双酚-A对Medaka的类雌激素效应研究

利用Medaka进行了双酚-A与17β-雌二醇的短期暴露研究.研究结果发现实验室建立的流水式暴露体系可为实验提供连续、恒定的污染物暴露浓度.利用肝肉指数可灵敏响应和指示污染物暴露.双酚-A可以剂量相关的方式诱导雄性Medaka肝脏产生卵黄蛋白原,从而有效证实了其类雌激素效应.

Comparison of toxicity effects of fuel oil treated by different dispersants on marine medaka(Oryzias melastigma) embryo

This study aims to evaluate the subacute toxic effects of oil under different treatments on marine organism by simulating natural contaminative processes. In this study, 120# （RMD15） fuel oil was selected as the pollutant and marine medaka （Oryzias melastigma） embryos as the experimental organism. The developmental toxicity of different volume concentrations （0.05%, 0.2%, 1% and 5%） of water-accommodated fractions, biologically-enhanced water-accommodated fractions, and chemically-enhanced water-accommodated fractions on the embryos in different exposure time （8, 15 and 22 d） were compared and the content of relevant polycyclic aromatic hydrocarbons （PAHs） was studied （in dispersion and in vivo）. The subacute toxic effects were assessed in terms of antioxidant activities of enzymes （superoxide dismutase, catalase and glutathione S-transferase） and the blue sac disease （BSD） indexes.The results showed that the BSD indexes of the treatment groups were significantly higher than the respective control groups and showed positive correlations with both concentration and exposure time. The experiments with three antioxidant enzymes indicated that enzymatic activities of the embryos changed dramatically under the oxidation stress of petroleum hydrocarbons, especially after adding the dispersants. With the increase of petroleum hydrocarbon concentration and exposure time, the three enzymes showed different degrees of induction and inhibition effects.

Hypoxic and temporal variation in the endocrine disrupting toxicity of perfluorobutanesulfonate in marine medaka(Oryzias melastigma)

Perfluorobutanesulfonate(PFBS)is an emerging pollutant capable of potently disrupting the sex and thyroid endocrine systems of teleosts.However,the hypoxic and temporal variation in PFBS endocrine disrupting toxicity remain largely unknown.In the present study,adult marine medaka were exposed to environmentally realistic concentrations of PFBS(0 and 10μg/L)under normoxia or hypoxia conditions for 7 days,aiming to explore the interactive behavior between PFBS and hypoxia.In addition,PFBS singular exposure was extended till 21days under normoxia to elucidate the time-course progression in PFBS toxicity.The results showed that hypoxia inhibited the growth and caused the suspension of egg spawn regardless of PFBS exposure.With regard to the sex endocrine system,7-day PFBS exposure led to an acute stimulation of transcriptional profiles in females,which,subsequently,recovered after the 21-day exposure.The potency of hypoxia to disturb the sex hormones was much stronger than PFBS.A remarkable increase in estradiol concentration was noted in medaka blood after hypoxia exposure.Changes in sex endocrinology of coexposed fish were largely determined by hypoxia,which drove the formation of an estrogenic environment.PFBS further enhanced the endocrine disrupting effects of hypoxia.However,the hepatic synthesis of vitellogenin and choriogenin,two commonly used sensitive biomarkers of estrogenic activity,failed to initiate in response to the estrogen stimulus.Compared to sex endocrine system,disturbances in thyroidal axis by PFBS or hypoxia were relatively mild.Overall,the present findings will advance our toxicological understanding about PFBS pollutant under the interference of hypoxia.

