芳香化酶抑制剂对暗纹东方鲀CYP19A、DMRT1基因表达及性腺分化的影响

采用组织学和分子生物学方法,研究了投喂芳香化酶抑制剂来曲唑(LE)后暗纹东方鲀(Takifugu obscures)初孵仔鱼CYP19A、DMRT1基因表达以及性腺的组织学变化,以期进一步了解P450芳香化酶(P450arom)在鱼类早期性别分化过程中的作用。RT-PCR结果显示,对照组样品CYP19A和DMRT1表达显示性二态,雌性表达CYP19A基因,雄性表达DMRT1基因。LE处理组在性别分化期间,雄性样品单一表达DMRT1,雌性样品则同时表达CYP19A和DMRT1。qRT-PCR结果显示:LE处理组雌性仔鱼CYP19A基因表达被显著抑;虽然在仔鱼出膜后22d(dph)的表达水平高于9 dph,但仅为同日对照组的2.11%。LE处理组雌性样品22 dph时DMRT1基因表达量上调,至150 dph时达对照组雄性水平。55 dph的性腺组织学结果表明,LE处理可导致暗纹东方鲀稚鱼原始卵巢退化,并向功能性精巢发育。150 dph的LE处理组性腺均为精巢,并与对照组精巢发育同步。结论认为,暗纹东方鲀性腺分化期间P450arom是卵巢形成和维持发育所必须的,抑制P450arom活性可导致雌性暗纹东方鲀发生雄性化逆转。     

芳香化酶抑制剂对暗纹东方鲀CYP19A、DMRT1基因表达及性腺分化的影响

摘要: 采用组织学和分子生物学方法, 研究了投喂芳香化酶抑制剂来曲唑(LE)后暗纹东方鲀(Takifugu obscures)初孵仔鱼CYP19A、DMRT1基因表达以及性腺的组织学变化, 以期进一步了解P450芳香化酶(P450arom)在鱼类早期性别分化过程中的作用。RT-PCR结果显示, 对照组样品CYP19A和DMRT1表达显示性二态, 雌性表达CYP19A基因, 雄性表达DMRT1基因。LE处理组在性别分化期间, 雄性样品单一表达DMRT1, 雌性样品则同时表达CYP19A和DMRT1。qRT-PCR结果显示: LE处理组雌性仔鱼CYP19A基因表达被显著抑; 虽然在仔鱼出膜后22 d(dph)的表达水平高于9 dph, 但仅为同日对照组的2.11%。LE处理组雌性样品22 dph时DMRT1基因表达量上调, 至150 dph时达对照组雄性水平。55 dph的性腺组织学结果表明, LE处理可导致暗纹东方鲀稚鱼原始卵巢退化, 并向功能性精巢发育。150 dph的LE处理组性腺均为精巢, 并与对照组精巢发育同步。结论认为, 暗纹东方鲀性腺分化期间P450arom是卵巢形成和维持发育所必须的, 抑制P450arom活性可导致雌性暗纹东方鲀发生雄性化逆转。

     

Morphology, endocrinology, and environmental modulation of gonadal sex differentiation in teleost fishes

Successful reproduction by an adult depends on the normal ontogenesis of the gonads, a complex process of cellular and histological differentiation that starts early in life. This process is theoretically predetermined by genetic factors and includes sensitisation of the bipotential gonads to endogenous endocrine factors prior to, during and even after commitment to maleness or femaleness. However, young fish are relatively vulnerable to a host of environmental (physical and chemical) factors that can affect this endogenous endocrine axis, disturbing or even overriding the putative developmental pathway. This sexually lability can be exploited to our advantage for the production of monosex fish populations of the most valuable sex for food production or aquarium fish trade. On the other hand, it represents also a potential path for undesirable influences from endocrine-disrupting chemicals and climatic factors, particularly environmental temperature. This paper provides a detailed account of the early histological process of gonadal sex differentiation, with special reference to gonochoristic species, and reviews the criteria employed to positively identify ovarian and testicular differentiation. It also reviews the development of endocrine competence and sensitivity of the differentiating gonads to exogenous influences in the context of the relative stability of genotypic sex determination in various fish species. Sex differentiation in some species seems to be under strong genetic control and may not require endogenous sex steroid production. Conversely, reliance on endogenous sex steroids for gonadal differentiation is observed in other species and this phenomenon is apparently associated with a higher incidence of environment (mainly temperature)-labile sex differentiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

鱼类性别决定的研究进展

有关鱼类性别决定的研究主要集中在温度、性激素、芳香化酶以及随机重复序列、核受体基因等对性别分化的调控方面。由于鱼类所处分类地位较低 ,生活环境千差万别 ,鱼类性别决定没有一个普遍的模式 ,目前研究的鱼类又各不相同 ,因此象哺乳动物那样的性别决定级联模式还没能阐述。本综述旨在阐述近几年有关鱼类性别决定机制方面的研究动态和进展 ,为系统研究鱼类性别决定机制提供参考     

