获能处理对山羊精子结合外源DNA能力的影响

鲜精离心洗涤除去精浆,然后用0.4 μmol/L钙离子载体(IA组)作用10 min或用10 mg/L肝素(HE组)作用90 min获能后,与DNA孵育,再用地高锌(DIG)末端标记的线形外源DNA进行转染,用免疫组化的方法分别检测它们的转染效率;用透射电镜观察精子获能前后的超微结构变化,并用碘化丙锭和羟化荧光素双探针检测获能对精子质膜完整性的影响,探讨精子获能影响精子结合及内化外源DNA变化的原因.结果显示,获能处理降低了精子结合和内化外源DNA能力,IA组和HE组结合率分别为(17.41±4.69)%和(15.00±4.36)%,都极显著低于未经获能处理的精子(32.97±4.58)%(P<0.01).IA组、HE组和对照组(未获能)内化率分别为(12.09±2.50)%、(8.93±1.79)%和(25.52±2.57)%,IA组、HE组均极差异小于对照组(P<0.01).超微结构观察和双荧光探针检测都发现精子获能后质膜完整性降低.用IA或HE获能后的精子质膜完整率是分别是(33.16±6.46)%和(33.90±4.14)%,与对照组差异极显著(P<0.01).结果表明,精子结合及内化外源DNA不是纯粹的分子渗透现象,而是受严格的分子调控的.     

猪精子结合和内化外源基因的影响因素研究

精子介导基因转移(SMGT)是目前转基因动物研究中简单而高效的方法之一,其中精子结合和内化外源基因的效率是精子介导转基因成功的关键.本试验以DIG标记的线性化EGFP作为示踪基因来检测猪精子结合和内化外源基因的影响因素,结果表明:猪精子能够自发结合外源基因,结合部位主要在精子顶体后区.精子结合外源DNA的阳性率随共孵育时间延长而增加,在37℃或39℃时孵育60 min后,阳性率不再增加;在17℃时孵育90min后,阳性率不再增加.在检查的15只公猪样本中,精子与外源DNA的结合率为6.57%～35.81%,内化率为2.99%～24.66%,个体间差异显著(P<0.01).精浆能够强烈抑制外源基因的结合和内化,脂质体及DMSO能够显著提高转染效率.死精子能够结合外源基因,但不能将其内化;反复冻融导致质膜破裂的精子对外源基因有更高的结合率,且不受个体影响.     

精子介导外源DNA转移的研究进展

精子因在受精过程中的独特作用,而被认为是转移外源ＤＮＡ的理想载体。本文对精子作载体进行转基因动物制作的方法、外源ＤＮＡ与精子作用的分子机制、导入动物个体或胚胎的外源ＤＮＡ的存在形式、影响精子与外源ＤＮＡ结合的因素进行了论述,并对精子作为载体的研究方向和发展前景给予了预测。     

精子作为外源基因载体的转基因方法研究进展

以精子为载体的转基因动物制作方法巳引起众多研究者的关注与青睐。近年来此法的研究巳取得较大进展，并在多种动物上获得成功。随着对精子载体法的深入研究，外源DNA与精子结合的方法也不断改进。作者就精子与外源DNA体内、体外的结合方法作一综述。     

应用精子载体法导入外源DNA影响家蚕遗传性的研究

我们曾经实验表明 ,无论采用直接注射幼虫方法 ,还是采用精子介导的“交配囊法” ,将外源DNA导入蓖麻蚕或家蚕体内 ,都有可能改变受体的某些遗传特性 ,有的变异还可以遗传并育成稳定的新品系。实验结果还表明 ,采用不同品种的家蚕DNA导入同一品种的蓖麻蚕体内 ,对遗传性有不同的影响。为了进一步探讨外源DNA对家蚕的遗传作用 ,也为了进一步验证精子介导的转基因技术在蚕上实际应用的可行性 ,本研究采用我们建立的“交配囊导入法” ,将不同种属的蓖麻蚕和柞蚕DNA导入家蚕处女蛾交配囊内 ,并与同种雄蛾交配 ,获得受精卵 ,观察由此受精卵…     

