节点文献
三倍体虹鳟卵巢败育的调控机制研究
Regulatory Mechanism of Triploid Female Rainbow Trout Gonad Abortion
【作者】 徐革锋;
【导师】 韩英;
【作者基本信息】 东北农业大学 , 动物遗传育种与繁殖, 2016, 博士
【摘要】 虹鳟(Onchorynchus mykiss),属于鲑形目(salmoniformes),鲑科(salmonidae),大麻哈鱼属(Oncorhynchus)。该鱼是FAO向世界推荐的高品质养殖对象之一,也是我国最主要的鲑鳟鱼引进品种和养殖对象。由于三倍体雌性虹鳟在鲑鳟鱼养殖业中显示了诸多优点,如性腺不发育、肌肉品质好等。因此,以全雌三倍体虹鳟作为鲑鳟鱼养殖业发展的主力品种已成为大势所趋。众所周知,鲑科鱼类即便多出一套甚至两套染色体也能够生存和繁殖,这是其他脊椎动物不具有的特性。四倍体和雄性三倍体虹鳟与二倍体虹鳟性腺具有相同结构与功能,然而,三倍体雌性虹鳟是一个例外,早期卵巢呈线状,并且缺少初级卵母细胞。因此,三倍体虹鳟的性别分化与发育机制之间的本质区别成为了研究焦点。尽管与性别决定和分化的相关基因在二倍体虹鳟中已被鉴定出来,如sdY、Dmrt1、Cyp19a1a、Foxl2和Sox9等,但关于三倍体雌性虹鳟独特性腺表型及其相关性别控制基因的表达规律仍未得到深入研究,尤其是早期卵巢发育阻滞导致的生殖细胞去分化特征。因此,本研究以三倍体雌性虹鳟性腺发育中起重要调控作用的特异候选基因作为重点研究对象,辅助性腺体细胞与生殖细胞分化方向的组织学证据和类固醇激素表达规律,并通过外源性激素诱导,对三倍体雌性虹鳟的性腺表型发育进行预期调控,验证各基因间的级联调控关系以及与性细胞重塑和性类固醇激素消长规律间的互作效应,揭示雌性三倍体虹鳟卵巢败育及其天然性转变的发生机制与调控机理。主要研究结果如下:(1)虹鳟三倍体与二倍体卵巢的分化特征基本一致。虹鳟二倍体与三倍体性腺分别在84dpf和98 dpf就已分化为卵巢。但三倍体虹鳟卵巢分化存在障碍,早期发育停滞,卵原细胞及其滤泡细胞数量有限。在154~574 dpf时期,二倍体虹鳟卵巢中充满卵母细胞,而三倍体卵巢缺少相当数量卵母细胞,卵巢呈线状,且在之后的发育期里一部分雌性生殖细胞开始去分化。在964 dpf时期,雌性三倍体性腺发生组织重构,并出现再分化现象,性腺中有类似于产精子样细胞囊存在,这些细胞显得比卵原细胞略小,与二倍体精巢分化早期生殖细胞相似。(2)在334~964 dpf时期,二倍体两性虹鳟血清中雌二醇和睾酮含量均呈逐渐升高的趋势变化,雌性血清雌二醇含量为50~650 pg/ml,较雄性高5~8倍;雄性血清睾酮含量为20~180ng/ml,较雌性高10~25倍。雌性三倍体虹鳟血清雌二醇含量呈先升高后降低趋势变化,但其含量始终没能达到二倍体水平,峰值为110 pg/ml(574 dpf);雌性三倍体睾酮含量呈逐渐升高趋势变化,在发育后期几乎达到了二倍体雄性水平。随着雌性三倍体虹鳟性腺发育,卵巢退化较为严重,导致雌激素代谢通路受阻,当精巢支持细胞出现,睾酮被转化为雌二醇的量大大降低。(3)在154~964 dpf时期,分别以二倍体雌、雄虹鳟作为同期基础对照,采用RT-PCR技术对雌性三倍体虹鳟性腺特异基因进行差异化表达分析。研究结果表明,除Dax1外,在二倍体和三倍体虹鳟性腺中均获得了Cyp19a1a、Foxl2、sdY、Dmrt1、Amh和Sox9的表达谱。在雌性三倍体虹鳟性腺中,Cyp19a1a和Foxl2表达量均呈先升高后降低趋势,并显著低于在二倍体卵巢中的表达水平(p<0.05),只有在154~574dpf时期,这两个基因表达量显著上调(p<0.05),但他们在整个发育期都显著高于在二倍体精巢中的表达水平(p<0.05);sdy和dmrt1表达量均呈先升高后降低趋势变化,amh和sox9表达量均呈逐渐上调趋势,这些基因在574~964dpf时期均显著高于在二倍体卵巢中的表达水平(p<0.05)。在发育早期,雌性三倍体虹鳟sdy和dmrt1表达量显著低于在二倍体精巢中的水平(p<0.05),但在574dpf时期他们的表达量达到最高,且sdy表达量显著高于二倍体雄性水平(p<0.05);尽管在574~964dpf时期,amh和sox9的表达量较二倍体雌性虹鳟高数百倍,但他们的表达仍极显著低于在二倍体精巢中的水平(p<0.05)。因此,本研究认为,cyp19a1a和foxl2不断被下调或抑制,是导致雌性三倍体卵巢败育主要因素之一。(4)三倍体雌性虹鳟经历了卵巢发育停滞、性腺结构重组和性转变过程。在幼鱼期,三倍体雌性虹鳟血清雌二醇含量最高,且foxl2和cyp19a1a表达量持续上调,而sdy、dmrt1、amh和sox9表达量均相对较低,血清睾酮含量也处于较低水平。但随着进入成年期发育(784~964dpf),仅靠雌激素已无法诱导foxl2持续高表达,而cyp19a1a的表达水平也随之急剧下调;该时期sdy、amh和sox9均持续高表达,sdy甚至达到了同期二倍体雄性的表达水平,而foxl2和cyp19a1a表达急剧下调直至微量表达。(5)本研究是第一次在三倍体雌性虹鳟上设计实验,外源性雄激素和雌激素采用虹鳟养殖业中的通常剂量。芳香化酶抑制剂(1,4,6-androstatriene-3,17-dione,atd)处理成功诱导了精巢分化,其发育模式与二倍体雄性的趋于一致,但精巢发育速度较慢,不过没影响其自然生理应答。17α-甲级睾丸酮(17α-methyltestosterone,met)处理不但没能产生雄性化诱导效应,还将遗传为雌性的性腺始终抑制在低分化水平,严重阻碍了性腺向任何一个方向发展的可能。因此本研究认为,三倍体化改变了雌性虹鳟自身雄性化诱导通路的作用机制。atd诱导与性腺重构的双重作用触发了雌性向雄性逆转,而之后进入精巢发育模式。在三倍体虹鳟卵巢去分化和再分化期进行雌激素回救处理,并不能有效促进或维持卵巢发育。(6)本研究依据雌-雄性转化生物模型来了解三倍体雌性虹鳟的性腺分化特点及其候选基因表达谱,探索当基因组被放大后,雄性化处理对某些在二倍体中微量表达的基因的影响效应,验证性腺特异基因在卵细胞发生去分化和再分化过程中的调控机制。通过总体表达谱可知,在整个发育过程中cyp19a1a和foxl2的表达均被atd所抑制,甚至在发育后期这两个典型的雌性基因也没能得到有效恢复。由于雌性基因不断被下调或抑制,导致了卵巢败育,并使卵细胞去分化返回到性原细胞时期;随着sdy和dmrt1表达量不断上调,激活了性原细胞再分化为雄性生殖细胞,并在sox9和amh协同作用下,开启了精巢分化模式。met在三倍体雌性分化过程中不具有对cyp19a1a和foxl2的抑制效应,反而是对其产生了增量调节,尽管同时也上调了其他雄性特异基因,但由于雌性分化通路并未被完全破坏,导致了雄性化处理失败。在雌性三倍体性腺去分化和再分化时期,外源性雌二醇处理破坏了foxl2与cyp19a1a级联调控关系,并始终抑制cyp19a1a表达。由于滤泡层退化或消失,雌性基因绝对数量骤减,进而导致内源性雌激素合成不足。由于原本cyp19a1a表达量就处于较低水平,再加之外源性雌激素强烈抑制,加剧了卵细胞的去分化进程,甚至在再分化期外源性雌激素有促进雄性特异基因表达的情况。说明雌性分化通路中雌激素与foxl2和cyp19a1a之间确实存在反馈调控关系。综上所述,本研究得到以下结论:(1)三倍体虹鳟卵巢分化同样由cyp19a1a与foxl2共同参与启动,而且受雌激素调控较为明显;在其卵巢败育时期,sd Y和Dmrt1联合调控作用将导致雌性化通路的Cyp19a1a与Foxl2的表达失调,而在性转变时期,Sox9和Amh显著上调将对sd Y和Dmrt1表达产生抑制效应。(2)三倍体虹鳟内源性雌激素缺乏是导致卵巢发育失败的关键因素。(3)三倍体化破坏了虹鳟雌性化通路的调控机制,导致其内源性雌激素合成能力不断下降,进而卵巢发育失败,并最终发生了天然雄性化逆转现象。(4)卵细胞去分化过程对于Cyp19a1a表达及雌二醇合成存在抑制效应。(5)芳香化酶抑制剂能够破坏雌性三倍体虹鳟性腺体细胞及生殖细胞的分化,并显著上调雄性特异基因表达,促进雌性向雄性转变。