贝类性别决定与分化相关基因研究进展

性别决定与分化是生命发育的基本事件,性别决定与分化机制一直是生命科学研究的热点问题。贝类具有雌雄同体、雌雄异体、雄性先熟和性转换等复杂的性别类型,是研究无脊椎动物性别决定与分化机制及其演化进程的理想动物类群。挖掘贝类性别决定与分化调控基因,阐明相关基因的调控作用,对于揭示贝类性别决定与分化的分子机制具有重要意义。本文就贝类性别决定与分化相关基因的研究进展进行了综述,并对该研究领域进行展望,以期为贝类的性别决定和分化机制、生殖操作和遗传改良等研究提供参考。     

基于转录组分析不同温度下池蝶蚌性别相关基因的表达

性别决定与分化是生命发育的基本事件,性别决定与分化机制一直是生命科学研究的热点问题。贝类具有雌雄同体、雌雄异体、雄性先熟和性转换等复杂的性别类型,是研究无脊椎动物性别决定与分化机制及其演化进程的理想动物类群。挖掘贝类性别决定与分化调控基因,阐明相关基因的调控作用,对于揭示贝类性别决定与分化的分子机制具有重要意义。本文就贝类性别决定与分化相关基因的研究进展进行了综述,并对该研究领域进行展望,以期为贝类的性别决定和分化机制、生殖操作和遗传改良等研究提供参考。     

Sex differentiation and sex determination in eels: consequences for management

The aim of this review is to present an overview of the sex differentiation and sex determination processes in eels in relation to the urgent need to provide scientific knowledge to better protect and manage the Anguilla genus. Indeed, the global decline of the three main temperate eel stocks, Anguilla anguilla, Anguillidae (Fisheries Management and Ecology, 2003, 10, 365); Anguilla japonica, Anguillidae (Casselman, Eel Biology, Springer Japan, 2003, 293) and Anguilla rostrata, Anguillidae (Tatsukawa, Eel Biology, Springer, Japan, 2003, 255), raises concerns about the necessity to better understand all stages of the life cycle of eels (Righton and Walker, Journal of Fish Biology, 2013, 83, 754). Little is known about the mechanisms involved in the production of males and females in this species with environmental sex determination. Previous reviews identifying the density of individuals as the major factor influencing sex determination were undertaken (Krueger and Oliveira, Environmental Biology of Fishes, 1999, 55, 381; Reviews in Fish Biology and Fisheries, 2005, 15, 37). Here, we review the current advances on the subject, focusing on the roles of early growth rate and interindividual relationships, which are mechanisms underpinned by density, as well as the sex differentiation process, and we question how this knowledge might influence global conservation measures.     

半滑舌鳎的性腺分化和温度对性别决定的影响

半滑舌鳎(Cynoglossus semilaevisGünther)是近年来新开发的一种理想的增养殖对象,雌性个体生长速度比雄性快2～3倍。由于温度可影响多种鱼类的性腺分化,因而探索温度对该鱼的性别决定和性腺分化的影响,获得性逆转雄性亲本,用于培育全雌后代,具有重要的应用前景和经济效益。通过石蜡组织切片,对半滑舌鳎早期性腺分化进行组织学观察。发现在24℃饲养条件下,孵化后30 d半滑舌鳎性腺开始分化,其中具有裂隙的性腺原基未来发育为卵巢,而不具裂隙的原基未来发育为精巢。在孵化后25～100 d对半滑舌鳎进行不同温度(16℃、20℃、24℃、28℃、32℃)处理,在9月龄时利用石蜡组织切片鉴定表型性别,观察温度对性腺分化的影响,结果表明,28℃和32℃高温能显著提高群体中的雄性比例,使其分别达到69.2%和66.7%;而低温16℃和20℃对性腺分化影响不大,群体中雄性比例分别为56.5%和57.1%;24℃处理群体中雌雄个体比例接近1∶1。半滑舌鳎雌性特异的遗传性别鉴定技术也检测到高温和低温处理组出现了由基因型雌性向表型雄性逆转的雄性个体。这些结果表明温度影响了半滑舌鳎性腺分化方向。     

Gonadogenesis in scallop Chlamys farreri and Cf‐foxl2 expression pattern during gonadal sex differentiation

Gonad development and sexual differentiation are important biological processes. More insight is needed in these processes to improve selection and breeding efficiency in the production of culture animals for monosex farm operations of commercially important shellfish species. Foxl2 is the earliest known sex dimorphic marker of ovarian differentiation in mammals, but studies on foxl2 in invertebrate species are rare. This study revealed the gonadogenesis and expression characteristics of foxl2 during the gonadal differentiation in the scallop Chlamys farreri, an important commercial mollusk in China. The result showed that C. farreri gonad becomes visible once juveniles reach a shell height of 5.5 mm. Sex can be distinguished based on gonad histology in animals with a minimum shell height of 9 mm. Expression of Cf‐foxl2 (C. farreri foxl2) was first observed in cytoplasm of ovarian germ and follicle cells, just after the initiation of ovarian differentiation. Results indicate that Cf‐foxl2 can be used as an early sex‐marker gene, and may have participated in the regulation of ovary differentiation in C. farreri.     

