Tumours and related lesions of the endocrine system of bony and cartilaginous fishes

The literature pertaining to the prevalence and histopathology of endocrine cell and tissue tumours and related lesions (TRL) in bony and cartilaginous fishes is catalogued and reviewed in the context of the embryological origin of the various components of the endocrine system. Reports of TRLs of endocrine cells or tissues of fishes are relatively rare, and of these, most describe thyroid neoplasms that appear to be simple hyperplasia. The vast majority of published reports of endocrine TRLs in fishes are case-studies, and thus, with a few notable exceptions, the aetiology of the lesions is unknown. The exceptions include the epizootics of thyroid neoplasms in introduced Pacific salmon in the Great Lakes, which appear to have an environmental aetiology, and epizootics of gonadal stromal cell neoplasms. The latter were all found in either hybrid species or highly inbred captive strains of fishes, suggesting a genetic involvement in the tumourigenesis. The possible aetiologies for the reported TRLs in wild and captive fish species are discussed, and various hypotheses proposing infectious agents, genetic factors and environmental contaminants are considered.     

An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish

Although there are almost thirty-thousand species of fish living in a great variety of habitats and utilizing vast reproductive strategies, our knowledge of morphofunctional and quantitative aspects of testis structure and spermatogenesis is still incipient for this group of vertebrates. In this review, we discuss aspects that are important to better understanding of testis structure and function, and of the development of germ cells (GC) during spermatogenesis. To achieve this, we have recently completed a number of studies presenting morphometric and functional data related to the numbers of GC and Sertoli cells (SC) per each type of spermatogenic cyst, the number of spermatogonial generations, the SC efficiency, and the magnitude of GC loss that normally occurs during spermatogenesis. We also investigated SC proliferation and the relationship of this important event to early spermatogenic cysts. The available data strongly suggest that SC proliferation in sexually mature tilapia is the primary factor responsible for the increase in testis size and for determination of the magnitude of sperm production. The influence of temperature on the duration of spermatogenesis in tilapia was also evaluated and we have used this knowledge to deplete endogenous spermatogenesis in this teleost, in order to develop an experimental system for GC transplantation. This exciting technique results in new possibilities for investigation of spermatogenesis and spermatogonial stem cell biology, creating also an entirely new and promising scenario in biotechnology—transgenic animal production and the preservation of the genetic stocks of valuable animals or endangered species.     

硬骨鱼肥大细胞的研究进展

有关肥大细胞，在某些啮齿动物及人类中研究已比较深入，但在鱼类及其他低等脊椎动物中研究较少。硬骨鱼(teleostean fish)肥大细胞(MC)，常被称为嗜酸性颗粒细胞(EGC)，或肥大细胞／嗜酸性颗粒细胞(MC／EGC)，本文对有关研究进展作一综述并对硬骨鱼肥大细胞的发生、组织化学性质、细胞颗粒介质及其在鱼类健康与疾病中可能的功能意义等进行讨论，旨为深入研究鱼类重大疫病的发生机理提供基础依据。     

鱼类粘液细胞研究进展

鱼类粘液细胞(ｍｕｃｏｕｓｃｅｌｌｓ)是普遍存在于鱼类上皮中的一种腺体细胞,主要分布在鱼的皮肤、鳃及消化道的上皮中,能分泌大量粘液。粘液中含有多种活性物质,如粘多糖、糖蛋白、免疫球蛋白及各种水解性酶类等,对鱼的许多生理功能有重要影响。对粘液细胞进行深入研究,不仅有助于对鱼类生长、发育以及自我保护等方面的一些基本理论问题的理解,而且在鱼类的养殖和病害防治中也具有重要的实践意义。有关鱼类粘液细胞的研究,国外开展得较早,也较广泛,已对多种鱼类的粘液细胞进行了研究。但是,国内有关这方面的研究却很少。本文结合本实验室…     

Mucosal responses of brown-marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) following intraperitoneal infection with Vibrio harveyi

