山羊精子结合和内化外源DNA的特征及影响因素

外源DNA与精子相互作用后的定位及内化率是精子载体法制备转基因动物的关键环节.实验以标记的DNA片段为示踪材料,就精子与外源DNA相互作用的基本特征及影响因素进行了研究.结果表明:山羊精子可自发性结合外源DNA,外源DNA最初结合于顶体后区质膜外表面,随后部分内化进入细胞内.精子对外源DNA的结合和内化能力随供体的不同而差异明显,在实验所检查的35只公羊中,结合率(DNaseⅠ消化前)波动于4.6%～62.4%,内化率(DNaseⅠ消化后)波动于2.1%～53.8%,个体间差异显著(P<0.01).对于同一供体的精子而言,阻止DNA结合的最主要因素是精浆,与射出的原精液相比,洗涤后精子的结合率和内化率分别提高了3倍和5倍;其次精子获能也将导致结合率和内化率降低(P<0.01).死精子不能完成外源DNA的内化过程,但反复冻融导致质膜破裂的死精子具有更高的结合率,而且阳性率与动物个体无关.上述结果提示,筛选合适的精子供体,采用优化的转染处理方法是提高精子载体方法效率的前提和保证.     

精子介导外源DNA转基因技术

转基因技术在现代生命科学研究、药物开发、临床治疗、遗传育种等方面发挥重要作用.精子介导转基因技术(sperm-mediated gene transfer,SMGT)是以精子细胞为载体,携带外源基因进入卵细胞受精,从而获得转基因动物.该文回顾了SMGT的历史发展,简述了SMGT的分子机制,包括外源DNA与精子的相互作用...     

山羊精子介导法转染外源基因影响因素及最适条件的研究

探讨山羊精子与外源DNA共孵育转染外源DNA效率的影响因素,优化精子转染程序.用DIG免疫组化方法检测和比较精子转染效率,因素为精子洗涤程度、转染缓冲液、精予活力、BSA、肝素、异种动物精浆等.离心洗涤3次以内的山羊精子DNase Ⅰ消化前、后阳性率随洗涤次数增加而显著增加(P＜0.05),而离心3次以上的转染效率略有下降;以mDM为共孵育缓冲体系可获得较高的转染效率;活力为0.55的精子转基因阳性率显著低于活力为0.9的精子(消化前、后阳性率分别为35.4±2.9%和21.8±5.3% vs 63.5±7.2%和50.3±2.8%,P＜0.os);共孵育体系中异种动物精浆可显著降低转染效率(P＜0.01),甚至与肝素可置换出已结合和内化转运到精子内的外源DNA,而共孵育体系中的BSA可抑制精子内化外源DNA.山羊共孵育法转染外源基因最适转染条件为mDM缓冲液洗涤3次后上浮收集高活力精子进行转染.     

精子结合外源DNA的特征及影响因素

外源DNA与精子相互作用后的定位及内化率是精子载体法制备转基因动物的关键环节。实验以标记的DNA片段为示踪材料,就精子与外源DNA相互作用的基本特征及影响因素进行了研究。结果表明：山羊精子可自发性结合外源DNA,外源DNA最初结合于顶体后区质膜外表面,随后部分内化进入细胞内。精子对外源DNA的结合和内化能力随供体的不同而差异明显,在实验所检查的35只公羊中,结合率（DNaseⅠ消化前）波动于4.6% ~ 62.4%,内化率（DNaseⅠ消化后）波动于2.1% ~ 53.8%,个体间差异显著（P<0.01）。对于同一供体的精子而言,阻止DNA结合的最主要因素是精浆,与射出的原精液相比,洗涤后精子的结合率和内化率分别提高了3倍和5倍;其次精子获能也将导致结合率和内化率降低（P<0.01）。死精子不能完成外源DNA的内化过程,但反复冻融导致质膜破裂的死精子具有更高的结合率,而且阳性率与动物个体无关。上述结果提示,筛选合适的精子供体,采用优化的转染处理方法是提高精子载体方法效率的前提和保证。     

