Glycoprotein hormone receptor family in medaka (Oryzias latipes)

Four types of GPHR cDNAs have been cloned from ovary and testis of medaka (Oryzias latipes) and their gene constructions have been determined. Two of them are closely related to known fish receptors for FSH and LH, respectively. Changes in their mRNA levels were examined during the course of oogenesis. FSH receptor mRNA could not be detected from 20 h before ovulation, whereas LH mRNA remained 5 h before ovulation.     

Effect of p-Nonylphenol on sperm motility in Japanese medaka (Oryzias latipes)

This study examined the effects of exposure to p-Nonylphenol (NP) on the motility of the spermatozoa of Japanese medaka (Oryzias latipes). Spermatozoa collected from adult males reared for 2 weeks in aqueous solutions of 20 and 100 μg/L NP showed similar swimming speeds but significantly lower percentages of motile spermatozoa than those of untreated controls. These results suggest that even a short exposure to NP might reduce significantly spermatozoan viability in fish.     

Molecular cloning and biochemical characterization of medaka (Oryzias latipes) lysosomal neu4 sialidase

Glycoconjugates are known to be involved in many physiological events in vertebrates. Sialidase is one of the glycosidases, which removes sialic acid from glycoconjugates. In mammals, the properties and physiological functions of sialidases have been investigated, while there is little understanding of fish sialidase. Here, to investigate the significance of fish neu4 sialidase, neu4 gene was cloned from medaka brain mRNA and identified. Sialidase-specific motifs (GPG, YRVP and Asp-Box) were well conserved in the medaka neu4 polypeptide. Optimal pH of medaka neu4 sialidase was 4.6, but its activity was sustained even at neutral and weak alkaline pH. The neu4 considerably cleaved sialic acid from 4-methylumbelliferyl-N-acetyl-α-d-neuraminic acid and sialyllactose, but not from ganglioside and fetuin, which are good substrates for human NEU4. neu4 activity was mostly detected in mitochondria/lysosome fraction after biochemical fractionation, and indirect immunofluorescence assays revealed neu4 localization in lysosome in neu4 overexpressed cells. Next, developmental change in medaka neu4 and other sialidase mRNA levels were estimated by real-time PCR. Each sialidases showed different expression patterns in embryonic development: neu4 was up-regulated at late developmental stage in embryo, and neu3a mRNA level was quite high in 0.5 dpf. On the other hand, neu3b expression was drastically increased after hatching, suggesting that each sialidase may play a different role in embryonic development.     

Towards obtaining long term embryonic stem cell like cultures from a marine flatfish, Scophtalmus maximus

Culturing pluripotent embryonic stem cells represents a unique model system for in vitro studies of embryo cell growth and differentiation, and represents a connection between in vitro and in vivo manipulation of genes. To further develop and refine stem cell technology for marine fish, we have established cultures of embryonic stem cells isolated from turbot blastulas. The pluripotent nature of our turbot-ES-like cells was supported by their morphology and elevated levels of alkaline phosphatase enzyme activity, their ability to remain undifferentiated for a prolonged culture period, their spontaneous differentiation potential in vitro and their ability to form embryoid bodies (EB) in response to changes in the extracellular environment. In addition, we show that turbot ES like cells express Oct-4 required for the maintenance of pluripotency of ES cells. Cells from 100 blastulas (>10⁵ cells/well) were seeded into gelatine coated 24 well cell culture clusters. The cells were polygonal in shape, with dense cytoplasm and large nuclei. The ES-like cells formed colonies within 24 h following seeding, multilayered in a pyramidal fashion, with maximum cell densities in the middle. The cells proliferated vigorously when seeding densities were high and the cells still had not attached to the gelatine-coated surface. Most of the cells became attached to the surface 48 h following seeding. Attached cells grew more slowly and 20% of the plated colonies could be kept stable for 60 days. Eventually, most of the cultures showed extensive differentiation or died. Only a few cultures (4–5%) survived prolonged culturing (>2 months). The cells were stained for alkaline phosphatase activity, a marker of pluripotency and showed intense staining. More specific, turbot ES like cells in culture expressed Oct-4, detected by immunofluorescence staining. Changing the medium conditions by adding retinoic acid and removing LIF, the proportion of embryoid bodies in our cultures increased. ES-like cells as well as fresh, intact fertilised eggs where successfully cryopreserved. ES cells from the cryopreserved eggs could be isolated and seeded into cultures, forming colonies like the cells from freshly fertilised eggs. Also cryopreserved ES-like cells could be successfully plated. The prolonged survival of these cryopreserved cells has not yet been investigated. The establishment of in vitro cultures of turbot ES-like cells represents a new experimental model for marine flatfish. This revised version was published online in July 2006 with corrections to the Cover Date.     

