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。

     

GnRH isoforms expression in relation to the gonadal cycle and to dominance rank in the Gilthead seabream, Sparus aurata

The manner in which behavior influences the gonadotropin-releasing hormone (GnRH) axis in hermaphroditic fishes is not understood. The Gilthead seabream, Sparus aurata, is a protandrous hermaphrodite with a complex gonadal cycle consisting of a quiescent, pre-spawning, spawning, and post-spawning stage. On two separate experiments, I used real-time quantitative PCR to measure the mRNA expression of three GnRH isoforms in homogenized seabream whole-brain extracts. In the first experiment, I measured the levels of GnRH-1, GnRH-2, and GnRH-3 mRNA throughout the gonad cycle. All three GnRH mRNAs increase around the peak of the spawning season (December). GnRH-3 mRNA expression is also elevated in August, which coincides with the beginning of gonad differentiation. All three GnRH mRNAs have the lowest expression levels in the month of September. There was no difference between males and females in the expression levels of any of the three GnRH mRNA. In the second experiment, I measured individual dominance ranks in six groups of fish, three during quiescence and three during spawning. GnRH-1 mRNA expression was positively correlated with dominance rank only during the quiescent period. The more dominant fish tended to have higher GnRH-1 mRNA expression. The existence of a quiescent-only correlation between GnRH-1 mRNA and dominance rank suggests a mechanism by which activation of gonad maturation could occur first in the most dominant ambisexual fish.     

三倍体僧帽牡蛎生殖腺发育观察

在僧帽牡蛎繁殖盛期,详细观察了二倍体,三倍体僧帽牡蛎生殖腺外部形态特征,比较了成熟卵母细胞卵径和核径。结果表明,外观上三倍体生殖腺发育较二倍体差,三倍本成熟卵母细胞卵径和核径分别比地倍体大１９．６％和１７．６％,体积分别比二倍体增加７０．３％和６４．２％,组织切片检查结果,二倍体生殖腺发育正常,三倍体生殖腺发育受阻,大都在增殖期和休止期,一部分个体可发育至生长期和成熟期,并能产生成熟的卵子和精子,     

石鲽仔、幼鱼性腺发育的组织学观察

利用组织学方法对人工培育的石鲽(Kareius bicoloratusBasiewsky)仔、幼鱼性腺发育进行研究。结果表明,性腺的发育与体长密切相关。刚孵化石鲽的原生殖细胞数目为2个,孵化3 d数目增至8个,之后经过迁移,至孵化9 d到达生殖嵴。在全长为7.2～8.5 mm(孵化9～11 d)的仔鱼中,性腺原基中的体细胞迅速增殖并包围原生殖细胞,后者在全长10～15 mm(孵化后10～35 d)的仔幼鱼中增殖成为生殖干细胞。原始性腺在全长15～30 mm(孵化40～60 d)的幼鱼中逐渐发育完善,呈细线状,位于腹腔后部中肾管下方紧贴体壁。雌性性腺最早在全长32.5 mm(孵化66 d)的个体中出现分化特征,至全长89～102 mm时雌性性腺特征完全分化。雄性性腺的分化较雌性性腺晚,最早在全长为91 mm的幼鱼中开始,至全长为114～118 mm时雄性性腺分化特征已经十分明显。     

