【作者】 吴风瑞；

【导师】 王德寿；

【作者基本信息】 西南大学 ， 水生生物学， 2010， 博士

【摘要】 近年来大量研究发现哺乳动物雌性性别决定和分化是在性腺体细胞Fox12和生殖细胞Wnt4/Rspo 1/β-catenin两条通路的严格控制之下而完成的,β-catenin是雌雄信号拮抗过程中的重要蛋白。对硬骨鱼类而言,其性别决定除受遗传基因控制外,雌激素(在硬骨鱼类由Cyp19a1a编码)在卵巢分化过程中起着决定性诱导作用,是卵巢分化的天然诱导剂。长期以来,Wnt4/Rspo 1/β-catenin通路在硬骨鱼类是否存在以及其在性别决定和分化过程中的作用、对Cyp19a1a否存在调控、与Fox12和Dmrt1控制的体细胞雌激素合成的关系未见报道。本研究首先从罗非鱼克隆了Rspo1和由两个不同基因编码的β-catenin(缩写β-cat,分别命名为β-cat-1和-2)。序列分析发现硬骨鱼类Rspo1、β-catenin氨基酸序列具有较高的保守性,系统发生分析表明两种β-catenin在硬骨鱼类是一种普遍现象,可能源于进化过程中硬骨鱼类特有的基因组复制。另外,我们采用RT-PCR的方法对罗非鱼Rspo1和β-catenin在各组织中的表达模式进行了研究,发现Rspo1呈泛表达的模式,β-cat-1和-2在脑、垂体、鳃、心脏、肾脏、肠、肾脏和性腺中表达。就性腺而言,β-cat-1只在卵巢中表达,Rspol和β-cat-2在卵巢中的表达要高于精巢。原位杂交研究发现罗非鱼Rspo1、β-cat-1和-2都表达于卵巢中3时相及其以前的卵母细胞,而不表达于卵巢体细胞,Rspo1还表达于精原细胞和精母细胞。在孵化后10天的罗非鱼卵巢中就能检测到β-cat-1和-2的表达信号,且伴随着卵巢发育二者表达量逐渐升高,直至3时相卵母细胞。通过免疫组化还发现罗非鱼β-catenin在卵巢中的蛋白表达水平远远高于精巢,从而证明在硬骨鱼类存在Rspo1/β-catenin介导的生殖细胞通路,暗示了其可能在硬骨鱼类卵巢分化和发育中具有重要作用。因此,本研究进一步采用荧光素酶报告基因系统研究了β-catenin是否影响Cyp19a1a转录活性以及生殖细胞Rspo1/β-catenin通路与已知性腺体细胞Fox12和Dmrt1控制的雌激素合成通路之间的关系。结果发现,在HEK293细胞中,罗非鱼β-cat-1和-2单独都不能激活Cyp19a1a的转录,但二者均可以增强Sf1激活的Cyp19a1a启动子活性；Fox12和β-cat-2协同增强Sf1激活的Cyp19a1a启动子活性,Dmrt1则能抑制β-cat-2增强和Sf1激活的Cyp19a1a启动子活性。以上结果表明Wnt/Rspo 1/β-catenin信号通路可能会和Fox12、Dmrt1一起通过调控Cyp19a1a转录和雌激素合成从而影响硬骨鱼类的性别决定和分化。在硬骨鱼类,雌激素不仅对卵巢分化和发育有重要作用,而且在后期卵巢功能维持上也必不可少,如硬骨鱼类卵巢周期性发育过程也同样需要大量雌激素的参与。细胞外基质蛋白凝血酶敏感素-1(Thromobospondin-1, TSP)是脊椎动物滤泡发生和卵巢周期性发育的重要因子,为了进一步研究TSP-1在硬骨鱼类性腺发育和产卵周期中的作用以及与雌激素的关系,本研究从罗非鱼和青鳉卵巢中均克隆了由两个不同基因编码的凝血酶敏感素TSP-1a和-1b,对脊椎动物TSP-1进行系统发生分析发现,两种TSP-1仅存在于硬骨鱼类。原位杂交结果发现TSP-1b从孵化后5天就开始在罗非鱼雌雄性腺体细胞中表达,在成体卵巢和精巢中则分别表达于鞘膜细胞和输出小管上皮细胞,TSP-1a从孵化后120天开始特异性表达于卵巢的颗粒细胞,这一结果暗示了TSP-1a可能参与了罗非鱼的卵黄发生,TSP-1b参与罗非鱼早期的性腺分化。在罗非鱼14天／产卵周期中,TSP-1a和-1b都在产卵后5天(卵黄发生期)和12天(卵子成熟期)出现两个峰值,这种表达变化规律与对罗非鱼产卵周期中激素水平如雌激素以及FSH和LH的变化研究一致,表明了它们可能通过影响卵巢体细胞中激素的合成而参与了罗非鱼卵巢周期性发育。此外,硬骨鱼类卵巢分化和发育离不开卵巢局部(包括体细胞和生殖细胞)因子的参与,它们连接成网,相互影响,通过自分泌或旁分泌的方式产生的雌激素。为了研究性腺局部因子在南方鲇卵巢分化和发育中的作用,我们还从南方鲇卵巢中克隆了促性腺激素GtHα、FSHβ和LHβ三个亚基的cDNA序列作为本研究的一个组成部分(附录部分)。这三个亚基除在垂体表达之外还在卵巢中特异性表达。在卵巢中,GtHα、FSHβ在孵化后25天就开始表达,LHβ在孵化后40天开始表达,与南方鲇原始生殖细胞有丝分裂和卵原细胞快速增殖的起始时间基本一致,说明性腺局部FSH和LH可能参与了南方鲇原始细胞增殖和早期卵巢发育。当用雌激素受体拮抗剂Tamoxifen对孵化后5天的南方鲇全雌幼鱼进行一定时间的处理后,发现三个亚基的表达水平出现了不同程度的下调,并出现了部分性逆转的现象。因而,我们推测南方鲇卵巢局部产生的促性腺激素很可能通过“脑-垂体-性腺”轴和其他性腺局部因子一起直接或间接影响雌激素合成从而影响南方鲇早期卵巢分化和发育。综上所述,本研究克隆了罗非鱼Rspo1/β-catenin信号通路重要分子Rspo1、β-catenin和细胞外基质主要成分TSP-1以及南方鲇促性腺激素三个亚基,研究了它们的组织和细胞表达模式,并通过荧光素酶报告基因首次研究了罗非鱼β-catenin对Cyp19a1a启动子活性的影响。本研究不仅初步阐明Rspo1/β-catenin所介导的生殖细胞信号通路在硬骨鱼类卵巢分化和发育中作用,也将其与已知的Fox12和Dmrt1体细胞控制的雌激素合成调控途径相偶联,进一步丰富了硬骨鱼类雌激素合成理论,并提出了以雌激素为核心的硬骨鱼类性腺体细胞和生殖细胞相互作用研究的新思路,为揭示脊椎动物特别是硬骨鱼类卵巢分化和发育以及体细胞和生殖细胞之间的相互作用的分子机制提供基础的理论依据。更多还原

【Abstract】 Recently, there are many reports light on the molecular mechanisms of mammalian female sex determination and suggest that the Wnt4/Rspol/β-catenin pathway and Foxl2 act in a complementary manner to determine ovarian differntiation and to antagonize testicular development, while P-catenin is a key protein in antagonism between the male and female pathways. In contrast, sex determination and differentiation of non-mammalian vertebrates are controlled by both genetic and environmental factors, e.g. hormones, especially estrogen which is a natural inducer for ovarian differentiation in non-mammalian vertebrates. However, studies on the roles of Wnt4/Rspol/p-catenin signaling pathway in sex determination and differentiation were mainly