外源基因对精子的影响及其在山羊早期胚胎中的表达

摘要: 在前期实验中发现, 山羊精子可自发结合外源DNA, 但结合能力在不同动物个体之间差异显著。挑选结合能力明显不同的3只公羊, 进一步探讨了外源DNA对精子的影响及其在早期胚胎中的表达, 结果发现: 外源基因与精子共同孵育后, 精子的活率、顶体反应发生率和受精能力均呈下降态势, 其降幅与精子的结合能力密切相关。利用与DNA共育后的精子进行体外受精, 外源基因可被导入卵母细胞并在早期胚胎中获得表达, 但胚胎阳性率因精子供体不同而差异显著(P<0.05); 其中来源于高、中结合能力供体生产的胚胎, 分别有16.2%(25/154)和5.3%(4/76)可检测到外源基因存在, 但表达仅见于高结合能力供体生产的早期胚胎, 表达率为6.5%(10/154); 低结合能力供体生产的胚胎无外源基因。研究表明, 在以精子载体方法生产转基因动物的实验过程中, 筛选对DNA结合能力较强的精子供体是提高转基因效率的前提, 但需要考虑外源DNA对精子受精能力的影响。     

提高精子转染外源DNA效率的山羊精液冷冻方法

目的筛选一种既提高精子转染外源DNA效率,又保持解冻后精子活力的山羊精液冷冻—解冻方法。方法应用正交设计L9(34),因素分别为稀释液种类、稀释比例、降温时间和解冻液,每个因素选择3个水平,检测和比较解冻后精子转染外源DNA效率和精子活力。结果所选冷冻—解冻各因素对精液转染效率影响不显著[F(8,18)=1.032,P=0.449];平衡时间对冷冻—解冻活力影响极显著[F(2,24)=9.972,P=0.001],平衡1h极显著小于平衡2 h和4 h的精液活力(P=0.003,P=0.000),以平衡4 h最好。用筛选的冷冻—解冻方法处理精液,解冻后精子的活力极明显降低(P=0.002);生存指数降低,GOT释放量增加,菌落数减少,与鲜精相比差异显著(P=0.018;P=0.016;P=0.018);精子畸形率增加,顶体完整率降低,与鲜精相比差异不显著(P=0.494;P=0.084)。结论优化了提高精子转染外源DNA效率的山羊精液冷冻—解冻方法。     

精子介导转基因动物的制备

近年来 ,通过显微注射DNA至孵育的卵母细胞原核或外源基因转染后的胚胎干细胞进行转基因动物的生产已取得了令人瞩目的成就。在过去的 1 0年中 ,以精子作载体制备转基因哺乳动物或脊椎动物也取得了一些不同程度的进展。这些技术主要包括 :直接将外源DNA与精子共孵育至成熟 ;提取分离的精子DNA或进行预处理至精子发育成熟 ;以及在辅助受精前分离精子细胞等。此外 ,一些显微注射技术 ,如在输精管内进行体内直接转染雄性生殖细胞 ;将体内转染的雄性生殖细胞植入已分离的雄性生殖细胞 ,再显微注射至受体的睾丸 ,这些技术也逐渐成熟起来。研究表明 ,通过体内、体外转染外源DNA的显微操作技术只需将雄性受体与野生型雌性交配就可产生出转基因的后代个体 ,同时也避免了辅助受精和胚胎操作带来的机械损伤 ,因此具有一定的优势。本文综述了精子介导转基因 (SMGT)技术的发展历程、研究现状及前沿进展。     

精子载体法制备转基因动物的研究进展

精子载体法是一种低成本、简单快速制备转基因动物的方法.外源基因主要通过两种方式实现基因整合,一种是直接整合到精子的基因组中;另一种是通过精子携带进入卵细胞,然后整合到受精卵的基因组中.尽管现在已经有很多利用精子载体法获得了转基因动物的研究,但是基于精子在受精过程中,结合、内化、以及转运外源基因等方面能力的差异,转染效率仍有待提高.就利用精子载体法制备转基因动物过程中,精子载体法制备转基因动物的分子机制和方法方面进行系统的阐述和分析.     

山羊精子结合外源DNA能力的年龄及品种依赖性

本文应用正交设计L16(44),优化精子和外源DNA处理条件;用建立的方法转染1-4岁川东白山羊和波南F1公羊、2岁波尔山羊和南江黄羊公羊共149只,用原位杂交法检测精子结合外源DNA的效率,比较不同品种、年龄的山羊(Capra hircus)精子结合外源DNA的能力。结果显示川东白山羊1岁时的阳性精子率为39.34%±13.76%,4岁时降为23.40%±19.37%,与1岁和2岁时相比,差异显著;南江黄羊的阳性精子率最高,波尔山羊最低;并筛选到一种山羊精子和外源DNA处理方法。表明实验山羊的精子结合外源DNA的能力有随着年龄的增长而减少的趋势,并表现出品种间差异。     

精子介导的转基因技术

精子介导的转基因技术是近十几年发展起来的一种新技术.从各个种的实验证明,精子有瞬间吸收外源DNA的能力.这一过程由一系列因子起重要调节作用.一种特异的DNA结合蛋白介导了外源DNA与精子的结合,同时精浆中存在一种因子起抑制两者结合的拮抗作用.CD4分子与外源基因的内化有关.内化的DNA可能与精子核支架结构(nuclear scaffold)结合,并整合或重排.但仍需要大量实验数据进一步证明是否产生真正可遗传的转基因后代,及如何提高转基因效率,以使这一方法得到普遍的推广及应用.     

