X、Y精子分离纯度评价方法的研究进展

对检测分析分离精液中X、Y精子纯度的方法进行了综述, 并将各种方法的原理、技术操作过程和方法的优缺点进行了比较分析。认为如能在技术上有所突破, 提高方法的灵敏度、精确性, 降低检测时间, 单精子巢式PCR方法将可能成为一种低成本、常规化的检测手段, 在精子分离方法优化研究中发挥更大的作用, 并推动其他单精子遗传检测技术取得新进展。     

二维电泳分离牛精子蛋白的技术研究

二维电泳是蛋白质分离技术并可由于对精子蛋白的分离。本研究旨在通过对双向电泳条件的研究摸索出一种适用于分离牛精子蛋白的二维电泳技术,并利用其对牛精子蛋白进行分离鉴定。在实验中,优化了等电聚焦程序,研究了精子蛋白的不同制备方法、不同上样量、不同胶条长度对电泳结果的影响。结果表明,采用尿素－盐酸胍两步裂解法裂解精子细胞制备蛋白,使用13cm非线性胶条进行蛋白二维电泳,能获得较好的电泳图谱。图谱经二维电泳软件分析,可检测出约800多个蛋白质点,分子量基本分布在10~100KD、等电点约为4~9的区域内。对精子蛋白二维电泳条件的摸索,为后续牛精子X、Y差异蛋白的检测和分析奠定了理论基础。     

用三色荧光原位杂交(Three-Color FISH)检测小鼠精子中染色体数目异常的研究

用小鼠X、Y和8号染色体特异的DNA探针,与经DTT（dithiotreitol）和LIS（lithium-3,5-diiodosalicylicacid）解聚的小鼠附单精子进行三色荧光原位杂交（fluoresceoceinsituhybridization,FISH）,以检测精子中的染色体数目异常,并与MMⅡ染色体分析比较．结果表明精子三色FISH具有以下优点和特点：（1）方法敏感稳定,且简便快速;（2）在每一个体至少分析10000尾精子的基础上计算非整倍体单,因此结果更为准确;（3）能检测多倍体即减数分裂停止的发生率及停止的时期;（4）不仅能测定发生于试数分裂Ⅰ（MI）的染色体分离异常,还能检测发生于减数分裂Ⅱ（MII）的不分离和丢失．并对探针的选用、分析标准的建立以及三色FISH用于精于染色体分析的必要性等进行了讨论．     

哺乳动物精子质量评定方法研究进展

哺乳动物精子质量的评估,对于人工授精技术和体外受精技术的应用具有重要意义。目前,可用于反映精子质量的指标和评估方法有：精子活力、精子活率、顶体膜完整性检测、顶体状态与获能的检测、精子线粒体活性、受精能力检测以及其他一些相关的精子质量检测方法。在生产上,进行多指标联合检测,是目前评定精子质量有效的方法。随着技术的进步,更加快速、准确、安全和高效的精子质量检测的新方法将被应用到人类生殖临床和畜牧生产中。     

流式细胞仪分离精子法的研究进展

以X精子和Y精子的DNA含量差别为基础的流式细胞仪分离精子技术是有效的哺乳动物性别控制方法。目前,流式细胞仪分离精子的速度与上世纪90年代初期相比已提高了30多倍,以3000个/秒的常规速度分离X精子或Y精子的准确率可达到85%～90%。迄今,用分离精子人工授精已产下了数万头的动物后代,而分离人的精子也开始应用到了避免X染色体连锁遗传疾病的生殖医学工程中。     

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。     

精子的运动特性

精子的运动特性与生育力有密切关系。本文从超微结构水平和分子生物学基础出发,阐述精子运动机制的微管滑动学说;介绍了精子在液体介质中的运动形式、检测精子活动力的主要技术、影响精子活动力的因素、从异常精液中选择正常精子的技术,以及精子活动力在雄性和雌性生殖道中的演变过程。     

哺乳动物精子性别特异膜蛋白的研究进展

在精子形成过程中,存在性染色体特异基因的表达,这是X、Y精子膜蛋白差异形成的基础。尽管精子形成过程中形成的细胞间桥可能使精子细胞间共享基因表达产物,但雄性传递偏移现象和性别偏移现象的发生又证明两类精子间存在非共享蛋白,同时H-Y抗原表位的成功鉴定、精子分离实验结果以及性别特异蛋白的检测结果都肯定了精子膜蛋白差异的存在,只是这种膜蛋白的差异很小。蛋白分离技术的进步及技术间的优化集成,为精子间细微差异膜蛋白的分离提供了可能。     

