【作者】 方弟安；

【导师】 王群；

【作者基本信息】 华东师范大学 ， 水生生物学， 2012， 博士

【摘要】 生殖生物学是生物学研究最为重要的内容之一,它涉及到许多重大的生物学问题,比如生殖细胞的发生、性别决定、遗传、发育和进化等,这些生物学过程的发生机制一直倍受国内外研究者的关注。目前对于生殖细胞发生的分子机制研究主要集中在人类和一些常见的模式生物中,而对于水生生物,在甲壳类虾蟹动物中,相关的生殖分子生物学研究报道较少,尤其是雄性生殖细胞发生的分子机制甚少。淡水红螯光壳螯虾原产于澳大利亚,自1985年以来逐渐成为一种水产养殖业的重要品种。红螯螯虾已经成为许多包括澳大利亚,美国,中国,厄瓜多尔,墨西哥和阿根廷等国家集约化或半集约化养殖的品种。目前对其雄性生殖生物学的研究仅仅局限于生殖系统的解剖结构和精子形成的生理过程相关研究,其精子发生的分子机制,已经成为雄性红螯螯虾生殖生物学研究迫在眉睫的热点问题。因此,本论文以淡水红螯光壳螯虾(Cherax quadricarinatus)为研究对象,开展精子发生相关分子机制的研究。本研究的主要内容包括：(1)、三个精子发生相关基因DDX5、HSP70和Prohibitin基因的分子克隆和基因表达谱分析；(2)、单侧摘除眼柄对红螯螯虾繁殖性能和DDX5基因表达谱的影响；(3)、免疫组织化学染色技术对精巢内HSP70蛋白进行了组织定位；(4)、免疫荧光染色技术对精巢内Prohibitin蛋白在精子发生过程中的定位研究；(5)、Western Blot免疫印迹方法对红螯螯虾精巢发生各个时期HSP70和Prohibitin蛋白进行了蛋白印迹的表达分析；(6)、对Prohibitin进行免疫胶体金实验,证实其在红螯螯虾精子线粒体膜蛋白中生物学功能,并探讨无脊椎动物的线粒体遗传机制。本研究的主要结果如下：1、通过同源克隆策略克隆了红螯光壳螯虾性腺DDX5、HSP70和Prohibitin的cDNA片段长度分别为368bp,493bp,302bp;通过RACE PCR技术获得了三个基因的全长序列分别为2,258bp,2,366bp,1,472bp;分别含有编码522(1,569bp),652(1,959bp),275(828bp)个氨基酸(amino acid, aa)的开放阅读框。2、BLAST分析发现,Cq-DDX5、Cq-HSP70和Cq-PHB都是高度保守的蛋白家族成员,Cq-DDX5蛋白与其他物种的同质体具有53-90%的同源性；Cq-HSP70与其他物种HSP70的同源性为73-95%；Cq-PHB与其他物种的PHB的同源性为55-92%。分别以各自蛋白家族成员的氨基酸序列为分子标记,用MEGA4.0软件中的邻接NJ法构建系统进化树均发现,红螯螯虾与无脊椎动物的虾蟹类亲缘关系最近,这些结果都支持了传统的进化理论。3、多序列比对和InterPro蛋白结构功能研究发现,Cq-DDX5所编码的蛋白含有典型的DEAD-box蛋白家族的9个保守结构,DEAD-box RNA解旋酶特有的Q基序和DEAD/DEAH结构域。Cq-HSP70蛋白序列中含有三个HSP70家族基本结构域,多个潜在的磷酸化位点等结构域,1个钙离子结合结构域,1个富含氨基乙酸区域,1个伴肌动蛋白重复结构和1个双向的核定位信号。然而Cq-PHB蛋白的一级结构缺乏典型的信号序列,核酸结合位点,ATP结合位点和相关的转录因子,只有8个高度保守的类似区域和4个与转录相关的控制蛋白基序。4、利用RT-PCR和实时荧光定量qRT-PCR技术进行各基因的组织表达谱分析发现,Cq-DDX5、Cq-HSP70和Cq-PHB在所有检测的各个机体组织中都有表达,在性腺等高分化的组织内表达量较高。实时荧光定量qRT-PCT分析各基因转录本的表达谱分析结果如下：(1)、在胚胎发育过程中,Cq-DDX5的表达量在受精卵期最低,在卵裂期迅速显著上升,在原肠期维持较高水平,到无节幼体期达到最高水平,在一龄幼体期也维持在较高的水平。在精巢发育各期中,其表达量在不育期和休止期非常低,在精巢发育期表达量最高,在休止期表达量最低。在雄性红螯螯虾年/季节的生殖周期中,在交配前期其转录本达最高并在交配期维持较高水平,之后逐渐下调至交配后期和休止期显著下降。眼柄摘除后,从0d到3d其表达量逐渐增加,在第6d明显上升,到12d达到峰值,到18d显著下降。(2)、Cq-HSP70mRNA的表达量在精巢发生期最高,并且在精子发生阶段高表达,在精巢发生成熟阶段,其表达量明显下调,并且在休眠时期/休止期一直维持显著的低表达水平(P<0.05)。在季节/年生殖周期中,在预备期(1月到3月),交配前期(4月)和交配期(5月到8月)与交配后期(9月到10月)/休止期(10月到次年1月)相比发现具有较高的表达量(P<0.05)。(3)、在整个红螯螯虾精巢发生周期中都可见Cq-PHB mRNA转录本的表达,其中在精巢发生期的表达量显著升高,到增殖期达到最高,在精巢发生的成熟阶段其表达量明显下调,并且在休眠时期/休止期维持较低的表达水平(P<0.05)。5、Western Blot结果发现蛋白提取物可与HSP70抗体免疫结合,在免疫印迹膜上有一条特异性条带,条带大小接近70kDa,与预测的Cq-HSP70的分子质量相匹配。免疫组织化学染色技术分析发现在精巢内各个阶段的生殖细胞中包括精母细胞,精细胞和成熟的精子等都可观察到HSP70的阳性信号,在精原细胞和初级精母细胞中信号最强,在次级精母细胞中信号较低,在成熟的精子细胞中信号很弱甚至没有,而且Cq-HSP70蛋白信号主要存在于发生中生殖细胞的细胞质中。6、从精巢提取的蛋白通过聚丙烯酰胺凝胶电泳SDS-PAGE分离后,Western Blot检测后结果发现蛋白提取物可与Prohibitin抗体免疫结合,在免疫印迹上有些明显的Cq-PHB大小不同的梯度条带,分子量大小在180kDa到30kDa不等。通过免疫荧光染色技术分析发现Cq-PHB蛋白的在精巢内各个阶段的生殖细胞中都可观察到Cq-PHB蛋白的阳性信号。其中最强信号出现在精巢发生期和增殖期的精原细胞和初级精母细胞中,在次级精母细胞中信号较低,在成熟的精子细胞中信号很弱。通过进一步的组织学鉴定发现,Cq-PHB蛋白主要存在于发生中生殖细胞的细胞质和生殖细胞周围。取精巢不同发育时期的生殖细胞,通过胶体金免疫电镜技术研究发现,免疫胶体金颗粒主要分布在精子细胞的线粒体内膜上,在精子细胞内还可见一些大的胶体金颗粒和一些游离的胶体金颗粒。通过以上研究结果可见,Cq-DDX5、Cq-HSP70和Cq-PHB分别是’DEAD-box’蛋白家族、HSP超家族和Band-7家族蛋白中新成员在红螯螯虾的首次报道。Cq-DDX5和其他物种的DDX5一样具有促进细胞分化的功能,我们的研究还证实了DDX5在红螯螯虾个体发育和精子发生过程中具有必要的促进或催化作用。在红螯螯虾精子发生过程中Cq-HSP70mRNA转录本表达水平的变化与精巢中各个时期的精子的HSP70免疫信号相一致。这些结果表明,Cq-HSP70蛋白在精子发生的起始阶段对于精母细胞的分化过程和精子细胞的形成过程是必不可少的,其可能通过调控生殖细胞分裂和分化过程中的胞质重组而调节红螯螯虾的精子发生过程,精子中HSP70蛋白的表达可能与红螯螯虾精子发生效率和精子质量密切相关。在红螯螯虾中Cq-PHB的研究结果表明,Cq-PHB参与了红螯螯虾精子发生的调控过程和精子发生的泛素化过程,不同大小的ladder条带提示PHB可能与不同大小的泛素分子结合参与精子发生的泛素化过程,而且Cq-PHB可能和在其他无脊椎动物的功能一样作为泛素的底物而参与其中的生物学过程。结合胶体金的实验还可以推断,PHB在甲壳动物甚至在无脊椎动物中可能也存在类似的参与线粒体母系遗传的生物学机制。综上所述,本研究首次在红螯螯虾中报道了精子发生密切相关的三个功能基因,分析了它们的基因结构,表达谱和相关蛋白的组织定位,这些研究方法对于进一步深入研究甲壳类生殖相关分子机制具有重要的借鉴意义,该研究的结果将充实并推动甲壳类生殖相关内容研究的进一步深化。这些研究成果将为虾类生殖分子生物学研究提供有用的信息并为后续有关无脊椎动物精子发生的分子生殖生物学研究奠定了坚实的研究基础。更多还原