精子获能的分子机制及研究进展

哺乳动物的精子在刚射入雌性生殖道时,或从附睾取出后,并不具备受精能力,必须在雌性生殖道内经过一段时间,发生生理及形态学的变化才能获得受精能力,这一过程被称为精子获能[1]。精子获能是精     

哺乳动物受精作用分子机制的研究进展

关于受精的研究已经有一百余年,有很多对受精过程的细致描述,但只有最近的研究才较为详细地阐明了受精作用的分子机制,具体地研究了参与受精作用的一些分子的结构和功能。现就精、孵细胞活化,精子获能,顶体反应,精卵细胞膜结合、融合,两性配子完全融合这一系列协同受精过程的部份分子机制的研究进展作简要概述。     

雄性生殖细胞介导外源DNA转移的新方法

介绍并评价在传统精子载体法的基础上的改进与发展，即辅以生精细胞显微授精的基因转移；输精管注射法体内转染成熟精子和曲细精管注寺转洒生精子细胞，这些方法的应用将为哺乳动力 为方便和高效的技术手段。     

精子载体法转基因技术研究进展

精子载体法是以精子作为外源基因的载体,在受精过程中将外源基因导入动物胚胎,从而使外源基因进入子代的基因组中。作者就精子结合外源基因的机制,精子与外源基因结合的方法、影响精子转染外源基因和生产转基因动物的精子因素进行简要介绍,并对发展起来的输精管内注射转染法、曲细精管微注射法和睾丸直接注射法等精子载体转基因新途径进行综述。     

调节精子功能的外源性物质及其作用机理

在人工授精及体外受精过程中,需要对精液进行稀释等处理,处理过程中外源性物质的添加会从不同的方面影响精液的质量和精子的受精力。本文阐述了氨基多糖、甲基黄嘌呤、无机离子、生殖激素、细胞因子、维生素、蛋白质等外源性物质对精子生理功能的调节及其作用机理。     

精子RNA的研究进展

1 精子RNA存在的争论 作为遗传物质的载体,精子的作用仅是将父本的基因组DNA成功地带入到卵子中,所以成熟精子不必含mRNA,这个观点被广泛接受.有关精子中存在RNA的报道,常被解释为有未成熟的精子细胞的存在,或是精子分析样本中混杂有体细胞,或是精子中含有未被支持细胞吸收的胞质小体造成~([1]),而胞质小体的存在又通常被认为是精子成熟度低的标志.     

Effect of freezing on sperm nuclear DNA

Sperm DNA damage has a significant impact on reproductive outcomes. In recent years, the search for optimal molecular markers for the evaluation of semen quality has resulted in the increased focus on sperm nuclear DNA assessment. The primary aim of this article was to review and summarize the effects of freezing-thawing procedure on nuclear DNA integrity of boar spermatozoa. Using different sperm DNA integrity assays, it has been confirmed that the sperm DNA undergoes structural changes during the freezing-thawing process. Evidence has been shown that a significant proportion of frozen-thawed spermatozoa with compromised chromatin integrity was highly susceptible to DNA fragmentation. Moreover, the possible mechanisms responsible for post-thaw sperm DNA damage could be because of cryo-induced oxidative stress and, to a lesser extent, to the activation of an apoptotic-like phenomenon. This review also highlights the ongoing effort employed to develop optimal strategies to reduce sperm DNA damage following freezing-thawing of boar semen.     

Antioxidant mechanisms and their benefit on post-thaw boar sperm quality

While being an important component of normal cellular function, excess levels of reactive oxygen species (ROS) cause cell damage and death. The ability to protect sperm against oxidative damage is of particular importance in the artificial reproduction industry because of the increased production of ROS by the sperm cell during processing. This review discusses the formation of ROS and the use of antioxidants in protecting boar sperm against oxidative damage.     