【Abstract】 Rainbow trout(Onchorynchus mykiss), belonging to the salmoniformes, salmonidae, Oncorhynchus, is one of the FAO recommended high quality fish to the world breeding objects, and is also the main introduced and cultured species in China. Due to its advantages of triploid female rainbow trout in aquaculture, such as gonad abortion, good muscle quality, it has become the general trend that the female triploid rainbow trout is developed as the main species in trout aquaculture.It is generally known that salmonidae fish is able to survive and reproduce even with a set or two sets of extra chromosomes, which does not exist in other vertebrates. The testes in tetraploid and triploid rainbow trout have the same structure and function as that of diploid. However, triploid female rainbow trout is an exception, since its ovarian is linear in early stage and lacks of primary oocyte. Therefore, the differences between sex differentiation and development mechanism in triploid female rainbow trout have become the focus of research. Although sex determination and differentiation related genes in diploid rainbow trout ha ve been identified, such as sd Y, Dmrt1, Cyp19a1 a, Foxl2 and Sox9, but the specific gonad phenotype and expression pattern of sex control genes in triploid female rainbow trout have not been studied thoroughly, especially the characteristics of germ cells differentiation in early stage ovarian due to retardation of development. The aim of this study was to reveal the mechanism of gonads abortion and natural sex reversal in triploid female rainbow trout. In order to obtain the results, the specific candidate genes which played an important regulatory role in gonad development in triploid female rainbow trout were researched as key objects. The histological evidence of somatic and germ cell differentiation and steroid hormone expression patterns were also determined. In addition, the gonad phenotypic development of triploid female has been controlled through the induction of exogenous hormones to verify the cascade regulation relationships among the genes and the interaction effects between sex cells reshape and the decline rules in sex steroid hormones. The main results were as follows:(1)The differentiation characteristics in ovary of diploid and triploid rainbow trout are basically identical. The diploid and triploid ovary differentiation began at 84 dpf and 98 dpf, respectively. But the ovary differentiation in triploid rainbow trout existed obstacle, which showed slow development in early stage and fewer oogonium and follicular cells. During the period of 154~574 dpf, large number of oocytes were in diploid ovary, while the oocytes were significantly decreased in triploid female rainbow trout ovarian. The ovary was linear, and a part of the female germ cells began to dedifferentiation during the later development. At 964 dpf, triploid female gonad began to reconstruction and redifferentiation. The sperm cell-like clusters appeared to distribute in gonads, which were slightly smaller than oogonium and similar to the germ cell in early stage of testis development in diploid trout.(2)During the period of 334~964 dpf, the contents of E2 and T in serum were gradually increasing in diploid rainbow trout. The E2 levels in serum of female rainbow trout were between 50~650 pg/ml, which was five to eight times higher than that of male. The T levels in serum of male rainbow trout were between 20~180 pg/ml, which was ten to twenty-five times higher than that of female. However, the E2 level increased at first and then decreased in triploid females with the peak value of 110 pg/ml(574 dpf), but always failed to reach the E2 level in diploid. The T level in triploid females gradually increased and almost reached the diploid male level in later development. With the development of gonad in triploid females, the oocyte degraded seriously, leading to blocked estrogen metabolic pathways. The amounts of T converting to E2 were reduced greatly when the testis sertoli cells appeared.