【Abstract】 Reproductive biology is one of the most important researches in biology, it involves many important biological problems, such as germ cells ontogenesis, development, sex determination, genetics and evolution, these mechanisms of biological processes has been arisen many researchers’concerns at home and abroad. Currently the molecular mechanism for germ cell ontogenesis mainly focused on humans and other common model animals, and there are little related molecular mechanism reports on the aquatic organism especially in crustaceans shrimps and crabs, and the molecular mechanisms on male germ cells is even rare. Therefore in our present study, we selected the freshwater crayfish Cherax quadricarinatus, which is a commercially important aquatic organism, as the experimental materials and to study the molecular mechanism on ontogenesis and spermatogenesis.The "red claw" crayfish Cherax quadricarinatus (von Martens,1898)(Astacida, Parastacidae) is an important species for culture since1985for consumption and acquarism. At present, it is cultured intensively and semi intensively in many countries including Australia, United States, China, Ecuador, Mexico and Argentina. As C. quadricarinatus is concerned, the knowledge about the reproductive biology of males is limited to the anatomy of the reproductive system and formation and transference of the spermatophore. Therefore, a better understanding of the molecular mechanism of spermatogenesis has become a research priority at the moment.Hence in this paper, the major contents are as follows:(1) molecular cloning of three spermatogenesis closely related genes DDX5, HSP70, Prohibitin, and their expression patterns at different spermatogenesis phase;(2) reproduction performance and temporal expression patterns of Cq-DDX5after unilateral eyestalk-ablation;(3) localization of the Cq-HSP70protein in testes was studied by immunohistochemical analyses;(4) immunofluorescence analysis on localization of the Cq-PHB protein in testes (5) HSP70and Prohibitin protein expression analysis on different testes development stage by Western Blot; and (6) localization of Cq-PHB in sperm mitochondria by immunogold electron microscopy to find out its biological function in mitochondria membrane proteins and explore the mitochondria maternal inheritance mechanism in invertebrate.The mainly results of this paper are as following:1. Using degenerated PCR amplification strategy, DDX5, HSP70and Prohibitin cDNA fragments were cloned from the crayfish testes, and their length are368bp,493bp,302bp respectively. After RACE PCR,2,258bp,2,366bp,1,472bp full-length cDNA of DDX5, HSP70and Prohibitin are obtained, which containing the open reading frame (ORF) encoded522aa (1,569bp),652aa (1,959bp),275aa (828bp) respectively.2. BLAST search against the databases revealed that the deduced amino acid sequence shows high similarity to other similar protein sequence. Cq-DDX5, Cq-HSP70and Cq-PHB are highly conserved multigene superfamily. The deduced amino acid sequence of Cq-DDX5has a53-90%similarity to DDX5of other eukaryotic species, and Cq-HSP70has73-95%similarity with others, and Cq-PHB has55-92%similarity. A neighbor-joining (NJ) phylogenetic tree was constructed using MEGA software version4.0. Phylogenetic analysis results also showed that C. quadricarinatus clustered with other invertebrates, and most closely to that of the gene products encoded by other shrimp or crab species, which supports the traditional taxonomic relationships.3. Using InterPro searches the predicted amino acid sequences showed:the Cq-DDX5comprise nine consensus sequence characteristic of the DEAD-box proteins, Q-motif of dead-box RNA helicase profile and DNA/RNA helicase (DEAD/DEAH box) domain. Three