Rspo1对雌性脊椎动物性别分化的功能

长期以来雌性脊椎动物的性别分化被认为是一个“默认”的程序.但是近些年研究发现,Rspo1基因的突变或缺失可导致哺乳动物XX型个体性反转为雄性.Rspo1在鱼类、两栖爬行类、鸟类和哺乳类动物性腺发育的不同阶段表达,其表达在雌雄个体性别分化时期有差异,是潜在的性别调控基因.Rspo1在性别发育早期可通过Wnt/β-catenin信号通路调控性腺分化相关因子的表达,影响原始生殖细胞分裂增殖、细胞周期和生长发育,参与调控性腺中体细胞的分化.本文总结了近年来Rspo1在脊椎动物中的表达调控及其在雌性性别决定方面功能的研究进展.     

鸡性别决定机制及相关基因研究进展

鸡性别决定虽然同哺乳动物一样受遗传控制,但其性染色体组成为ZZ／ZW,同哺乳动物相反呈现雌异型,并且鸡性腺性别分化同一些低等脊椎动物一样易受性激素影响。目前参照哺乳动物性别决定相关基因已获得了一些鸡同源基因序列(AMH,SF1,DAX1,SOX9)和3个可能与鸡性别决定有重要关联的候选基因(DMRT1,ASW和FET1)。对这些基因的表达模式及其在层次调控中的功能比较分析结果显示,鸡性别决定的遗传机制同其它脊椎动物相对一致,但也有明显的不同。     

Rspo1在雌性脊椎动物性别分化中的功能

长期以来雌性脊椎动物的性别分化被认为是一个"默认"的程序.但是近些年研究发现,Rspo1基因的突变或缺失可导致哺乳动物XX型个体性反转为雄性.Rspo1在鱼类、两栖爬行类、鸟类和哺乳类动物性腺发育的不同阶段表达,其表达在雌雄个体性别分化时期有差异,是潜在的性别调控基因.Rspo1在性别发育早期可通过Wnt/β-catenin信号通路调控性腺分化相关因子的表达,影响原始生殖细胞分裂增殖、细胞周期和生长发育,参与调控性腺中体细胞的分化.本文总结了近年来Rspo1在脊椎动物中的表达调控及其在雌性性别决定方面功能的研究进展.     

鸟类性别决定候选基因在性反转鸡胚中的表达

DMRT1、PKCIW和FET1是鸟类性别决定过程中重要的候选基因。以芳香化酶抑制剂处理的鸡胚为实验材料, 对这3个基因的表达变化进行了研究。结果表明, 在整个性别决定关键时期(E4.5 ~ E10.5), DMRT1在雄性的表达量显著高于雌性, 并且在ZW性反转鸡胚中表达大幅上升, 表明DMRT1的上调表达是与睾丸形成相关的。PKCIW基因在雌性特异表达并在性反转鸡胚表达上升, 这可能与其特殊作用模式有关, 即使性反转鸡胚PKCIW代偿性的表达升高, 却也未能阻止睾丸的形成。此外, FET1为雌性特异表达, 但在性反转鸡胚中表达无变化。综上, 实验结果支持了DMRT1是鸟类睾丸发育决定因子的假说。     

雌激素对中华鳖性腺分化及DMRT1、SOX9基因表达的影响

中华鳖（Pelodiscus sinensis）性别决定的方式一直存在较大的争议,分子机制更是不清楚。在大部分脊椎动物中,雌激素在性别决定和性腺分化中扮演重要的调控作用。实验通过对性别分化前胚胎进行雌二醇（E2）和芳香化酶抑制剂（AI）处理,研究雌激素在中华鳖性腺分化中的作用及机理。实验结果显示,与对照组（雌性比例49%）相比,E2处理组中雌性中华鳖仔鳖比例显著增加,高达92.3%;而在AI处理组中,雌性比例显著下调至13.1%。HE染色分析表明,ZZ（雄性）和ZW（雌性）胚胎分别经过E2和AI处理后,ZZ和ZW性腺结构呈现明显的雌性化和雄性化特征。同时,通过RT-PCR和免疫荧光染色发现,E2能显著降低雄性性别关键因子DMRT1和SOX9 mRNA和蛋白表达水平;AI则表现相反的调节作用。综上所述,雌激素通过抑制雄性性别关键因子DMRT1和SOX9的表达来抑制雄性分化,促进雌性分化,揭示雌激素在中华鳖雌性性别分化中起着重要的调控作用。       

中华鳖Dmrt1基因的克隆、表达及在性逆转中的变化分析

在大部分脊椎动物中,Dmrt1基因在雄性性别决定和性腺分化中起重要的调控作用.本文从m RNA和蛋白水平分析Dmrt1基因的组织差异性表达、在不同发育阶段性腺中的细胞定位及在性逆转中的表达变化,研究Dmrt1基因在中华鳖性别分化中的调控作用.Rapid-amplification of c DNA ends(RACE)结果显示,Dmrt1基因c DNA序列全长2409 bp,其中5′非编码区为230 bp,3′非编码区为1072 bp,开放阅读框为1107 bp,编码368个氨基酸,具有一个高度保守的DM结构域.荧光定量PCR和免疫组化结果显示,Dmrt1在性腺分化之前的第16期雄性性腺中开始表达,先于Amh和Sox9基因表达.随着性腺的发育,Dmrt1蛋白主要定位于性腺Sertoli细胞的细胞核上,在雌性性腺发育过程中并未见其表达.此外,在雌二醇诱导的雄性转雌性性逆转胚胎性腺中,Dmrt1表达显著下调;在芳香化酶抑制剂诱导的雌性转雄性性腺中,Dmrt1表达则显著上升.上述研究表明,Dmrt1基因是中华鳖雄性特异性基因,参与雄性性腺的发育过程,可能在中华鳖早期性别决定中起重要的调控作用.     