雌雄同体鱼类性别分化及性转变研究进展

在脊椎动物中,鱼类具有多样的性别分化方式,大致可分为雌雄异体、雌雄同体以及单性生殖3类.一般情况下,鱼类性别决定后,性腺可分化为卵巢或精巢,并且在整个生命周期内保持不变.而在雌雄同体鱼类,其性别可以从雌性转变为雄性、雄性转变为雌性或者在雌雄两种性别间进行多次转变.雌雄同体鱼类具有多种性别转变形式,是研究脊椎动物性别决定...     

Histological characterization of gonadal sex differentiation in Nibea albiflora

The aim of this study was to investigate the period of sex differentiation and the pattern of gonadal differentiation in Nibea albiflora using histological techniques. The fish were raised in temperature between 19.5 and 21.5°C. The promordial gonads formed at the posterior region of the abdomen and the fish remained undifferentiated till the 36 day post hatching (dph) with a total length (TL) of 21.20 ± 3.90 mm. Ovarian differentiation precedes testicular differentiation, as usual in teleost fish. Oocytes undergoing meiosis at zygotene (bouquet) and pachytene stages were firstly observed when the total length reached 33.05 ± 4.61 mm at 40 dph, and the formation of the ovarian cavity began simultaneously at 46 dph with total length of 49.40 ± 4.83 mm. When the mean total length reached 78.05 ± 5.24 mm (61 dph), ovarian cavity grew bigger, and oocytes were firstly detected. By contrast, signs of morphological differentiation in the presumptive testis are not observed until their total length reached 69.18 ± 5.26 mm at 55 dph. The unrestricted lobular organization of the testis is evident at the stage of TL = 78.05 ± 5.24 mm (61 dph). These findings indicate that N. albiflora is a differentiated gonochorist and the critical period of sex differentiation in N. albiflora occurs at the stage of TL = 21.20 ± 3.90 mm (36 dph).     

鱼类性别决定的遗传基础研究概况

动物从受精卵发育到具有不同性别特征的个体是一个奇妙而又严谨的过程,是人类长期以来试图揭示的自然现象。上世纪90年代初在人类Y染色体上发现了性别决定基因SRY[1],进而发现了一个新的Sox基因家族[2]。上述基因的发现,促进了以哺乳类为代表的动物性别决定和分化机制研究。由于鱼类在脊椎动物中的特殊进化地位、庞大的种类数量以及显著的社会经济价值,鱼类的性别决定研究一直受到遗传和发育学者的重视。尽管离最终阐明鱼类性别决定的机制还有距离,但近20多年来鱼类性别决定的遗传基础研究已取得不少重要进展。本文试图根据现有文献资料,…     

Role of sex steroids in gonadal differentiation of the mussel Choromytilus chorus (Bivalvia Mytilidae) (Molina 1782)

In Chile, the species Choromytilus chorus stands out for its ecological and economic importance. This mussel has a very particular condition in the colour of the gonad, being cream‐yellow colour the male gonad and dark brown the female which is hardly desired by the consumers. In this context, the aim of this research is to determine the role that sex steroids have in gonadal differentiation of the mussel C. chorus. For this, juveniles with sizes 15–22 mm were selected, which were subjected to acute exposure in the form of bath for 60 days to four treatments: T1: dihydrotestosterone (DHT); T2: 17β‐estradiol (E2), T3: fadrozole (F) and T4: DHT‐F (DF), plus a control without chemicals. Each treatment as well as control included three replicates with 90 individuals each. The mussels undergoing treatment with E2 had a sex ratio (male:female) of 0.47 compared to 1:1 in the control group. In contrast, in the groups treated with DHT, F and DF the sex ratio changed to 2.0, 1.60 and 1.70 respectively. In the fertilization trials, all the mussels produced functional gametes, as they were able to generate gametes that were developed to morula, and veliger larva. Nevertheless, it should be noted that the size of oocytes from the group exposed to estradiol (61.12 ± 2.40 μm) was significantly higher than in other groups. These results support the hypothesis that sex steroids would be involved in sexual differentiation of marine bivalves.     