半滑舌鳎(Cynoglossus semilaevis) gnrh2基因克隆、组织分布及卵巢成熟过程中表达分析

为了研究下丘脑神经肽促性腺激素释放激素(Gonadotropin-releasing hormone 2,GnRH2)在半滑舌鳎(Cynoglossus semilaevis)卵巢成熟过程中的生理作用,本研究通过RT-PCR及RACE方法获得了半滑舌鳎GnRH2全长cDNA序列;通过实时荧光定量PCR(qPCR)对gnrh2 mRNA的组织分布以及卵巢成熟过程中的时空表达特性进行了分析.结果显示,半滑舌鳎GnRH2全长cDNA序列为538 bp(不包括polyA尾),其中,5'非编码区(Untranslated region,UTR)为154 bp,3'UTR为126 bp,开放阅读框(Open reading frame,ORF)为258 bp,编码85个氨基酸的前体多肽,其分子量及等电点分别为9.69 kDa和8.55.GnRH2前体多肽由信号肽、GnRH2十肽、酶切位点(GKR)以及GnRH相关肽共4部分组成.序列比对分析发现,GnRH2在鱼类中同源性极高,尤其是十肽(QHWSHGWYPG)在所有硬骨鱼类中完全相同.半滑舌鳎GnRH2与鲈形目同源性最高(89.41％-90.5 9％),其次为鲽形目、鲑形目和鲍形目(78.82％-85.88％),与鲤形目同源性最低(61.18％-71.76％).gnrh2 mRNA主要在脑中表达,在垂体及其他外周组织中表达量极低.此外,组织学分析显示,半滑舌鳎卵巢发育共分为5个时期(Ⅱ、Ⅲ、Ⅳ、Ⅴ和Ⅵ期).在卵巢成熟过程中,脑gnrh2 mRNA表达量在卵黄生成期(Ⅲ期)显著性增加,达到峰值;随后表达量急剧下降,在成熟期(Ⅴ期)达到最小值;在排卵后期(Ⅵ期)又显著性增加.然而,在卵巢成熟过程中,垂体gnrh2 mRNA表达量在卵黄生成后期(Ⅳ期)显著性降低,随后在成熟期(Ⅴ期)有所增加,但在排卵后期(Ⅵ期)又急剧下降.上述研究结果表明,脑GnRH2可能参与了半滑舌鳎卵巢发育过程.     

西南大西洋公海阿根廷滑柔鱼渔业生物学初探

根据2012年1-4月西南大西洋公海鱿钓渔船随机采集的666尾阿根廷滑柔鱼样本,对其鱿钓渔获的群体组成、形态特征、雌雄性比、缠卵腺及性腺发育等渔业生物学进行分析。结果显示：调查期间鱿钓渔业阿根廷滑柔鱼渔获群体胴长为91～351 mm,呈双峰分布,雌、雄性平均胴长分别为243.6 mm 和214.5 mm,雌性个体显著大于雄性;雌雄性比为1.26：1,以雌性个体为主;雌性群体性成熟以Ⅱ期为主（57.9%）,Ⅲ期以上个体为42.1%;雌性性成熟度、缠卵腺发育程度与个体大小密切相关,进一步研究认为缠卵腺长与其重量存在幂函数关系,且雌性性成熟个体缠卵腺指数明显大于未成熟个体;雄性个体性腺随着个体生长逐步发育。根据个体大小和性成熟状况推测,调查期间捕获的阿根廷滑柔鱼群体以南巴塔哥尼亚种群（ SPS）为主,混有少量的夏季产卵（ SSS）群体。     

仿洄游培育美洲鲥亲本的性腺发育

为解决美洲鲥(Alosa sapidissima)人工培育亲本性腺发育不佳的问题,开展了美洲鲥亲本仿洄游的培育,研究观察了美洲鲥亲本卵巢和精巢不同发育阶段(II^V期)的形态特征及相关参数,确定了仿洄游培育美洲鲥亲本性腺初次发育的特征。结果显示:在美洲鲥仿洄游培育过程中,在卵巢、精巢发育前期(II期),雌雄亲本生长迅速;而性腺发育到III期后,雌雄亲本的生长滞缓,雌鱼亲本甚至出现负生长。仿洄游培育美洲鲥性腺初次发育(II^V期)过程中,雌雄亲本均有较高的肥满度,分别为1.55~2.07和1.56~1.71;卵巢发育Ⅴ期雌鱼亲本的肥满度(2.07)显著高于在卵巢II^IV期亲本(1.55~1.67);而雄鱼亲本肥满度在精巢发育各阶段(II^V期)均无显著性差异。仿洄游培育美洲鲥性腺发育良好,雌雄亲本的性腺指数均较高,性腺发育Ⅴ期雌雄亲本的性腺指数(GSI)分别为(26.10±6.46)(18.88~33.62)和(5.18±0.68)(4.49~6.11)。研究表明,仿洄游培育的水温(10.8~30.8℃)和盐度(1~15)的周年变化符合美洲鲥亲本性腺发育的要求。     

