Studies on the extracellular ubiquitin-proteasomes system involved in fertilization

我们以前曾报道，精子泛素-蛋白酶体系统在受精过程中起着关键作用，特别是在精子穿透卵黄层的过程中，海鞘，Halocynthia roretzi。在这项研究中，我们试图确定精子酶参与泛素化的卵黄外套在海鞘受精。首先，我们试图从精子渗出液中分离泛素结合酶复合物，精子反应或精子活化后的上清液部分。然而，纯化的酶的量不足以分析分离的酶的N-末端序列。然后，我们感兴趣的同源酶或蛋白质表达的海鞘海鞘，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有关的泛素糖基化酶，我们在海鞘精巢表达的基因模型中探索了一个含有RING结构域的蛋白。我们认为 关于我们 筛选出2个基因模型，经RT-PCR证实在睾丸组织中高表达。其中一个基因模型（ci 100130063）具有86 kDa的分子量，除了RING结构域之外还含有TRP结构域和LON结构域。另一个基因模型（ci 100137400）的分子量为57 kDa，除RING结构域外，还含有一个FERM结构域。为了研究这些蛋白质在受精过程中的生物学功能，在兔中对各自的融合蛋白，这是在大肠杆菌中表达的多克隆抗体。通过使用这些抗体，我们发现，这些蛋白质的表达水平的程度似乎是低的，并有几个交叉反应的条带在精子中的C. acidinalis。另一方面，我们检测了与罗氏盐辛西娅精子中表达的蛋白质的交叉反应性，我们发现罗氏盐辛西娅精子中存在交叉反应条带，这意味着含有RING结构域的E3样蛋白可能在罗氏盐辛西娅精子中表达，并且可能参与海鞘受精。我们探索了在C. tumor基因模型中的E2样泛素缀合酶。我们发现，几个E2样蛋白在该物种的睾丸中高度表达。进一步的研究是必要的，以澄清这些蛋白质是否参与受精的海鞘C. roretzi，以及是否congate酶参与受精的表达在H.roretzi精子。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