Effects Of Tributyltin On The Gametogenesis And Reproductive Behaviors Of Zebrafish

Tributyltin(TBT),an organotin acting as aromatase(Cyp19a1)inhibitor,has been widely used as a biocide,antifouling paints for ships and wood preservation.In the past,limited research showed that TBT can cause abnormalities in gametogenesis and reproduction behavioral regulation in several fish species,however,the mechanisms underlying the disrupted gametogenesis and reproductive behavior is still unclear.To further understand the mechanism underlying the toxicity of TBT on fish gametogenesis and endocrine regulatory disorders leading to abnormal reproductive behaviors,adult zebrafish(Danio rerio)were continuously exposed to two nominal concentrations of TBT(100 and 500 ng/L)for 28 days.In the present study,1.TBT decreased the total egg number,reduced the hatchability,elevated the mortality of the larvae and the gonadosomatic index(GSI)of the female fish.Moreover,TBT decreased the plasma level of 17β-estradiol(E2)and suppressed the expressions of cyp19a1 a in the ovary,indicating a disrupted ovarian development and oogensis.Furthermore,TBT increased the percentage of early-stage follicles(PG),and upregulated the mRNA levels of gene involved in meiosis(such as aldh1a2,sycp3,and dmc1),indicating that the enhanced meiosis might be responsible for the increased ratio of early follicles after exposure.In addition,reduced percentage of mid/late-stage follicles(EV,MV and FG stages),which is consistent with the reduced vtg1 mRNA level in the liver,suggesting that blocked VTG synthesis in the liver might be one of the main reasons for reduced mid/late-stage follicles.TBT exposure to zebrafish males led to significantly reduced sperm count,fertilization rate,and significantly increased mortality of embryos.Increased number of spermatocytes,reduced number of spermatozoa and lacunas possibly resulting from the loss of spermatozoa in several spermotocysts were further observed in the testis of exposed fish.In addition,increased level of 11-KT and declined level of E2 were also present in the plasma of exposed fish,however it was inconsistent with the decreased sperm count,suggesting that other pathway(s)might be responsible for the disrupted spermatogenesis.Fish spermatogenesis consists of various cellular processes,such as germ cell proliferation,meiosis,and apoptosis.In this study,TBT exposure to zebrafish resulted in down-regulated mRNA levels of genes for proliferation(such as cyclind1 and pcna),suggesting that TBT may interfere with germ cell proliferation in the testis of treated fish.In addition,up-regulated mRNA levels of genes involved in meiosis(such as aldh1a2,sycp3 and dmc1)were also observed in the testis of treated fish indicating that TBT might have enhanced the early stages of meiosis.In contrast,the expression of cyp26a1,which encodes the key factor responsible for the endpoint of meiosis,was dramatically down-regulated,indicating the blocked meiosis process and sperm formation.These data were in consistence with the increased number of spermatocyte and decreased of spermatozoa in the testis.On the other hand,the expressional ratio of bax/bcl-2 and tnfrsf1a/tnfrsf1 b,which are the markers for intrinsicand extrinsic-apoptosis respectively,were both elevated in the testis of treated fish,suggesting an enhanced apoptosis through intrinsic and extrinsic apoptotic pathways in the treated fish.This may be one of the pivotal reasons for the decreased number of spermatozoa and lacunas in the testis of treated fish.2.TBT significantly suppressed the reproductive behaviors(such as frequency of meeting,duration in spawning area and visits in spawning area)of both sexes of zebrafish.In consistence with the disrupted reproductive behaviors,the plasma level of E2 and the expressions of genes(such as cyp19a1 b,gnrh-3 and kiss2)involved in the regulation of reproductive behaviors were also significantly suppressed in the brain of treated fish,suggesting that TBT may disrupt reproductive behaviors through affecting E2 production and the Gnrh/Kisspeptin system.Besides,disrupted non-reproductive behavior may also give rise to altered reproductive behaviors.In teleosts,serotonin(5-HT)plays pivotal role in the mood control and the regulation of non-reproductive behaviors.In the present study,TBT exposure significantly suppressed the expressions of genes(tph2,pet1 and kiss1)in the 5-HT synthesis and regulation of 5-HT neurons in the female,suggesting that TBT exposure may lead to enhanced non-reproductive behavior of zebrafish females,thereby interfering with reproductive behavior indirectly.In addition,the mRNA level of ompb in TBT-treated male fish was dramatically suppressed,Consistent with abnormal reproductive behavior.In summary,TBT not only seriously interferes with the gametogenesis of zebrafish,leads to gametogenesis disorder,which affects the quantity and quality of gametes,but also disrupted reproduction behavior.These alterations in response to TBT exposure may unavoidably give rise to serious ecological consequences in zebrafish.The ecological risks of TBT are worthy of attention.