Groupers are popular aquaculture species in South-East Asia, but their cultivation is affected by infectious disease outbreaks. Mucosa-associated lymphoid tissues provide a first-line defence against pathogens; however, few studies are available relating to cellular or proteomic responses of mucosal immunity in grouper. Skin, gill and intestine were sampled from brown-marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) at 4 and 96 hr post-infection (hpi) and 7 days post-infection (dpi) following intraperitoneal infection with Vibrio harveyi, and stained with haematoxylin/eosin and Alcian Blue/periodic acid–Schiff. Skin mucus was analysed by 2D-gel electrophoresis, and proteins modulated by the bacterial infection identified. In the infected fish, significant increases in sacciform cells in skin and increased levels of nucleoside diphosphate kinase in mucus were detected at 4 hpi. At 96 hpi, goblet cells containing acidic mucins significantly increased in the intestine, while those containing mixed mucins increased in skin and gills of infected fish. Proteasome subunit alpha type-I and extracellular Cu/Zn superoxide dismutase levels also increased in mucus. Rodlet and mast cells did not appear to respond to the infection. Mucosal tissues of grouper appeared actively involved in response to Vibrio infection. This information may help future research on improving grouper health, production and vaccine development.     

日本新糠虾卵巢和肝胰腺主要细胞内5-HT的免疫组织化学定位

运用免疫组化方法,观察了5-HT阳性物质在日本新糠虾不同发育期的卵巢卵细胞和滤泡细胞及肝胰腺细胞中的分布和变化。结果表明:随卵细胞的发育,滤泡细胞逐渐迁移至其近旁。各期卵巢卵细胞和滤泡细胞及肝胰腺细胞中均存在大量5-HT阳性细胞,阳性物质呈深棕色;各期卵巢中5-HT在滤泡细胞内均呈阳性;在Ⅰ、Ⅱ和Ⅲ期卵巢中卵细胞胞质和胞核均呈阳性,且阳性强度呈递减;Ⅳ期卵巢中5-HT在卵细胞中呈阴性,而在Ⅴ期卵巢中5-HT在卵细胞中又呈现弱阳性。5-HT在发育各期的肝胰腺细胞中均呈阳性。同一期卵巢中,不同类型肝胰腺细胞间5-HT的分布没有差别。除Ⅳ期卵巢外,在其余各期卵巢中5-HT阳性强度在卵细胞胞质和肝胰腺细胞胞质中表现一致。     

大西洋鲑卵泡颗粒细胞和鞘膜细胞释放性激素的研究

本文通过对大西洋鲑的完整卵泡(包括卵母细胞、外周的鞘膜细胞和颗粒细胞)、手术分离的鞘膜细胞以及分离的颗粒细胞分别进行离体培育,测定它们在促性腺激素(GTH)刺激下,释放性激素的能力。实验证明,完整卵泡具有释放孕酮,17α—羟孕酮,雄烯二酮,睾酮,雌二醇和17α20β—双羟孕酮的能力。分离的鞘膜细胞只能释放前四种性激素。分离的颗粒细胞不能释放以上任何一种性激素。结果说明,单独的鞘膜细胞或单独的颗粒细胞都不具有合成雌二醇和17α20β—双羟孕酮的能力。     

Surplus spermine does not protect salmon cells against lipopolysaccharide (LPS)‐induced stress,but excess spermine is eliminated through spermidine/spermine N1‐acetyltransferase (SSAT)

The present trials tested the efficiency of surplus spermine to reduce inflammation and oxidative stress following LPS‐induced stress using an in vitro model of head kidney and liver cells isolated from Atlantic salmon. Spermine did not protect cells from LPS‐induced inflammatory response at either 0.3, 0.6 or 0.9 mM. However, as the gene expression of spermidine/spermine N1‐acetyltransferase (SSAT) increased with increasing spermine concentration, we addressed possible oxidative effects of the increased SSAT using its activator DENSPM or inhibitor of polyamine oxidation of the acetylated polyamines using MDL72527 at a spermine concentration of 0.6 mM. There was no significant effect of DENSPM, but MDL72527 decreased gene expression of GPX‐3 (p = .04), while gene expression of catalase and MnSOD was unaffected by treatment (p = .30 and p = .48, respectively). In conclusion, spermine did not protect cells from LPS‐provoked inflammation. The higher the spermine concentration, the more SSAT producing acetylated spermine occurred. Inhibiting the acetylated polyamine oxidases by MDL72527 improved oxidation status as expected due to a lower endogenous production of H₂O₂ by polyamine and acetylated polyamine oxidases. Probably care should be taken using polyamines or arginine as functional ingredients to avoid any increased oxidation within cells.     