西伯利亚鲟性别及性腺发育的超声波鉴定

利用超声波检查仪对人工养殖西伯利亚鲟的性别及性腺发育进行超声波扫描,并利用微创手术检查和组织学观察法来验证超声波对西伯利亚鲟性别及发育时期扫描的鉴定结果。通过对比研究表明:超声波方法对西伯利亚鲟性别鉴定的准确率较高,对2～5龄雌性鉴定的准确率为95%,对雄性鉴定的准确率为87%。超声波技术鉴定性腺发育的准确率随性腺的发育而提高,鉴定早期性腺(Ⅰ～Ⅱ期)发育的准确率(65.25%)低于对晚期性腺(Ⅲ～Ⅴ期)发育鉴定的准确率(88.65%)。超声波技术鉴定西伯利亚鲟性别及性腺发育速度较快,根据鱼的发育状况,平均每尾鱼所用的时间约30s。     

mRNA expression of GnRH variants and receptors in the brain,pituitary and ovaries of pejerrey (<Emphasis Type="Italic">Odontesthes bonariensis</Emphasis>) in relation to the reproductive status

The present study examined the differential mRNA expression levels of three forms of GnRH (sGnRH, pjGnRH and cGnRH-II) and two forms of GnRH receptor (pjGnRH-R I and pjGnRH-R II) in the brain, pituitary, and ovaries of pejerrey in relation to the reproductive status. The analysis revealed the presence of significant amounts of mRNA of the three GnRH forms while the ovaries showed only two (sGnRH and pjGnRH). The GnRH receptor II was found ubiquitously in the brain, pituitary, and ovaries while the form I was detected only in the brain. The levels of pjGnRH mRNA in the brain and pjGnRH-R II in the pituitary gland varied in correlation with the ovarian condition. However, brain sGnRH and pjGnRH-R I mRNA levels reached a maximum during early stages of ovarian development. In contrast, the brain levels of cGnRH-II mRNA showed no variation. The present study also shows a good correlation of ovarian sGnRH and pjGnRH-R II mRNA levels with the reproductive condition, suggesting that these molecules are may be involved in the regulation of pejerrey ovarian function.     

The existence of gonadotropin-releasing hormone (GnRH) immunoreactivity in the ovary and the effects of GnRHs on the ovarian maturation in the black tiger shrimp Penaeus monodon

Immunocytochemical localization using antibodies against five isoforms of gonadotropin-releasing hormone (GnRH), namely, luteinizing hormone-releasing hormone (LHRH), salmon (s)GnRH, octopus (oct)GnRH, lamprey (l)GnRH-I, and lGnRH-III, showed that only lGnRH-I immunoreactivity (ir-lGnRH-I) was localized in follicular cells of proliferative, vitellogenic, and mature ovaries. The effects of exogenous GnRHs on the ovarian maturation cycle of Penaeus monodon were compared by treating female broodstocks with LHRH, sGnRH, and lGnRH-I. The cycle of ovarian maturation in both eyestalk-ablated and eyestalk-intact shrimp administered with the three isoforms of GnRH was significantly shorter than that of the control animals. Moreover, administrations of all GnRH isoforms showed similar numbers of spawned eggs and the percentage of successful fertilization as in the control animals. These findings suggest that GnRHs may be highly conserved peptides that play an important role in inducing the ovarian maturation in the shrimp.     

Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus

The three-spot wrasse (Halichoeres trimaculatus), which inhabits the coral reefs of Okinawa, changes sex from female to male. Sex change in this species is controlled by a social system. Oocytes disappear completely from the ovary, and male germ cells and somatic cells comprising testicular tissue arise a new during the sex change process. However, little is known of the fate and origin of the gonadal tissue-forming cells during sex change. In particular, the fate of ovarian somatic cells has not been determined, although the ovarian tissue regresses histologically. To approach this question, we analyzed apoptosis and cell proliferation in the sex-changing gonads. Unexpectedly, we found that few apoptotic somatic cells were present during sex change, suggesting that ovarian somatic cells might survive during the regression of the ovarian tissue. On the other hand, cell proliferation was detected in many granulosa cells surrounding the degenerating oocytes, a few epithelial cells covering ovigerous lamella and a few somatic cells associated with gonial germ cells at an early stage of sex change. Then, we found that proliferative ovarian somatic cells remained in the gonads late in the sex change process. Based on these results, we concluded that some functional somatic cells of the ovary are reused as testicular somatic cells during the gonadal sex change in the three-spot wrasse.     

