Alternative splicing and differential expression of P450c17 (CYP17) in gonads during sex transformation in the rice field eel

Several mechanisms were used in determination of the development of the male or female of vertebrates. The genes for determination of sequential hermaphrodite sex are unknown. Here, we reported cloning, alternative splicing, and expression patterns of the CYP17 gene of the rice field eel, a teleost fish with a characteristic of nature sex reversal. The CYP17 gene of the rice field eel was clustered into the CYP17 gene group of all the other vertebrates, especially into the fish subgroup. Four isoforms of the CYP17 were generated in gonads by alternative splicing and polyadenylation. Alternative splicing events of all these isoforms occurred in 3(') regions, which encoded three different sizes (517, 512, and 159aa) of proteins. RT-PCR results indicate specific expression in gonads of these isoforms. Northern blot analysis shows that expression patterns of the CYP17 (dominantly expressed in testis, less in ovary, and the least in ovotestis) are consistent with the sex reversal process of the rice field eel. In situ hybridization further shows its specific expression in germinal lamellae, the gonadal epithelium of the gonads. These findings indicate that CYP17 is differentially regulated in a sex- and developmentally specific manner, suggesting that the CYP17 potentially has important roles in gonad differentiation during sex reversal of the rice field eel.     

黄鳝Nup93基因的分子克隆及其在性腺和肾的显著表达

核孔蛋白（Nucleoporins,Nups）是核孔复合体（Nuclear pore complexes,NPC）的重要组成成分,核孔复合体可以控制细胞内信号分子在核质问的双向转运,从而控制基因表达、细胞增殖和分化。在构建的黄鳝精巢SMART cDNA文库中,采用差异筛选的方法得到黄鳝核孔蛋白家族中Nup93基因的3’端片段,根据此段序列设计引物,使用兼并PCR和5＇RACE方法克隆得到此基因的全长cDNA。序列比对显示该基因与酵母Nic96、斑马鱼Nup93和人类Nup93的同源性分别为36．5％、94．6％和90．5％。进化树分析显示,黄鳝Nup93与其他鱼类的Nup93归为一支。采用荧光定量PCR方法对不同性别黄鳝的性腺和其他组织内该基因的表达作定量分析发现,Nup93在性腺和肾中的表达量远高于其他组织,而且表达量存在一定的性别差异。这一结果提示Nup93可能与性腺发育相关。     

黄鳝β-actin基因的克隆及其在鱼类中的系统发生分析

β-actin是actin家族的一员,在维持细胞结构,细胞内运动,细胞分裂等细胞生理活动方面发挥着重要的作用。克隆的黄鳝β-actin基因的cDNA全长1860bp,编码375个氨基酸,在脊椎动物中不同物种的β-actin基因之间的序列同源性超过了98％。RT-PCR表明克隆的黄鳝β-actin基因在睾丸、卵巢、心、肝、脾、脑等组织中广谱表达。基于目前已知的全部鱼类β-actin cds,构建了进化树。星形辐射的树型结构一致支持将鱼类β-actin基因划分为4类。到目前为止,所有的鱼都没有发现拥有全部4个β-actin基因。这暗示伴随着鱼类的辐射式进化历程,可能发生了种系特异性的β-actin的丢失。     

Molecular Cloning of the Rice Field Eel Nup 93 with Predominant Expression in Gonad and Kidney

Nucleoporins (Nups) are important components of nuclear pore complexes (NPCs). NPCs control gene expression, cells proliferation and differentiation by mediating exchange of cellular signal molecules on both nuclear and cytoplasmic sides. Using subtractive screening, 3'end fragment of Nup 93 from the testis cDNA library of the rice field eel was obtained. Full-length cDNA of the gene was further cloned by degenerate PCR and 5'RACE methods. Sequence analysis indicated that the homology of the rice field eel Nup 93 were 36.5% with yeast Nic 96, 94.6% and 90.5% with Nup 93 of zebrafish and human, respectively. Phylogenetic analysis showed that the rice field eel Nup 93 fits with Nup 93 of the other fishes. Real-time PCR result showed that expression of Nup 93 in gonads and kidney were much higher than in other tissues, and different expression quantities among gonads of three sexes were also observed, suggesting that Nup 93 may involve in gonad development.     