Separation and characterization of connective tissue cells expressing 43-kDa zinc-binding protein from common carp

To separate the connective tissue cells expressing the 43-kDa zinc-binding protein from the common carp, mucosa of the digestive tract of the fish was removed by scraping with a glass slide, and the de-mucosa tissue was digested with a collagenase type IV solution. The cells collected from the collagenase-treated suspension were rather homogeneous, and more than 90% of the cells were round with a diameter of approximately 6 μm. Significant quantities of the 43kDa zinc-binding protein were shown to be present on the cell surface of the approximately 6-μm cells. The mean zinc concentration in the cells was found to be 2.21 μg zinc/10⁶ cells, which is approximately 20—30 times higher than that found in the other three fish species (grass carp, silver carp, tilapia), studied. The present work may provide a basis for the culture of these cells.     

Effects of dietary cholesterol on hepatopancreas associated with morphotypic differentiation in male Amazon River Prawns,Macrobrachium amazonicum (Heller, 1862)

Cholesterol is a necessary element in the diet of prawns and it is involved in the metabolic processes of sexual maturation and synthesis of the molting hormone. Freshwater prawns are not able to synthesise cholesterol from specialised metabolic pathways. The aim of this study was to describe changes in structural and morphometric patterns of hepatopancreas associated with morphotypic differentiation of males Macrobrachium amazonicum when fed with different levels of cholesterol. Considering the four morphotypes of this species, Translucent Claw morphotype specimens were split into experimental units and divided into four groups, fed with different levels of cholesterol (0%, 0.5%, 1%, 2%) for 50 days. They were collected as they reached Green Claw (GC) morphotype, weighed to determine the hepatosomatic index, and the hepatopancreas was processed in histological routine for morphologic and morphometric analysis. In this study, it was possible to observe that the 2% cholesterol treatment exhibited more animals that reached the GC morphotype, and there was no significant weight gain in all treatments. Histologically, B‐cell abundance and hypervacuolized as well as F‐ and R‐cells shortages were observed as the percentage of cholesterol increased. This alteration in cellular profile correlates with the morphometric analyses that exhibited an increase in epithelial area and alterations in the lumen shape. This imbalance observed in the lumen‐epithelium relationship may be associated with a low digestive performance and possibly congested the functions of hepatopancreas. Thus, high doses of cholesterol promoted morphotypic differentiation without weight gain, due to alterations on hepatopancreas.     

不同盐度和驯养时间中华鲟子二代幼鱼鳃的显微结构变化

采用光学显微镜和组织切片方法,研究了不同盐度和驯养时间中华鲟幼鱼鳃及鳃中泌氯细胞的结构变化特征。试验用鱼为1+龄中华鲟子二代,平均全长50.7 cm,平均体重约441.9 g。试验前在养殖池中暂养1周。试验设2个盐度梯度(10、15)组和1个对照组(淡水)。盐度驯养为将中华鲟子二代从暂养池转入盐度10海水中驯养,分别取驯养1、3、6、9、12、24、48、72、96、120、144、168、192、216、240和264 h的幼鱼各1尾用于试验;将剩下的鱼再转入盐度15海水中驯养,分别取驯养24、48、72 h的幼鱼各1尾用于试验。结果显示,在淡水组中,中华鲟子二代幼鱼鳃丝发达,细胞饱满,鳃小片宽且长。鳃丝上皮的泌氯细胞数量较少,通常靠近鳃小片基部,呈近椭圆状。与淡水组相比,盐度10组的幼鱼鳃丝主干部明显变窄,鳃小片基部区域及鳃小片上泌氯细胞的数量略有增加,胞体变大,且随驯养时间的延长,出现了明显的细胞膜间隙。幼鱼在盐度15组中鳃丝主干明显变窄变短,鳃小片也显著缩短,鳃泌氯细胞数量显著增加,主要分布在鳃小片基部,有明显的分泌腔。中华鲟子二代幼鱼能够快速适应外界水体盐度的变化,这与鳃及鳃上皮中调节渗透压功能细胞的数量和结构变化相适应。     