Transgenerational bone toxicity in F3 medaka (Oryzias latipes) induced by ancestral benzo[a]pyrene exposure: Cellular and transcriptomic insights

Benzo[a]pyrene(BaP),a ubiquitous pollutant,raises environmental health concerns due to induction of bone toxicity in the unexposed offspring.Exposure of F0 ancestor medaka(Oryzias latipes)to 1μg/L BaP for 21 days causes reduced vertebral bone thickness in the unexposed F3 male offspring.To reveal the inherited modifications,osteoblast(OB)abundance and molecular signaling pathways of transgenerational BaP-induced bone thinning were assessed.Histomorphometric analysis showed a reduction in OB abundance.Analyses of the miRNA and mRNA transcriptomes revealed the dysregulation of Wnt signaling(frzb/ola-miR-1–3p,sfrp5/ola-miR-96–5p/miR-455–5p)and bone morphogenetic protein(Bmp)signaling(bmp3/ola-miR-96–5p/miR-181b-5p/miR-199a-5p/miR-205–5p/miR-455–5p).Both pathways are major indicators of impaired bone formation,while the altered Rank signaling in osteoclasts(c-fos/miR-205–5p)suggests a potentially augmented bone resorption.Interestingly,a typical BaP-responsive pathway,the Nrf2-mediated oxidative stress response(gst/ola-miR-181b-5p/miR-199a-5p/miR-205),was also affected.Moreover,mRNA levels of epigenetic modification enzymes(e.g.,hdac6,hdac7,kdm5b)were found dysregulated.The findings indicated that epigenetic factors(e.g.,miRNAs,histone modifications)may directly regulate the expression of genes associated with transgenerational BaP bone toxicity and warrants further studies.The identified candidate genes and miRNAs may serve as potential biomarkers for BaP-induced bone disease and as indicators of historic exposures in wild fish for conservation purposes.

Medaka fish stem cells and their applications

Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental processes such as pluripotency control and cell fate decision and for regenerative medicine by stem cell-based therapy. Stem cell cultures have been best studied in 3 vertebrate organisms. These are the mouse, human and a small laboratory fish called medaka. Specifically, medaka has given rise to the first embryonic stem (ES) cells besides the mouse, the first adult testis-derived male stem cells spermatogonia capable of test-tube sperm production, and most recently, even haploid ES cells capable of producing Holly, a semi-cloned fertile female medaka from a mosaic oocyte created by microinjecting a haploid ES cell nucleus directly into a normal oocyte. These breakthroughs make medaka a favoring vertebrate model for stem cell research, the topic of this review.

青鳉nup 58的表达及其在性别分化中的潜在作用

核孔复合体是物质穿梭核膜的通道。核孔蛋白能影响核质和胞质间的物质交换,而核质运输具有调控生殖细胞进入雌性或雄性分化途径的能力。性腺体细胞衍生因子(gonadal soma-derived factor,gsdf)能调控青鳉Oryzias latipes雄性生殖细胞,并在有丝分裂最后一轮之前开启或关闭配子发生中起关键作用。通过RT-qPCR实验发现nup 58(nucleoporin 58)在野生型青鳉精巢中的表达高于卵巢。对青鳉性腺转录组数据分析发现,nup 58在gsdf敲除后精巢中的表达会显著升高。免疫荧光实验结果显示,nup 58在配子发生不同阶段的所有生殖细胞中都有表达。我们的研究显示Nup58作为一种核孔蛋白,受gsdf信号的调控,并与雄性生殖细胞的增殖密切相关。由Nup58核孔蛋白协调的核质运输在生殖细胞性别分化中的作用和动态变化值得进一步探讨。

Genetic Diversity of Chinese Medaka(Oryzias sinensis)in Dian Lake Drainage Area by ISSR Analysis

Chinese medaka(Oryzias sinensis),widely distributed in the plateau lakes,Yunnan Province,which was the indicative specie for the habitat quality of riparian zone in the wetland ecosystems and also was the key specie in the lake ecosystems,and also was one of the fishes who suffered the severe invasion of alien species.There,we investigated that the genetic diversity and population structure of the wild Chinese medaka by using inter-simple sequence repeat(ISSR)analysis.The samples(20 individuals in total)of Chinese medaka in three populations in the drainage area of Dian Lake were obtained.The 7 ISSR primers were picked out to assess the genetic diversity.The ISSR primers combinations revealed 82.69%polymorphism,among 52 genetic loci amplified.The sum of effective number of alleles and observed number of alleles were 1.492 4 and 1.826 9,respectively.The Shannon genetic diversity and Nei genetic diversity indexes of all the populations were 0.434 5 and 0.289 7,respectively.The gene differentiation(GST)was 0.198 9,and the gene flow(NM)was 2.013 2.The AMOVA demonstrated that only 6.65%of genetic variance came from inter-population,and 93.35%of genetic variance came from intra-population.Phylogenetic tree showed that 20 individuals from 3 populations were clustering mixed each other.Also,the principal coordinate analysis(PCA)supported the cluster analysis results.These results indicated that the genetic diversity of Oryzias sinensis was at the middle level,and there was no obvious genetic differentiation among the three populations of Oryzias sinensis.