(3)During the period of 154~964 dpf, RT-PCR was carried out to detect the differential expression of gonad specific genes in female triploid rainbow trout. And the diploid female and male rainbow trout were used as the control. The results indicated that the gene expression patterns of Cyp19a1 a, Foxl2, sdY, Dmrt1, Amh and Sox9 were obtained in the gonads of diploid and triploid rainbow trout. In triploid female gonad, Cyp19a1 a and Foxl2 expressions decreased after the first increase, which were significantly lower than in ovarian of diploids(P<0.05). The expression of Cyp19a1 a and Foxl2 were significantly up-regulated(P<0.05) only during the period of 154~574 dpf, but they are significantly higher than that in the whole growth period in the diploid testis(P<0.05). The expressions of sd Y and Dmrt1, Amh and Sox9 were continuously up-regulated, in triploid females showed a trend of decrease after the first increases, which were expressed significantly higher than in diploid during the period of 574~964 dpf(P<0.05). The expression of sdY and Dmrt1 was significantly lower than that in diploid testis in the early development(P<0.05), but reached maximum at 574 dpf. And the expression of sd Y was significantly higher than that of diploid male(P<0.05). Although Amh and Sox9 expressions in triploid female were more hundreds of times of diploid female in the period of 574 ~964 dpf, but their expressions were still significantly lower than that in diploid testis( P <0.05).(4)Triploid female rainbow trout had experienced the stagnation of the ovaries, oocyte degradation, germ cells dedifferentiate gonads restructuring and sex reversal. The E2 level in serum of triploid female rainbow trout was highest in the larval stage. During this stage, Cyp19a1 a and Foxl2 were continuously up-regulated, while the expression level of sd Y, Dmrt1, Amh and Sox9 were lower, and the T level was relatively lower. As the development into adulthood(784-964 dpf), estrogen alone had been unable to induce continuous high expression of Foxl2 and the expression of Cyp19 a also decreased sharply. During this period, sdY, Amh and Sox9 were continuously up-regulated, and sdY expression even reached the level of diploid male at the same tim, while Foxl2 and Cyp19a1 a expression were sharply decreased to tiny. The estradiol levels decreased at the same period, while testosterone levels increased continuously and even reached the levels of male diploid.(5)This study is the first design experiments on triploid female rainbow trout. The exogenous androgen and estrogen were used here at the usual dosage in rainbow trout aquaculture. ATD group successfully induced testis differentiation in triploid female rainbow trout and its development mode was consistent with diploid male. The testis in ATD group developed slowly but had no effect on the natural physiological response. MET treatment could not induce the same results as in diploid male-like processing, and MET genetic for female gonad treatment were also always at the lower level of differentiation, which seriously hampered the development of the gonads in any direction. Therefore, this study suggested that the dual role of ATD induction and gonad reconstruction triggered the sex reverse of female to male and the testis development. The estrogen treatment during the dedifferentiation and redifferentiation of gonad in female triploid rainbow trout could not effectively promote or maintain the ovary developmen t.