【作者】 董然然；

【导师】 王德寿；

【作者基本信息】 西南大学 ， 动物学， 2014， 博士

【摘要】 南方鲇(Silurus meridionalis, Chen)广泛分布于长江流域,是近年来开发的名特优养殖新品种:并且西南大学在南方鲇的移养、驯化和人工繁殖上进行了开创性工作。在野生条件下,雌雄比列约为1:1；而人工繁殖的南方鲇却为全雌。为找到其雌化的原因,本实验室开展了十余年的研究。其性腺分化发生在孵化后7天左右,并且卵巢和精巢中的生殖细胞分别在55和135dah左右进入减数分裂。前期通过对水质,光照,不同的人工授精方法、孵化温度、孵化后养殖温度和饵料的研究,初步证明开口饵料水蚯蚓可能是引起人工繁殖南方鲇全雌化的主要原因。但仍有许多问题有待阐明,比如,投喂水蚯蚓多久可以引起全雌化?水蚯蚓诱导全雌化的根本原因是什么?全雌化的机制是什么?鱼类性别与减数分裂起始的时间有关,投喂水蚯蚓是否会影响南方鲇减数分裂的起始时间?围绕上述问题,本论文主要开展了以下研究：为找到水蚯蚓投喂时间与雌性化的关系,本研究将孵化的鱼苗分为五组,分别投喂水蚯蚓0(对照组)、5、]0、30和90天,商业饵料投喂至3月龄；以投喂0天为对照组；性腺组织学检测表明,投喂水蚯蚓5天,雌鱼所占的比例为73%,投喂水蚯蚓10,30和90天雌鱼所占的比例均为100%。而对照组雌、雄性比约为1:1。以上结果初步说明人工繁殖南方鲇全雌化是由于早期投喂水蚯蚓所致,水蚯蚓影响性别的关键时期是在孵化后5-10天,这与南方鲇性别决定和分化的关键时期一致。为找到水蚯蚓诱导南方鲇人工繁殖全雌化的真正原因,课题组从嘉陵江3个不同的小溪中分别采集水蚯蚓,样品经过前处理之后,利用高效液相色谱法检测水蚯蚓体内的环境内分泌干扰物质(EDCs),结果表明,水蚯蚓体内含有双酚A、己烯雌酚、辛基酚和壬基酚。并且在3个采样点,壬基酚的平均含量最高。为进一步验证这4种EDCs,利用半制备高效液相分别进行收集,经过纯度分析后,通过质谱对其进行初步定性,结果表明,这四种物质确为双酚A、己烯雌酚、辛基酚和壬基酚。为进一步验证水蚯蚓在南方鲇全雌化过程中的作用,本研究分别设计不同的饵料实验组：(1)投喂高温灭活的水蚯蚓；(2)含有EDCs cooktail的商业饵料；(3)鱼苗自孵化后一直投喂正常商业饵料(对照组)。孵化后90天解剖取出性腺,肉眼观察初步判定雌雄；同时,性腺用波恩氏液固定,常规石蜡包埋、切片、HE染色、显微观察,进一步核实性别,并统计性比。结果显示：投喂高温灭活水蚯蚓组和喷洒EDCs cooktail(双酚A、己烯雌酚、辛基酚和壬基酚的混合物)的商业饵料组的实验鱼为全雌；因此,水蚯蚓体内的EDCs是诱导人工繁殖南方鲇全雌化的主要原因。对已知的与性别决定分化相关基因在1龄转录组中的表达情况进行分析,结果显示在鱼类性别决定和分化过程中起关键作用且在雌性表达高的Foxl2和在雄性表达高的Dmrt1, Sf1和Wtl在1龄南方鲇性腺中仍然有表达,且此时它们在雌、雄性腺的表达模式与其在性别决定分化时期在性腺的表达模式一致,说明这些基因不仅参与了鱼类的性别决定与分化,而且对性腺的发育和维持也具有重要作用。通过Real time PCR检测了性别决定关键基因Dmrt1、 Sf1、Foxl2和Cyp19a1a在南方鲇雌性化过程中的表达变化情况。结果显示：在孵化后30天和90天,Foxl2和Cyp19a1a在卵巢中是高表达的,在精巢中的表达量很低；而Dmrtl和Sfl在精巢中高表达,在卵巢中低表达；并且这4个基因在投喂水蚯蚓的南方鲇性腺中的表达模式与其在正常卵巢中表达模式是一致的。因此,南方鲇性别分化相关转录因子以及Cyp19a1a的表达在投喂水蚯蚓后都受到了影响,最终导致Cyp19a1a高表达。与此相吻合,对3月龄南方鲇血清中雌、雄激素和卵黄蛋白原水平进行检测,结果显示,投喂水蚯蚓的南方鲇血清中含有高水平的E2和卵黄蛋白原以及低水平的11-KT,这与正常雌鱼血清中这三种物质的表达水平类似。以上结果表明,南方鲇性别分化相关转录因子以及Cyp19a1a的表达模式在投喂水蚯蚓后发生了变化,最终导致Cyp19a1a高表达,使雌激素维持在高水平,从而产生全雌化现象。本研究通过Real-time PCR检测Stra8的表达,结果表明,RA信号通路关键因子Stra8在50dah卵巢和130dah精巢中表达量最高,60dah卵巢和150dah精巢表达量开始急剧降低。细胞表达模式研究表明Stra8特异表达于性腺的生殖细胞中,而且在50dah卵巢和130dah精巢中检测到的信号最强,60dah卵巢和150dah精巢中检测到的信号较弱。这些数据初步表明,Stra8可能与减数分裂起始有密切联系。利用RA浸浴南方鲇后,Real-time PCR和免疫组化检测Stra8的表达,结果均显示,RA可以上调Stra8的表达,表明RA信号通路在脊椎动物减数分裂起始过程中的作用可能是相对保守的。通过免疫组化检测正常精、卵巢和投喂水蚯蚓的南方鲇性腺中Stra8的表达表明,水蚯蚓投喂组性腺中Stra8的信号强度与其在正常卵巢中信号强度一致,而在正常精巢中几乎没有检测到Stra8阳性信号。因此,在投喂水蚯蚓的南方鲇中,EDCs可能是通过影响Stra8的表达来控制减数分裂的起始时间,从而引起南方鲇的全雌化。综上所述,本研究结果表明,南方鲇人工繁殖全雌化是由于在性别决定的关键时期投喂水蚯蚓所致,并且水蚯蚓体内富集的EDCs是引起全雌化的主要原因,这些EDCs可能通过影响性别决定与分化关键基因Dmrt1、Sf1、Foxl2和Cyp19a1a和减数分裂起始关键基因Stra8的表达模式,使南方鲇内源雌激素水平升高,减数分裂起始时间提前,最终导致南方鲇的全雌化。本研究首次提出投喂一种活的生物饵料可以引起鱼类的雌性化。更多还原

【Abstract】 Southern catfish (Silurus meridionalis, Chen), which is endemic to China and widely distributed in the Yangtze River basin, is a special economic fish in China. Sexual differentiation of southern catfish took place at around7dah (day after hatching). Meiosis is initiated at about55dah and135dah in germ cells of ovary and testis, respectively. The artificial reared fries were all female, while the sex ratio of the feral catfish was1:1. Previous studies from our group have demonstrated that the feminization of the Southern catfish by artificial propagation may be attributed to the feeding of annelids(Limnodilus spp.) through studying of water, photoperiodicity, hatching temperature, rearing temperature after hatching, artificial insemination and feed. How many days did annelids need to induce feminization? Why annelids could