Subchronic toxicological effects of aquatic nitrobenzene on Medaka and Chinese rare minnow

Based on the simulative exposure experiments of nitrobenzene to two small experimental fishes, Medaka (Oryzias latipes) and Chinese Rare Minnow (Gobiocypris rarus), it was found that nitrobenzene could decrease in a linear way in the static aquatic system. Both fishes could accumulate dose-related levels of nitrobenzene quickly and eliminate the compound rapidly when they were transferred to clean water. The Chinese rare minnow showed more sensitivity to the acute toxicity exposure of nitrobenzene than Medaka. Typical molecular biomarkers in oxidative defense system including superoxide dismu- tase (SOD), catalase (CAT) showed the chemical induced alterations. The changes of acetylcholi- nesterase (AChE) activities indicated that nitrobenzene might affect the normal neural function. Nitro- benzene exposure could also lead to obvious damaging effects on the target organs, such as gill and liver by hitopathological studies.

Characterization and expression analysis of gonad specific igf3 in the medaka ovary

The gonad specific expression of insulin-like growth factor 3(igf3)has suggested an important role of igf3 in fish reproduction.In this study,medaka igf3 was isolated and its expression patterns were compared with igf1,and germ cell gene vasa in adult medaka ovary.Molecular cloning and sequencing showed that the open reading frame(ORF)of medaka igf3 was comprised of 501 nucleotides and encoded 166 amino acid residues.Reverse transcription polymerase chain reaction(RT-PCR)analysis showed that igf3 RNA was specifically expressed in developing embryos and adult gonads of both sexes.Real time quantitative PCR(q-PCR)indicated that igf3 expression was gradually increased during oogenesis,and reached its highest level at stage V.Using Chromogenic and fluorescent in situ hybridization,igf3 was shown to be present in the germ cells of oogonia and in oocytes at stages I-III,as well as abundant in granulosa cells and theca cells of oocytes at stages IV-V.In addition,igf3 and igf1 were expressed mutually in the outer theca cells of stage V oocytes in ovary.Collectively,we demonstrated that gonad specific igf3 could mark medaka ovarian somatic cells and germ cells.These findings highlight the importance of igf3 during ovarian development.

Exposure to difenoconazole inhibits reproductive ability in male marine medaka(Oryzias melastigma)

Difenoconazole（DFZ） is a triazole fungicide which has been detected in the aquatic environment, including estuaries and embayments. However, few studies addressing the reproductive toxicity and transgenerational effects of DFZ on marine fishes are available.The present study was conducted to investigate the effects of DFZ on male marine medaka（Oryzias melastigma）. After exposure of the embryo to 1, 10, 100 and 1000 ng/L DFZ for180 days, the gonadosomatic index was significantly decreased in the 1000 ng/L treatment.The number of sperm was reduced while the abundances of spermatocytes and spermatogonia in the testes were increased in all the treatments. The m RNA levels of salmontype gnrh（sgnrh）, the luteinizing hormone（lhβ） and the follicle-stimulating hormone（fshβ）genes in the brain all exhibited a significant down-regulation, the expression of androgen receptors（arα and arβ） was decreased and that of estrogen receptor β and cytochrome P450 aromatase（cyp19 B） was increased in the testes. The expression levels of cyp19 A and cyp19 B were increased in the liver. The decrease of ars m RNA levels might be one of the reasons causing the reduction of sperm. The down-regulation of sgnrh, lhβ and fshβ m RNA levels suggested that DFZ might impact the spermatogenesis via the brain–pituitary–gonad pathway. The decrease of the fertilization success, the hatch ability and the swim-up success in the F1 generation indicated that DFZ pollution at environmental levels might cause a decrease of wild fish populations.

