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.

Neuropeptide Y receptor Y8b(npy8br)regulates feeding and digestion in Japanese medaka(Oryzias latipes)larvae:evidence from gene knockout

Neuropeptide Y receptor Y8(NPY8R)is a fish-specific receptor with two subtypes,NPY8AR and NPY8BR.Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation;this has been found in only a few fish,at present.In order to better understand the physiological function of npy8br,especially in digestion,we used clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology to generate npy8br-/-japanese medaka(Oryzias latipes).We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability,ultimately affecting their growth.Specifically,npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes(npy and agrp).npy8br-/-medaka larvae fed for 10 d(10th day of feeding)still had incompletely digested brine shrimp(Artemia nauplii)in the digestive tract 8 h after feeding,the messenger RNA(mRNA)expression levels of digestion-related genes(amy,lpl,ctra,and ctrb)were significantly decreased,and the activity of amylase,trypsin,and lipase also significantly decreased.The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes(gh and igf1).Hematoxylin and eosin(H&E)sections of intestinal tissue showed that npy8br-/-medaka larvae had damaged intestine,thinned intestinal wall,and shortened intestinal villi.So far,this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.

Medaka factor in the germline alpha(figα)is indispensable for oogenesis but not for gonadal sex differentiation

A basic helix-loop-helix transcription factor,figα,is one of the earliest marker genes of oocyte differentiation in vertebrates.In the present study,we made figαknockout medaka by CRISPR/Cas9,expecting aborted progress of oogenesis,to see if differentiation of somatic ovarian tissues is affected.Figαknockout male gonads differentiated normally into testes with functional sperm.The females,on the other hand,were sterile;there are oocytes only up to pachytene.No growing oocytes in diplotene were found.The phenotype was already apparent at 10 days after hatching,when diplotene oocytes start to develop in the control.Furthermore,several putative target genes of figαwere not expressed in the mutant female gonads.Previous studies showed that medaka lacking germ cells have morphologically abnormal gonads and female to male sex reversal occurred.Figαknockout female gonads differentiated morphologically into ovaries,suggesting that in medaka,figαknockout prevents oogenesis progress into diplotene stage,and ovarian differentiation do not need developed oocytes beyond pachytene stage.This ovarian phenotype reminded us of"pachytene checkpoint",which blocks meiotic progress into diplotene when chromosomal abnormalities are present.Therefore,we further analyzed phenotype of oocytes in the mutant ovaries.The number of oocytes at each meiotic stage suggested that oogenesis normally proceeded up to pachytene stage.The number of apoptotic oocytes in the knockout ovaries was small.Finally,we did not detect any abnormal pairings or unrepaired double-strand breaks by immunostaining.Taken together,figαknockout likely stop oocyte growth at or around pachytene via unknown mechanisms other than"pachytene checkpoint"and arrested oocytes remained in the ovary.This is in contrast to figαknockout mice where all oocytes underwent apoptosis quickly after birth.

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.

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.

布洛芬对海洋青鳉鱼生命初期毒性效应

为探究布洛芬(Ibuprofen,IBP)对海洋青鳉鱼(Oryzias melastigma)早期生命阶段的毒性效应,将海洋青鳉鱼胚胎在不同浓度(0、1、10、100、1000μg·L^(-1))IBP溶液中暴露至孵化后的第30天(未孵化胚胎暴露20 d).观察统计胚胎的孵化率、孵化时间、心率、仔鱼30 d存活率以及体长体重等发育参数,并检测胚胎的抗氧化酶(超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽巯基转移酶(Glutathione S-transferase,GST))活性、抗氧化剂(还原型谷胱甘肽(glutathione,GSH))、氧化产物(丙二醛(malonic dialdehyde,MDA))浓度和神经递质降解酶(乙酰胆碱酯酶(acetylcholinesterase,AChE))活性变化,以评估IBP对海洋青鳉鱼早期生命阶段的发育毒性、抗氧化系统以及神经递质系统的影响.结果表明,1μg·L^(-1)IBP对海洋青鳉鱼胚胎及仔鱼不存在发育毒性,其余各浓度导致胚胎孵化率、孵化时间、心率和仔鱼存活率降低,所有浓度下仔鱼体长体重均未变化;在第5天时,随浓度升高,SOD活性和GSH含量呈下降趋势,CAT、GST活性和MDA含量先上升后下降,第8天时各指标趋于正常;IBP浓度的升高导致AChE活性先增加后降低.综上,环境相关浓度下的IBP对海洋青鳉鱼胚胎及仔鱼不存在发育毒性,各浓度IBP均会导致胚胎产生氧化应激和神经毒性效应.

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。

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.

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.

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。