岩虫的性腺发育和生殖周期

根据性腺发育的组织切片观察，岩虫（Marphysa sanguinea）性腺发育可分为增生期、成熟期、排放期和休止期4个阶段。通过连续两年每月对岩虫性腺组织切片观察可以确定，在山东省乳山海区的岩虫性腺发育为1年1个周期。12月至翌年6月为增生期；7～8月为成熟期；7月下旬至8月为排放期；9～11月为休止期，其中7月下旬至8月中旬为繁殖盛期。岩虫的性腺起源于体腔腹面生殖上皮。生殖细胞发生时不出现滋养细胞。除休止期外，生殖细胞持续发生，成熟时多次排放。雄性生殖细胞形成合胞体（syncytium），精子为原始类型。雌性生殖细胞不形成合胞体，卵母细胞在发育的早期阶段，卵黄发生期问从卵巢（生殖上皮）排入体腔中，并在体腔中发育。卵细胞成熟时，有卵外膜，结构复杂。     

Characterization of β‐catenin 1 during the gonad development in the common carp (Cyprinus carpio)

β‐catenin gene is a pivotal gene for gonad development and maintenance of ovarian function in mammals. However, little is known about its expression and function in gonad development of fish. In this study, a complete cDNA (3342 bp) sequence of β‐catenin 1 was cloned from the common carp, Cyprinus carpio, by RACE PCR, which encodes a 780‐amino‐acid protein. Quantitative real‐time PCR demonstrated that β‐catenin 1 mRNA expressions were high in the testis and ovary tissue and the expression increased as the testes developed and the early stage ovaries developed. Western blot results revealed a single immunoreactive band with an estimated molecular weight of 90 kDa in testes. Immunohistochemistry analysis revealed that the β‐catenin 1 protein was concentrated mainly in the cytoplasm of early development stage of oocyte cells and in the cytomembrane of developing and mature sperm cells. 17β‐Ethinylestradiol injecting intraperitoneally into the fish decreased the relative β‐catenin 1 mRNA expression level except 1 μg/g 72 hr and 5 μg/g 48 hr of treatments in the ovary by real‐time PCR. These results suggest, for the first time, that β‐catenin 1 is an essential protein in gonad development and might be involved in ovarian early development of C. carpio.     

Dimorphic expression of sex-related genes in different gonadal development stages of sterlet, <Emphasis Type="Italic">Acipenser ruthenus</Emphasis>, a primitive fish species

Molecular mechanism of sex determination and differentiation of sturgeon, a primitive fish species, is extraordinarily important due to the valuable caviar; however, it is still poorly known. The present work aimed to identify the major genes involved in regulating gonadal development of sterlet, a small species of sturgeon, from 13 candidate genes which have been shown to relate to gonadal differentiation and development in other teleost fish. The sex and gonadal development of sterlets were determined by histological observation and levels of sex steroids testosterone (T), 11-ketotestosterone (11-KT), and 17β-estradiol (E2) in serum. Sexually dimorphic gene expressions were investigated. The results revealed that gonadal development were asynchronous in 2-year-old male and female sterlets with the testes in early or mid-spermatogenesis and the ovaries in chromatin nucleolus stage or perinucleolus stage, respectively. The levels of T and E2 were not significantly different between sexes or different gonadal development stages while 11-KT had the higher level in mid-spermatogenesis testis stage. In all the investigated gonadal development stages, gene dmrt1 and hsd11b2 were expressed higher in male whereas foxl2 and cyp19a1 were expressed higher in female. Thus, these genes provided the promising markers for sex identification of sterlet. It was unexpected that dkk1 and dax1 had significantly higher expression in ovarian perinucleolus stage than in ovarian chromatin nucleolus stage and in the testis, suggesting that these two genes had more correlation with ovarian development than with the testis, contrary to the previous reports in other vertebrates. Testicular development-related genes (gsdf and amh) and estrogen receptor genes (era and erb) differentially expressed at different testis or ovary development stages, but their expressions were not absolutely significantly different in male and female, depending on the gonadal development stage. Expression of androgen receptor gene ar or rspo, which was supposed to be related to ovarian development, presented no difference between gonadal development stages investigated in this study whenever in male or female.     