漠斑牙鲆胚胎细胞系的建立与鉴定

漠斑牙鲆(Paralichthys lethostigma)是中国近年新开发的重要海水养殖鱼类,本研究以漠斑牙鲆囊胚期胚胎为材料,探索了鱼类胚胎细胞分离和培养的条件,建立了漠斑牙鲆胚胎细胞培养技术。建立的漠斑牙鲆胚胎细胞系(SFEC)至今已经培养了240多天,传至80多代。SFEC的完全培养基采用添加抗生素、胎牛血清(FBS)、花鲈血清(SPS)、成纤维生长因子(bFGF)的DMEM。SFEC形态小而呈圆形、多边形,在培养基中生长迅速。本实验检测了温度、胎牛血清浓度、成纤维生长因子对SFEC生长的影响。在24~30℃之间SFEC生长良好,但当温度低于18℃时细胞生长速度明显减慢。在含15?S的培养基中,SFEC生长速度明显比在含7.5?S的培养基中快,在培养基中添加bFGF可以使细胞生长速度显著提高。SFEC的二倍体核型为2n=6st 42t。将GFP报告基因通过脂质体介导的方法转入SFEC中并成功地获得了表达,转化率为20%。     

Effect of temperature on the efficiency of feminization of medaka (Oryzias latipes) by hormonal (estradiol) manipulation

During the critical time of sex determination, fish gonads are liable to chemicals and environmental factors that can disrupt the normal course of sex differentiation. This study was conducted to examine the possibility of a combined effect of water temperature and an exogenous steroid hormone (estradiol-17β, E₂) on the process of sex differentiation of Japanese medaka (Oryzias latipes) of the d-rR strain. Sex reversal experiments conducted at different temperatures (14, 18, 22, 26, 30, and 34 °C) and E₂ concentrations (0, 1 and 10 ppb) showed that the percentage of feminization of genotypic males into phenotypic females decreased with increasing temperature for both E₂ concentrations, suggesting a marked synergism between the two factors.     

A morphological study off a thyroid carcinoma in a medaka, Oryzias latipes (Temminck & Schlegel)

A large mass protruding beyond the left operculum was found in an adult male medaka, Oryzias latipes (Temminck & Schlegel). Histologically, the mass was mainly composed of thyroid follicles measuring up to 292 μm in diameter. The follicular epithelium consisted of cuboidal or squamous cells. The follicles lacked colloidal material in the lumen. The thyroid tissue infiltrated into the kidney. Electron microscopically, the tumour cells were characterized by dilation and vesiculation of rough endoplasmic reticulum, which extended throughout the cytoplasm, and an irregular outline of the nucleus. Mitotic figures and partly degenerate cells were frequently observed. Based on the above features, the tumour was diagnosed as a follicular carcinoma of the thyroid, the first reported case in the medaka.     

Establishment and characterization of a continuous cell line (GF-1) derived from grouper, Epinephelus coioides (Hamilton): a cell line susceptible to grouper nervous necrosis virus (GNNV)

A new continuous cell line (GF-1) was established and characterized. The GF-1 cell line, derived from the fin tissue of a grouper, Epinephelus coioides (Hamilton), was maintained in L15 medium containing 5% foetal bovine serum (FBS) at 28 °C, and has been subcultured more than 160 times since 1995. The majority of GF-1 cells are fibroblast-like, together with some epithelioid cells. Spontaneous transformation of GF-1 cells occurred during subculture 50 to subculture 80, and led to an increase of plating efficiency, less requirement of FBS and de novo susceptibility to grouper nervous necrosis virus (GNNV). Cytopathic effects (CPEs) could be observed in GF-1 cells 3–5 days post-infection with pancreatic necrosis virus (IPNV), hard clam reovirus (HCRV), eel herpes virus Formosa (EHVF) and GNNV. In addition, abundant GNNV particles were found in the cytoplasm of GNNV-infected GF-1 cells using electron microscopy and nucleic acids of GNNV virus were detected by polymerase chain reaction in the culture medium of GNNV-infected cells after CPE appeared. The experimental results indicated that GF-1 can effectively proliferate fish nodavirus and is a promising tool for studying fish nodavirus.     