The rice field eel as a model system for vertebrate sexual development

Complex developmental mechanisms of vertebrates are unraveled using comparative genomic approaches. Several teleosts, such as zebrafish, medaka and pufferfish, are used as genetic model systems because they are amenable to studies of gene function. The rice field eel, a freshwater fish, is emerging as a specific model system for studies of vertebrate sexual development because of its small genome size and naturally occurring sex reversal. Data presented here support the use of the rice field eel as another important fish model for comparative genome studies, especially in vertebrate sexual development. This model system is complementary rather than redundant.     

Ubiquitin C-terminal hydrolase-L1 (Uch-L1) correlates with gonadal transformation in the rice field eel

The ubiquitin-proteasome pathway is crucial for a variety of biological processes, including spermatogenesis. Ubiquitin C-terminal hydrolase-L1 (Uch-L1) is thought to associate with monoubiquitin to control ubiquitin levels. Here, we report the identification of Uch-L1 cDNA from the testis of the rice field eel, a natural sex reversal vertebrate, by using cDNA microarray analysis. Uch-L1 encodes a protein of 220 amino acids that shows high homology to Uch-L1 of vertebrates, especially fish species. Both mRNA and protein are mainly expressed in testis, ovotestis and ovary, as well as in the brain. Immunohistochemistry analysis revealed differential expression of Uch-L1 in three kinds of gonads. In the ovary, expression of Uch-L1 was observed mainly in the developing ovary and slightly in the mature ovary. In ovotestis during the intersex stage, Uch-L1 was expressed in the male gonad epithelium and degraded ovary. In testis, expression was observed in developing germ cells, including spermatogonia and spermatocytes. Furthermore, Uch-L1 was upregulated during gonadal transformation, especially from the beginning of the intersex stage onwards. Native-PAGE showed that Uch-L1 underwent dimerization and oligomerization in gonads, and that the relative level of dimerization/oligomerization decreased during gonadal transformation. Simultaneously, ubiquitin polypeptide expression was upregulated during this process. These results suggest that Uch-L1, via the ubiquitin-proteasome system, may play an important role not only in gametogenesis, but also in the gonadal transformation process in the rice field eel.     

Characterization of estrogen receptor beta2 and expression of the estrogen receptor subtypes alpha, beta1, and beta2 in the protandrous black porgy (Acanthopagrus schlegeli) during the sex change process

Estrogens play an important role in many physiological processes in both female and male vertebrates, mediated by specific nuclear receptor, estrogen receptors (ERs). We have isolated a third ER (ERbeta2), which was found to contain 2004 nucleotides including an open reading frame that encodes 667 amino acids. We have also cloned ERalpha and ERbeta1 from the published information (GenBank accession nos. AY074780 and AY074779) and investigated the expression pattern of these ER subtypes in the gonads during gonad sex change of black porgy by quantitative polymerase chain reaction. Maturity stages can be divided into five stages during the sex change process from immature male to female (immature male, mature male, male of mostly testis, male of mostly ovary and mature female). The expression of ERalpha mRNA was highest in the ovary of mature female, followed by the testis of mature male and testicular portion of mostly testis. ERbeta1 expression was higher in the mature testis and ovary than in the gonads of other maturity stages. In contrast to that, ERbeta2 was highest in the ovary of mature female, and significantly lower levels of ERbeta2 expression were observed in the gonads of the other maturity stages. The present study describes the molecular characterization of ERbeta2, and documents the expression changes of three ER subtypes during sex change process of the protandrous black porgy.     

