哺乳动物受精过程中精子和透明带的初级及次级结合

在哺乳动物的受精过程中,获能的精子穿过卵丘细胞层到达卵子透明带后,精子头部便结合在透明带的表面上.精子和透明带的结合可以分为初级结合和次级结合两个阶段,初级结合是指二者在顶体反应之前的结合,次级结合是指二者在顶体反应之后的结合.精子和透明带的结合是精子顶体反应和精子入卵的先决条件,是哺乳动物受精过程中非常重要的环节,涉及到精子以及透明带的多种蛋白质分子之间的相互作用.这方面的研究工作主要是以小鼠为实验对象进行的,在其他哺乳动物中也有很多报道.本文将主要以小鼠为例,概述哺乳动物受精过程中精子和透明带结合的分子机制,并兼述在二者的结合过程中透明带糖蛋白ZP3对精子顶体反应的诱导作用.     

人卵透明带蛋白ZP3a和ZP3b肽段的免疫原性及其抗血清体外抑制人精子-半透明带结合

分析大肠杆菌表达的重组人卵透明带-3（r-huZP3）蛋白两个肽段（r-huZP3a^22-176及r-huZP3b^177-348）的免疫原性,比较两者抗血清体外抑制人精子-半透明带结合的能力。以制备性聚丙烯酰胺凝胶电泳（PAGE）纯化的大肠杆菌表达蛋白为抗原,主动免疫新西兰兔产生抗huZP3a^22-176及huZP3b^177-348抗血清：通过ELISA测定比较两者的抗体应答水平;以蛋白印迹和免疫组化方法测定两者抗血清同重组表达蛋白、大然人卵ZP以及卵巢组织的反应性;通过竞争性半透明带结合试验（hemizona assay,HZA）观察两者抗血清体外抑制人精子-ZP结合能力。结果显示：未与人分子蛋白载体耦联的r-huZP3a^22-176和r-huZP3b^177-348抗原都在免疫兔中产生了较高的抗体滴度,而且它们的抗血清可识别人肠杆菌表达的各自重组ZP3肽以及人卵细胞表面天然ZP,两者抗血清也都能在体外抑制人精子-ZP结合。由此可见,r-huZP3^22-176及r-huZP3b^177-348蛋白具有良好免疫原性,所产生抗血清也都显示出细胞和组织特异性。因此,单一或合并两个huZP3肽段均可作为抗原研制检测不明原因性不孕妇女中是否存在抗自身ZP抗体的诊断试剂盒,另外它们的抗血清也能用于鉴定已知huZP3表位肽段的最小B-细胞表位基序。     

猪精子中与卵透明带糖蛋白ZP3结合的蛋白质

依次经ＰＳＬ－Ｓｅｐｈａｒｏｓｅ亲和层析柱和纤维素ＣＭ－５２离子交换层析柱,从猪精子的ＣＨＡＰＳ抽提液分离得４个蛋白质组分。用固相透明带精蛋白结合试验（ＩＺＰＧＢＡ）检测;表明精子蛋白ＳＰ１和ＳＰ２具有结合透明带糖蛋白ＺＰ３的活性,ＳＰ２并显示凝集血球的活性。精子蛋白ＳＰ１与卵预温育明显抑制精卵结合,抑制活性与加入的精子蛋白的浓度呈正相关。用生物素标记的ＺＰ３和蛋白质印迹技术,证明ＳＰ１中的６８ｋＤ精子蛋白与ＺＰ３结合,提示６８ｋＤ精子蛋白参与精卵结合。     

重组人卵透明带ZP3蛋白在毕赤酵母中的表达

利用P.pastoris表达人卵透明带ZP3蛋白。设计特定引物从全长hZP3 cDNA上扩增含跨膜区序列的人卵透明带ZP3基因片段,并在N末端接上串联组氨酸编码序列的重组基因序列;扩增片段插入表达载体pPIC9K中;线性化后的重组质粒转入P.pastoris中,用高浓度G418筛选高拷贝菌株,然后甲醇诱导目的蛋白表达。用SDS-PAGE和Western blot分析表达产物。结果发现P.pastoris表达的人ZP3蛋白可以分泌到培养液中,并且可溶性好。纯化前后的重组人ZP3蛋白均能与兔抗猪ZP3蛋白抗体发生交叉反应,证实表达的目的蛋白具有反应原性。     

