【作者】 沈志刚；

【导师】 谢从新； 樊启学； 王汉平；

【作者基本信息】 华中农业大学 ， 水产养殖， 2014， 博士

【摘要】 鱼类性别控制与性别决定机制的研究相辅相成,性别控制的结果有助于深入进行性别决定机制的研究,而性别决定机制的阐明将有利于更好的进行性别控制。鱼类性别决定机制的多样性和性别分化的可塑性,是众多具有性别特异性(如生长差异、观赏价值差异、性腺价值差异、繁殖价值差异等)鱼类单性种群生产的理论基础和前提条件,比如用三系配套法(正常XY雄鱼、XY雌鱼和YY超雄鱼)生产全雄鱼种群便是利用性别决定机制生产单性种群最好的例子。鱼类性别决定机制的原始性(异形性染色体与非异形性染色体)、多样性(遗传型性别决定与温度依赖型性别决定、雌核发育、雌雄同体与雌雄异体等)和易变性(受温度、外源激素及其他环境条件等的影响)也突显鱼类在脊椎动物性别决定机制进化、性别选择、种群性比适应性等研究领域的重要性。从另一方面来讲,鱼类性别控制是性别决定机制研究最重要的手段,外源激素、性激素生成酶抑制剂(如芳香化酶抑制剂)、性激素受体拮抗剂、温度等处理诱导鱼类性逆转是研究性激素或温度等条件在性别决定及性别分化过程中作用的极好方法,也是研究性别决定基因、性别分化相关基因及性别决定分子路径的重要手段,同时性激素诱导性逆转的个体(比如雌性)与正常发育的、相反生理性别的个体(比如正常雄性)交配产生的后代性比是初步认定性染色体类型的重要手段(比如XX/XY、ZW/ZZ等),温度处理诱导性逆转也是研究及区分温度依赖型性别决定、遗传型性别决定及遗传型性别决定加温度影响机制的唯一方法。本论文主要研究黄颡鱼和蓝鳃太阳鱼的性别控制及性别决定机制。黄颡鱼不仅在国内,在亚洲其他国家如韩国、日本、越南等地亦是受市场欢迎的重要经济鱼类。蓝鳃太阳鱼则是北美地区最受欢迎的垂钓鱼类之一,也是美国北部五大水产养殖种类之一,其作为重要的经济食用及休闲鱼类,也已引入国内,在江浙一带、广州等地形成初具规模的养殖。黄颡鱼和蓝鳃太阳鱼在生长上都表现性别二态性,雄性都比雌性生长快,较雌性达到更大规格,而且性成熟时间早,能在养殖条件下自然繁殖,特别是蓝鳃太阳鱼,属于一年多次产卵鱼类,这给大规模生产带来了一系列问题。鱼类生理性别的可塑性使得单性养殖成为可能,单性养殖也是解决性成熟早、繁殖快、密度大、生长缓慢、规格不均匀等一系列阻碍大规模生产问题的理想方法。蓝鳃太阳鱼具有极为特别的性别决定机制及繁殖生物学,比如遗传型性别决定与温度依赖型性别决定同时存在,存在选择性繁殖策略(两种雄性：亲本型雄鱼与卫星型雄鱼)。本论文借鉴前人在性别控制方面的研究,对黄颡鱼雄性化药物及其浓度进行了筛选；在蓝鳃太阳鱼性别控制的研究基础上,以及在其性别决定机制极其复杂的背景下,阐明了蓝鳃太阳鱼温度依赖型性别决定机制,并通过分子生物学手段研究了性别分化过程中与性分化相关基因的表达特性。主要研究成果简要如下：(1)17α-甲基睾丸酮或芳香化酶抑制剂Letrozole诱导黄颡鱼雄性化研究研究了两种雄性诱导药物：17α-甲基睾丸酮或芳香化酶抑制剂Letrozole不同浓度口服处理对黄颡鱼性逆转、存活和生长的影响及药物处理停止后性腺组织结构的变化。在10-59日龄进行17α-甲基睾丸酮口服处理并没有显著改变黄颡鱼幼鱼性比,但所有处理组都诱导了一定比例的间性性腺；三种浓度的17α-甲基睾丸酮处理以浓度依赖性的方式抑制了黄颡鱼幼鱼的生长,但这种生长抑制作用在药物处理结束后45天时消失。三种浓度的Letrozole口服处理分别生产了75%、83.3%和75%的雄性,显著高于对照组37%的雄性率,说明Letrozole具有诱导黄颡鱼雄性化的潜力,但处理浓度可能还需进一步优化。低浓度的Letrozole处理显著地促进了黄颡鱼幼鱼的生长,但在两个高浓度处理组并未发现生长促进作用；Letrozole处理还促进了精巢的发育,如较对照组大的精小叶,在两月龄幼鱼性腺中发现大量精子细胞。药物处理停止后性比的显著改变说明黄颡鱼雌性性腺细胞在性分化完成之后仍然具有双性分化的潜力。(2)芳香化酶抑制剂诱导黄颡鱼性逆转的研究在前面研究结果的基础上,我们进一步研究了各种浓度Letrozole处理对黄颡鱼性逆转的影响,试图诱导100%黄颡鱼性逆转,同时研究了各处理浓度对黄颡鱼幼鱼生长的影响。四种浓度的Letrozole处理诱导了浓度依赖型的性逆转,分别生产了68.3%、80.00%、88.3%和96.7%的雄性,都显著高于对照组38.3%的雄性率。Letrozole处理促进了精子生成,在60日龄时的性腺中发现大量精子细胞。性比数据结合组织学的研究表明,芳香化酶在卵巢分化中起重要作用,芳香化酶的抑制可能是诱导鱼类雄性化的必要条件之一。(3)蓝鳃太阳鱼温度依赖型性别决定研究越来越多的证据表明,温度对鱼类性比的影响是极为普遍的。蓝鳃太阳鱼隶属于鲈形目,棘臀鱼科,太阳鱼属,分布于北美各大水系、湖泊和水库,属广温性鱼类,繁殖季节水温19-33℃。本实验室之前的研究表明,温度处理影响蓝鳃太阳鱼的性别比例。本研究在前面研究结果的基础上,使用了四个蓝鳃太阳鱼地理种群,即Hebron, Jones, Hocking和Missouri,开展了基因型-温度交互作用对其性别决定的影响。我们发现了三种性比对温度反应类型,其中两种正好相反：一种是高温产生更多雄性,一种是低温产生更多雄性,还有一种是性比对温度不敏感类型,即温度处理没有显著改变性比。Hebron种群中,6-90日龄之间高温处理(24℃和32℃)相对低温处理(17℃)产生了更多的雄鱼；而在Jones种群中,相同处理时间内低温处理却相对两个高温处理产生了更多的雄鱼。其他两个种群的性比却对温度处理不敏感,即三个温度处理组之间性比无显著差异。结果还发现,在幼鱼1-90、6-90和25-90日龄进行温度处理显著影响Hebron种群性比,但40-90日龄的温度处理并没有显著改变性比,表明蓝鳃太阳鱼性分化的温度敏感期位于40日龄之前。结合存活率数据及性比结果,本研究表明温度依赖型性别决定与遗传型性别决定同时存在于蓝鳃太阳鱼。因此,如果能通过选育,选择温度能诱导高雄性率的种群,便能开发一条环境友好型、消费者友好型的全雄鱼生产方法。本研究结果结合前人研究成果也说明蓝鳃太阳鱼是研究性别决定机制和性别选择进化的极好材料。(4)蓝鳃太阳鱼性分化相关基因的表达研究很多与性别分化相关的基因先于组织形态学性分化最早标志表达,而且这些基因的表达通常是性别特异性的。对性分化相关基因表达的研究,有助于开发能在早期发育阶段鉴定个体性别或种群性比的分子标记,而且能进一步阐明鱼类性分化过程的分子机制。我们研究了与雌性性分化相关的基因foxl2和cypl9ala及与雄性性分化相关的基因dmrtl在性分化前及性分化过程中不同温度条件下的表达特性,发现这些与性分化相关的基因早在蓝鳃太阳鱼7日龄时就已开始表达,远早于其形态学性分化的最早标志。幼鱼7-17日龄时,与雌性性分化相关基因foxl2的表达急剧升高,27-37日龄维持高表达水平,之后稍有下降,表明雌性个体在7-27日龄之间发生性分化。foxl2基因的表达也呈现极大的个体差异,表明foxl2基因有潜力成为性别鉴定的分子标记。foxl2的表达在27日龄时还受到温度的影响,说明该基因可能在温度诱导性逆转过程中起作用。本论文开展了两种雄性化药物对黄颡鱼性逆转的影响研究,并对浓度和药物进行了筛选,获得全雄鱼种群,研究结果为黄颡鱼全雌种群、全雄种群生产和进一步阐明黄颡鱼性别决定机制提供了材料,也为鱼类性别决定机制增添了内容。研究结果还发现,黄颡鱼性分化完成后雌性生殖细胞仍然具有双性分化潜能,这为今后鱼类单性种群的生产提供了新的思路和指导。本论文研究了蓝鳃太阳鱼温度依赖型性别决定机制,以及与性分化相关基因的表达特性,第一次报道了在同一种鱼类中存在不同的子代性比对温度敏感类型,为蓝鳃太阳鱼性别决定机制增添了新的内容,为鱼类温度依赖型性别决定机制提供了材料和新的研究方向,也为开展环境友好型、消费者友好型单性种群生产提供了借鉴。更多还原