Studies on feminization,sex determination,and differentiation of the Southern catfish, <Emphasis Type="Italic">Silurus meridionalis</Emphasis>—a review

The sex ratio of the feral Southern catfish was reported to be about 1:1, while the fish obtained by artificial fertilization were always female. Hence, we examined the possible influence of the micro-environment during artificial insemination (pH of the ovarian fluid and concentration of the semen) and early development (feed, hatching temperature, and water) on the sex ratio of Southern catfish fry. In order to examine the possibility of the occurrence of gynogenesis during artificial propagation, cytological observations on the insemination processes and the artificial induction of gynogenesis were also performed. However, no male fish were obtained even in these experiments, excluding the possibilities of these micro-environmental changes on catfish sex ratio and the occurrence of gynogenesis during artificial propagation. Female-to-male sex reversal was achieved by treatment with fadrozole (an aromatase inhibitor) and tamoxifen (an estrogen receptor antagonist). Histological analyses on the gonadal development of both female and induced male fish were subsequently performed. Moreover, several genes involved in sex differentiation, such as dmrt1, foxl2, and cyp19, and three subunits of gonadotropin (gth), i.e., gthα, lhβ, and fshβ, were isolated. Their expression patterns were studied under normal gonadal development and sex reversal conditions. The results revealed that dmrt1, foxl2, and cyp19a were closely related to catfish sex differentiation, and the gth subunits were possibly related to ovarian differentiation and oocyte development. Taken together, we hypothesized that estrogen was highly responsible for the ovarian differentiation and feminization of catfish fry under artificial propagation, although the mechanism involved remains elusive.     

皱纹盘鲍脯氨酰内肽酶的分离纯化及性质

脯氨酰内肽酶(Prolyl Endopeptidase,PEP)广泛分布在动植物和微生物体内,参与细胞生长和代谢调控。PEP在哺乳动物生殖器官的生长发育过程中起着重要作用,但贝类PEP与性腺发育是否有关尚未有相关报道。本研究以皱纹盘鲍为对象,采用荧光定量PCR技术(qRT-PCR)在性腺检测到有最高的基因表达量和较高的酶活性。通过硫酸铵分级沉淀和连续柱层析,从皱纹盘鲍性腺中分离纯化得到分子量约为82 ku,等电点约为5.5的PEP,其最适温度和最适pH分别为25 °C和6.0,在15~25 °C和pH 5~8能够保持较高的酶活性。利用LC/MS/MS分析纯化蛋白,得到182个氨基酸残基,与皱纹盘鲍脯氨酰内肽酶一致。圆二色谱分析显示,温度对PEP二级结构有较大的影响,拟合热变性温度为51.4±0.2 °C。为了进一步探究PEP在鲍鱼性腺发育过程中的作用,对不同发育时期(生长初期、前期、中期和后期)的雌雄鲍鱼,采用免疫印迹(Western Blot)和qRT-PCR分析性腺中PEP的蛋白和基因表达水平。结果表明,PEP在雄性和雌性性腺的各个时期均能被检测到,在雄性性腺的成熟中期和雌性性腺的成熟后期表达量最高。PEP在鲍鱼性腺不同发育时期时表达量的差异表明其可能参与到性腺发育的过程。     

The effects of a prolonged photoperiod and light source on growth,sexual maturation,fin condition,and vulnerability to fungal disease in brook trout Salvelinus fontinalis

The effects of an artificially prolonged photoperiod on growth, survival rate, colouration, and sexual maturation in brook trout Salvelinus fontinalis during pre‐ and post‐spawning periods from 21 June to 06 November were investigated. Fish of mean initial weight ?150 g were reared at ambient photoperiod as well as with an artificially prolonged photoperiod produced by either a light‐emitting diode or a metal‐halide light. The fish groups subjected to a prolonged period of artificial light grew significantly larger and had a higher survival rate (p < 0.05), regardless of sex, and showed lower occurrence of fungal disease compared to controls reared in the natural photoperiod. We found a significantly higher number of sexually mature fish in the control groups compared with experimental groups. The increased photoperiod effectively delayed gonad development and increased somatic growth in both male and female brook trout, and also increased resistance to fungal disease. The increased photoperiod produced an observable difference in fish colouration, with control groups exhibiting more intensive spawning colouration.