Molecular Characterization, Expression Pattern and Transcriptional Regulation of Figla During Gonad Development in Japanese Founder (Paralichthys olivaceus)

The factor in the germline alpha(figla), as a member of the basic helix-loop-helix family, has been reported to be involved in ovary development in mammals and teleosts. However, the regulatory mechanisms of figla in teleosts remain unclear. Here,figla in P. olivaceus(Pofigla) was characterized with encoding a 202 amino acid protein that contains a conserved basic region and helix-loop-helix(HLH) domain. Amino acids alignment and synteny analysis revealed that Pofigla was conserved with the orthologous gene sequences in other vertebrates. The results of qRT-PCR showed Pofigla was maternally inherited during embryonic development. For tissue distribution, Pofigla showed a sexually dimorphic gene expression in the gonad of different genders, with a higher expression in ovary than in testis. In situ hybridization(ISH) results demonstrated Pofigla was specifically expressed in germ cells including oocytes, spermatogonia and spermatocytes. By screening and analyzing two proximal regions(-2966/-2126 and-772/-444) with high promoter activity, we found SOX5, LEF1, FOXP1 and GATA1 may play important roles in the transcriptional regulation of Pofigla. Furthermore, we observed the co-localization between Figla and LEF1 in HEK 293T cells. And the significant up-regulation effect of the canonical Wnt signaling pathway on the expression of Pofigla was found in cultured ovarian cells. This study provided the first evidence that figla not only has an important function in ovary development, but also plays some potential roles in testis development and/or male germ cell differentiation during early testis development in P. olivaceus. The results provide valuable reference in exploring the regulatory network of figla in teleost.

Gonad traits and age structure of the sea urchin Heliocidaris crassispina in central range:a comparison with rangeextenders

The sea urchin Heliocidaris crassispina has northern expanded in geographic range to Toga Bay,Oga Peninsula in the Sea of Japan due to the global warming.To demonstrate the gonad traits and age structure of H.crassispina in the central range and compare the difference with those in northern extended range,we collected 100 H.crassispina individuals from a barren off Shitsumi,Fukui(central range),in August 2018.Their growth,gonad traits(size,development and color),and sex ratio were investigated and compared with those in Toga Bay,in August 2014(extended range).The successive 2012–2016 year classes of H.crassispina indicated successive juvenile recruitment in the central range,as found in the extended range.The individuals at three years old in central range were obviously larger than those in the extended range,showing large test diameter and body weight,possibly due to the higher temperature in central range than that in northern extended range.In addition,a balanced sex ratio in the central range was found,in contrast to the female-skewed sex ratio found in the extended range.Gonadal development was sex-and site-dependent.The testicular development in the central range was not only delayed in comparison to ovarian development,but also less delayed than those in the extended range,which possibly attribute to the higher water temperature in the central range.A significantly lower male gonad index and L*(lightness)value was recorded in central ranges compared to that in extended range,possibly due to the releasement of sperm of H.crassispina in central ranges.

Morphological and histological changes in the brains of turbot(Scophthalmus maximus)with gonadal development

The brain plays a critical role in controlling reproduction through the hypothalamus-pituitary-gonadal(HPG)axis in vertebrates.Turbot(Scophthalmus maximus)has become an economically important marine fish in Europe and North China.Previous research investigating turbot reproduction has focused on the role of the HPG axis in regulating egg and sperm production.However,the morphology and histology of the organs in the HPG axis have not been studied.In this study,we investigated the morphology and histology of brains in female and male turbot at different stages of gonadal development.The results showed that the brains of both female and male turbot were composed of seven parts that are typical of advanced teleosts:the telencephalon,diencephalon,cerebellum,hypothalamus,pituitary gland,myelencephalon,and olfactory bulbs.The telencephalon was well-developed and contained five distinct lobes,with the contiguous diencephalon at the caudal portion.The torus longitudinales and rostral torus semicircularis of the mesencephalon flattened along the dorsal surface,and the rostral corpus cerebellum was located in the dorsal portion.The actual total brain volume in mature males was significantly greater(p<0.05)than that of females with gonadal development.Notably,the pituitary volume in male turbot significantly increased(p<0.05)from immature to mature stage,but this difference did not occur in females.The data together illustrate a distinct sex difference in the turbot brain during gonadal development,providing insight into their HPG axes.