Cloning of rat sp56,the homologue of mouse sperm ZP3 receptor—sp56

Mouse sp56 is considered as one of the candidates for mouse zona pellucida 3(mZP3)receptor,Up to date,its homologue has only been cloned from guinea pig,namely,AM67.Based on the cDNA sequence of mouse sp56,we designed a pair of primer to amplify its homologue from rat testis cDNA.Using RT-PCR, two tragments of 743 bp and 938 bp were amplified.The PCR products show very high homology to mouse sp56.However,the 743 bp product completely lacks one of the seven Sushi domains of mouse sp56.Using the 743 bp product as the probe to detect the expression profile of sp56 in rat tissues,Northern blot shows that a-2.0kb mRNA expresses specifically in testis.Employed the RACE method,two full cDNA sequences of rat sp56 were obtained.A Mr-42KD band was detected in denatured and non-reducing protein sample of rat testis and sperm with anti-mouse sp56 monoclonal antibody by Western blot method.Rat sp56 was localized on rat sperm head by the indirect immunofluorescence method.Rat sp56 immunoreactivity was detected from the early pachytene spermatocytes and throughout the spermatogenesis.Its cloning will further our understanding of the mechanism of the sperm-egg recognition and binding.     

N—糖链在纤连蛋白分泌及纤连蛋白受体与配体结合的作用

应用能断糖蛋白Ｎ－糖链合成的衣霉素（ＴＭ）,研究了Ｎ－糖链缺失对ＨＴ１０８０细胞分泌纤连蛋白（Ｆｎ）以及纤连蛋白受体（ＦｎＲ）与配体结合的影响。结果表现,１μｇ／ｍｌ的ＴＭ可抑制Ｎ－糖链的合成（此时,３Ｈ－甘露糖掺入下降６３％）,但细胞分泌Ｆｎ的量仅下降３％,这主要是由于蛋白合成受ＴＭ抑制（２５％）而引起。因而,Ｎ－糖链缺失可能并不影响Ｆｎ的分泌,而在同样条件下,单个细胞结合１２５Ｉ－Ｆｎ的量显著     

卵透明带ZP3的研究及其应用

卵透明带蛋白围绕在卵母细胞外,在受精过程中起着重要作用。ZP3蛋白是卵透明带蛋白家族中的重要成员,在功能上,ZP3作为初级精子受体,起始精卵结合和顶体反应。由于ZP3在受精中的重要作用,它成为免疫避孕的有效靶点。ZP3蛋白疫苗和DNA疫苗可以诱导机体产生较强的免疫反应,导致生育降低,同时带来一定程度的副作用。本文重点介绍了ZP3的免疫特性及其应用。     

转铁蛋白糖链结构对其受体亲和性的影响

用伴刀豆凝集素,小扁豆凝集素和欧曼陀罗凝集素亲和层析法,分别从正常人血清转铁蛋白和孕妇血清Ｔｆ中获得二天线糖链Ｔｆ和多天线糖链的Ｔｆ,用于研究其对胎盘中纯化得到的转铁蛋白受体和亲和性。经Ｓｃａｔｃｈａｒｄ作图结果发现,表明含多天线糖链的Ｔｆ对受体的亲和性比二天线糖链的Ｔｆ降低１倍,而ＴｆＲ的结合位点数不变。     

O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity

Previously, we reported that ZP3, one of three different glycoproteins present in the mouse egg's zona pellucida, serves as a sperm receptor. Furthermore, small glycopeptides derived from egg ZP3 retain full sperm receptor activity, suggesting a role for carbohydrate, rather than polypeptide chain in receptor function. Here, we report that removal of O-linked oligosaccharides from ZP3 destroys its sperm receptor activity, whereas removal of N-linked oligosaccharides has no effect. A specific size class of O-linked oligosaccharides, recovered following mild alkaline hydrolysis and reduction of ZP3, is shown to possess sperm receptor activity and to bind to sperm. The results presented strongly suggest that mouse sperm bind to eggs via O-linked oligosaccharides present on ZP3.     

