【作者】 胡重江；

【导师】 王德寿；

【作者基本信息】 西南大学 ， 水生生物学， 2008， 博士

【摘要】 性别决定和分化一直是生物学、生理学、内分泌学研究的热点问题之一。鱼类的性别在很大程度上受到环境因素如:温度、光照、外源性激素水平的影响,甚至还能导致性逆转。过去,对于性别分化的研究主要集中在对性腺体细胞的分化方面,相继克隆了许多在鱼类性别决定和分化中起重要作用的基因,并对其功能进行了深入研究。近年来,由于对性别分化的认识不断加深,加上鱼类养殖的要求,对生殖细胞的分化和发育逐渐成为一个研究的热点。对鱼类生殖细胞标记(germ cell marker)基因进行研究将为深入开展对鱼类生殖细胞分化和发育分子机制研究,拓展对这一领域的认识奠定坚实基础,也对水产养殖业具有指导意义。本研究所用模式动物尼罗罗非鱼(Oreochromis niloticus)具有生长速度快、性成熟时间快、繁殖周期短等特点,是世界最重要的养殖鱼类之一。利用现代生物学手段对其进行单性化的养殖提出了新的要求。因此对罗非鱼性别分化,特别是生殖细胞的分化和发育进行研究具有重要的理论意义和应用价值。本实验从尼罗罗非鱼克隆了几种生殖细胞标记,研究了它们的组织表达模式和在个体发生中的表达。同时我们还利用fadrozole(芳香化酶抑制剂)、雌二醇处理,人工诱导雌雄性逆转罗非鱼,研究了生殖细胞标记在这些性逆转鱼性腺中的表达变化情况。采用RT-PCR和RACE相结合的方法,克隆了两个尼罗罗非鱼早期发育阶段的生殖细胞标记:Figla和Oct4。克隆得到的Figla序列长度为1254 bp,开放阅读框(open reading frame,ORF)长度为594碱基(base pair,bp),编码197个氨基酸(amino acid,aa)。Oct4序列长度为1787 bp,ORF长度为1380 bp,编码459个aa。它们在卵巢中的表达均高于精巢。罗非鱼Figla(ntFigla)m RNA从孵化后第5天(days after hatching,dah)开始表达于雌性和雄性性腺中,并从10 dah开始呈现性别二态性表达,RT-PCR和定量RT-PCR(quantitative RT-PCR,qRT-PCR)都显示,直到70 dah ntFigla在精巢的mRNA水平都维持较低水平。而性成熟后,它在精巢的表达明显增加。ntOct4 mRNA在性腺发育的早期高表达,这暗示它在这个阶段可能具有重要功能。但在孵化后1个月通过原位杂交可以检测到它的性别二态性表达,之后它在卵巢中持续高水平表达,而在精巢中表达量逐渐降低,直到检测不到。采用RT-PCR和RACE相结合的方法,我们从尼罗罗非鱼克隆了两个与生殖细胞减数分裂相关的基因:Spo11和Scp3。Spo11是最早分离得到的减数分裂重组基因之一,而Scp3是减数分裂联会复合体(Synaptonemal complex,SC)的主要结构蛋白,它们均为减数分裂特异的标记。Scp3的cDNA序列为1070 bp,ORF为717 bp,编码238个aa;尼罗罗非鱼Spo11序列为1468 bp,ORF为1173 bp,编码390个aa。组织分布结果表明,两者都在罗非鱼性腺中特异表达,ntScp3主要表达于精巢,而ntSpo11主要表达于卵巢。它们都能用作检测处于减数分裂的生殖细胞。在个体发生研究结果表明,Scp3 mRNA的表达总是精巢高于卵巢,这种差异一直持续到性成熟。在卵巢中,ntScp3 mRNA主要在Ⅰ、Ⅱ、Ⅲ时相的卵母细胞中表达,在第Ⅳ时相的卵母细胞中表达量低,且主要集中在细胞边缘。ntSpo11主要在卵巢中表达,这种性别差异均能在不同时期(10 dah、30 dah、40 dah、5个月和成体)检测到。Vasa蛋白是DEAD-box蛋白家族成员中一种ATP依赖的RNA解旋酶,它在高等后生动物的生殖细胞形成中起着关键的作用。Vasa基因特异性地表达于许多后生动物的生殖细胞中,并被广泛确定为许多脊椎动物和无脊椎动物生殖细胞的特异标记。采用RT-PCR和RACE相结合的方法,从南方鲇中分离得到Vasa基因的两个亚型,scVasa和scVasa-s。通过RACE方法获得了它们的全长。核苷酸序列分析显示,scVasa cDNA序列全长2,563bp,其中开放阅读框ORF长1,989bp,可以编码含662个氨基酸的蛋白;scVasa-s cDNA序列全长2,475bp,其中开放阅读框ORF长1,926bp,可以编码含641个氨基酸的蛋白。scVasa-s缺乏scVasa N末端区域的一段序列。这两种推导出的氨基酸序列均具有DEAD-box蛋白家族成员的特征,即含有4个精氨酸-甘氨酸-甘氨酸(RGG)基序和另外8个保守基序。它们和银鲫的Vasa同源蛋白有很高的相似性(75.2%和73.8%)。原位杂交结果表明:在卵巢,scVasa主要在Ⅰ、Ⅱ时相的卵母细胞表达,而在精巢,主要在精原细胞和初级精母细胞表达。半定量PCR结果显示,在生殖周期中,两种亚型在以Ⅱ时相卵母细胞为主体的卵巢恢复期表达均高于以Ⅲ-Ⅳ时相卵母细胞为主体的卵黄生成期。本研究首次从硬骨鱼类罗非鱼和南方鲇系统的克隆了生殖细胞和减数分裂的几种分子标记。尽管目前国内外众多学者对生殖细胞和减数分裂的分子标记进行了深入广泛的研究,但是大都集中在模式生物如小鼠、斑马鱼和青鳉上,而对具有重要经济意义的水产养殖鱼类的研究却很少,甚至是空白,因而本研究通过对我国重要的养殖鱼类罗非鱼和南方鲇的生殖细胞和减数分裂的分子标记的表达模式以及它们在药物处理性逆转过程中的表达变化,为最终通过生物技术手段解释性别分化的机制、对减数分裂起始的调控机制、体细胞和生殖细胞的相互关系的研究奠定基础,而且在未来的水产养殖业中有非常广阔的应用前景。更多还原

【Abstract】 Sex determination and differentiation have always been the focus in the researches of biology, physiology and endocrinology.In mammals,sex was controlled by genetic factors,especially the sex determining gene,SRY/Sry.While in lower vertebrates,including fish,sex is also influenced by environmental factors,such as temperature,photoperiod and exogenous sex steroids.These factors might even cause sex reversal in many species.Sex differentiation includes the differentiation of both somatic cells and germ cells.In the past,much attention has been drawn to the differentiation of somatic cells,genes closely related to the sex determination and differentiation have been cloned and their functions further investigated.Recently,researches on the differentiation of germ cells have gradually become a new hotspot based on the better