为了研究绒山羊Izumo基因3′端未知序列在内蒙古白绒山羊精卵融合中的分子机制,研究根据已知的内蒙古白绒山羊Izumo基因的部分序列,采用Primer Premier 5.0软件,按照3′-Full RACECore Set Ver.2.0试剂盒的要求设计1对PCR引物,用RACE技...为了研究绒山羊Izumo基因3′端未知序列在内蒙古白绒山羊精卵融合中的分子机制,研究根据已知的内蒙古白绒山羊Izumo基因的部分序列,采用Primer Premier 5.0软件,按照3′-Full RACECore Set Ver.2.0试剂盒的要求设计1对PCR引物,用RACE技术对Izumo基因3′端未知序列进行克隆和序列分析。结果表明:扩增获得长度为429 bp的Izumo基因cDNA序列。展开更多
精卵融合是受精过程中最为重要的步骤。目前公认IZUMO1、JUNO和CD9(cluster of differentiation antigen 9)为精卵融合的必需蛋白质,其中IZUMO1与JUNO在精卵识别时会形成复合体。有研究表明IZUMO1、JUNO和CD9主要参与精卵融合最初的黏...精卵融合是受精过程中最为重要的步骤。目前公认IZUMO1、JUNO和CD9(cluster of differentiation antigen 9)为精卵融合的必需蛋白质,其中IZUMO1与JUNO在精卵识别时会形成复合体。有研究表明IZUMO1、JUNO和CD9主要参与精卵融合最初的黏附过程,且它们之间是相互关联发挥作用的。近年来由于X射线晶体学相关技术的发展,IZUMO1-JUNO晶体结构基本被阐明,然而精卵融合的具体分子机制仍未被完全揭示。所以,对哺乳动物精卵融合必需蛋白质IZUMO1-JUNO/CD9的结构、功能和分子机制进行阐述是十分必要的。展开更多
An average human ejaculate contains over 100 million sperm, but only a few succeed in accomplishing the journey to an egg by migration through the female reproductive tract. Among these few sperm, only one participate...An average human ejaculate contains over 100 million sperm, but only a few succeed in accomplishing the journey to an egg by migration through the female reproductive tract. Among these few sperm, only one participates in fertilization. There might be an ingenious molecular mechanism to ensure that the very best sperm fertilize an egg. However, recent gene disruption experiments in mice have revealed that many factors previously described as important for fertilization are largely dispensable. One could argue that the fertilization mechanism is made robust against gene disruptions. However, this is not likely, as there are already six different gene-disrupted mouse lines （Calmegin, Adam Ia, Adam2, Adam3, Ace and Pgapl）, all of which result in male sterility. The sperm from these animals are known to have defective zona-binding ability and at the same time lose oviduct-migrating ability. Concerning spermzona binding, the widely accepted involvement of sugar moiety on zona pellucida 3 （ZP3） is indicated to be dispensable by gene disruption experiments. Thus, the landscape of the mechanism of fertilization is revolving considerably. In the sperm-egg fusion process, CD9 on egg and IZUMO1 on sperm have emerged as essential factors. This review focuses on the mechanism of fertilization elucidated by gene-manipulated animals.展开更多
Background: Cryopreservation is currently the most efficient method for long-term preservation of mammalian gametes and is extensively used in swine artificial insemination(AI) centres. However, it is well-known that ...Background: Cryopreservation is currently the most efficient method for long-term preservation of mammalian gametes and is extensively used in swine artificial insemination(AI) centres. However, it is well-known that cryopreservation procedures induce changes in the water phase in both intra and extracellular compartments,which alter the content and localisation of several proteins and ends up curtailing the structural integrity of functional sperm(i.e., cryoinjuries). Alterations and deficiencies of sperm-oocyte binding proteins during gamete recognition are one of the causes of reproductive failure both in vitro and in vivo. In this sense, characterisation of cryopreservation effects upon oocyte-binding proteins of sperm, such as IZUMO1 and GSTM3, is essential when assessing the impact of this technique in swine reproduction.Results: Cryopreservation was found to induce changes in the localisation of IZUMO1 and GSTM3 in boar sperm.However, the relative content of both proteins was not altered after thawing. Furthermore, whereas IZUMO1 content was found not to be related to the cryotolerance of boar sperm, GSTM3 content was observed to be higher in poor(PFE) than in good(GFE) freezability