focused on mammals. There is no such report on non-mammalian vertebrates. In the present study, Rspol, twoβ-catenin (named asβ-catenin-1 and-2, abbreviated asβ-cat-1 and-2) encoded by two different genes were identified and cloned from the Nile tilapia (Oreochromis niloticus) ovary by RT-PCR and subsequent RACE. Phylogenetic analysis of P-catenin from various vertebrates indicated that the co-existence of twoβ-catenins is an unique phenomenon in teleosts and are probably derived from the additional genome duplication (the fish-specific genome duplication, FGSD) that occurred at the emergence of modern fish. The tissue distribution analysis by RT-PCR revealed that tilapia Rspol was ubiquitously expressed in all examined tissues,β-cat-1 and-2 were expressed in brain, pituitary, gill, heart, kidney, intestine and gonads. In the gonads, the expression levels of Rspol and P-cat-2 were higher in the ovary than in the testis, whereasβ-cat-1 was exclusively expressed in the ovary. Moreover, the gonadal cell types where Rspol and two types ofβ-cat express were further analyzed by in situ hybridization. Both of them were expressed in oocytes at phaseⅠ,ⅡandⅢof ovary, not in somatic cells, while Rspol was also expressed in spermatogonium and spermatocytes. It is worth noting that the expression level ofβ-cat gradually increases with the ovarain development from 10 days after hatching to latter stage in primary oocytes at phaseⅠ,ⅡandⅢImmunochemistry ananlyses further confirmed that tilapiaβ-catenin was localized in the germ cells of ovary, suggesting that P-catenin might be an important factor involved in teleost ovary differentiation and development. Furthermore, Luciferase assays were performed to study the effect of P-catenin on the Cyp19ala gene transcriptional activity and the relationship between P-catenin and the known pathway of estrogen synthesis which was controlled by Foxl2 and Dmrtl. The results showed thatβ-cat-1 and-2 alone was unable to activate Cyp19ala transcription, however, both of them enhanced Sfl-mediated Cypl9ala transcription in HEK293 cells. Moreover, Foxl2 and P-cat-2 synergistically enhanced the Sfl-mediated Cypl9ala gene transcription, while Dmrtl suppressed the P-cat-2 enhanced and Sfl activated Cypl9ala promoter activity. The results indicate that both Rspol/β-catenin-mediated germ cell pathway and Foxl2/Dmrtl-mediated somatic cell pathway might control the production of estrogen in the gonad through transcriptional regulation of aromatase gene, which in turn, determine the gonadal differentiation and maintain the gonadal function.Estrogen and factors within the extracellular matrix (ECM) have been reported to. be important in the vertebrate follicular development and ovarian cycle. Thromobospondins (TSPs 1-5) are important members of the ECM proteins. In order to address the roles of TSP-1 in the teleost gonadal development and spawning cycle, two types of TSP-1 (named TSP-1a and TSP-1b) were cloned from Nile tilapia. Phylogenetic analysis of these TSP-1 sequences, together with those available from other vertebrates further demonstrated that two types of TSP-1 exist only in teleosts, extending the finding in fugu and tetraodon to two additional fish species. The expression of both genes was examined using tilapia at