understanding of sex differentiation and requirement of fishery industry.To date,researches of germ cells have been mostly restricted in mammals,while in lower vertebrates,especially in fish,the germ cells were poorly investigated.One of the most important reasons responsible for this is due to the lack of germ cell markers. Investigations on the germ cell marker genes in fish will lay foundations for the researches on the mechanisms of germ cell differentiation and development,and expanding the understanding of sex differentiation in fish.It also has practical implications for aquaculture.The Nile tilapia(Oreochromis niloticus),because of its fast growth rate and short reproductive cycle,is developed as one of the most important species for aquaculture and experiment globally.As the growth of male is much faster than female by 50%,males are always favored.Because the masculinization methods generally utilized was of much disadvantages,new techniques by modern molecular biology is highly required in growing monosex progeny.As a result,investigations on the sex differentiation,especially on the differentiatioin and development of germ cells,are of decisive theoretical significance and practical implications.In the present study,several germ cell markers were cloned and their expressions in various adult tissues and ontogenic expressions in gonad were investigated.Sex reversals were successfully obatained by treatment with aromatase inhitor(fadrozole) and estrogen(17-βestrodial).The expressions of these marker genes in the gonads of sex reversed fish were also examined.First of all,two cDNAs of germ cell markers(Oct4 and Figla) responsible for early development of tilapia gonad were isolated by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends(RACE).The length of Figla cDNA is 1254bp, containing an open reading frame(ORF) of 594bp,which encode a 198aa protein.The length of Oct4 cDNA is 1787bp,containing an ORF of 1380bp,which encode a 460aa protein.The expression of Figla mRNA initiated from the 5 days after hatching(dab) and showed a sexual dimorphic pattern from 10 dah onwards.The expression level of Figla mRNA in testis remained low till 70 dah,while increased significant when sexually matured detected by both RT-PCR and quantitative RT-PCR(qRT-PCR).High expression of ntOct4 was detected during early gonadal development in both sexes,indicating its decisive role in this period.In situ hybridization(ISH) results revealed that ntOct4 showed a sexual dimorphic expression pattern, expressing higher in ovary than in testis from 1 month after hatching,after which,its expression level decreased significantly and became undetectable in the adult male.cDNAs of Spo11,one of the earliest marker related to recombination process in meiosis and Scp3 encoding the structural protein of synaptonemal complex during meiosis were also isolated by RT-PCR and RACE methods in the Nile tilapia.Both of them are markers closely related to the meiosis of germ cells.Full length cDNAs of Scp3 and Spo11 were 1070 and 1468 bp,which contain ORFs of 717 and 1173 bp,encoding proteins of 239 and 390 aa,respectively.Expressions of both markers were restricted to the gonads of the tilapia,with higher level in testis for Scp3 and higher