cause feminization of Southern catfish? What is the mechanism of feminization? Meiotic initiation affects sex determination of fish. Therefore, whether meiotic initiation could be influenced by annelids? Methods and results based on above questions as follow:To determine the treatment length needed for feminization, the experimental fish were divided into five groups, fed with living Limnodilus spp. for0(control),5,10,30and90days, respectively. According to the previous studies on the morphology and microstructure of Southern catfish, the gender of gonads was identified. The results showed that fish fed with living annelids for5days only displayed73%female, while fish fed with living annelids for10,30and90days were all females. The control fish displayed a1:1sex ratio. Even feeding of annelids for ten days is sufficient for feminization of Southern catfish. Therefore, feminization of catfish fry was resulted from feeding of annelids during the early development stage.Why annelids could cause feminization of Southern catfish? In this study, annelids were collected from three different streams of the Jialing River in Chongqing. After sample pretreatment, EDCs in annelids were detected by HPLC. The results showed that the extraction of annelids contained EDCs, including Bisphenol A (BPA), Diethylstilbestrol (DES),4-tert-octylphenol (4-t-OP) and4-nonylphenol (4-NP), which were further confirmed by LC-MS (Liquid chromatography-mass spectrometry). All of the identified EDCs were detected in the three samples collected from different streams receiving different effluents of Jialing River. Moreover, average content of NP is the highest in the three samples. Therefore, feeding of annelids from the three sample sites resulted in all female.In order to verified annelids on feminization of Southern catfish, different food and time treatment were applied to catfish:1. To confirm that feminization was caused by EDCs in annelids, the experimental fish were divided into two groups, one fed with heat inactivated annelids, the other with commercial diet containing EDCs cocktail.2. The last group was fed with commercial fish diets as control.Gonads of90dah (days after hatching) Southern catfish were dissected and genders of these fish were preliminarily judged by the gonadal shape. The gonads were fixed in Bouin’s solution respectively and then embeded in paraffin and sectioned at5μm thickness. Statistical analysis of sex ratio was performed by microscopic observations of the sections. According to the previous studies on the morphology and microstructure of Southern catfish, the gender of gonads was identified. The results showed that fish fed with heat inactivated annelids resulted in100%female. In addition, diets containing EDCs cocktail also led to all females when checking histologically at90dah. Whereas the control fish displayed a1:1sex ratio.Therefore, complete feminization of catfish was caused by EDCs in annelids.Genes, such as Cyp19a1a, Foxl2, Dmrt1, etc., which were known to play important roles in sex determination and differentiation in teleosts, were found to be expressed in the1-year gonads according to transcriptome analyses, indicating that these genes play important roles not only in early sex determination and differentiation, but