奶山羊DDX3Y蛋白多克隆抗体的制备

【目的】制备针对奶山羊DDX3Y蛋白的特异性多克隆抗体,为奶山羊H-Y抗原蛋白DDX3Y的功能研究奠定基础。【方法】以奶山羊睾丸组织提取的RNA反转录所得的cDNA为模板,采用PCR方法扩增得到奶山羊DDX3Y基因CDs区全序列,测序后通过生物信息学方法比对分析确定DDX3Y蛋白N端1-120氨基酸及C端570-660氨基酸为抗原片段,然后用PCR分别克隆这2个片段的基因,最后利用搭桥PCR方法得到奶山羊DDX3Y截短抗原基因序列。将DDX3Y截短抗原基因序列连接pET32a(+)质粒,构建pET32a(+)-DDX3Y原核表达载体,将该载体导入到大肠杆菌BL21中诱导表达。通过Ni-NTA Resin纯化重组蛋白后免疫3月龄雌性新西兰大耳兔,采用间接ELISA(iELISA)法测定抗体效价,用Western blot测定抗体特异性。【结果】PCR扩增得到1 983 bp奶山羊DDX3Y基因CDs区全长序列及CDs区5′端360 bp(1-360 bp)序列和3′端273 bp(1 710-1 983 bp)序列。搭桥PCR获得了633 bp的DDX3Y截短抗原基因序列。成功构建了pET32a(+)-DDX3Y载体,并表达出分子质量约为42 ku的重组蛋白。制备出奶山羊DDX3Y截短抗原蛋白多克隆抗体,iELISA法检测抗体效价为1∶10~5;Western blot检测表明,制备的多克隆抗体能够特异性识别原核表达的奶山羊DDX3Y截短抗原蛋白以及公羊睾丸组织DDX3Y蛋白。【结论】成功制备出了奶山羊DDX3Y截短抗原蛋白多克隆抗体。

下调猪精子DDX3Y mRNA水平对其IVF胚胎体外发育的影响

为了研究猪精子中DDX3Y mRNA的保留情况,探讨其对受精后胚胎体外发育的影响,试验采用定量PCR分析猪精子中DDX3Y mRNA,并利用转染抗DDX3Y小干扰RNA(siRNA)的猪精子进行体外受精,统计其体外受精(IVF)胚胎体外发育效率及性别比例。结果表明:猪精子中DDX3Y mRNA有较高水平的表达;利用有效抗DDX3Y的siRNA转染猪精子,可将其DDX3Y mRNA显著下调至对照组的12.4%(P<0.05),精子活力显著下降到0.44(P<0.05);利用转染抗DDX3Y siRNA的猪精子进行体外受精,产生的胚胎在卵裂率、囊胚率及性别比例等方面与对照组相比差异不显著(P>0.05)。

Cloning,Sequencing and Analysis of DDX3Y Gene in Five Male Equus Animals

[ Objective] This study analyzed the differences in DDX3 Y gene sequences among male horse, male donkey, male mule, male hinny and male foal to investigate the relationship between DDX3Y gene structure and the mechanism of infertility in male Equus fetus × asirms. [ Method] Partial sequences of DDX3Y gene in male horse, male donkey, male mule, male hinny and male foal were obtained by genome comparison, sequencing and cloning, and compared by bioinformatics methods. [ Result] The CDS regions varied in 5 base pairs and two amino acids. DDX3Y protein was predicted to be an unstable protein. In addition, the results also showed that DDX3 Y gene in male horse displayed a closer phylogenetic relationship to male mule and male foal; DDX3Y gene in male donkey displayed a closer phylogenetic relationship to male hinny. [ Conclusion] This study provided scientific basis for further exploring the function of DDX3 Y gene and the mecha- nism of reproductive regulation in male Equus ferus × asinus.

醋酸铅对雄性小鼠附睾Y染色体基因Ddx3y蛋白表达的影响

[目的]通过观察亚慢性铅暴露对小鼠附睾组织Y染色体基因Ddx3y表达的影响,为探讨铅的雄性生殖毒作用机制提供依据。[方法]昆明种小鼠48只,按体重将小鼠随机分为对照组、0.5 g/L、1.0 g/L和1.5 g/L醋酸铅染毒组。通过自然饮用含不同浓度醋酸铅的方式使小鼠染铅,连续染毒60 d后取附睾组织。HE染色观察组织病理学变化,Western blotting和免疫组化方法检测附睾组织Ddx3y蛋白的表达及组织分布。[结果]与对照组比较,染铅组附睾管内精子数和分泌液均减少,尤其1.5 g/L染铅组最为明显。Western blotting检测结果显示,染铅组的小鼠附睾组织中Ddx3y蛋白表达明显低于对照组,且有剂量-反应关系。免疫组化结果显示,随着染铅剂量的增加,附睾组织中抗Ddx3y蛋白免疫反应明显降低。[结论]亚慢性铅暴露显著下调小鼠附睾组织Y染色体基因Ddx3y蛋白表达。