various developmental stages. Tilapia TSP-1a and TSP-1b were each expressed in a wide range of tissues examined. The early expression of TSP-1b in both XX and XY gonads from 5 dah onwards suggested an important role in the formation of gonads, while the expression of TSP-1a only in ovaries during later stages of development (from 120 dah onwards) may suggest that TSP-1a is involved in vitellogenesis. In situ hybridization analyses revealed differential expression in adult fish, with TSP-1a occurring in granulosa cells and TSP-1b in theca cells and in the epithelial cells of the efferent duct of the testis. During the 14-day spawning cycle, the expression of both types of TSP-1 exhibited distinct peaks at day 5 (peak of vitellogenesis) and day 12 (oocyte maturation), corresponding to active estrogen production and vitellogenesis and to the synthesis of FSH, LH and oocyte maturation, suggesting that they have important roles in spawning cycle of the Nile tilapia.Fish gonad differentiation and development are mediated by local factors, such as a variety of hormones and growth factors, produced in an autocrine and paracrine fashion by gonad somatic cell and germ cell. They form an intimate regulatory network and interact with each other. In present study, three gonadotropin subunit cDNAs, GtHa, FSHfβand LHβ, were isolated from Southern catfish (Silurus meridionalis Chen) ovary. RT-PCR revealed that GtHa, FSHβand LHβmRNA were expressed in ovary, female and male pituitaries, but not in testis. Ontogenic study showed that GtHa and FSHβexpressed in ovary from 25 dah and LHβexpressed from 40dah onwards. These results indicated the involvement of the local gonadotropin in the proliferation of Southern catfish primordial germ cell and early ovrian development. All-female Southern catfish fry were treated with estrogen receptor tamoxifen (TAM) from 5 to 25dah). The expressions of the three subunits were down-regulated but not vanished in TAM treated gonad as compared with control. Some partial and complete sex reversed fish was observed in TAM group. Together with other findings reported in other fish, it is conceivable for us to presume that the secretion of gonadotropin in both pituitary and gonads might have important roles on sex steroids synthesis and interacted with those factors directly or indirectly in undifferentiated gonad and in turn on sex differentiation through brain-pituitary-gonad axis.In summary, Rspol, two types ofβ-catenin and TSP-1 were cloned from tilapia. Tissue distribution, cell-specific expressions and expression profile during the spawning cycle were studied by RT-PCR, real time PCR, in situ hybridization and immunochemistry. Moreover, effects of tilapiaβ-catenin on Cypl9ala promoter activity were further investigated by luciferase assay. Our results indicate that these genes might have important roles in fish ovarian differentiation and development. These findings highlight the relationship between theβ-catenin-mediated germ cell pathway and the known Foxl2/Dmrtl-mediated somatic cell pathway in the control of estrogen synthesis and sex differentiation; provide basic data for the research on the molecular mechanism of ovarian differentiation and development, and the crosstalk between germ cell and somatic cell in fish.更多还原