also in the later development and maintenance of gonads. Real-time PCR were performed to determine the expression levels of Sfl, Foxl2, Dmrtl and Cypl9ala in gonads from annelids fed fish, female and male control fish. Foxl2and Cypl9ala exhibited significantly higher expression levels in female control gonads; while Sf1and Dmrt1exhibited significantly higher expression levels in male control gonads both at30dah and90dah. In annelids fed gonads, the expression levels of the four genes were similar to those of control female gonads both at30dah and90dah. To determine whether ECDs influence the estrogen, androgen and vitellogenin production, we collected blood samples from the annelids fed and control fish, then measured their serum E2(estradiol-17β),11-KT (11-ketotestosterone) and vitellogenin levels. High E2(5.4ng/mL), vitellogenin (5.7μg/μL) and low11-KT (0.12ng/mL) levels, similar to those of the control female fish (4.3ng/mL and0.17ng/mL), were detected in samples from annelids fed fish; while low E2(0.18ng/mL) and high11-KT (0.89ng/mL) levels were detected in samples from control male fish. Therefore, EDCs could significantly affect expression of key genes involved in sex differentiation in catfish. Up-regulation of Foxl2and Cyp19a1a and down-regulation of Dmrtl could result in up-regulated aromatase expression and estrogen production, which in turn lead to the feminization of catfish.Stra8mRNA expression analysis for different meiotic stages of ovaries and testes were conducted via Real-time PCR. The results showed that the relative expression of Stra8mRNA were at the highest level in ovaries at50dah and testes at130dah, corresponding to the premeiotic stage of germ cells, and at lower levels in ovaries at40and60dah and testes at110and150dah. To identify the localization of Stra8/Stra8in gonads, in situ hybridization and immunohistochemistry were performed using ovaries at40,50and60dah and testes at110,130and150dah. The results showed that specific signals of Stra8/Stra8were observed in germ cells in gonads of both sexes. Weak mRNA and protein signals were detected in40dah ovaries and110dah testes. Strong signals were found in premeiotic germ cells in50dah ovaries and130dah testes. The signals became weaker in the60dah ovary and150dah testes. In addition, RA could upregulate the expression of Stra8. Hence, these results suggest that Stra8might participate in meiotic initiation in teleosts. Moreover, RA signal pathway might also play important roles in meiotic initiation in teleosts. Expression of Stra8in ovary, testis and gonad from annelids fed catfish were detected by immunohistochemistry. The results showed that strong signals were detected in ovary and gonad from annelids fed catfish. While few signal was detected in the testis. Therefore, EDCs might be control meiotic initiation through affecting the expression of Stra8.In summary, our data showed that feeding of annelids for ten days is sufficient for feminization of southern catfish. It is the EDCs in annelids resulted in the feminization through up-regulation of the endogenous estrogen level and altering meiotic initiation. This is the first report showing that feeding of any living organism for such short time resulted in complete feminization of a vertebrate.更多还原