HSP70family signature motifs were identified in the Cq-HSP70protein sequence, many potential phosphorylation sites, a calcium-binding domain profile (EF-HAND-2), a glycine-rich region profile motif (GLY-RICH), a nebulin repeat profile (NEBULIN) and a bipartite nuclear localization signal profile (NLS-BP) were also identified. But the primary structure of Cq-PHB protein lacks motifs typical for signal sequences, nuclear localization signals, ATP-binding sites or transcription factors. The comparison between PHB genes in other species discovers eight highly conserved regions with four of them corresponding to binding sites of known transcriptional control proteins (CCAAT box,’SV40’sites and two Spl sites) were identified in the Cq-PHB protein sequence.4. The mRNA transcript of Cq-DDX5, Cq-HSP70and Cq-PHB were expressed universally in all the organs investigated, including the brain, eyestalk, gill, thoracic ganglia, heart, muscle, hemocytes, hepatopancreas, stomach, intestine, testes and ovaries. Expression was at the high level in high differentiation organizations such as gonads, hepatopancreas and hemocytes. Further, the temporal expression patterns on each gene transcripts are as follows:(1). During the course of embryonic development, the expression of Cq-DDX5is very low in the fertilized egg stage but significantly up-regulate at the cleavage stage, and maintain at high level at gastrula stage and sharply up-regulate to peak at the nauplii stage and maintained at high level even in one-day larvae stage. In testes developmental period, the expression of Cq-DDX5was significantly low during absence period and resting phases when compared to the developmental period and multiplication phase, and a highest level was seen at the developmental phase and a lowest level at the resting level. In the mature male testes seasonal/annual cycle, the Cq-DDX5transcript up-regulate sharply and peak at the prespawning phase and maintain high level at the spawning phase, and then down-regulate and decline drastically during post-spawning/regressed phase. After eyestalk ablation, the relative testicular weight of destalked males was higher than that of intact males, but decreased sharply after the second week and these differences were not statistically significant. Interestingly, a gradual increase in Cq-DDX5expression is seen from0d to3d time interval, and significantly increase at6d and peak at12d after eyestalk ablation treatment group then declined drastically at18d.(2). In the testes developmental cycle, Cq-HSP70mRNA was up-regulated to peak expression during the developmental phase, and high expression was maintained during the spermatogonial multiplication period. Then the expression was down-regulated during the mature sperm stage, and significantly lower expression levels were found during the resting phase (P<0.05). In the seasonal/annual reproductive cycle, significantly greater expression (P＜0.05) was found in the preparatory (January-March), pre-spawning (April) and spawning (May-August) phases compared with the post-spawning/regressed phases (November-January). Overall, Cq-HSP70mRNA transcripts were maintained at high levels during the spermatogenesis and spawning stages.