中华蜜蜂精子介导egfp基因转移

中华蜜蜂Apis cerana cerana是一种真社会性昆虫,也是我国重要的经济昆虫。本实验目的是为了检测精子是否可以作为载体将外源egfp基因介导转入中华蜜蜂。首先将雄蜂精子与线性化的质粒DNA共浴,然后通过人工授精技术将精子导入处女王,再对实验蜂群后代进行分析。结果显示EGFP蛋白在一群实验组蜂的1～2日龄小幼虫中表达较强,能检测到0.01%～0.02%荧光阳性小幼虫个体;通过PCR和RT-PCR技术分析,证实转入的外源egfp基因获得表达。实验结果表明精子载体法能够用于中华蜜蜂外源基因的转移和表达。     

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。     

猪单个精子单倍型分析方法研究

应用引物延伸预扩增,内嵌引物设计策略进行了ＰＣＲ扩增并结合聚丙烯酰胺凝胶电泳及银染技术,对猪单个精子１２号染色体上４个微卫星位点进行了ＰＣＲ扩增。结果表明,上述技术的应用可以清晰地鉴定出每个精子的单倍型,单精子分型技术的应用为猪高精度遗传作图及需要样本量足够的大遗传现象研究提供了独特的工具。     

Selection and separation of X- and Y- chromosome-bearing mammalian sperm

Preselection of the gender of offspring is a subject that has held man's attention since the beginning of recorded history. Most scientific hypotheses for producing the desired sex of offspring address separation of X- and Y-bearing sperm, and most have had limited, if any success. Eight of these hypotheses and their experimental verifications are discussed here. Three hypotheses are based on physical characteristics of sperm, one on supposed differences in size and shape, another on differences in density, and a third on differences in surface charge. There has been no experimental verification of differences based on size and shape, and the results from attempts to verify separation of X- and Y-bearing sperm based on density have been mixed. Electrophoresis may provide a method for separating X-and Y-bearing sperm, but it is currently unproven and would be of little practical utility, since sperm motility is lost. A fourth hypothesis employs H-Y antigen to select preimplantation embryos. This method reliably produces female offspring, but does not permit the selection of male offspring and does not work on sperm. There are two applications of the theory that X- and Y-bearing sperm should be separable by flow fractionation. Flow fractionation using thermal convection, counter-streaming sedimentation, and galvanization is highly promoted by its originator but has not gained wide acceptance due to lack of independent confirmation. Flow fractionation by laminar flow is said to provide up to 80% enrichment of both X- and Y-bearing sperm; however, this method also has not been confirmed by other workers or tested in breeding trials. The sixth theory discussed is that of separation through Sephadex gel filtration. This method may provide enrichment of X-bearing sperm, but, again, other experimenters have not been able to adequately confirm the enrichment. The best-known approach to sperm separation is that employing albumin centrifugation, yet even with this method, not all researchers have been able to confirm a final fraction rich in Y sperm, and trials in animals have given contradictory results. The most reliable method for separating X- and Y-bearing sperm is use of flow cytometric and flow sorting techniques. These techniques routinely separate fractions with a purity greater than 80% and can be above 90%. Unfortunately, these methods do not always work for human samples. Furthermore, as with electrophoretic approaches, the methods identify and separate only chemically fixed sperm and provide limited biological applications. Generally accepted experimental laboratory procedures for verification of proportions of X- and Y-bearing sperm are lacking. Staining of sperm with the fluorochrome dye quinacrine will identify a structure known as the “F-body” in human sperm and sperm from a few primates. The dye does not work other mammalian sperm. Its validity as a measure of sperm genotype is the topic of debate. We have used two methods to verify claims of separation of sperm. flow cytometry, and in vitro fusion. One can use flow cytometry to test the efficiency of separation of sperm samples. We tested seven commercial methods for the separation of bovine sper, and none were found of result in enrichment. We also used in vitro fusion of human sperm to denuded hamster ova to test enrichment of Y-bearing sperm from the albumin separation process. out results demonstrated no Y-bearing-sperm enrichment from this process. Scientific problems impeding the success of separation seem to be under investigation with an ever-increasing rate. Hybridization probes for DNA sequences specific to the X or Y chromosome may be the next appropriate technology to test of the selection and separation of X- and Y-chromosome-bearing mammalian sperm.     

Evolution of sperm structure and energetics in passerine birds

Spermatozoa exhibit considerable interspecific variability in size and shape. Our understanding of the adaptive significance of this diversity, however, remains limited. Determining how variation in sperm structure translates into variation in sperm performance will contribute to our understanding of the evolutionary diversification of sperm form. Here, using data from passerine birds, we test the hypothesis that longer sperm swim faster because they have more available energy. We found that sperm with longer midpieces have higher levels of intracellular adenosine triphosphate (ATP), but that greater energy reserves do not translate into faster-swimming sperm. Additionally, we found that interspecific variation in sperm ATP concentration is not associated with the level of sperm competition faced by males. Finally, using Bayesian methods, we compared the evolutionary trajectories of sperm morphology and ATP content, and show that both traits have undergone directional evolutionary change. However, in contrast to recent suggestions in other taxa, we show that changes in ATP are unlikely to have preceded changes in morphology in passerine sperm. These results suggest that variable selective pressures are likely to have driven the evolution of sperm traits in different taxa, and highlight fundamental biological differences between taxa with internal and external fertilization, as well as those with and without sperm storage.     