Hypothalamic-Pituitary-Gonadal(HPG)Axis and Transcriptional Regulatory Elements Regulate piwil2 Gene Expression During Gametogenesis and Gonadal Development in Japanese Flounder(Paralichthys olivaceus)

The P-element induced wimpy testis(Piwi)proteins,which are associated with PIWI-interacting RNAs(piRNAs),play important roles in meiosis,germ cell division,and germline maintenance.In this study,we identified and characterized the Paralichthys olivaceus piwil2 gene,a constituent factor of the piRNA pathways involved in the biogenesis of reproductive development.The biological analysis indicated that piwil2,which contains PAZ and PIWI domains,was highly conserved between teleosts and tetrapods.The piwil2 distribution profile in different tissues confirmed a sexually dimorphic expression pattern,with a higher expression level in testis.In situ hybridization demonstrated that piwil2 was expressed in the oogonia and oocytes of the ovaries as well as in the Sertoli cells and spermatocytes of the testes.Gene piwil2 showed a maternally inherited expression pattern during embryonic development,and was highly expressed during the early embryonic development.Different luciferase reporters were constructed to determine the transcriptional regulatory mechanisms of piwil2.The piwil2 core promoter region was located at−360 bp to−60 bp.Furthermore,some representative sex hormones,including human chorionic gonadotropin,17α-methyltestosterone,and estradiol-17βhad distinct regulatory effects on piwil2.In a summery,these results indicate that piwil2,regulated by sex hormones and transcriptional elements,has vital functions in the reproductive cycle and gonadal development.

克氏原螯虾PcCRCN-L基因的克隆鉴定及在低氧胁迫下的表达响应

虾青蛋白(Crustacyanin,CRCN)在甲壳动物脂类代谢及低氧胁迫应激调控等方面具有重要的功能。为获取虾青蛋白在克氏原螯虾(Procambarus clarkii)性腺发育和低氧胁迫应答中的作用,本研究在克氏原螯虾肝胰腺组织中鉴定出1个类虾青蛋白PcCRCN-L基因的c DNA序列,分析了PcCRCN-L基因的结构特征和进化模式,研究了PcCRCN-L基因在不同组织与性腺不同发育时期的表达特征,探讨了Pc CRCN-L在低氧–复氧胁迫下的表达响应模式。结果显示,PcCRCN-L基因c DNA序列长2700 bp,其开放阅读框(ORF)长度为1587 bp,编码528个氨基酸残基;DNA序列长6130 bp,位于克氏原螯虾基因组的第12号染色体,包含5个外显子和4个内含子,内含子/外显子剪接方式符合GT-AG规则。Pc CRCN-L具有1个完整的lipocalin结构域,包含典型的序列保守区Ⅰ(SCR1)序列G-X-W、保守区Ⅱ(SCR2)序列T-D-Y和保守区Ⅲ(SCR3)序列精氨酸R。多序列比对与系统进化分析结果显示,PcCRCN-L独立于传统虾青蛋白A和C亚族,单独聚为一支。Pc CRCN-L在克氏原螯虾多个组织中均有表达,在肝胰腺中表达量最高;在性腺不同发育时期,肝胰腺以及卵巢组织中的Pc CRCN-L基因表达量显著降低;低氧胁迫下,肝胰腺组织中Pc CRCN-L表达量显著降低,复氧后显著升高。研究结果表明,PcCRCN-L与克氏原螯虾性腺发育密切相关,并参与了克氏原螯虾的低氧–复氧胁迫应激调控。

黄海北部虾夷扇贝性腺发育及繁殖规律研究

通过性腺指数周年监测及性腺的组织切片观察,对黄海北部虾夷扇贝性腺发育及繁殖规律进行了研究。试验结果表明,虾夷扇贝性腺发育可划分为5期,即增殖期(Ⅰ期)、生长期(Ⅱ期)、成熟期(Ⅲ期)、排放期(Ⅳ)和休止期(Ⅴ期)。性腺周年变化规律是,11—12月为增殖期,1—2月为生长期,3月为成熟期、4—5月为排放期,6—10月为休止期。水温对性腺发育和产卵期的影响是,水温低性腺指数峰值高,产卵期相对滞后;水温高性腺指数峰值低,产卵期相对提早。