Immunolocalization of Na+, K+-ATPase-rich cells in the gill and urinary system of Persian sturgeon, Acipenser persicus, fry

Localization of Na⁺, K⁺-ATPase-rich cells in the gill and urinary system of Acipenser persicus fry was performed through immunofluorescence light microscopy using a mouse monoclonal antibody IgGα₅ raised against the α-subunit of chicken Na⁺, K⁺-ATPase. Different types of epithelia were clearly identified in the gill epithelium: epithelia of branchial arch, interbranchial septum, filament and lamellar epithelium. The Na⁺, K⁺-ATPase-rich cells were found in the epithelia of branchial arch, interbranchial septum, filament, interlamellar region and also in the lamellae. Histologically, the urinary system is divided into head kidney, trunk kidney and short caudal kidney. The head kidney is composed of the pronephric tubules and the haemopoietic tissues, while the trunk kidney is composed of a large number of glomeruli and convoluted nephrons. Each nephron consisted of a large glomerulus and tubules (neck, proximal, distal and collecting tubules) which connected to ureters. Posteriorly, ureters extended and joined together to form a small urinary bladder. In the urinary system, no specific fluorescence staining was observed in the glomerulus, neck segment and proximal tubules. The distal tubule cells and collecting tubule cells showed a strong immunostaining of Na⁺, K⁺-ATPase. Epithelia of ureters and urinary bladder also showed several isolated immunofluorescent cells. Immunofluorescent cells were rich in Na⁺, K⁺-ATPase enzyme which is very important for osmoregulation.     

一种虹彩病毒感染大菱鲆的病理学研究

对我国虹彩病毒感染的大菱鲆Scophthalmus maximus进行的组织病理和超微病理学研究发现,该病典型的病理学特点是在病鱼的脾脏、肾脏、肠、肝脏、鳃、心脏和皮肤等器官组织内出现嗜碱性的肿大细胞。病毒感染导致患病大菱鲆多个器官组织发生了不同程度的病理变化,其中以脾脏组织的病理变化最为显著,表现为造血组织的严重坏死。此外,肾脏造血组织发生坏死、肠固有膜和黏膜下层出血和水肿、肝细胞水样变性、心肌局灶性坏死以及皮肤真皮层出血并伴有水肿和炎性渗出也是该病常见的组织病理学变化。超微病理研究表明,肿大细胞内有虹彩病毒粒子存在。病毒分布于受感染细胞的胞质、组织间隙以及血管腔内。受感染细胞出现线粒体和内质网等细胞器肿胀、崩解等细胞病理变化。研究认为,病毒感染造成皮下组织血管损伤出血,是虹彩病毒感染的大菱鲆发生＂红体病＂的原因所在。虹彩病毒感染所致的机体严重贫血是患病大菱鲆死亡的主要原因,而主要器官组织的病变使得病鱼器官功能衰竭则可加速鱼的死亡。     

牡蛎糖胺聚糖对损伤的血管内皮细胞功能的影响

采用人脐静脉内皮细胞株ECV304体外培养的方法,复制过氧化氢(H2O2终浓度为50μg/ml)诱导的血管内皮细胞氧化损伤模型,应用硝酸还原酶法检测一氧化氮(NO)的含量,采用化学比色法分别检测谷胱甘肽过氧化物酶(GSH-PX)活性、总抗氧化能力(T-AOC)、总一氧化氮合酶(T-NOS)和诱导型一氧化氮合酶(iNOS)的活性。结果显示,与正常对照组相比,过氧化氢损伤的血管内皮细胞其GSH-PX活性、T-AOC、NO含量及T-NOS活性均明显降低(P<0.01),iNOS活性明显增加(P<0.01)。与损伤模型组相比,O-GAG各浓度保护组(50、100和200μg/ml)细胞的GSH-PX活性、T-AOC、NO含量及T-NOS活性均明显升高(P<0.01),而iNOS活性则显著降低(P<0.01)。表明牡蛎糖胺聚糖可以提高受损血管内皮细胞的抗氧化能力以及合成释放NO的功能,对H2O2诱导的血管内皮细胞氧化损伤有保护作用。     