小鼠性别决定过程中性腺体细胞分化的调控

哺乳动物的性腺由生殖细胞和体细胞共同形成,性别决定前的性腺具有双向分化的潜能,性腺中体细胞的分化决定其发育为睾丸或卵巢。这一分化过程受到多种因子的精细调控。其中SRY、SOX9、SOX3、SOX8、SOX10、FGF9/FGFR2、PGD2、AMH和DMRT1等参与睾丸的发育和分化,而FOXL2、CTNNB1、RSPO1、WNT4、Follistatin、ERα/β和BMP2则在卵巢发育过程中发挥关键作用。如果这些分子调控网络受到内源性或外源性因子的破坏,则会引起两性发育紊乱,甚至导致雄性向雌性或雌性向雄性的性别逆转。本文以小鼠模型为例,阐述了在性别决定过程中体细胞命运决定以及谱系分化的分子调控网络。     

奥利亚罗非鱼DMRT1和DMRT4抗体制备及组织表达谱分析

DMRT1和DMRT4是DMRT基因家族的成员,该家族成员与果蝇的性别决定基因和线虫性别决定基因一样,所编码的蛋白质都包含一个具有DNA结合能力的保守基序,即DM结构域,并以锌指结构与特异DNA序列相结合,在性别决定和分化发育中起调控作用。采用RT-PCR方法分别从奥利亚罗非鱼卵巢和精巢中扩增克隆出DMRT1和DMRT4全长cDNA片段,构建表达载体,在大肠杆菌中表达了BMP-DMRT4和BMP-DMRT1蛋白。经Xa切割、Amylose-sepharose柱层析纯化后作为抗原免疫新西兰白兔制备了DMRT1和DMRT4多克隆抗体,并进行纯化。对纯化多抗进行Western blot分析,结果表明获得了高特异性的DMRT1和DMRT4抗体。为了观察DMRT1和DMRT4在组织中的表达谱,首先,我们通过实时荧光定量RT-PCR检测雌雄奥利亚罗非鱼多种组织mRNA的表达,仅在卵巢和脑中检测到DMRT4,在精巢中检测到DMRT1;其次,制备了多种组织匀浆蛋白,使用纯化的抗体进行Western blot分析,仅分别在卵巢和精巢中检测到DMRT4和DMRT1蛋白的表达;制备多种奥利亚罗非鱼组织切片,使用纯化的DMRT4和DMRT1多抗进行免疫组织化学分析,发现DMRT4仅在卵巢表达,而DMRT1仅在精巢表达。这些结果有助于阐明DMRT4和DMRT1的功能及在鱼类性别调控中的作用。     

黄河鲤性别决定时间和相关基因表达研究

鱼类性别决定和分化机制极为复杂,通过性腺组织切片鉴定得出黄河鲤从未分化性腺发育为Ⅱ期精巢、卵巢的时间为受精后第40天到第80天。选取一些可能参与黄河鲤性别决定分化相关的基因（amh、ar、cyp19a、cyp19b、dax1、dmrt1、er、foxl2、nobox、sox9a、sox9b、zp2）进行实时荧光定量PCR分析各个基因在受精后40d、45d、50d、55d、65d和80d的表达情况。结果显示性别决定相关基因在50d都有高表达,推测45-50 d为性别决定的关键时间。ar、amh、dax1、dmrt1、sox9a、sox9b六个基因在80d雄性表达量升高,且雄性明显高于雌性,推测这些基因参与精巢分化发育过程。cyp19a、cyp19b、foxl2、nobox、zp2五个基因在80d雌性表达升高,且高于雄性,推测其可能参与卵巢分化发育。     

Sex determination in the chicken embryo.

The chicken embryo represents a suitable model for studying vertebrate sex determination and gonadal sex differentiation. While the basic mechanism of sex determination in birds is still unknown, gonadal morphogenesis is very similar to that in mammals, and most of the genes implicated in mammalian sex determination have avian homologues. However, in the chicken embryo, these genes show some interesting differences in structure or expression patterns to their mammalian counterparts, broadening our understanding of their functions. The novel candidate testis-determining gene in mammals, DMRT1, is also present in the chicken, and is expressed specifically in the embryonic gonads. In chicken embryos, DMRT1 is more highly expressed in the gonads and Müllerian ducts of male embryos than in those of females. Meanwhile, expression of the orphan nuclear receptor, Steroidogenic Factor 1 (SF1) is up-regulated during ovarian differentiation in the chicken embryo. This contrasts with the expression pattern of SF1 in mouse embryos, in which expression is down-regulated during female differentiation. Another orphan receptor initially implicated in mammalian sex determination, DAX1, is poorly conserved in the chicken. A chicken DAX1 homologue isolated from a urogenital ridge library lacked the unusual DNA-binding motif seen in mammals. Chicken DAX1 is autosomal, and is expressed in the embryonic gonads, showing somewhat higher expression in female compared to male gonads, as in mammals. However, expression is not down-regulated at the onset of testicular differentiation in chicken embryos, as occurs in mice. These comparative data shed light on vertebrate sex determination in general.     

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.