马粪海胆卵巢发育周年变化研究

利用光镜和电镜观察研究了马粪海胆(Hemicentrotus pulcherrimus)卵巢组织的周年变化,同时逐月测定了海胆的生殖腺指数.在此基础上将生殖腺的发育周期划分为五期:恢复期、生长期、成熟前期、成熟期、排放后期.实验结果表明,青岛近岸马粪海胆的产卵期为4～6月.

Study on Early Propagation Technology of Yellow Catfish (Pelteobagrus fulvidraco)

[Objectives] This study was conducted to study early reproduction techniques of yellow catfish(Pelteobagrus fulvidraco) in Jianghan Plain. [Methods] Parents of P. fulvidraco were cultured by the methods of artificial temperature control and nutrition enhancement. [Results] After culture, the body weight, gonad weight, maturity coefficient and egg diameter of P. fulvidraco increased gradually. Up to April 30, the gonad weight of the female parents of P. fulvidraco cultured in greenhouses was significantly higher than that of the parents under natural conditions(P<0.05), 54% higher than that of the parents naturally cultured at the same period;the maturity coefficient of the parents cultured under strengthened conditions was also significantly higher than that of the parents under natural conditions at the same period(P<0.05);and the ovaries of the strengthened parents were orange yellow and full of eggs, and the number of eggs per fish was relatively high. In early May, the parents cultured under strengthened conditions were induced to spawn artificially. The average induced spawning rate and fertilization rate reached 86.7% and 71.6%, respectively, reaching the respective normal level. The gonad maturity of P. fulvidraco cultured under strengthened conditions was 15 d earlier than that under natural conditions, resulting in mass production of early-propagated larvae of P. fulvidraco. [Conclusions] In Jianghan plain area, artificial propagation can be achieved in early May by means of artificial temperature control and strengthened culture, which is 20 d ahead of natural conditions.

牙鲆Lhx1基因的克隆和表达分析

Lhx1基因对于哺乳动物卵巢缪勒氏管的形成具有重要作用,但鱼类中的报道仅限于其在体轴和肾脏形成中的作用,尚未见到其与性别及性腺发育相关的报道。本研究克隆获得了牙鲆(Paralichthys olivaceus)Lhx1a和Lhx1b开放阅读框(ORF)序列,长度分别为1224 bp和1206 bp(GenBank注册号为:JX999940、JX999941),同源序列比较显示牙鲆Lhx1a和Lhx1b基因均具有两个LIM及一个HOX结构域,符合Lhx1进化保守性。这两个基因在牙鲆雌、雄成鱼各组织RT-PCR差异表达谱不尽相同,Lhx1a在精巢中弱表达,卵巢中不表达,在其他组织中雌、雄表达谱差异不大,都只在脑和眼组织有弱表达;而Lhx1b的表达在性腺中相对较高,且卵巢的表达量高于精巢。进一步检测牙鲆Lhx1a和Lhx1b在雌、雄性腺发育各期的表达谱,发现这两个基因表达集中于Ⅲ、Ⅳ期性腺,Lhx1a在精巢中弱表达,Lhx1b卵巢中的表达明显高于精巢,而在Ⅰ、Ⅱ、Ⅱ、Ⅴ期性腺几乎均不表达。由此推断牙鲆Lhx1a和Lhx1b均为性别相关基因,但在两性性腺发育中的作用可能各有不同。

Impacts of different feeding modes on the gonadal development, total edible yield, and nutritional composition of male Chinese mitten crab (Eriocheir sinensis)