精子DNA损伤检测技术的研究进展

精子DNA完整性与男性生育力之间的关系是近些年来生殖医学研究领域的热点之一,精子DNA损伤已成为反映男性生育力的一个新指标。精子DNA的损伤原因有很多,有时可能是多种因素共同作用的结果。生殖系统疾病、环境污染、吸烟、微量元素及各种理化因素等原因都可能导致精子DNA完整性受损。常见的精子DNA完整性检测技术有原位末端标记法、精子染色体扩散实验、精子染色质结构分析试验、单细胞凝胶电泳、荧光原位杂交技术和8-羟基脱氧鸟苷测定法等。随着检验技术的不断发展,关于精子DNA损伤的检测技术也在不断更新改进。本文主要就近十年来精子DNA损伤机制、检测技术的相关研究进展作一综述,提示现有的精子DNA完整性检测技术尚不能满足临床和科研需要,急需找到一种理想的检测方法为男性不育的诊断和治疗提供重要依据。     

哺乳动物精子分离方法的研究进展

分离X、Y精子,用于人工授精或体外受精,是目前实现哺乳动物性别控制的最有效手段。本文对哺乳动物精子分离及分离精子纯度评估方法的研究历史及现状作一回顾和总结。     

利用卵胞浆精子注射（ICSI）技术生产转基因小鼠

在掌握小鼠ICSI技术的基础上,进行了ICSI技术生产转基因小鼠的研究。来自成年KM小鼠附睾尾的精子,使用未加抗冻剂的HEPES-CZB溶液,在液氮中冻融1次后,用于本实验。解冻精子与DNA混匀1min后,精子头被显微注射到B6D2F1小鼠成熟卵母细胞质中。精子头与pEGFP-N1环状DNA共注射生产的ICSI受精卵,在CZB溶液中培养至囊胚期时,39.1%(9/23)的囊胚表达GFP基因。精子注射后6h,直接移植ICSI受精卵后,7只妊娠受体一共产仔30只,效率为23.8%(30/126)。Southernblot分析其中16只小鼠发现,3只(18.8%)转基因小鼠同时整合了GFP和Neomycin基因,它们全部来自精子和线性DNA混合的实验组(阳性效率为33.3%,3/9),相反,精子与环状质粒DNA共注射生产的7只ICSI后代中,没有检测到外源基因。转基因小鼠整合的外源基因能够传递给它们后代。结果说明,利用ICSI技术可以高效地生产转基因小鼠,宿主基因组可能更容易整合线性化的外源基因。     

Sperm chromatin damage associated with male smoking

Cigarette smoke is a rich source of mutagens and carcinogens; thus, we have investigated the effects of male smoking on the DNA of human sperm. This was performed using the sperm chromatin structure assay (SCSA), which measures the sensitivity of sperm DNA to acid induced denaturation, and the terminal deoxynucleotidyl transferase assay (TdTA), which measures DNA strand breaks by addition of the biotinylated nucleotide dUTP to 3'-OH ends of DNA, sites of DNA breakage, using the enzyme terminal deoxynucleotidyl transferase. Sperm from subjects who smoked were significantly more sensitive to acid induced denaturation than non-smokers (P<0.02) and possessed higher levels of DNA strand breaks (P<0.05). We hypothesise that smoking damages the chromatin structure and produces endogenous DNA strand breaks in human sperm. These changes may result in sperm DNA mutations, that predispose offspring to greater risk of malformations, cancer and genetic diseases.     