青鳉gsdf调控Igf2bp2/Igf2bp3-m6A阅读器在雌雄生殖细胞的差异表达

青鳉gsdf敲除导致生殖细胞异常增殖,产生XY巨大精巢或XY巨大卵巢。正常精巢、卵巢及gsdf缺失型XY巨大精巢或巨大卵巢的mRNA转录组比对分析和qPCR验证结果显示,igf2bp2表达可能受gsdf信号调控。脊椎动物igf2bp2的分子结构高度进化保守,igf2bp2 mRNA在胚胎发育各时期均有表达,在成体精巢表达量最高,提示igf2bp2可能在早期胚胎发育、性别分化及精子生成发育中起重要作用。免疫荧光检测显示XY精巢的抗Igf2bp2免疫反应信号强于XX卵巢、gsdf缺失型XY精巢和XY卵巢。不同于gsdf缺失激活igf2bp3的表达,gsdf缺失反而减弱igf2bp2表达。Gsdf对igf2bp2和igf2bp3的正负调控影响其介导的m6A甲基腺苷修饰,以及其靶向的mRNA转录后修饰和翻译,这一分子调控机制可能在脊椎动物性别分化和精子生成过程中普遍存在。

含吡啶类腈类医药中间体废盐对海洋青鳉鱼胚胎的毒性效应

工业废盐是化工生产过程中产生的副产结晶盐。农药、医药等精细化工行业的生产中往往伴随着大量废盐的产生,《国家危险废物名录》中明确将多种化学制药、农药生产过程中产生的废盐列为危险废物。然而,由于处理成本高昂和处置能力不足,废盐限制了企业的健康发展。在《危险废物环境管理指南化工废盐》中明确了工业废盐无害化排海的相关要求,但目前仍缺少相关领域的实验研究。海洋青鳉鱼(Oryzias melastigma)具有与斑马鱼类似的研究特征优势,是国际生命科学学会(ILSI)与健康和环境科学研究所(HESI)认定的海洋生态毒理学的重要研究工具。研究了含吡啶类腈类医药中间体废盐和经过实验室处理后的除杂盐对海洋青鳉鱼胚胎的急性毒性、发育毒性以及对抗氧化系统、免疫系统的影响。结果表明,废盐暴露对海洋青鳉鱼胚胎的生存和发育有显著影响并造成系统性损伤,而除杂盐对海洋青鳉鱼胚胎没有较大影响。本研究为废盐排海提供了基础的理论支持,也为后续实验开展提供了良好的思路引导。

Efficient gene editing in a medaka(Oryzias latipes)cell line and embryos by SpCas9/tRNA-gRNA

Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRNA)or protein and transcribed guide RNA(gRNA).However,the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present.In this study,we employed a poly-transfer RNA(tRNA)-gRNA(PTG)system driven by cytomegalovirus(CMV)promoter to target the medaka(Oryzias latipes)endogenous gene tyrosinase(tyr)or paired box 6.1(pax6.1)and illustrated its function in a medaka cell line and embryos.The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka.This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.

Characterization and expression analysis of sox3 in medaka gonads

Numerous studies have showed that sox3 is involved in neurogenesis and sex differentiation in vertebrates.However,the accurate expression pattern is still unclear in fish.In this study,medaka sox3 was isolated and its expression patterns were compared with germ cell gene vasa in adult gonads.By reverse transcription-polymerase chain reaction(RT-PCR)analysis,sox3 RNA expression was detected in the brain,eyes and gonads.By chromogenic and fluorescent in situ hybridization,sox3 was present in the oogonia and early stages of oocytes as well as granulosa cells and theca cells in the later stages;in the testis,sox3 was occurred in the Sertoli cells and sperm with a specific signal in the location of chromatid body of sperm;in the embryos,sox3 was expressed in the central nervous.These results suggest that medaka sox3 gene was occurred in somatic and germ cells in adult gonads of both sexes,involved in the process of spermatogenesis,as well as the development of the nervous system.This study provides a precise expression pattern of sox3 and suggests that sox3 may be involved in gonadal and nervous system development of fish.