This study analyzed the tissue indices,proximate composition,fatty acid and amino acid composition of edible tissues of male E.sinensis under three feeding modes(formulated diets(FD),natural diets(ND),and traditional diets(TD)).The results showed that different feeding modes had no impacts on the overall gonadal development,as no significant difference was found in gonadsomatic index among three treatments.For edible yield,three modes all had similar levels of the meat yield,hepatosomatic index,and total edible yield.Although different feeding modes did not affect the individual edible yield and gonadal development,significant differences were detected in the nutritional composition of males from three modes.For proximate composition,significant differences were found in the total lipids of gonads and the carbohydrate of hepatopancreas and meat.For fatty acids,crabs from ND had a higher proportion of monounsaturated fatty acids.In gonads and hepatopancreas,FD had a higher proportion of long-chain polyunsaturated fatty acids.For amino acids,FD had higher amino acid contents,while ND had the lowest.Except tryptophan,the essential amino acids scores of the meat were all greater than 100.As there was no significant difference on meat quality and meat yields between three feeding modes,this study suggests that the formulated diet could replace the natural diet and traditional diet towards sustainable crab farming in future.

暗纹东方鲀芳香化酶基因的结构及表达分析

性腺型芳香化酶基因是一种调节雌雄激素平衡的基因。为初步探究暗纹东方鲀性腺型芳香化酶基因在性腺发育和性别分化过程中的作用,通过RACE及荧光定量PCR技术,成功克隆出暗纹东方鲀性腺型芳香化酶基因的cDNA全长序列,共1786 bp,编码514个氨基酸,与红鳍东方鲀及星点东方鲀同源性最高。氨基酸序列分析显示,该基因编码的蛋白是一种稳定的亲水性蛋白,但不存在信号肽序列;性腺型芳香化酶含有38个磷酸化位点、4个N-糖基化位点、2个跨膜保守结构域;其氨基酸序列在哺乳动物和鱼类中均十分保守,且含有跨膜区、Ⅰ-螺旋区、Ozol′s肽区、芳香化酶特异性保守区和亚铁血红素结合区等功能保守区。表达分析显示,暗纹东方鲀性腺型芳香化酶基因主要在肌肉和卵巢中表达,其次在其他组织中也有少量表达,而且在幼鱼不同发育期卵巢中的表达量呈现出逐渐升高再降低的表达趋势,在精巢中则基本不表达。研究结果表明,性腺型芳香化酶基因很可能参与了暗纹东方鲀雌鱼性腺分化后卵巢的发育、雌性特征的维持和其他组织的发育等过程。

Environmental hypoxia:A threat to the gonadal development and reproduction in bony fishes

Vast stretches of open water bodies are gradually becoming hypoxic as a result of depletion of oxygen levels mainly due to various human anthropogenic activities.This problem of hypoxic stress on the fish population is likely to be exacerbated soon since the aquatic hypoxic environment is continuously spreading over vast areas worldwide.In recent years,various harmful effects of hypoxia to bony fishes have been reported,such as the restriction of energy-consuming metabolic processes,arrest of the growth of ovary and testes that are associated with endocrine disruption,loss of sperm and egg quality,inhibition of fertilization,hatching success,and also the reduction of larval survivability,thereby impairment of overall reproductive and developmental processes in fish.Disruption of the brain-pituitary-gonad axis,and certain enzymes related to steroidogenesis and vitellogenesis in fish have also been reported as the primary targets for an endocrine malfunction during hypoxia.Hypoxia-sensitive downregulation of key genes responsible for controlling sex hormones’synthesis has been documented in certain bony fishes.Further,continuous exposure to hypoxia was reported to induce early expression of pro-apoptotic/tumor suppressor p53 genes,thereby causing immense cell death in hypoxic embryos.However,the cellular responses to long-term hypoxia exposure and the degree of reproductive impairments in bony fishes are still not adequate to figure out the actual underlying mechanisms.The present review intends to highlight the current knowledge about the detrimental impact of chronic/acute hypoxia at different stages of fish reproduction and the associated underlying molecular mechanisms.