温度对杂合克隆牙鲆性别分化、性比和生长的影响

以有丝分裂雌核发育牙鲆(Paralichthys olivaceus)中的雄性和雌性作为父母本,进行人工授精获得杂合克隆牙鲆。从35日龄起,实验鱼类分别在16、19、22、25、28和31℃水温下培育至110日龄,观察其性别分化、性比和生长的差异。结果表明,性别分化与培育水温密切相关,在22、25和28℃水温下,60日龄开始性别分化;在16℃和19℃下,分别在70日龄和90日龄开始性别分化;而31℃下,110日龄仍未发生性别分化。通过180尾标本的组织切片观察,各温度下的杂合克隆牙鲆均为雌鱼,未发现雄鱼,表明高水温没有改变雌性性别。对于全长和体质量的生长,除31℃外,其他各实验组随着水温升高而生长加快;但是,55日龄后,25℃下的生长超过28℃,成为生长最快的温度。本实验结果证明杂合克隆牙鲆性别分化随水温升高而加快;性别比例不受温度影响,全部为雌性;25℃下牙鲆生长最快。     

采用RAPD技术筛选马口鱼性别差异相关分子标记

选用SBSA和SBSB两组随机引物（各20条）分别对马口鱼卵巢DNA和精巢DNA进行RAPD扩增,并用1．5％的琼脂糖凝胶电泳检测扩增结果。结果表明,在40条随机引物中,有11条引物可以扩增出明显的雌雄性别差异条带,条带范围以SBSA5、SBSA6、SBSA9、SBSA16、SBSA19、SBSA20、SBSB2、SBSB3、SBSB9、SBSB16、SBSB17为主。运用RAPD筛选获得马口鱼性别DNA标记将有助于了解其调控雌雄性别发生的差异基因,为进一步的遗传育种提供基础。     

芳香化酶抑制剂来曲唑对胡子鲇性腺分化及相关基因表达的影响

采用组织学和荧光实时定量PCR方法, 检测饲喂不同剂量芳香化酶抑制剂来曲唑(letrozole, 50、100和200 μg/g)对胡子鲇(Clarias fuscus)性腺组织学和性别比率的影响, 以及200 μg/g 来曲唑对性分化前后脑型芳香化酶基因(Cyp19a1b)和翼状螺旋/叉头转录因子2(Foxl2)基因表达的影响, 结果表明, 50 μg/g 剂量的来曲唑对胡子鲇性腺分化无显著影响; 但100 μg/g和200 μg/g剂量的来曲唑可促进胡子鲇精巢分化, 抑制卵巢分化, 卵巢腔最早出现时间和初级卵母细胞出现时间均分别推迟3 d和6 d, 而初级精母细胞最早出现时间则分别提前2 d和5 d, 且雄性率分别达65.8%和71.3%, 显著高于50 μg/L组和对照组(P<0.05)。胡子鲇性腺分化前(出膜后12 d), Cyp19a1b和Foxl2基因即开始表达, 性腺分化前后Cyp19a1b相对表达量无显著差异(P>0.05), 但Foxl2相对表达量则随性分化而逐渐降低, 且来曲唑显著抑制性腺分化过程中Cyp19a1b和Foxl2的表达(P<0.05)。结果表明, 100 μg/g 以上剂量的来曲唑可有效诱导胡子鲇分化为雄性; Cyp19a1b可能不直接参与胡子鲇性腺分化, 但Foxl2直接参与此过程。     

Gonadal morphogenesis and sex differentiation in cultured chub mackerel,Scomber japonicus

The timing of primordial germ‐cell (PGC) migration with regard to the gonadal anlagen, gonad formation and sex differentiation was examined histologically in the chub mackerel (Scomber japonicus) at 5–190 days post hatching (dph). At 5 dph, PGCs appeared on the peritoneal epithelium surface or in the mesentery, on the dorsal side of the abdominal cavity. By 10 dph, stromal cells around the PGCs proliferated. The gonadal primordium was formed by 15 dph. The gonadosomatic index was 0.01% at 30 dph and increased thereafter (0.32% in females and 0.04% in males at 160 dph). Ovarian differentiation occurred at 30–40 dph, indicated by ovarian cavity formation (elongation and fusion of the upper and lower ovarian edges). Meiosis was subsequently initiated. A few meiotic oocytes surrounded the cavity at 50 dph; most were in the perinucleolus stage at 60 dph and attained a diameter of 60–70 μm at 190 dph. Testicular differentiation occurred at 30 dph, indicated by the formation of the sperm duct primordium. Spermatogonia gradually proliferated, developing into spermatocytes at the chromatin–nucleolus stage (after 90 dph) and subsequently into spermatids and spermatozoa (160 dph). These data could aid the development of seeding and cell‐engineering technologies for scombrid fish.