【Abstract】 Generally, in teleost fish, production of mono-sex population and investigation of sex-determining mechanism supplement each other. The results during the mono-sex production will be beneficial for the investigation of sex-determining mechanism, meanwhile clear sex-determining mechanism will provide theoretical guide for sex control. The diversity of sex-determining mechanism and lability of sex differentiation of fish are theoretical basis and prerequisite for mono-sex production in the fish species which display sexual specificity, e.g. different in growth, ornamental value, gonadal value, and reproductive value. Applying three mating strains (normal XY male, sex reversed XY female, and YY super-male) produce all-male population is the best example of using the knowledge of sex-determining mechanism to produce mono-sex population. Primitiveness (heteromorphic and homomorphic sex chromosomes), diversity (genetic sex determination and temperature-dependent sex determination, gynogenesis, hermaphrodite and gonochorism), and lability (affected by temperature, exogenous hormones, and other environmental factors) of sex-determining mechanism in fish highlight the importance of fish in the research field of sex-determining mechanism evolution, sexual selection, population sex ratio adaptability in vertebrate. In the other hand, sex control is one of the most important methods to study sex-determining mechanism. For example, sex reversal induction by exogenous hormones, hormonogenic enzyme inhibitor (aromatase inhibitor), antagonist of sex hormone receptor, and temperature etc., are unexceptionable approach to investigate the function of sex hormones or temperature conditions in the processes of sex determination and sex differentiation, and are important method to explore the sex-determining genes, sex differentiation related genes, and molecular pathway in sex determination. Meanwhile, offspring sex ratio result by mating sex reversed individual (e.g. phenotypic female) and normally developed, opposite phenotypic sex individual (e.g. male) is important approach to preliminarily distinguish the type of sex chromosome (e.g. XX/XY, ZW/ZZ). Sex inversion induced by temperature treatment is the single way to differentiate temperature-dependent sex determination, genetic sex determination, and genetic sex determination plus temperature effects.The present dissertation are mainly focus on the sex control and sex determination mechanism in yellow catfish and bluegill sunfish. Yellow catfish is a popular and economically important fish species in China, but in other Asian countries, e.g. North Korea, Japan, and Vietnam as well. Bluegill sunfish is one of the most popular sport fish in North America, as well as one of the five aquaculture species in northern Unite State; as important food fish, it has been introduced into China. The growth pattern of yellow catfish and bluegill shows sexual dimorphism, that the males grow faster and reach bigger ultimate size than females. Both of them reach sexual maturation early and could propagate in rearing conditions. Bluegill, especially, could reproduce multiple times during spawning season in a single year. Plasticity or lability of phenotypic sex allow us to produce mono-sex population of fish. A series of problems in large-scale industry of yellow catfish and bluegill, e.g. early sexual maturation, high reproductive ability, high density, slow growth rate, and high size variation could be solved theoretically and practically by mono-sex production. Bluegill sunfish display extraordinary sex determination mechanism and reproductive biology, e.g. coexist of genetic sex determination and temperature-dependent sex determination, displaying alternative mating tactics (two types of male:parental males and cuckolder males).In the present study, we investigated the effects of different concentrations of two masculinizing chemicals on sex reversion of yellow catfish; base on previous results of sex control in bluegill, and in the background of complicated sex-determining mechanism in bluegill, we clarified temperature-dependent sex determination in bluegill, and studied sex differentiation related genes prior to and during sex differentiation in different rearing temperatures. Main results are shown as follows.