(3). In the testes developmental cycle, Cq-PHB mRNA was up-regulated during the developmental phase, and high expression was maintained during the spermatogonial multiplication period. Then the expression was down-regulated during the mature sperm stage, and lower expression levels were found during the resting phase (P<0.05).5. Western Blot results showed that a distinct single band characteristic of Cq-HSP70was observed on the immunoblot when the protein extracts were transferred to a nitrocellulose membrane and immunoprobed with anti-HSP70. The anti-HSP70recognized bands of approximately70kDa, which matches well with the calculated molecular mass for CqHSP70. Furthermore the Immunohistochemistry (IHC) results showed that immunoreactive positive signals (in brown color) for the HSP70protein were detected in spermatocytes, spermatids and spermatozoa of normal mature testes. Within the testes, the strongest signals for Cq-HSP70were found in spermatogonia, with lower positive signals in secondary spermatocytes, and weak or absent signals in mature sperm. Moreover, the Cq-HSP70protein was concentrated mainly in the cytoplasm of developmental sperm cells.6. When the protein extracts were transferred to a nitrocellulose membrane and immunoprobed with anti-PHB, a series of ladder bands characteristic of Cq-PHB was observed on the immunoblot. The anti-PHB recognized bands of approximately from180kDa to30kDa. Further the Immunofluorescence (IF) results showed that immunoreactive positive signals (blue fluorescence) for the PHB protein were detected in spermatocytes, spermatids and spermatozoa of normal mature testes. Within the testes, the strongest signals for Cq-PHB were found in spermatogonia, with lower positive signals in secondary spermatocytes, and weak or absent signals in mature sperm. Moreover, the Cq-PHB protein was concentrated mainly in the cytoplasm of developmental sperm cells and the peripheral’sertoli cells’. The Immunogold Electron Microscopy (IEM) results showed that Prohibitin was found mainly on the mitochondrial inner membrane of the spermatozoa, with some labeling in the matrix and free mitochondria particles. Prohibitin immunoreactivity was also observed on the spermatozoa nucleus but not any signals in negative preparations with omission of first antibody.Taken together the above results, we first reported Cq-DDX5, Cq-HSP70and Cq-PHB are new members in’DEAD-box’protein family, HSP superfamily and Band-7protein family respectively. Our findings demonstrated that Cq-DDX5might have an essential role in ontogenesis and spermatogenesis. The Cq-HSP70mRNA transcript levels changed during the sperm development process, which was in accordance with the immunoreactive signals of Cq-HSP70in the testes. These observations suggest that Cq-HSP70is critical for spermatogonial multiplication and spermatogenesis. The high levels of Cq-HSP70mRNA observed in the spermatogonial multiplication period may imply that Cq-HSP70is also an essential promoter in crayfish spermatogenesis, in turn, could be involved in high spermatogenic efficiency, which would support earlier findings in vertebrates. The results in Cq-PHB showed that it is related to the maternal inheritance of mtDNA and Cq-PHB also has a similar biological function involved in ubiquitination process in crustacean even in the invertebrate animals.Overall, this study provides the forwarding step towards understanding molecular mechanism about ontogenesis and spermatogenesis in marine invertebrates, which will contribute towards improving the quality and quantity of aquaculture. In the future, the molecular mechanism(s) linking DDX5or HSP70functions to spermatogenesis and ontogenesis need to be determined, particularly if these genes are to be exploited as a molecular biomarker in further studies of development. As to the Cq-PHB’s function, it is also therefore necessarily to find out the mitochondrial inheritance mechanism in invertebrate animals for further research.更多还原