牦牛ZP3基因的克隆及蛋白质结构的预测

本研究对牦牛ZP3基因的编码区进行了克隆,在此基础上对ZP3蛋白的分子结构预测,为研究牦牛受精生物学提供基础。根据GenBank中普通牛的ZP3核苷酸序列设计特异性引物,以牦牛卵巢组织总RNA为模板,通过RT-PCR技术扩增牦牛ZP3基因cDNA序列(GenBank登录号为GQ856646),利用DNAMAN生物软件进行核苷酸和氨基酸序列分析、蛋白质专家系统ExPASy进行ZP3蛋白质分子结构预测。结果表明,扩增出的牦牛ZP3基因编码序列长1 266 bp,编码421个氨基酸。牦牛与牛、猪、狗、人、鼠和鸡ZP3基因核苷酸相应序列的同源性分别为98.42%、96.73%、79.67%、78.71%、69.15%和56.61%,氨基酸同源性分别为98.10%、83.85%、74.24%、70.26%、62.62%和46.12%,符合物种进化规律。预测的ZP3蛋白二级和三级结构显示它是一个具有22个氨基酸信号肽的亲水性β-桶状跨膜蛋白。牦牛ZP3基因编码区的成功克隆,为进一步研究该基因的结构与功能及其在受精过程中的作用提供了基础。     

Porcine zona pellucida glycoprotein ZP4 is responsible for the sperm-binding activity of the ZP3/ZP4 complex

Summary The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-egg interactions. Porcine and bovine ZPs consist of glycoproteins ZP2, ZP3, and ZP4. In both pig and bovine a heterocomplex consisting of ZP3 and ZP4 binds to sperm, however it is not clarified whether ZP3 or ZP4 in the complex is responsible for the sperm binding. Previously, we have established a baculovirus-Sf9 cell expression system for porcine ZP glycoproteins. A mixture of recombinant ZP3 (rZP3) and rZP4 displayed sperm-binding activity toward bovine sperm but not porcine sperm, probably due to differences in carbohydrate structure between the native and recombinant ZP glycoproteins. In this study, a mixture of porcine rZP3 and native ZP4 (nZP4) inhibited the binding of porcine sperm to the ZP. In contrast, a mixture of porcine nZP3 and rZP4 did not inhibit the binding of porcine sperm, although the mixture inhibited the binding of bovine sperm. The porcine rZP3/nZP4 mixture bound to the acrosomal region of porcine sperm, in a manner similar to that of the nZP3/nZP4 mixture. nZP3 was precipitated with rZP4, and nZP4 was precipitated with rZP3 by utilising the N-terminal tags on the recombinant proteins. These results indicated that nZP4, but not rZP4, is necessary for binding activity of porcine ZP3/ZP4 complex towards porcine sperm and further suggested that the carbohydrate structures of ZP4 in the porcine ZP3/ZP4 complex are responsible for porcine sperm-binding activity of the complex.     

N-Linked Oligosaccharides of Sea Urchin Egg Jelly Induce the Sperm Acrosome Reaction

The sea urchin egg jelly coat (EJ) induces the acrosome reaction (AR) of sperm. We previously demonstrated that a fraction of EJ containing two glycoproteins of 82- and 138-kDa possess the AR inducing activity (8). Here we show that Peptide-N-Glycosidase-F treatment of EJ followed by precipitation and washing in 70% ethanol results in a substantial loss of AR inducing activity in the ethanol insoluble material. When a PNGase-F digest of EJ is chromatographed on a Sepacryl-200 gel filtration column, an AR inducing fraction elutes within the partitioning volume. Acrosome reaction inducing activity of undigested EJ does not elute within the partitioning volume. The chromatographed AR inducing fraction of the PNGase-F digest reacts strongly in the phenol-sulfuric assay demonstrating carbohydrate is present; silver stained gels do not detect the presence of protein. Harsh alkaline hydrolysis of EJ in an excess of NaBH₄, preserves a substantial amount of AR inducing activity. These data show that N-linked oligosaccharides released from EJ by PNGase-F digestion are capable of inducing the sperm acrosome reaction.     