Molecular Karyotyping of Human Single Sperm by Array- Comparative Genomic Hybridization

No valid method is currently available to analyze the entire genome of sperm, including aneuploidies and structural chromosomal alterations. Here we describe the optimization and application of array-Comparative Genomic Hybridization (aCGH) on single human sperm. The aCGH procedure involves screening of the entire chromosome complement by DNA microarray allowing having a molecular karyotype, and it is currently used in research and in diagnostic clinical practice (prenatal diagnosis, pre-implantation genetic diagnosis), but it has never been applied on sperm. DNA from single human sperm isolated by micromanipulator was extracted, decondensed and amplified by whole-genome amplification (WGA) and then labeled, hybridized to BAC array, and scanned by microarray scanner. Application of this protocol to 129 single sperm from normozoospermic donors identified 7.8% of sperm with different genetic anomalies, including aneuploidies and gains and losses in different chromosomes (unbalanced sperm). On the contrary, of 130 single sperm from men affected by Hodgkin lymphoma at the end of three months of chemotherapy cycles 23.8% were unbalanced. Validation of the method also included analysis of 43 sperm from a man with a balanced translocation [46,XY,t(2;12)(p11.2;q24.31)], which showed gains and losses corresponding to the regions involved in the translocation in 18.6% of sperm and alterations in other chromosomes in 16.3% of sperm. Future application of this method might give important information on the biology and pathophysiology of spermatogenesis and sperm chromosome aberrations in normal subjects and in patients at higher risk of producing unbalanced sperm, such as infertile men, carriers of karyotype anomalies, men with advanced age, subjects treated with chemotherapy, and partners of couples with repeated miscarriage and repeated failure during assisted reproduction techniques.     

Sperm DNA damage and its role in IVF and ICSI

While the semen analysis has traditionally been relied upon to differentiate fertile and infertile men, its utility has been questioned in the current era of assisted reproductive technologies. The desire for more sophisticated diagnostic and predictive tools has led to increased use of sperm DNA damage in the management of male infertility. Despite the availability of numerous assays to measure sperm DNA damage, our understanding of the etiology, measurement, and clinical implications of sperm DNA damage remains incomplete. While the current evidence is fraught with heterogeneity that complicates attempts at comparison and meta-analysis, there does appear to be a role for sperm DNA damage in the development and maintenance of pregnancy in the era of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). However, as noted by the American Society for Reproductive Medicine, the routine and widespread use of sperm DNA damage testing is not yet supported. Further studies are needed to standardize the measurement of sperm DNA damage and to clarify the exact role of sperm DNA damage within the myriad of other male and female factors contributing to reproductive outcomes in IVF and ICSI.     

Sperm DNA adducts impair fertilization during ICSI but not during IVF

Many studies emphasize the influence of the status of spermatozoal nucleus on fertilization, mainly with regard to DNA fragmentation. This study was undertaken to analyze the influence of DNA adducts content in spermatozoa on fertilization during assisted reproduction. Ovarian hyperstimulation, oocyte retrieval and laboratory work-up in 61 IVF (in vitro fertilization) and 118 ICSI (intracytoplasmic sperm injection) first cycles were performed according to the same protocol. Semen analysis was made according to WHO Manual (1999). DNA adducts assay in spermatozoa was performed by 32Ppostlabeling method. In total 331 fertilizable oocytes were obtained during IVF and 659 during ICSI. Both groups differed significantly by sperm count, motility and morphology but not by the concentration of DNA adducts in spermatozoa (0.0306 +/- 0.0217 in IVF versus 0.0373 +/- 0.0321 in ICSI). The fertilization rate during IVF was significantly influenced by sperm count (p=0.0002) and motility (p=0.0037) but not by DNA adducts concentration (p=0.30528), whereas during ICSI was positively influenced by sperm motility (p=0.04669) and negatively by DNA adducts concentration (p=0.00796). DNA adducts concentration in spermatozoa significantly negatively influences fertilization rate during ICSI, but not during IVF.     

Sperm DNA fragmentation and oxidation are independent of malondialdheyde

Background  

There is clinical evidence to show that sperm DNA damage could be a marker of sperm quality and extensive data exist on the relationship between DNA damage and male fertility status. Detecting such damage in sperm could provide new elements besides semen parameters in diagnosing male infertility. We aimed to assess sperm DNA fragmentation and oxidation and to study the association between these two markers, routine semen parameters and malondialdehyde formation.     

精子DNA甲基化异常与男性不育

DNA甲基化／去甲基化是表观遗传学最重要的内容并可以控制基因的表达和印迹,越来越多的研究显示DNA甲基化异常与不育男性精子发生异常、特定肿瘤的发生、神经系统疾病、Rett综合征等有关。文章通过总结近来的相关研究资料来阐述精子发生过程中的DNA甲基化状态的改变,探讨精子DNA的甲基化异常与男性不育之间的联系,旨在为男性不育的治疗提供新的临床思路。