Effects of exogenous lactoferrin on characteristics and functions of bovine epididymal,ejaculated and frozen-thawed sperm

The purpose of this study was to investigate effects of addition of lactoferrin on characteristics and functions of bovine epididymal, ejaculated, and frozen-thawed sperm. The addition of lactoferrin was significantly (p < .05) effective on increasing values of progressive motility, straightness, and linearity in caput epididymal sperm and values of motility in cauda epididymal sperm. When ejaculated sperm were incubated in capacitation medium, percentages of motile and progressively motile sperm decreased largely within the first period of 30 min, followed by only minor changes. However, the addition of lactoferrin significantly lessened the early decreases of these parameters and additionally promoted capacitation-dependent changes of chlortetracycline staining patterns (from F pattern to B pattern). In other experiments, when ejaculated sperm were exposed to oxidative stress with 100-µM H₂O₂, the addition of lactoferrin partially protected them from dysfunction of flagellar movement and loss of progressive movement. In final experiments with frozen-thawed samples incubated in the capacitation medium, the addition of lactoferrin effectively survived dying sperm and suppressed occurrence of sperm agglutination. These results may suggest biological and biotechnological potentials of lactoferrin for modulation of bovine sperm viability, motility, capacitation state, and preservation in vitro.     

Disappearance of the PHA-E lectin binding site on the surface of ejaculated sperm and sperm capacitation in the dog

Ejaculated semen and cross sections of the cauda epididymides collected from 3 normal dogs were smeared or stamped on glass slides, and the sperm on the slides were stained with 7 different FITC-lectins (Con A, DBA, GS-1, PHA-E, PSA, UEA-1, WGA) to examine the relation between sperm-binding glycoprotein derived from the canine prostate and sperm capacitation. The only lectin that stained the ejaculated sperm but not the cauda epididymal sperm was PHA-E. The sperm ejaculated by 5 other dogs were incubated for 4 hr in fluid flushed from the uterine horns or oviducts of estrous bitches, and then the percentages of actively motile sperm and hyperactivated sperm (HA-sperm) were determined. The percentages of PHA-E-labeled sperm and sperm labeled with fluoresceinated Ca indicator to assess the influx of Ca into the sperm were also evaluated. The mean percentages of actively motile sperm, HA-sperm, and Ca-labeled sperm after 4 hr of incubation in the uterine flush fluid and oviductal flush fluid were significantly higher than in control medium (P<0.05, 0.01), but the mean percentages of PHA-E-labeled sperm were lower (both P<0.01). The percentages of PHA-E-unlabeled sperm correlated with the percentages of both HA-sperm and Ca-labeled sperm (r(2)=0.787 and 0.812, respectively). The results indicate that loss of the glycoprotein secreted by the canine prostate on the sperm surface induces the influx of Ca into the sperm, and then hyperactivation of the sperm.     

生物素的生理功能及其分子作用机制

生物素是动物机体内维持正常生理机能所必需的一种维生素。它作为4种羧化酶辅酶成分,在哺乳动物体内的葡萄糖、氨基酸和脂肪酸代谢中起着重要作用。越来越多的研究表明,生物素对基因表达的调控起着重要的作用。本文综述了生物素的营养生理作用及其对基因表达调控的影响。     

Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos

Epigenetic reprogramming confers totipotency even during somatic cell nuclear transfer (SCNT), which has been used to clone various animal species. However, as even apparently healthy cloned animals sometimes have aberrant epigenetic status, the harmful effects of these defects could be passed onto their offspring. This is one of the biggest obstacles for the application of cloned animals for livestock production. Here, we investigated the DNA methylation status of four developmentally regulated genes (PEG3, XIST, OCT4, and NANOG) in sperms from a cloned and a non‐cloned bull, and blastocysts obtained by in vitro fertilization using those sperms and SCNT. We found no differences in the methylation status of the above genes between cloned and non‐cloned bull sperms. Moreover, the methylation status was also similar in blastocysts obtained with cloned and non‐cloned bull sperms. In contrast, the methylation status was compromised in the SCNT blastocysts. These results indicate that sperm from cloned bulls would be adequately reprogrammed during spermatogenesis and, thus, could be used to produce epigenetically normal embryos. This study highlights the normality of cloned bull offspring and supports the application of cloned cattle for calf production.