level in ovary for Spo11.Tissue distribution analyse showed that the the expression of Scp3 was much higher in testis than in ovary.Ontogeny analysis by RT-PCR also revealed this pattern basically.It was also revealed that Scp3 was expressed mainly in theⅠ,ⅡandⅢphase oocytes and its expression level decreased in oocytes ofⅣphase,in which mainly concentrating on the edge.Spo11 expression also showed sexual dimorphic in tissue distribution analysis,being higher in ovary than in testis.Ontogeny analysis by qRT-PCR revealed the same pattern in 10 dah,30 dah,40 dah and 5 months fish and adults.Vasa protein,a member of the DEAD(Asp-Glu-Alu-Asp) box protein family of ATP-dependent RNA helicases,plays key roles in germ cell formation in higher metazoans.The Vasa gene,found exclusively expressed in germline cells of many metazoans,has been extensively characterized as germ cell specific marker in many vertebrate and invertebrate species.Two isoforms of Vasa cDNA (scVasa and it’s short form scVasa-s) were isolated and characterized in a teleost fish,southern catfish by RT-PCR and RACE methods.Analysis of the nucleotide sequences revealed that the full length cDNA of scVasa comprises 2,522 bp with an ORF of 1,989 bp encoding 662 amino acids, while that of scVasa-s comprises 2,475 bp with an ORF of 1,926 bp encoding 641 amino acids. scVasa-s lacks a part of the N-terminal region found in the scVasa.Both of the two deduced amino acid sequences contain all of the known characteristics belonging to the DEAD-box protein family, four arginine-glycine-glycine(RGG) motifs and other eight conserved motifs.They show high similarity to Vasa homolog of Giebel carp(75.2%and 73.8%).Tissue distribution analysis by RT-PCR revealed that these two isoforms were exclusively expressed in the gonads of both female and male.In adult fish,scVasa was found to be mainly expressed in the primary oocytes atⅠandⅡphases in the ovary while in the spermatogonia and primary spermatocytes in the testis by in situ hybridization.Semi-quantitative RT-PCR analysis showed that the expressions of both scVasa isoforms were much higher in ovarian recrudescent stage(mainly with phaseⅡoocytes) than in vitellogenic stage(mainly with phaseⅢandⅣoocytes).This is the first time to clone so many germ cell markers in teleosts,the Nile tilapia and Southern catfish,both of which are important for aquaculture.Although investigations on these markers have been taken by reseachers all over the world extensively,most studies were focused only on a few model species,such as mouse,zebrafish and medaka,while little attentions have been paid to the important aquacultural speices.In order to lay foundations for growing monosex progeny and improving the growth rate of the aquacultural fishes by biologcial techeniques,expression patterns and changes during sex reversal by drug treatment of these germ cells and meiosis markers were investigated in the Nile tilapia and Southern catfish,two important aquacultural fish species in our country.The results of the study are important not only for researches on the mechanisms of sex differentiation,meiosis regulation and the cross talk between germ cells and somatic cells,but also important for practical implications in future aquaculture.更多还原