ejaculates in both pre-frozen(1.00 INT·mm^2± 0.14 INT·mm^2 vs.0.72 INT·mm^2± 0.15 INT·mm^2;P < 0.05) and post-thawed(0.96 INT·mm^2± 0.20 INT·mm^2 vs. 70 INT·mm^2± 0.19 INT·mm^2;P < 0.05) samples. Moreover, GSTM3 levels were found to be higher in those spermatozoa that exhibited low mitochondrial activity, high reactive oxygen species(ROS) production, and high membrane lipid disorder postthaw(P < 0.05).Conclusions: The difference in GSTM3 content between GFE and PFE, together with this protein having been found to be related to poor sperm quality post-thaw, suggests that it could be used as a cryotolerance marker of boar spermatozoa. Furthermore, both IZUMO1 and GSTM3 relocate during cryopreservation, which could contribute to the reduced fertilising capacity of frozen-thawed boar sperm.展开更多
Background:JUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for spermoocyte fusion;their interaction is indispensable for fertilization.Methods:PCR was used to clone the full-leng...Background:JUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for spermoocyte fusion;their interaction is indispensable for fertilization.Methods:PCR was used to clone the full-length DNA sequence of the Juno gene in sheep.The single nucleotide polymorphism(SNP)loci of Juno were genotyped by Sequenom MassARRAY®.PCR combined with rapid amplification of cDNA Ends were used to clone the full-length cDNA sequence of Juno and Izumo1.Reverse transcriptase-PCR(RT-PCR)and real time-quantitative-PCR(RT-qPCR)were used to analyze the genes’expression in tissues of sheep,and single cell RNA-seq was used to analyze the genes’expression in oocytes,granulosa cells and follicular theca of polytocous and monotocous Small Tail Han ewes.Bioinformatics was used to analyze advanced structure and phylogeny of JUNO and IZUMO1 proteins.Results:The full-length DNA sequence of the Juno gene in sheep was cloned and nine SNPs were screened.We found a significant association between the g.848253 C>A locus of Juno and litter size of Small Tail Han sheep(P<0.05).The full-length cDNA sequence of Juno and Izumo1 genes from Small Tail Han sheep were obtained.We found a new segment of the Izumo1 CDS consisting of 35 bp,and we confirmed the Izumo1 gene has 9 exons,not 8.RT-qPCR showed that Juno and Izumo1 genes were highly expressed in ovarian and testicular tissues,respectively(P<0.01).Single cell RNA-seq showed Juno was specifically expressed in oocytes,but not in granulosa cells or follicular theca,while Izumo1 displayed little to no expression in all three cell types.There was no difference in expression of the Juno gene in oocyte and ovarian tissue in sheep with different litter sizes,indicating expression of Juno is not related to litter size traits.Bioinformatic analysis revealed the g.848253 C>A locus of Juno results in a nonconservative missense point mutation leading to a change from Phe to Leu at position 219 in the amino acid sequence.Conclusions:For the first time,this study systematically analyzed the expression,structure and function of Juno and Izumo1 genes and their encoded proteins in Small Tail Han sheep,providing the basis for future studies of the regulatory mechanisms of Juno and Izumo1 genes.展开更多
受精是一个复杂的过程,其中精卵融合是受精过程的关键步骤。本文详细阐述精卵质膜融合过程中精子膜上相关蛋白成分,如:Izumo蛋白、ADAMs(a distintegrin and metalloprotease)基因家族蛋白、富含半胱氨酸分泌蛋白(Crisp)基因家族蛋白的...受精是一个复杂的过程,其中精卵融合是受精过程的关键步骤。本文详细阐述精卵质膜融合过程中精子膜上相关蛋白成分,如:Izumo蛋白、ADAMs(a distintegrin and metalloprotease)基因家族蛋白、富含半胱氨酸分泌蛋白(Crisp)基因家族蛋白的作用。这对于深入了解精卵融合的机制,提高临床上男性不育的诊治水平,并对避孕药物的研制具有现实意义。展开更多
文摘为了研究绒山羊Izumo基因3′端未知序列在内蒙古白绒山羊精卵融合中的分子机制,研究根据已知的内蒙古白绒山羊Izumo基因的部分序列,采用Primer Premier 5.0软件,按照3′-Full RACECore Set Ver.2.0试剂盒的要求设计1对PCR引物,用RACE技术对Izumo基因3′端未知序列进行克隆和序列分析。结果表明:扩增获得长度为429 bp的Izumo基因cDNA序列。