(1) Effects of17a-methyltestosterone or aromatase inhibitor Letrozole on masculinization in yellow catfishThe masculinization of yellow catfish (Pelteobagrus fulvidraco) by oral administration of various doses of17a-methyltestosterone (MT) and aromatase inhibitor letrozole (LZ) was investigated and their effects on the survival, growth performance, sex ratio and changes of gonadal structure were evaluated. Three doses of MT treatments from10days post-hatching (DPH) to59DPH failed to alter the male percentage; however, a certain proportion of intersex were detected in all MT treatments. Growth performance in MT treatments was depressed significantly compared with control group in a dose-dependent manner at the end of the masculinization experiment, and the suppression effect discontinued45days after the termination of treatments. LZ treated groups produced75.0%,83.3%, and75.0%male respectively, which were significantly higher than the control (37.5%). Low dose LZ treatment significantly advanced growth performance compared with the control, with no advancement in two high-dose treatments. Enlarged lobule lumens and a large amount of spermatozoa in male histological sections suggested the promoting potential of testicular development in early gonad development stage following the treatments of LZ in male yellow catfish. The changes of sex ratios after the termination of LZ treatments indicated that female germ cells possess some degree of bipotentiality. The present study demonstrated that LZ possesses the ability of masculinization in yellow catfish, and LZ treatment at low levels could promote growth performance and advance testicular development. Oral MT administration (from20to100mg kg-1) did not reverse the sex from phenotypic female to male, but repressed growth during treatments.(2) Effects of non-steroidal aromatase inhibitor Letrozole on sex inversion in yellow catfishThe effects of Letrozole (LZ), a potent non-steroidal aromatase inhibitor (AI), on growth performance, sex inversion and sex changes after the termination of the treatments were investigated in yellow catfish which displays sexual dimorphic growth. Growth performance was promoted significantly in the low dose LZ treatment compared with the control. Four LZ treatments produced dose-dependent male proportions, which were significantly higher than the control. Histological examination of testes treated by LZ displayed a large amount of spermatozoa and enlarged lobule lumens, indicating that LZ treatments have the stimulation potential in the process of spermatogenesis. Changes of sex proportions45days after the end of the LZ treatment prove that the female germ cells possess a certain extent of bipotentiality. These results suggest that aromatase activity plays a vital role in sex differentiation with inhibition of aromatase activity by AI bringing about sex inversion.