醋酸铅对雄性小鼠睾丸Y染色体基因Ddx3y蛋白表达的影响

目的通过观察亚慢性铅暴露对小鼠睾丸组织Y染色体基因Ddx3y蛋白表达的影响,为探讨铅的雄性生殖毒作用机制提供新的靶标依据。方法:昆明种小鼠48只,按体重将小鼠随机分为对照组,0.5、1.0和1.5 g/L醋酸铅染毒组。通过自然饮用含不同浓度醋酸铅的方式使小鼠染铅,连续染毒60 d。取小鼠睾丸组织,用苏木素-伊红(HE)染色法进行组织病理学观察,用Western blotting和免疫组化方法检测睾丸组织Ddx3y蛋白的表达及组织分布。结果与对照组比较,染铅组睾丸曲细精管内细胞排列松散、紊乱、层次不清,精原细胞层和初级精母细胞层之间间隙变宽,且随着染铅浓度的增加精子细胞明显减少。Western blotting检测结果显示,染铅组的小鼠睾丸组织中Ddx3y蛋白表达显著低于对照组,且有剂量-反应关系。免疫组化结果显示,随着染铅剂量的增加,睾丸组织中抗Ddx3y蛋白免疫反应明显降低。结论亚慢性铅暴露可抑制小鼠精子的发生、发育和下调睾丸组织Y染色体基因Ddx3y蛋白的表达。

砷对大鼠睾丸组织Ddx3y基因及其蛋白表达的影响

目的探讨砷对大鼠睾丸组织Ddx3y基因及其蛋白表达的影响。方法40只雄性SD大鼠随机分成5组,每组8只。分别是空白对照组、阳性对照组(100μg/kg雌二醇)和0.15、0.75、1.5 mg/kg砷染毒组,每天灌胃一次,连续染毒4周后,对大鼠精子数量和精子存活率、睾丸组织Ddx3y基因及其蛋白的表达进行测定。结果阳性对照组的精子数量、精子存活率均低于空白对照组(P<0.05);各砷染毒组精子数量、精子存活率均低于空白对照组(P<0.05),0.75、1.5 mg/kg砷染毒组精子存活率低于阳性对照组(P<0.05)。阳性对照组和0.15、0.75、1.5 mg/kg砷染毒组相对于空白对照组Ddx3y基因的表达量分别为61.06%、66.37%、54.08%、36.25%,1.5 mg/kg砷染毒组Ddx3y基因的表达量低于空白对照组(P<0.05)。与空白对照组相比,阳性对照组和各砷染毒组Ddx3y蛋白的表达下调(P<0.05);与阳性对照组相比,各砷染毒组Ddx3y蛋白的表达下调(P<0.05)。结论砷可抑制大鼠精子的生成,下调Ddx3y基因及其蛋白的表达,砷的雄性生殖毒性可能与其抑制Ddx3y基因及蛋白表达有关。

Absolute copy number differences of Y chromosomal genes between crossbred (Bos taurus×Bos indicus) and Indicine bulls

Background： The Y chromosome in mammal is paternally inherited and harbors genes related to male fertility and spermatogenesis. The unique intra-chromosomal recombination pattern of Y chromosome and morphological difference of this chromosome between Bos taurus and Bos indicus make it an ideal model for studying structural variation, especially in crossbred （Bos taurus x Bos indicus） bulls. Copy Number Variation （CNV） is a type of genomic structural variation that gives information complementary to SNP data. The purpose of this study was to find out copy number differences of four Y chromosomal spermatogenesis-related candidate genes in genomic DNA of crossbred and purebred Indicine bulls. Result： Four Y chromosomal candidate genes of spermatogenesis namely, sex determining gene on Ychromosome （SRY）, DEAD box po/ypeptide 3-Y chromosome （DDX3 Y）, Ubiquidn specific peptidase 9, Y-linked （ usPgY）, testis-specific protein on Y chromosome （TSPY） were evaluated. Absolute copy numbers of Y chromosomal genes were determined by standard curve-based quantitative real time PCR. Copy numbers of SRYand TSPYgenes per unit amount of genomic DNA are higher in crossbred than Indicine bulls. However, no difference was observed in DDX3Yand usPgYgene copy numbers between two groups. Conclusion： The present study demonstrates that the structural organization of Y chromosomes differs between crossbred and Indicine bulls which are reproductively healthy as observed from analysis of semen attributes. The absolute copy numbers of SRY and TSPY genes in unit mass of genomic DNA of crossbred bulls are significantly higher than Indicine bulls. No alteration in absolute copies ofDDX3Yand usPgYgene was found between the genome of crossbred and Indicine bulls. This study suggests that the DDX3Yand USPgYare likely to be single copy genes in the genome of crossbred and Indicine bulls and variation in Y chromosome length between crossbred and Indicine bulls may be due to the copy number variation of SRY gene and TSPYarray.

徐州地区部分人群Y染色体微缺失现状分析

目的分析徐州地区部分人群Y染色体微缺失类型分布及其临床表征。方法将2018年1月至2021年8月在本科室进行Y染色体微缺失检测的男性患者408例纳入研究范围,采其外周血提取DNA,进行多重荧光PCR,必要时进行电泳分析及染色体微阵列分析(CMA)。结果共检出Y染色体微缺失39例,除去不符合研究要求1例,总缺失率为9.34%(38/407),缺失类型中AZFc、AZFa+b+c、AZFb+c、AZFb、AZFa sY86缺失率依次为57.89%(22/38)、18.42%(7/38)、15.79%(6/38)、5.26%(2/38)、2.63%(1/38)。缺失患者除精子异常、夫妻不孕不育为主要表现外,睾丸发育不良也占据较大比例。结论鉴于徐州地区发生Y染色体微缺失的概率相对偏高,建议将Y染色体微缺失检测纳入男性不育、精子异常、睾丸发育不良人群的临床检测首选项目。