Medaka gcnf is a component of chromatoid body during spermiogenesis

Germ cells of many organisms exhibit a unique perinuclear cytoplasmic cloud-like structure called the chromatoid body(CB).Specific components of the CB,such as vasa,are crucial for germline development.Herein we identify another component of the CB,the Oryzias latipes gcnf homolog(Olgcnf).By reverse transcription polymerase chain reaction(RT-PCR),Olgcnf transcript was restricted to gonads in the adult tissues and was maternally provided and persisted throughout embryogenesis.By in situ hybridization on gonadal sections,Olgcnf transcript was restricted to germ cells.In the ovary,Olgcnf was predominantly expressed in the early stages of oocytes.In the testis,Olgcnf showed dynamic expression in round spermatids and sperm,accumulated in the CB during spermatid morphogenesis and concentrated in elongated spermatids of acrosome cap region and flagellar manchette.Moreover,Olgcnf was colocalized with vasa,which is the best-characterized component of the CB.Therefore,Olgcnf is specific to germ cells of both sexes and is a component of the CB during spermiogenesis.

Knockout of lipoprotein lipase with CRISPR/Cas9 causes severe developmental defects and affects lipid deposition in Japanese medaka(Oryzias latipes)

In mammals,lipoprotein lipase(LPL)has been found to play an important role in lipid mentalismand deposition.LPL deficiency in humans(Homo sapiens)and mice(Mus musculus)tends to cause hypertriglyceridemia.The lpl gene is not expressed in adult mammalian liver,but is in adult fish liver.The functions provided by the lpl gene are diverse in vertebrates.Here,we knocked out the lpl gene in Japanese medaka(Oryzias latipes)with the CRISPR/Cas9 system.The lpl-knockout(KO)homozygous individuals showed severe developmental defects with an extremely emaciated and deformedbody and onlyaccounted for about5%of the F2fish.This is consistentwiththefindings inmicebut disaccords with the results for zebrafish(Danio rerio).Compared with wild-type(WT)madaka,the mRNA level of lpl in lpl-KO heterozygous mutant was significantly higher in the muscle,showed no significant difference in the liver,and was significantly lower in the heart.Under lpl heterozygous deficiency,the relative area of Oil RedO and triglycerides(TG)level in the liver,heart and muscle tissue covaried with levels of lpl mRNA in medaka.The lpl heterozygous deficiency did not affect the levelsofTG,low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and total cholesterol(TC)in the plasma of medaka,which is inconsistent with the findings in mammals.In general,the lpl gene plays an important role in the growth and development and is closely related to lipid deposition of medaka.

硒代蛋氨酸对青鳉亲本繁育和子代发育的影响

本试验旨在研究硒代蛋氨酸(Se-Met)对青鳉亲本繁育和子代发育的影响。把17条4~5月龄青鳉随机分配到4个组,每组3~5条鱼。对照组投喂基础干饲料,试验组分别投喂含2、10和50μg/g Se-Met的干饲料。试验期7 d。结果表明,与对照组相比:1)10μg/g Se-Met组卵巢硒浓度显著升高(P<0.05);2)10、50μg/g Se-Met组的接近、求偶方向和求偶圆舞发生频率显著降低(P<0.05);3)50μg/g Se-Met组的Ⅲ期卵细胞计数显著升高(P<0.05),10和50μg/g Se-Met组的精子计数显著降低(P<0.05);4)10和50μg/g Se-Met组大脑中细胞色素P45019b(CYP19b)的mRNA相对表达水平显著升高(P<0.05),50μg/gSe-Met组卵巢中细胞色素P45019a(CYP19a)、卵泡刺激素b(FSHb)、促性腺激素释放激素1b(GnRH1b)和黄体生成素b(LHb)的mRNA相对表达水平显著升高(P<0.05);5)50μg/g Se-Met组胚胎的未受精率显著升高(P<0.05),10和50μg/g Se-Met组胚胎的畸形率显著升高(P<0.05)。由此可见,过量补充Se-Met会导致青鳉的求偶行为活动降低,雄性青鳉精原细胞数量减少,雌性青鳉卵巢及卵细胞形态紊乱,以及子代未受精率和畸形率增加,提示水产鱼类饲料添加Se-Met时应注意硒的实际需求和适当的补充剂量。