The relationship between `deep-hole' mitochondria-rich cells and salinity adaptation in the euryhaline teleost, Oreochromis mossambicus

The present study elucidates the relationship between deep-hole MR cells (Lee et al. 1996) and salinity adaptation in tilapia (Oreochromis mossambicus). Freshwater tilapia were transferred to salt water of various salinities, i.e., 5 (hypotonic), 10 (isotonic), 20 (hypertonic), and 30 (hypertonic), for 2 weeks. The density of MR cells, protein content, activity, and localization of the sodium pump were examined. There was no significant difference in serum osmolarity and Na⁺, K⁺, Cl^– levels in fish of the various treatment groups. The amounts of protein and activity of Na,K-ATPase were elevated in fish from SW with the highest salinity. MR cells observed by scanning electron microscope revealed small pits (0.5–1.0 m in diameter) in groups from hypotonic and isotonic water and large crypts (2.4–3.8 m in diameter) in fish from hypertonic water. Moreover, the density of these deep-hole MR cells increased significantly in fish adapted to hypertonic SW. Larger and more numerous deep-hole MR cells of euryhaline tilapia may account for higher protein amounts and activities of Na,K-ATPase, probably to meet the physiological demand of euryhaline teleosts engaged in hyporegulation.     

New methods for the primary culture of gill epithelia from freshwater rainbow trout

Gill epithelia from freshwater rainbow trout can be grown in primary culture in Leibovitz's L-15 medium, by seeding freshly isolated gill cells on two successive days, from two different fish, directly onto permeable filter supports (DSI technique). This preparation allows the measurement of transepithelial resistance (TER) and exposure of the apical surface to freshwater, as in vivo. New culture methods were developed and evaluated, using TER as an indicator of epithelial integrity, in an effort to improve the utility of the preparation for proteomic and toxicological research. TER was not related to cell density or protein content in DSI epithelia. To eliminate bovine proteins, the 5% foetal bovine serum (FBS) normally required for epithelial development was replaced with trout plasma. While previously frozen trout plasma proved toxic, freshly collected heparinized plasma, provided by chronically cannulated adult trout, was not. The use of 5% fresh trout plasma supported a TER development curve identical to that with 5% FBS, a useful advance for proteomic research because foreign (bovine) proteins are eliminated. However, 10% plasma reduced TER development, and 100% plasma abolished it. The inhibitory effect on TER of high plasma levels was seen only early in epithelial development, and was exerted from the apical side, likely an effect on tight junction formation. Mature plasma-supplemented preparations mounted a TER rise in response to apical freshwater exposure comparable to that of FBS-supplemented epithelia. Yolk-sac fry extract was inhibitory to TER development, even in the presence of 5% FBS. Transfer of mature epithelia from 18 °C to 4 °C maintained stable TER and extended the useable lifespan by at least ten days, thereby facilitating storage of preparations for toxicity testing. A new method of growing epithelia, involving only a single seeding of cells from a single fish, directly onto filter inserts (SDSI technique), provided mature epithelia with much lower TER, a smaller TER response to apical freshwater, and lower cell density and protein content than DSI epithelia. These SDSI epithelia offer the advantage of multiple preparations grown directly from unique individuals for in vitro toxicity testing. This revised version was published online in July 2006 with corrections to the Cover Date.     