聚苯乙烯微塑料长期暴露对海水青鳉 (Oryzias melastigma) 亲代生长、繁殖及子代发育的影响

海水青鳉 (Oryzias melastigma) 作为雌雄异体的模式动物,在研究外来污染物毒性效应性别差异上具有优势。运用转录组学技术系统研究了雌、雄海水青鳉肝脏组织中的差异表达基因,结果显示雌、雄青鳉肝脏中共有1 351个显著差异表达基因,其中683个在雌鱼肝脏中高表达,668个在雄鱼肝脏中高表达。雌鱼肝脏中高表达的差异基因主要涉及生殖和性激素合成相关通路,如卵黄蛋白原和雌激素受体。雄鱼肝脏中高表达的差异基因主要涉及能量代谢、细胞骨架和肌肉收缩等相关生物过程,如丙酮酸激酶、肌酸激酶、肌球蛋白和肌钙蛋白等。实时荧光定量PCR验证结果显示,除DNA错配修复蛋白基因以外,其他17个基因差异表达倍数与RNA-seq对应基因差异表达趋势基本一致,表明转录组分析数据基本可靠。研究表明,雌、雄海水青鳉肝脏中基因表达具有差异调控模式,研究获得的差异基因和调控通路将为海水青鳉对外来污染物性别差异响应分子机制研究提供一定的理论基础。     

Growth-independent effects of a fluctuating thermal regime on the life-history traits of the Japanese medaka (Oryzias latipes)

Abstract –  Temperature fluctuation has been shown to affect somatic growth rates and metabolic functions of fish, but little is known about the effects of fluctuating temperatures on life-history traits. We examined these effects on female Japanese medaka ( Oryzias latipes ) reared from hatch to maturity at two mean temperatures (27 and 30 °C) with diel temperatures either held constant or allowed to fluctuate ±5 °C. Feeding regimes were manipulated to equilibrate medaka growth rates in all treatments. Age and length at maturity decreased as a result of thermal fluctuation, whereas there was no effect of temperature fluctuation on the proportion of fish attaining maturity or on the number of eggs produced by a mature female. We hypothesise that short-term high temperatures associated with temperature fluctuation decreases development time, whereas reduced energy conversion efficiency at higher temperatures does not allow for increased egg production.     

Establishment and characterization of an ovarian cell line from Southern catfish (Silurus meridionalis)

An ovarian cell line was successfully developed from the juvenile ovary of Southern catfish (SCO1) (Silurus meridionalis), which was designated as SCO1. The cell line multiplied preferentially in L-15 medium with 15 % fetal bovine serum at 28 °C for more than 70 passages over a period of 420 days. SCO1 showed fibroblast-like morphology and predominantly retained a diploid karyotype of 58 chromosomes. From the gene expression patterns, SCO1 showed a characteristic of ovarian granulosa cells. After the cells were transfected with the green fluorescent protein expression vector, bright fluorescent signals could be observed in approximately 30 % cells. This cell line may be valuable for the evaluation of endocrine disruptors and studying interactions between somatic cells and germ cells.     

Development of an efficient bioreactor system for delivering foreign proteins secreted from liver into eggs with a vitellogenin signal in medaka Oryzias latipes

In this study, we developed a novel bioreactor system to deliver and accumulate foreign proteins in eggs using medaka fish Oryzias latipes with the aid of a partial sequence of vitellogenin (Vtg). In teleost fish, Vtg, the hepatically generated precursor of egg yolk proteins, is secreted into the bloodstream and then taken up into eggs. We predicted in silico a probable region (Vtg signal) of Vtg that mediates transportation of proteins from the liver into eggs. Then, we established two transgenic lines expressing the fused proteins including the Vtg signal and each reporter gene, enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC)-fused EGFP, in the liver driven by a liver-specific choriogeninH (chgH) promoter. Each reporter signal was detected from the fertilized eggs spawned by the transgenic females, showing successful transportation of the proteins into the eggs with the Vtg signal. This is the first report demonstrating that the Vtg signal has capability to deliver exogenous proteins into eggs. Because Vtg is a highly conserved protein among most of oviparous organisms, our findings hold promise for establishing bioreactor systems viable in a wide range of organisms.

     

不同因子对花鲈胚胎干细胞增殖的影响

胚胎干细胞(ES细胞)是从动物早期发育胚胎中分离出来的具有发育全能性的一种未分化细胞系。维持花鲈胚胎干细胞(LJES1)的体外生长、增殖及未分化状态需要在培养基中添加一些生长因子。实验通过配制省略1种或多种因子的培养基PESM0～10，计数LJES1细胞在各培养基中增殖的数量，确定各种生长因子对LJES1细胞的增殖作用。同时，对LIF因子和bFGF因子的作用进行了重点的研究，发现LIF因子对早期的LJES1细胞增殖几乎没有作用，其主要作用是维持LJES1细胞的未分化状态，但对晚期LJES1细胞的增殖有一定的作用；bFGF因子对LJES1细胞有强烈的刺激增殖的作用；鲈鱼胚胎抽提液(PEE)以及鲈鱼血清(FS)也促进了LJES1细胞的增殖，2-巯基乙醇(2-ME)具有还原血清中的含硫化合物、防止过氧化物对LJES1细胞的损害及促进贴壁的作用，因而也促进了LJES1细胞的增殖。     