文摘精卵融合是受精过程中最为重要的步骤。目前公认IZUMO1、JUNO和CD9(cluster of differentiation antigen 9)为精卵融合的必需蛋白质,其中IZUMO1与JUNO在精卵识别时会形成复合体。有研究表明IZUMO1、JUNO和CD9主要参与精卵融合最初的黏附过程,且它们之间是相互关联发挥作用的。近年来由于X射线晶体学相关技术的发展,IZUMO1-JUNO晶体结构基本被阐明,然而精卵融合的具体分子机制仍未被完全揭示。所以,对哺乳动物精卵融合必需蛋白质IZUMO1-JUNO/CD9的结构、功能和分子机制进行阐述是十分必要的。
文摘An average human ejaculate contains over 100 million sperm, but only a few succeed in accomplishing the journey to an egg by migration through the female reproductive tract. Among these few sperm, only one participates in fertilization. There might be an ingenious molecular mechanism to ensure that the very best sperm fertilize an egg. However, recent gene disruption experiments in mice have revealed that many factors previously described as important for fertilization are largely dispensable. One could argue that the fertilization mechanism is made robust against gene disruptions. However, this is not likely, as there are already six different gene-disrupted mouse lines （Calmegin, Adam Ia, Adam2, Adam3, Ace and Pgapl）, all of which result in male sterility. The sperm from these animals are known to have defective zona-binding ability and at the same time lose oviduct-migrating ability. Concerning spermzona binding, the widely accepted involvement of sugar moiety on zona pellucida 3 （ZP3） is indicated to be dispensable by gene disruption experiments. Thus, the landscape of the mechanism of fertilization is revolving considerably. In the sperm-egg fusion process, CD9 on egg and IZUMO1 on sperm have emerged as essential factors. This review focuses on the mechanism of fertilization elucidated by gene-manipulated animals.
基金support from Ministry of Science,Innovation and Universities,Spain(Grants:RYC-2014-15581,AGL2017–88329-R and FJCI-2017-31689)European Commission(H2020-MSCA-IF-79212)
文摘Background: Cryopreservation is currently the most efficient method for long-term preservation of mammalian gametes and is extensively used in swine artificial insemination(AI) centres. However, it is well-known that cryopreservation procedures induce changes in the water phase in both intra and extracellular compartments,which alter the content and localisation of several proteins and ends up curtailing the structural integrity of functional sperm(i.e., cryoinjuries). Alterations and deficiencies of sperm-oocyte binding proteins during gamete recognition are one of the causes of reproductive failure both in vitro and in vivo. In this sense, characterisation of cryopreservation effects upon oocyte-binding proteins of sperm, such as IZUMO1 and GSTM3, is essential when assessing the impact of this technique in swine reproduction.Results: Cryopreservation was found to induce changes in the localisation of IZUMO1 and GSTM3 in boar sperm.However, the relative content of both proteins was not altered after thawing. Furthermore, whereas IZUMO1 content was found not to be related to the cryotolerance of boar sperm, GSTM3 content was observed to be higher in poor(PFE) than in good(GFE) freezability ejaculates in both pre-frozen(1.00 INT·mm^2± 0.14 INT·mm^2 vs.0.72 INT·mm^2± 0.15 INT·mm^2;P < 0.05) and post-thawed(0.96 INT·mm^2± 0.20 INT·mm^2 vs. 70 INT·mm^2± 0.19 INT·mm^2;P < 0.05) samples. Moreover, GSTM3 levels were found to be higher in those spermatozoa that exhibited low mitochondrial activity, high reactive oxygen species(ROS) production, and high membrane lipid disorder postthaw(P < 0.05).Conclusions: The difference in GSTM3 content between GFE and PFE, together with this protein having been found to be related to poor sperm quality post-thaw, suggests that it could be used as a cryotolerance marker of boar spermatozoa. Furthermore, both IZUMO1 and GSTM3 relocate during cryopreservation, which could contribute to the reduced fertilising capacity of frozen-thawed boar sperm.