(3) Temperature-dependent sex determination in bluegill sunfishIncreasing evidence shows that temperature effects on sex ratio in fish species are ubiquitous. In the present study, effects of genotype-temperature interactions on sex determination in bluegill sunfish (Lepomis macrochirus) were investigated using four geographic strains, Hebron, Jones, Hocking, and Missouri. Two opposite sensitive response patterns of sex ratio to temperature and non-sensitive pattern were found in a single fish species for the first time. In the Hebron strain, higher temperature treatment groups (24℃and32℃) produced more males compared with the low temperature group (17℃) from6days post-hatching (dph) to90dph. On the contrary, low temperature treatment produced more males than that of the other two higher temperature treatments in the Jones strain during the same treatment period as Hebron strain. No significant effects of temperature on sex ratio were detected in the other two strains. Combining sex ratio and survival data, it is strongly suggested that both temperature-dependent sex determination and genetic sex determination exist in this fish species, meanwhile, genotype-temperature interactions influence sex determination in bluegill. Therefore, a consumer-and environment-friendly approach to significantly increase the proportion of males could be achieved through selection of temperature sensitivity in bluegill. The evolutionary implications of our findings on sex determining mechanisms were discussed. Results suggested bluegill could be a unique candidate model species for investigations of sex-determining mechanisms and evolution of sexual selection.(4) Expression of foxl2during early gonadal development in bluegill sunfishMany genes associated to sex differentiation express prior to the first sign of morphological sex differentiation. Expression of some of these genes are sex specific. Investigation of these genes allow us to develop molecular markers for identification of individual sex or population sex ratio in early development stage, and will unveil molecular mechanisms of sex differentiation. We investigated the expression profiles of ovary differentiation related genes foxl2and cyp19a1a, as well as testis differentiation related gene dmrtl during early gonadal development. Results show that these sex differentiation related genes expressed as early as7days post-hatching (dph). The mRNA level of foxl2increased sharply during7to17dph, and kept high during17to27dph, then decreased slightly thereafter, suggesting ovary differentiation of bluegill occurred between7and27dph. The expression of foxl2displayed huge individual difference, indicating this gene could be a molecular marker for identification of sex. The expression of foxl2was modulated by temperature on27dph of bluegill, indicating that this gene may involve in temperature induced sex reverse.We studied the effects of two masculinizing chemicals on sex inversion, and optimized the concentrations, produced all-male population of yellow catfish. Results of the present dissertation will provide guidance for mono-sex production, be beneficial for further clarification of sex-determining mechanisms of yellow catfish, as well as increase content of sex-determining mechanism in teleost fish. We also found that female germ cells of yellow catfish possess a certain degree of bipotentiality after the completion of sex differentiation, which will offer new ideas and guidance for future mono-sex production.We investigated temperature-dependent sex determination mechanism in bluegill sunfish, analyzed expression profiles of sex differentiation related genes. We found different response patterns of sex ratio to temperature in a single fish species. These results will enrich sex-determining mechanism of bluegill, provide materials and new research field for temperature-dependent sex determination of fish, and be a reference for environment-and customer-friendly mono-sex production.更多还原