海洋微塑料对海水青鳉毒性效应研究进展分析

海水青鳉是研究微塑料对海洋生物毒理作用的理想模式生物。文章围绕微塑料在海水青鳉中毒性研究,通过查阅相关文献,对已发表文献年度变化和地区分布进行了统计分析;通过分析实验所用微塑料种类、颗粒大小和实验浓度、暴露时间和方式以及不同生长阶段海水青鳉实验对象选择,总结归纳微塑料在海水青鳉体内代谢、肠道损伤、生长和发育毒性、生殖毒性、遗传毒性以及对其他海洋污染物的放大或缓解效应6个方面的研究结果,并以分析结果为基础,建议在后期研究海洋微塑料对海水青鳉毒性效应,应重点考虑实验时间长短、实验材料选择和实验设计、毒理作用机制分析等因素。

氰戊菊酯和氯化镉暴露下日本青鳉的行为反应差异

以天然河流中的日本青鳉(Oryzias latipes)行为变化规律为基础,采用水质安全在线生物预警系统(BEWs-1.1)对氰戊菊酯和氯化镉暴露下日本青鳉的行为反应进行了探讨.结果表明:1)在源水中,日本青鳉行为反应具有明显规律性,并表现出生物钟现象;2)氰戊菊酯和氯化镉连续暴露和间断暴露中,日本青鳉行为反应与污染物浓度和暴露时间均直接相关,并表现出相似的行为变化规律性,行为变化均符合生物行为的环境胁迫阈模型;3)高浓度(10mg·L-1)氯化镉间断暴露下的日本青鳉行为强度变化与低浓度(2mg·L-1)连续暴露下的行为强度变化差别不明显,而高浓度(10mg·L-1)氰戊菊酯间断暴露下的日本青鳉行为强度变化与低浓度(2mg·L-1)连续暴露时具有明显差异,高浓度暴露时产生了明显的行为毒性效应;4)器官损伤性污染物(如镉)引起的行为毒性效应在重新加入源水后可以得到部分恢复,而神经损伤性污染物(如氰戊菊酯)不可恢复,污染物不同的作用机理会影响水生生物的行为反应.

Cd^(2+)、Hg^(2+)、Cr^(6+)和Pb^(2+)对黑点青鳉(Oryzias melastigma)早期生活阶段的毒性效应研究

近岸海域重金属污染不容乐观,作为"一种潜在的海洋模式鱼种"黑点青鳉(marine medaka,Oryzias melastigma)对不同重金属胁迫的响应水平却鲜有报道。为探究并比较海水鱼种黑点青鳉早期生活阶段对Cd2+、Hg2+、Cr6+和Pb2+等不同重金属胁迫的响应,在实验室通过半静态方式,对黑点青鳉受精卵和初孵仔鱼分别进行了14d短期毒性和96h急性毒性实验。结果显示:当Cd2+、Hg2+、Cr6+和Pb2+浓度分别高于147μg·L-1、24μg·L-1、235μg·L-1和200μg·L-1时,4种重金属离子对黑点青鳉胚胎发育具有显著的毒性效应,可显著降低胚胎的孵化能力和心脏跳动,并导致初孵仔鱼的发育畸形;Cd2+、Hg2+、Cr6+和Pb2+对黑点青鳉初孵仔鱼的96h-LC50分别为1.12(0.496～2.306)mg·L-1、0.097(0.042～0.196)mg·L-1、1.456(0.547～3.242)mg·L-1和>20mg·L-1;与其他海洋鱼种相比,4种重金属离子对黑点青鳉初孵仔鱼的LC50相对较低,表明该鱼种对重金属胁迫具有较强的敏感性。此外,该鱼种具有个体小,易于实验室长期培养,世代周期短和对内分泌干扰物敏感等多项优势,因此,笔者推荐将黑点青鳉作为潜在的海洋模式鱼种用于生态毒理学研究。