管角螺胚胎对卵黄蛋白利用规律及机制的研究

Reproduction and embryonic development are important research aspects of reproductive biology of shellfish, they also play a fundamental role in the areas of the shellfish exploitation and conservation activities. The tuba false fusus Hemifusus tuba (Gmelin) is one of the most commercially important shellfish resources which distributed along the southeastern coast of our country. The embryos of H. tuba hatch as benthic juveniles with the observation of nurse eggs, which exhibit no difference in appearance compared with normal eggs at laying, but the development of nurse eggs was aborted and they will be ingested by embryos as one of the important extracapsular nutrition resources. Vitellin is the main component of the yolk, which deposited in the oocyte during vitellogenesis as nutritional reserves for the development of oviparous embryos. The Ubiquitin Proteasomes Pathway (UPP) was proved to play an important role in the degradation of vitellin cleavage products of lower molecular weights. In this project, we plan to observe the ingestion process of nurse eggs, compare the change in vitellin content and molecular weights, analyse the degradation pattern of low molecular weight vitellin connected with ubiquitin, together with the expression level and location of relative genes in the UPP during the development of H. tuba embryos to investigate the pattern and mechanisms of vitellin utilization in H. tuba embryos, and serve as a foundation for further understanding the origin, evolution of nurse eggs and the reproductive strategy of nurse egg-producing species.

繁殖与胚胎发育是贝类生物学研究的重要内容，也是经济贝类开发利用和资源保护活动的基础。管角螺为浅海较大型的经济腹足类，在我国浙江、福建、广西、广东和海南各省的沿海都有分布。在管角螺胚胎的直接发生过程中有保育卵现象，保育卵在产出时外观与正常卵没有差别，但是并不发育并且会被卵囊中正常发育的胚胎摄入，成为胚胎发育重要的外部营养来源之一。卵黄蛋白(Vn)是卵黄的主要成分，泛素-蛋白酶体途径被证明在低分子量的卵黄蛋白裂解产物(Vp)的完全降解过程中发挥了重要作用。本项目拟从管角螺保育卵吸收过程的显微观察开始，通过比较胚胎不同发育阶段卵黄蛋白含量和分子量的变化、泛素化低分子量卵黄蛋白的分解规律，结合泛素-蛋白酶体途径相关基因表达量以及组织定位信息对胚胎卵黄蛋白营养的摄取/利用规律及机制进行研究，为进一步了解保育卵在胚胎发育中的产生和进化机制以及产保育卵亲本在繁殖阶段的策略及其影响因素奠定基础。

本项目运用荧光显微镜、普通光镜、组织切片和电镜技术对管角螺的受精细胞学及胚胎发育过程进行了观察，阐明了管角螺胚胎对保育卵的摄取方式和规律。观察发现管角螺同个卵荚内多个发育阶段共存，受精率约43.14%，发育率仅5.70%。不受精或者受精卵停止分裂的卵子成为保育卵，两者比率约为3:2。正常发育胚胎从第6d开始通过口凹吞食保育卵，吞食过程至第24d，被吞食的保育卵在体内呈块状。采用ELISA法检测不同发育阶段的管角螺胚胎卵黄蛋白含量发现，胚胎中Vn含量呈现先增后减趋势，6d时的卵黄蛋白含量达到最高峰。同时对胚胎发育的外环境-卵囊液成分进行了GC-TOF-MS 分析，结果表明卵囊液从一开始为单一的糖类物质，随着胚胎的发育降解为氨基酸单元，同时胚胎代谢物也排放其中，成分逐渐复杂，较难进行外环境模拟。泛素途径关键基因的QPCR检测结果显示，管角螺整个发育过程中，泛素(UBB)的大量合成发生在1-6d，之后相对表达水平较平稳。而HECT结构型的泛素连接酶在发育至18d的相对表达量达到最高，30d最低。两种泛素连接酶HT Ring和HT HECT中，Ring型表达水平相对较高。卵黄蛋白WB、泛素基因原位杂交及WB结果表明，刚产出的卵子中含有来自于母体的26kDa卵黄蛋白裂解产物，卵囊液中含有少量母源泛素；发育过程中，随着卵黄蛋白降解，呈现大小片段共存的现象，30d时泛素大面积存在于膜结构周围，保育卵已基本消化完，但胚胎发育1-6d卵黄蛋白的降解并不依靠泛素蛋白酶体途径(UPP)，UPP途径从第12天开始参与降解12,26,55,95kDa的卵黄蛋白，且在30天之前已完成卵黄蛋白的泛素化降解。