ZFX基因同源序列在黄鳝基因组中的检出及其染色体定位

以大熊猫锌指蛋白基因Zfx为探针 ,在黄鳝基因组DNA中检测到一条长约 9 5kb的杂交带。依据哺乳类和爬行类动物锌指蛋白基因 (ZFX/Zfc)编码第 7～ 13个锌指结构的DNA序列保守性设计引物 ,在黄鳝基因组DNA中仅扩增到一条 5 12bp的DNA片段。将此片段克隆至载体 pBS中 ,从雌性、雄性个体中分别挑选 4个含有插入片段的白色克隆进行测序。测序结果表明 ,这些克隆中插入片段的核苷酸序列一致。该DNA片段在核苷酸水平上与人类ZFX和ZFY分别具有 88%和 87%同源性 ,但其与美洲鳄鱼Zfc的同源性可达 90 % ,而在氨基酸水平上则分别存在 95 9%、95 9%和 93 5 %的同源性 (170个氨基酸 )。该基因命名为黄鳝锌指蛋白基因Zfa ,并运用FISH将其定位于黄鳝 1号染色体 ,距离着丝粒的相对位置为 6 0 1± 0 38。通过进一步研究证明 ,黄鳝 1号染色体上存在有真兽类哺乳动物X染色质同源的保守片段 ,该保守片段有可能就是哺乳动物X染色体起源和进化的原始物质基础之一。应用哺乳动物X染色体连锁的其他基因在鱼类开展染色体比较定位研究 ,将有望促进脊椎动物性染色体进化的深入研究     

黄鳝P-450芳香化酶基因的克隆及序列分析

P-450芳香化酶(P450arom)是催化雄激素生物合成雌激素的关键酶。本文采用RT-PCR和RACE(Rapid amplifi- cation of cDNA ends)法,首次分离和克隆了雌雄同体鱼黄鳝卵巢中P450 arom基因。该基因cDNA全长1802bp(不包 括poly(A)),5'端非翻译区有49bp,3'端202bp(不包含poly(A)),阅读框(Open reading frame,ORF)1551bp,翻译成517 个氨基酸,计算的蛋白质分子量58．2kDa。同源性分析显示,黄鳝卵巢P450arom的氨基酸序列与其他鱼卵巢 P450arom具有63％-80％同源性,与其他鱼脑P450arom为58％-60％同源,与人胚盘和鸡卵巢P450arom则为 50％-52％同源;但在芳香化酶高保守区(包括1-螺旋区,芳香化酶特异保守区和血红素结合区)的同源性高达 76％-92％。系统发育分析表明芳香化酶基因是单起源,黄鳝卵巢芳香化酶基因与鳉鱼卵巢的关系最近,与鱼类卵 巢P450arom属于同一分支的,与鱼类脑及鸡和人的属于不同分支。     

黄鳝Hprt基因的克隆及表达分析

次黄嘌呤鸟嘌呤磷酸核糖转移酶(Hprt)参与嘌呤核苷酸的补救合成。采用RACE技术克隆了黄鳝的次黄嘌呤鸟嘌呤磷酸核糖转移酶基因,它的全长cDNA 为1 452 bp,预测编码218个氨基酸,与人类、小鼠、鸡和斑马鱼等脊椎动物Hprt氨基酸序列之间的同源性超过76.7％。基于该基因氨基酸序列构建了进化树,显示与斑马鱼Hprt基因更同源。RT-PCR表明黄鳝Hprt基因在多种组织中广谱表达,表明黄鳝该基因在功能和进化上的保守性。      

Cloning and Characterization of a Rice Field Eel vasa-Like Gene cDNA and Its Expression in Gonads During Natural Sex Transformation

The vasa (vas)-related gene encodes an RNA helicase protein member of the DEAD-box family and plays key roles in germ-cell formation in higher metazoans. Using degenerate PCR and RACE, we cloned the vasa gene of the rice field eel (Monopterus albus), which is homologous to the Drosophila vasa gene. We named it ma-vas (Monopterus albus vas). Ma-vas encodes a protein of 618 amino acids, which contains all of the known characteristics of vasa homologs. RT-PCR analysis revealed that ma-vas was exclusively expressed in the gonads of the female, intersex, and male. During gonadal natural sex reversal, ma-vas is expressed in oocytes at all stages of oogenesis, in degenerating oocytes of ovotestis, and in spermatogonia and spermatocytes at early stages. The vasa positive signal was also observed in the peripheral layer of late ovary. It was not found, however, in that layer of the testis. Alkaline phosphatase (AKP) staining on the ovary and testis also indicated that some cells had differentiational potential in the peripheral layer of the ovary, suggesting that spermatogonia might arise from cells with AKP and vasa-positive staining in the peripheral layer of the female gonad.     