军曹鱼稚鱼外周血细胞及其形态学观察

以出膜后第60天的军曹鱼外周血液为材料,利用光镜显微技术研究了其血细胞的种类和形态。外周血液包括5种类型的血细胞,红细胞、淋巴细胞、单核细胞、嗜中性粒细胞和血栓细胞。血细胞计数测得红细胞密度为(2.97±0.82)×109ind.mL-1,白细胞密度为(1.39±0.94)×106ind.mL-1,淋巴细胞、嗜中性粒细胞、单核细胞和血栓细胞在白细胞中所占的比例分别为(53.00±8.51)%、(17.59±4.28)%、(4.78±1.37)%和(24.63±4.08)%。未见到嗜酸性粒细胞和嗜碱性粒细胞。血细胞计数结果表明,红细胞数量远远大于白细胞,白细胞中淋巴细胞数量最多,单核细胞数量最少。实验还观察到处于分裂状态的红细胞、细胞表面有伪足样胞突的小淋巴细胞和细胞表面有许多微绒毛突起的大淋巴细胞、核呈多种形态的单核细胞和中性粒细胞及不同形态的血栓细胞等。     

日本黄姑鱼血细胞发生的观察

用Wright’sGiemsa混合染料对日本黄姑鱼外周血涂片以及头肾、肾脏、肝脏和脾脏组织印片染色,观察其中各种血细胞的大小、形态特征,研究了日本黄姑鱼血细胞的发生发育模式。观察发现,在外周血涂片中,除红细胞外,还观察到多种白细胞,主要有单核细胞、嗜中性细胞、嗜酸性细胞和淋巴细胞,而嗜碱性细胞没有被发现。红细胞、淋巴细胞和单核细胞的发生主要在肾脏和头肾,其次是脾脏;粒细胞的发生主要在头肾和脾脏。红细胞的发育经历3个阶段:原红细胞、幼红细胞和成熟红细胞阶段。红细胞在成熟过程中,细胞核的体积逐渐变小,细胞与细胞核的体积比由大变小然后再变大。成熟红细胞除了由幼稚细胞发育而来以外,还可以进行一分为二的方式进行分裂;粒细胞的发育经历5个阶段:原粒细胞、早幼粒细胞、中幼粒细胞、晚幼粒细胞和成熟粒细胞;淋巴细胞经历了3个阶段:原淋巴细胞、幼淋巴细胞和成熟淋巴细胞;单核细胞的发育和淋巴细胞相似,也经历了3个阶段:原单核细胞、幼单核细胞和成熟单核细胞。     

Long-term culture of a cell population from Siberian sturgeon (Acipenser baerii) head kidney

In vitro cultures of native fish cell lines are of great importance, both for basic research and applied science. In particular, there is strong demand for long-term growable cell lines from breeding fish, like sturgeon. Here, we describe the culture of cells from Siberian sturgeon (Acipenser baerii) head kidney. The cells have so far been cultured over a period of 12 months (24 passages). Cytochemical and immunocytochemical examination suggests that, in vitro, the cells exhibit markers that are indicative for different cell types. In particular, fat storing cells (adipocytes) were observed, and the expression of cytokeratins and glial fibrilar acidic protein (GFAP) can be concluded on the basis of immuncytochemical analysis. The observation of different morphologies additionally underlines the heterogeneity of the cell population and matches the typical behaviour of in vitro cultures of stem/progenitor cells. Different applications can be imagined.     

驼背鲈血细胞的超微结构

利用电镜技术对驼背鲈（Cromileptes altivelis）外周血细胞的超微结构进行了研究。结果表明,电镜下可区分6种类型的细胞：红细胞、粒细胞、单核细胞、淋巴细胞、血栓细胞和浆细胞。红细胞呈圆形或椭圆形,可见线粒体、少数囊泡;根据细胞中的颗粒形态大小和细胞核的形态,可将粒细胞分为4种类型：I型粒细胞、II型粒细胞、III型粒细胞和IV型粒细胞。单核细胞圆形,表面较平整,偶有伪足伸出,空泡多见;淋巴细胞的胞质少,仅在细胞核边缘处形成薄薄的一层,细胞器少;血栓细胞异染色质丰富,沿核膜呈带状分布,胞质中有较多大小不等的空泡和少量的线粒体;浆细胞中粗面内质网成层分布包绕在核周围。此外可见到嗜曙红细胞吞噬红细胞、血栓细胞成群聚集分布的现象。