中华绒螯蟹首次排卵后卵巢发育的组织学研究

利用组织切片技术,结合外观特征,在光学水平对江苏南通地区第一次排卵后18天内(即卵巢的二次发育)的中华绒螯蟹的卵巢发育进行组织学和细胞学的观察研究.本实验可以观察到卵细胞发育的四个不同时期:初级卵母细胞小生长期、初级卵母细胞大生长期、成熟期以及退化时期的卵细胞.排卵后的卵巢在营养贫乏的情况下,出现直接退化现象;而在营养丰富的情况下,处于大生长前期的卵细胞则会迅速生长,为二次排卵做好准备.

Gonadal development and silvering of the Japanese Eel(Anguilla japonica)in the Yangtze River during downstream migration

The Yangtze River and its branches are important habitats for the Japanese Eel(Anguilla japonica),however,there are few data on gonadal development and silvering indices of eels during the downstream spawning migration.This study investigates gonadal development in 121 Japanese eels caught downstream from the Jingjiang Reach of the Yangtze River in October 2012.We found that in females the ovaries of most of the out-migrating population developed earlier than the testes in males,that is,the ovaries reached stage 3(F3)or early stage 4(eF4),while the testes were at stage 2(M2)or stage 3(M3).Gonadal development in eels collected in 2012 was earlier than that of samples caught from the same region in 2007e2008 and from the Pearl River Estuary in 1972e1973.The average age,body size and gonadal-somatic index of females were significantly greater than those in males.For both females and males,there were no significant differences between the two stages of gonadal development in any of the morphological indices,which indicates that gonadal development of the Japanese Eel is independent of body size.These results help us to understand the spawning population of Japanese Eels in the Yangtze River.

Full sexual maturity-cycle exposure to environmentally relevant concentrations of 17β-estradiol decreases reproductive capacity of zebrafish

17β-Estradiol(E2)has been widely detected in natural water and treatment with E2 induces potential endocrine disrupting effects in fish.However,effects on fish fecundity and steroid system after treatment with environmentally relevant concentrations of E2 for the full sexual maturation cycle remain unclear.In this study,zebrafish were treatment with 0,10 or100 ng/L E2 from embryo to adult stage,and effects on gonadal development and differentiation,steroid hormone levels,transcription of genes associated with the hypothalamic-pituitary-gonadal-liver(HPG)axis in adults and fertilization rate of offspring were assessed.The results showed that treatment with E2 lead to increased number of feminization in zebrafish.In females,E2 decreased cumulative amount of spawning and inhibited the maturation of oocyte.In males,E2 inhibited the maturation and motility of sperm,as well as decreased the movement speed of sperm.These adverse effects on sperm might be responsible for the reduced fertilization observed in offspring.In addition,treatment with E2 changed the levels of steroid hormones in zebrafish gonad and altered the transcriptional levels of genes associated with HPG axis,which is responsible for the regulation of germ cells maturation and gonadal development in zebrafish.Overall,these results suggested that treatment with environmentally relevant concentrations of E2 for the full sexual maturity cycle resulted in adverse effects on reproduction in zebrafish.

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.

A critical role of foxp3a-positive regulatory T cells in maintaining immune homeostasis in zebrafish testis development

Suppressive regulatory T cells(Treg cells)play a vital role in preventing autoimmunity and restraining excessive immune response to both self-and non-self-antigens.Studies on humans and mice show that the Forkhead box p3(Foxp3)is a key regulatory gene for the development and function of Treg cells.In zebrafish,Treg cells have been identified by using foxp3a as a reliable marker.However,little is known about the function of foxp3a and Treg cells in gonadal development and sex differentiation.Here,we show that foxp3a is essential for maintaining immune homeostasis in zebrafish testis development.We found that foxp3a was specifically expressed in a subset of T cells in zebrafish testis,while knockout of foxp3a led to deficiency of foxp3a-positive Treg cells in the testis.More than 80%of foxp3a^(-/-)mutants developed as subfertile males,and the rest of the mutants developed as fertile females with decreased ovulation.Further study revealed that foxp3a^(-/-)mutants had a delayed juvenile ovary-to-testis transition in definite males and sex reversal in about half of the definite females,which led to a dominance of later male development.Owing to the absence of foxp3a-positive Treg cells in the differentiating testis of foxp3a^(-/-)mutants,abundant T cells and macrophages expand to disrupt an immunosuppressive milieu,resulting in defective development of germ cells and gonadal somatic cells and leading to development of infertile males.Therefore,our study reveals that foxp3a-positive Treg cells play an essential role in the orchestration of gonadal development and sex differentiation in zebrafish.