Analysis of medaka sox9 orthologue reveals a conserved role in germ cell maintenance

The sex determining gene is divergent among different animal species. However, sox9 is up-regulated in the male gonads in a number of species in which it is the essential regulator of testis determination. It is therefore often discussed that the sex determining gene-sox9 axis functions in several vertebrates. In our current study, we show that sox9b in the medaka (Oryzias latipes) is one of the orthologues of mammalian Sox9 at syntenic and expression levels. Medaka sox9b affects the organization of extracellular matrices, which represents a conserved role of sox9, but does not directly regulate testis determination. We made this determination via gene expression and phenotype analyses of medaka with different copy numbers of sox9b. Sox9b is involved in promoting cellular associations and is indispensible for the proper proliferation and survival of germ cells in both female and male medaka gonads. Medaka mutants that lack sox9b function exhibit a seemingly paradoxical phenotype of sex reversal to male. This is explained by a reduction in the germ cell number associated with aberrant extracellular matrices. Together with its identified roles in other vertebrate gonads, a testis-determining role for Sox9 in mammals is likely to have been neofunctionalized and appended to its conserved role in germ cell maintenance.     

Sex determination and sexual differentiation in the avian model

The sex of birds is determined by the inheritance of sex chromosomes (ZZ male and ZW female). Genes carried on one or both of these sex chromosomes control sexual differentiation during embryonic life, producing testes in males (ZZ) and ovaries in females (ZW). This minireview summarizes our current understanding of avian sex determination and gonadal development. Most recently, it has been shown that sex is cell autonomous in birds. Evidence from gynandromorphic chickens (male on one side, female on the other) points to the likelihood that sex is determined directly in each cell of the body, independently of, or in addition to, hormonal signalling. Hence, sex-determining genes may operate not only in the gonads, to produce testes or ovaries, but also throughout cells of the body. In the chicken, as in other birds, the gonads develop into ovaries or testes during embryonic life, a process that must be triggered by sex-determining genes. This process involves the Z-linked DMRT1 gene. If DMRT1 gene activity is experimentally reduced, the gonads of male embryos (ZZ) are feminized, with ovarian-type structure, downregulation of male markers and activation of female markers. DMRT1 is currently the best candidate gene thought to regulate gonadal sex differentiation. However, if sex is cell autonomous, DMRT1 cannot be the master regulator, as its expression is confined to the urogenital system. Female development in the avian model appears to be shared with mammals; both the FOXL2 and RSPO1/WNT4 pathways are implicated in ovarian differentiation.     

Both cell‐autonomous mechanisms and hormones contribute to sexual development in vertebrates and insects

The differentiation of male and female characteristics in vertebrates and insects has long been thought to proceed via different mechanisms. Traditionally, vertebrate sexual development was thought to occur in two phases: a primary and a secondary phase, the primary phase involving the differentiation of the gonads, and the secondary phase involving the differentiation of other sexual traits via the influence of sex hormones secreted by the gonads. In contrast, insect sexual development was thought to depend exclusively on cell‐autonomous expression of sex‐specific genes. Recently, however, new evidence indicates that both vertebrates and insects rely on sex hormones as well as cell‐autonomous mechanisms to develop sexual traits. Collectively, these new data challenge the traditional vertebrate definitions of primary and secondary sexual development, call for a redefinition of these terms, and indicate the need for research aimed at explaining the relative dependence on cell‐autonomous versus hormonally guided sexual development in animals.