三种脂质体介导的花鲈胚胎干细胞转化效率的比较

通过3种脂质体(genejammer, genejuice和metafectene)介导绿色荧光蛋白基因转化花鲈胚胎干细胞,探讨不同脂质体介导的花鲈胚胎干细胞（LJES1）的转化效率,以及影响外源基因转化效率的因素,如细胞接种时间、初始接种密度、DNA和脂质体用量以及它们之间的比例等。实验发现,genejammer转化LJES1细胞效率最高,适合于该细胞系的转化。对于同一种脂质体,DNA与脂质体用量的比例对于绿色荧光蛋白基因转化LJES1最为重要,DNA与genejammer的比例为1∶6时,转化效率最高,为27.3%;DNA与genejuice的比例为1∶4时,转化效率最高,为12.1%;而DNA与metafectene的比例为1∶3时,转化效率达到最高,为5.3%。随着DNA、脂质体量的增加,转化效率有所提高,但达到一定的用量后,转化效率反而下降。     

An astacin-like protease, alveolin, is released from cortical vesicles and induces changes in egg envelope proteins during fertilization of medaka (Oryzias latipes) eggs

Upon fertilization, two major protein groups, ZI-1,2 and ZI-3, composing the egg envelope are polymerized to insoluble forms via temporary formation of 132 kDa and 61–62 kDa intermediate proteins. We have shown that an astacin-like protease, `alveolin' is released from cortical vesicles into the perivitelline space after egg activation and induces initiation of the polymerization of egg envelope proteins. To clarify initial reactions of the polymerization, the primary structure of the 132 kDa protein was analyzed.     

The effect of cyclooxygenase (COX) inhibitors on Japanese medaka (Oryzias latipes) reproduction parameters fed with high level of arachidonic acid (20:4 n-6)

Broodstock of mature Japanese medaka (Oryzias latipes Temminck and Schlegel, 1846) (mean weight 0.45 ± 0.03 g; age 6 months) were maintained in glass aquaria (8 males and 8 females in each aquarium). The fish were fed commercial feed (group C) or this feed was supplemented with arachidonic acid ARA (2 % of total fatty acids in feed; group A) and one of the COX inhibitors: resveratrol (group AR) or NS-398 (group ANS) with amount 20 mg kg^?1 BW day^?1 of one of the COX inhibitors. The effect of 2 weeks of feeding experimental diets on reproductive parameters (fecundity, embryo and larval survival, hatching rate, and larvae body weight) was analyzed. Larvae (obtained from experimental dietary treatments) were reared for 2 weeks. The percent of spawning female and fecundity was similar in dietary treatments. COX inhibitors in broodstock diet caused a decrease in embryo survival rate. Eggs of medaka from group ANS and AR revealed the lowest hatchability rate. In contrast, group A revealed a higher hatchability rate at the same time. Larval body weight after hatching was significantly the highest in this group. At the end of the experiment, larvae body weight ranged from 1.62 ± 0.43 mg in group AN to 2.89 ± 0.74 mg in group A. These results show that the COX inhibitors decreased gamete and larval quality regardless of high levels of ARA in broodstock diet.     

Correction to: Development of an efficient bioreactor system for delivering foreign proteins secreted from liver into eggs with a vitellogenin signal in medaka Oryzias latipes

Fisheries Science - The article "Development of an efficient bioreactor system for delivering foreign proteins secreted from liver into eggs with a vitellogenin signal in medaka Oryzias...     

青鱼鳍条组织细胞系的建立及其生物学特性

采用组织块移植培养技术,对来源于青鱼(Mylopharyngodon piceus)的鳍条组织细胞进行原代培养,建立了青鱼鳍条组织细胞系,定名为BC-Fin。青鱼鳍条组织细胞为成纤维样细胞,已稳定传代培养50多代,其最适培养温度为25℃,最佳培养基为L-15,最适血清浓度为15%,在最适培养条件下,青鱼鳍条组织细胞的群体倍增时间为60.6 h。青鱼鳍条组织细胞液氮冷冻保藏6个月后,经台盼蓝染色,约(90.09±4.65)%的细胞具有细胞活性,复苏后的细胞生长旺盛。细胞染色体分析显示,第16代青鱼鳍条组织细胞的染色体数目为正常二倍体(2n=48),第41代细胞染色体众数为46。通过对离体培养细胞的线粒体中的16S rRNA基因进行特异性扩增,获得长度为320 bp的核酸片段,核酸序列比对分析结果表明,其与青鱼16S rRNA基因序列的一致性达98%,表明该细胞来源于青鱼。病毒敏感性试验结果显示,在感染草鱼呼肠孤病毒(GCRV)后BC-Fin细胞系可产生典型细胞病变效应,病毒滴度为105.33±0.21TCID50/m L,且PCR检测可检测出细胞培养的草鱼呼肠孤病毒,表明BCFin细胞系对草鱼呼肠孤病毒较敏感。