regulated kinases(ERK)were elevated in ovaries from zebrafish exposed to 5 and 20 ?g/L MC-LR,and cyclin B phosphorylation level was also upregulated notably in the 20 ?g/L MC-LR group.However,MC-LR exposure did not cause any change in the levels of c AMP-dependent protein kinase(PKA)protein and cdc2 phosphorylation among all the treatments.All the doses of MC-LR reduced the number of eggs and increased the abnormal rate of offspring generation.In summary,the present study demonstrates that MC-LR promotes oocyte maturation by activating the ERK1/2 and MPF signaling pathways,and c AMP is involved in this process.2.In this study,the experimental treatment is as above.Exposure of female zebrafish to 5,20 ?g/L MC-LR caused a significant decrease in gonadal index(GSI).Our data suggested that MC-LR significantly increased the transcription levels of eif2s1 a,atf4 and eif2ak3 in the ovary.The m RNA levels of atf4,atf6,bcl-2,hspa5,eif2s1 a and eif2ak3 upregulated notably in the offspring.After treatment with 20 ?g/L MC-LR in female zebrafish,c-Jun N-terminal kinase(JNK)phosphorylation level and cleaved-caspase3 protein expression elevated obviously in the ovary,but presented no remarkable change in the offspring.Furthermore,TUNEL results showed that 5,20 ?g/L MC-LR significantly induced apoptosis in the ovary,while acridine orange(AO)staining indicated that MC-LR did not cause abnormal apoptosis in offspring.Concisely,the present study indicates that MC-LR leads to apoptosis through different ERS pathways in the ovary and offspring,and also provides a new perspective for understanding the apoptosis caused by MC-LR.In conclusion,the results show that MC-LR affects the maturation of zebrafish oocytes through the ERK1/2-MPF signaling pathway,and moreover induces apoptosis in zebrafish oocytes through ERS.The toxic effects of MC-LR on oocytes leads to damage of female zebrafish ovarian tissue,decreased egg production,increased offspring mortality and malformation rate,thus exerting reproductive toxicity on female zebrafish.