Variation of lipids and fatty acids composition in the tissues of wild devil stinger(Inimicus japonicas)during sexual maturation

The present study was conducted to characterize the lipid and fatty acid profiles in different tissues(gonad,liver and muscle)of the wild-caught devil stinger during sexual maturation.Wild-caught devil stinger were divided into six groups according to gonad development and gender.Total lipid content was up to 40%dry weight in liver and was significantly higher than in other tissues(ovary,testis and muscle)in both male and female stinger during sexual maturation.During maturation,total lipid content in the female stinger liver decreased,but in males significantly increased in all tissues.Higher proportions of neutral lipids(NL)were observed in the stinger liver,while polar lipids(PL)were mainly quantified in the gonad and muscle of both male and female stinger.The PL content of the ovary significantly decreased with sexual maturation,but NL were significantly increased.The fatty acids composition of the stinger broodstocks were tissue-and lipid class-specific.In the stinger gonad high-unsaturated fatty acids(HUFA),such as ARA,DHA and EPA,are selectively transferred and conserved,indicating the crucial role of these fatty acids during sexual maturation of this fish species.

Higher dietary protein increases growth performance,anti-oxidative enzymes activity and transcription of heat shock protein 70 in the juvenile sea urchin(Strongylocentrotus intermedius)under a heat stress

This study was conducted to investigate the effect of dietary protein concentration(12%,18%,24%,30% and 36%)on the growth performance,activity of anti-oxidative enzymes and heat shock protein 70(HSP70)transcription in the sea urchin(Strongylocentrotus intermedius)under a heat stress.After 112 days of feeding trial the sea urchins were heat stressed(26
C)and the coelomic fluid and intestine sampled at time 0 and 15 min,2 h and 6 h.The results showed that an increase in dietary protein(12%-24%),significantly increased(p<0.05)the sea urchin weight gain rate(WGR).As dietary protein increased(from 18% to 36%),the gonadosomatic index(GI)of juvenile sea urchins also significantly increased(p<0.05)from 18.0%to 22.6%.Superoxide dismutase(SOD)activity increased with dietary protein increase(12%-30%)and the enzyme activity was significantly higher(p<0.05)in the coelomic fluid of sea urchins that were fed with 30% diets when compared to 12% and 36% protein diets at all time points after the heat stress.Catalase(CAT)activity showed a similar tendency with the increase in dietary protein concentration at time 0 and 15 min after the heat stress(p<0.05).Transcription of HSP70 in the intestine also showed a similar trend to SOD and was highest in the animals that were fed with 30% protein diets(p<0.05).Our results suggest that 24% protein diets could meet the requirements for growth performance but a 30% protein diet resulted in improved gonad development and anti-heat stress capacity in this sea urchin species.

池养美洲西鲱的性腺发育进程

采用解剖学、组织学、形态测量等技术方法,研究了池塘培育美洲西鲱亲本初次性成熟性腺发育的进程。结果显示,池塘培育美洲西鲱雌雄鱼2龄开始初次性成熟,卵巢和精巢的发育进程呈现明显的阶段性,即等待期(12月5日前)、启动期(12月5日后)和快速期(雄鱼在3月7日后,雌鱼在4月20后)。雌雄亲本的体重与性腺重均呈良好的指数函数关系(y=a ebx,P<0.01)。雄性成熟期早于雌性成熟期约半个月,即Ⅴ期精巢出现在4月20日,而Ⅴ期卵巢则出现在5月6日,表明池塘培育美洲西鲱亲本催产的交配窗口期开始于5月6日。本文建议,长三角地区美洲西鲱亲本的池塘培育,应将雌雄亲本分开饲养,以减少互相干扰,同时适当降低雄鱼的培育水温;当亲本性腺发育进入快速期,应强化饲料营养,增加投喂频率;亲本的人工催产可选择5月初开始。