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Studies on the extracellular ubiquitin-proteasomes system involved in fertilization

细胞外泛素-蛋白酶体系统参与受精的研究

基本信息

批准号：
15390023
负责人：
SAWADA Hitoshi
金额：
$ 9.86万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

We have previously reported that the sperm ubiquitin-proteasome system plays a key role in fertilization, particularly in the process of sperm penetration of the vitelline coat, of the ascidian, Halocynthia roretzi. In this studies, we attempted to identify sperm enzymes involved in ubiquitination of the vitelline coat during ascidian fertilization. First, we tried to isolate the ubiquitin-conjugating enzyme complex from sperm exudate, a supernatant fraction upon sperm reaction or sperm activation. However, the amount of the purified enzyme was not sufficient for the analysis of the N-terminal sequence of the isolated enzymes.Then, we were interested in the cognate enzymes or proteins expressed in the ascidian Ciona intestinalis, since the draft genomic DNA sequence in this species has already been published. In order to identify ubiquitin lygases involved in ascidian fertilization, we explored a RING-domain-containing proteins among gene models exressed in C.intestinalis testis. We se … More lected 2 gene models, which were highly expressed in testis as revealed by RT-PCR. One (ci100130063) of the gene models with a molecular mass of 86 kDa contains a TRP domain and a LON doman in addition to a RING domain. The other gene model (ci100137400) with a molecular mass of 57 kDa contains a FERM domain in addtition to a RING domain. In order to investigate the biological functions of these proteins during fertilization, polyclonal antibodies were raised in rabbits against the respective fusion proteins, which were expressed in E.coli. By using these antibodies, we found that the extent of the expression level of these proteins in C.intestinalis sperm appears to be low and that there are several cross-reacted bands in sperm of C.intestinalis. On the other hand, we examined the cross-reactivity toward the proteins expressed in sperm of Halocynthia roretzi, and we found that there are cross-reactable bands in H.roretzi sperm, implying that RING-domain-containing E3-like protein may be expressed in H.roretzi sperm and may be involved in ascidian fertilization.In addition to investigate candidate E3-like proteins, we explored E2-like ubiquitin-conjugating enzymes in C.intestinalis gene models. We revealed that several E2-like proteins are highly expressed in testis of this species. Further studies are necessary to clarify whether these proteins are involved in fertilization of the ascidian C.intestinalis and also whether the congate enzymes involved in fertilization are expressed in H.roretzi sperm. Less

我们以前报道过精子泛素-蛋白酶体系统在受精过程中起关键作用，特别是在精子穿透海鞘（Halocynthia roretzi）卵黄膜的过程中。在本研究中，我们试图鉴定在海鞘受精过程中参与卵黄被泛素化的精子酶。首先，我们试图从精子渗出液中分离出泛素结合酶复合物，这是精子反应或精子活化后的上清部分。然而，纯化酶的量不足以分析分离酶的n端序列。然后，我们对海鞘中表达的同源酶或蛋白质感兴趣，因为该物种的基因组DNA序列草案已经发表。为了确定参与海鞘受精的泛素气体，我们在c.n testinalis表达的基因模型中探索了一个含有ring结构域的蛋白。我们选择了2个基因模型，经RT-PCR证实，这2个基因模型在睾丸中高表达。其中一个分子量为86 kDa的基因模型（ci100130063）除了含有一个RING结构域外，还含有一个TRP结构域和一个LON结构域。另一个分子量为57 kDa的基因模型（ci100137400）除了含有一个RING结构域外，还含有一个FERM结构域。为了研究这些蛋白在受精过程中的生物学功能，我们在家兔中培养了针对这些融合蛋白的多克隆抗体，这些融合蛋白在大肠杆菌中表达。利用这些抗体，我们发现这些蛋白在c.n testinalis精子中的表达水平似乎很低，并且在c.n testinalis精子中存在几个交叉反应带。另一方面，我们对海鞘鞘精子中表达的蛋白进行了交叉反应性检测，发现海鞘鞘精子中存在可交叉反应的条带，提示海鞘鞘精子中可能表达含有ring结构域的e3样蛋白，并可能参与海鞘受精。除了研究候选的e3样蛋白外，我们还在大肠杆菌基因模型中探索了e2样泛素结合酶。我们发现几个e2样蛋白在该物种的睾丸中高度表达。这些蛋白是否参与海鞘c.n untinalis的受精，以及参与受精的聚合酶是否在H.roretzi精子中表达，还需要进一步的研究。少

项目成果

期刊论文数量（19）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

New impact on protein modifications in the regulation of reproductive system

蛋白质修饰对生殖系统调节的新影响

DOI：
发表时间：
2007
期刊：
影响因子：
0
作者：
Satoh;A.;Sakamaki;K.;Ide;H.;Tamura;K.;Celina Haraguchi;Toshinobu Tokumoto;Celina Haraguchi;Toshinobu Tokumoto;Celina Haraguchi;Toshinobu Tokumoto;Mika Tokumoto;Ryo Horiguchi;Yuki Ohsaki;Mika Tokumoto;Mika Tokumoto;Ryo Horiguchi;Yuki Ohsaki;Mika Tokumoto;Mika Tokumoto;Mika Tokumoto;Ryo Horiguchi;Ryo Horiguchi;Norihiko Nakamura;Toshinobu Tokumoto;Ryo Horiguchi;Ryo Horiguchi;Norihiko Nakamura;Toshinobu Tokumoto;Ryo Horiguchi;Toshinobu Tokumoto 編
通讯作者：
Toshinobu Tokumoto 編

N.Sakai, M.T.Sawada, H.Sawada: "Non-traditional roles of ubiquitin-proteasome system in fertilization and gametogenesis"International Journal of Biochemistry and Cell Biology. 36. 776-784 (2004)

N.Sakai、M.T.Sawada、H.Sawada：“泛素蛋白酶体系统在受精和配子发生中的非传统作用”国际生物化学和细胞生物学杂志。