(6)This study was aimed to understand the characteristics of the triploid female gonad differentiation of rainbow trout and those involved candidate gene expression profiles on the basis of the female-to-male biological model, to explore the effects of masculine treatment on the trace expression of genes in diploid when the genome is enlarged, and to verify the regulatory mechanism of gonad specific genes in the process of oocytes dedifferentiation and redifferentiation. The overall expression patterns showed that Cyp19a1 a and Foxl2 were all suppressed by ATD in the whole development process. These two typical female genes didn’t express effectively even in the late development. Because the female genes were down regulated or suppressed, the ovary was abortive and the oocytes dedifferentiated to be the primary germ cell. With the up-regulated expression of sd Y and Dmrt1, the primary germ cell was stimulated to redifferentiate into male germ cells and the testis development was activated under the synergy of Sox9 and Amh. During the triploid female differentiation process, MET could not inhibit the expression of Cyp19a1 a and Foxl2. In contrast, MET induced the up-regulated expression of these tow genes and also other male specific genes at the same time. The exogenous estradiol treatment destroyed the cascade control of Foxl2 and Cyp19a1 a and always inhibited the expression of Cyp19a1 a during the period of gonad dedifferentiation and redifferentiation in triploid female. Due to the degradation or disappear of the follicular layer, the absolute amounts of female genes decreased sharply, which led to the synthesis deficiency of endogenous estrogen. As cyp19a1 a expression was at a low level originally and the exogenous estrogen was severely suppressed, the dedifferentiation of the oocytes was aggravated. The exogenous estrogen even could promote the expression of male specific genes in the differentiation period. The results indicated that there was a feedback regulation relationship between estrogen and Foxl2 and Cyp19a1 a in the female differentiation pathway.To sum up, this study got the following conclusions:(1) The ovary differentiation of female triploid rainbow trout was activated by Cyp19a1 a and Foxl2 expression, and was obviously regulated by estrogen. In the gonadal abortive period, the combined regulation of sdY and Dmrt1 will lead to disorder expression of Cyp19a1 a and Foxl2 in the female pathway. The significant up-regulation of Sox9 and Amh will have an inhibition effect on the expression of sdY and Dmrt1 during the transition period.(2) The deficiency of endogenous estrogen in female triploid rainbow trout was the key factor to cause the failure of the ovary development.(3) The regulatory mechanism of rainbow trout feminization pathways was destroyed, leading to the decline of endogenous estrogen synthesis ability, which caused the failure of ovarian development and eventually the natural male-like reversal phenomenon.(4) Oocytes differentiation has inhibition effect on Cyp19a1 a expression and the synthesis of estradiol.(5) The aromatase inhibitors could destroy the differentiation of gonads somatic cell and germ cell, significantly up-regulate the expression of male specific genes and promote the female to male differentiation.
【Key words】 Rainbow trout; All-female triploid; Ovary abortion; Sex differentiation; Development mechanism;