基金This research was funded by National Natural Science Foundation of China,grant number 31501941Central Public-interest Scientific Institution Basal Research Fund,grant number 2018-YWF-YB-1,and 2015ywf-zd-2+1 种基金the Earmarked Fund for China Agriculture Research System,grant number CARS-38the Agricultural Science and Technology Innovation Program of China,grant number ASTIP-IAS13.
文摘Background:JUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for spermoocyte fusion;their interaction is indispensable for fertilization.Methods:PCR was used to clone the full-length DNA sequence of the Juno gene in sheep.The single nucleotide polymorphism(SNP)loci of Juno were genotyped by Sequenom MassARRAY®.PCR combined with rapid amplification of cDNA Ends were used to clone the full-length cDNA sequence of Juno and Izumo1.Reverse transcriptase-PCR(RT-PCR)and real time-quantitative-PCR(RT-qPCR)were used to analyze the genes’expression in tissues of sheep,and single cell RNA-seq was used to analyze the genes’expression in oocytes,granulosa cells and follicular theca of polytocous and monotocous Small Tail Han ewes.Bioinformatics was used to analyze advanced structure and phylogeny of JUNO and IZUMO1 proteins.Results:The full-length DNA sequence of the Juno gene in sheep was cloned and nine SNPs were screened.We found a significant association between the g.848253 C>A locus of Juno and litter size of Small Tail Han sheep(P<0.05).The full-length cDNA sequence of Juno and Izumo1 genes from Small Tail Han sheep were obtained.We found a new segment of the Izumo1 CDS consisting of 35 bp,and we confirmed the Izumo1 gene has 9 exons,not 8.RT-qPCR showed that Juno and Izumo1 genes were highly expressed in ovarian and testicular tissues,respectively(P<0.01).Single cell RNA-seq showed Juno was specifically expressed in oocytes,but not in granulosa cells or follicular theca,while Izumo1 displayed little to no expression in all three cell types.There was no difference in expression of the Juno gene in oocyte and ovarian tissue in sheep with different litter sizes,indicating expression of Juno is not related to litter size traits.Bioinformatic analysis revealed the g.848253 C>A locus of Juno results in a nonconservative missense point mutation leading to a change from Phe to Leu at position 219 in the amino acid sequence.Conclusions:For the first time,this study systematically analyzed the expression,structure and function of Juno and Izumo1 genes and their encoded proteins in Small Tail Han sheep,providing the basis for future studies of the regulatory mechanisms of Juno and Izumo1 genes.
文摘受精是一个复杂的过程,其中精卵融合是受精过程的关键步骤。本文详细阐述精卵质膜融合过程中精子膜上相关蛋白成分,如:Izumo蛋白、ADAMs(a distintegrin and metalloprotease)基因家族蛋白、富含半胱氨酸分泌蛋白(Crisp)基因家族蛋白的作用。这对于深入了解精卵融合的机制,提高临床上男性不育的诊治水平,并对避孕药物的研制具有现实意义。