Heterozygous mutations in ZP1 and ZP3 cause formation disorder of ZP and female infertility in human

The human zona pellucida (ZP) is a highly organized glycoprotein matrix that encircles oocytes and plays an essential role in successful reproduction. Previous studies have reported that mutations in human ZP1, ZP2 and ZP3 influence their functions and result in a lack of ZP or in an abnormal oocytes and empty follicle syndrome, which leads to female infertility. Here, we performed whole‐exome sequencing in two probands with primary infertility whose oocytes lacked a ZP, and we identified a heterozygous mutation in ZP1 (NM_207341:c.326G>A p.Arg109His), which is situated in the N‐terminus, and a heterozygous mutation in ZP3 (NM_001110354:c.400G>A p.Ala134Thr), which is situated in the ZP domain. The effects of the mutations were investigated through structure prediction and in vitro studies in HeLa cells. The results, which were in line with the phenotype, suggested that these mutations might impede the function of cross‐linking and secretion of ZP proteins. Our study showed that the two mutations in ZP1 and ZP3 influenced the formation of the ZP, causing female infertility. Meanwhile, these data highlight the importance of the ZP1 N‐terminus in addition to the conserved domains for ZP1 function and ZP formation. Additionally, the patient with the ZP1 mutation delivered a baby following intracytoplasmic sperm injection (ICSI); thus, we suggest the targeted genetic diagnosis of ZP genes to choose appropriate fertilization methods and improve the success rate of assisted reproductive technology (ART) treatments.     

Disulfide linkage patterns of pig zona pellucida glycoproteins ZP3 and ZP4

Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays major roles in fertilization and consists of three or four glycoproteins. Primary structures, and especially the positions of cysteine (Cys) residues in the zona glycoproteins, are well conserved among mammals. In this study, we analyzed the disulfide linkages of pig ZP3 and ZP4 purified from ovaries. While disulfide linkage patterns of four Cys residues in the N-terminal halves of the ZP domains of ZP3 and ZP4 were identical to those previously reported for mice, rats, humans, and fish, the disulfide linkage patterns of six Cys residues in the C-terminal half of the ZP domain in ZP4, as well as eight Cys residues in the C-terminal region of the ZP domain and a following region unique to ZP3, were different from those previously reported. Thus, higher-order structures of zona glycoproteins might not be conserved in the C-terminal regions.     

Egg's ZP3 structure speaks volumes

Binding of mammalian sperm to eggs depends in part on ZP3, a glycoprotein in the egg's extracellular coat, the zona pellucida. In this issue, Han et?al. (2010) describe the structure of an avian ZP3 homolog, providing insights into ZP3 processing and polymerization and the roles of the ZP3 polypeptide and its carbohydrate in sperm binding.     

银鲫ZP3的表达特征和卵壳ZP3蛋白的分离

克隆得到银鲫(Carassius auratus gibelio)ZP3基因全长cDNA,在体外表达出银鲫的ZP3融合蛋白,并制备出多抗血清;通过免疫印迹和RT-PCR分析,研究了银鲫ZP3在卵子发生过程中的表达特征和在早期发育胚胎中的状态变化。研究结果表明,银鲫ZP3的转录发生在卵黄发生以前,而ZP3蛋白的翻译起始于卵黄形成阶段,且随着卵母细胞进一步成熟,其含量不断增加。ZP3蛋白的存在状态在受精后和早期胚胎发育过程中发生了明显变化。在受精后5-30min期间,抽提液中原始的ZP3蛋白带迅速消失,取而代之的是分子量大约为90KD以及更高分子量的蛋白带;而在受精后80min和8-16胞期的胚胎抽提液中,二聚体和多聚体复合体蛋白带也相继消失。这种状态变化意味着ZP3蛋白在卵子受精后有可能与自身或其他蛋白共价结合转变成了二聚体和多聚体。接着,用获得的ZP3抗体作为检测指标,通过卵壳蛋白的凝胶分离,从卵壳中分离纯化出银鲫ZP3蛋白。