Studies on the extracellular ubiquitin-proteasomes system involved in fertilization

我们以前曾报道过精子泛素-蛋白酶体系统在受精过程中起着关键作用，特别是在海鞘中精子穿透卵黄外壳的过程中。在这项研究中，我们试图确定在海鞘受精过程中与卵黄外壳泛素化有关的精子酶。首先，我们试图从精子渗出物中分离泛素结合酶复合体，精子渗出物是精子反应或精子激活的上清部分。然而，纯化的酶的量不足以分析分离的酶的N-末端序列。于是，我们对在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序列草案已经公布。为了鉴定与海鞘受精相关的泛素裂解气体，我们在肠圆线虫睾丸表达的基因模型中探索了一个含有环状结构域的蛋白质。我们看到了…共筛选出2个基因模型，RT-PCR结果显示其在睾丸中高表达。其中一个基因模型(Ci100130063)的分子质量为86 kDa，除环区外，还含有Trp结构域和LON结构域。另一个基因模型(Ci100137400)的分子质量为57 kDa，除一个环状结构域外，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，在兔体内制备了针对各自融合蛋白的多克隆抗体，并在大肠杆菌中进行了表达。通过使用这些抗体，我们发现这些蛋白在肠衣原体精子中的表达水平似乎很低，并且在肠衣原体精子中存在几条交叉反应条带。另一方面，我们检测了罗氏梭鱼精子中表达的蛋白质的交叉反应性，我们发现在罗氏梭鱼精子中存在可交叉反应的条带，这意味着含有环状结构域的E3蛋白可能在罗氏梭鱼精子中表达，并可能参与海鞘受精。除了研究候选的E3蛋白外，我们还探索了肠衣原体基因模型中的E2样泛素结合酶。我们发现，在该物种的睾丸中，有几个类E2蛋白高度表达。这些蛋白是否参与了肠囊虫的受精，以及参与受精的凝固酶是否在罗氏海鞘精子中表达，还需要进一步的研究。较少

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