Identification of biomarkers for bull fertility using functional genomics

Prediction of bull fertility is critical for the sustainability of both dairy and beef cattle production. Even though bulls produce ample amounts of sperm with normal parameters, some bulls may still suffer from subpar fertility. This causes major economic losses in the cattle industry because using artificial insemination, semen from one single bull can be used to inseminate hundreds of thousands of cows. Although there are several traditional methods to estimate bull fertility, such methods are not sufficient to explain and accurately predict the subfertility of individual bulls. Since fertility is a complex trait influenced by a number of factors including genetics, epigenetics, and environment, there is an urgent need for a comprehensive methodological approach to clarify uncertainty in male subfertility. The present review focuses on molecular and functional signatures of bull sperm associated with fertility. Potential roles of functional genomics (proteome, small noncoding RNAs, lipidome, metabolome) on determining male fertility and its potential as a fertility biomarker are discussed. This review provides a better understanding of the molecular signatures of viable and fertile sperm cells and their potential to be used as fertility biomarkers. This information will help uncover the underlying reasons for idiopathic subfertility.     

Current knowledge of the human sperm proteome

The knowledge of the mature sperm proteome is undoubtedly the basis for understanding sperm function, the mechanisms responsible for fertilization, the reasons for infertility and possible treatments. The methods of sperm protein extraction depend mainly on the proteins of interest and the protein separation techniques that will be employed. The isolation of the membrane proteins appears to be most problematic step. Nevertheless, two-dimensional electrophoresis and mass spectrometry have become the main techniques used in human sperm protein analysis. We outline the present techniques used to examine the sperm proteome and data generated from studies on the human sperm and different types of male infertility. We present the most characteristic proteins that are involved in sperm function. Their value as biomarkers for diagnosis and treatment of infertility would require further validation. We focus on selected and critical studies of the human sperm proteome to present our subjective view of this fast-moving field.     

大熊猫精子体外获能的研究

本文通过１６次大熊猫精子体外获能和精子穿透试验,建立起一套大熊猫精子体外获能的有效方法,特别是促获能物质的筛选,将为今后开展大熊猫胚胎移植提供可靠的参考资料。     

Observations on the acrosome reaction of human sperm in vitro

Human sperm were incubated in vitro in serum or the defined medium TMPA and were periodically assessed for acrosome reactions using two new methods of assay. The first method, FITC-RCA labeling, was previously shown to be valid for estimating the percentage of normal acrosome reactions of human sperm. The second method, a triple staining technique, is shown in this study to give results comparable to those obtained with FITC-RCA labeling. The percentage of acrosome-reacted sperm was determined at 0, 2.5, 5, and 7 hr of incubation. In both media, some sperm had reacted by 2.5 hr; a maximum percentage of reactions occurred between 5 and 7 hr. The maximum percentage never exceeded 20–25%, which represents only one-third of the live sperm, ie, those potentially able to undergo normal acrosome reactions. It will be important in future studies to determine if this low-peak percentage is due to the fact that: (1) Commonly used culture media are suboptimal or (2) only about 25% of the sperm in a human ejaculate are capable of undergoing normal acrosome reactions.     

Competition Between the Sperm of a Single Male Can Increase the Evolutionary Rate of Haploid Expressed Genes

The population genetic behavior of mutations in sperm genes is theoretically investigated. We modeled the processes at two levels. One is the standard population genetic process, in which the population allele frequencies change generation by generation, depending on the difference in selective advantages. The other is the sperm competition during each genetic transmission from one generation to the next generation. For the sperm competition process, we formulate the situation where a huge number of sperm with alleles A and B, produced by a single heterozygous male, compete to fertilize a single egg. This “minimal model” demonstrates that a very slight difference in sperm performance amounts to quite a large difference between the alleles’ winning probabilities. By incorporating this effect of paternity-sharing sperm competition into the standard population genetic process, we show that fierce sperm competition can enhance the fixation probability of a mutation with a very small phenotypic effect at the single-sperm level, suggesting a contribution of sperm competition to rapid amino acid substitutions in haploid-expressed sperm genes. Considering recent genome-wide demonstrations that a substantial fraction of the mammalian sperm genes are haploid expressed, our model could provide a potential explanation of rapid evolution of sperm genes with a wide variety of functions (as long as they are expressed in the haploid phase). Another advantage of our model is that it is applicable to a wide range of species, irrespective of whether the species is externally fertilizing, polygamous, or monogamous. The theoretical result was applied to mammalian data to estimate the selection intensity on nonsynonymous mutations in sperm genes.     

Effect of male age on sperm traits and sperm competition success in the guppy (Poecilia reticulata)

Deleterious mutations can accumulate in the germline with age, decreasing the genetic quality of sperm and imposing a cost on female fitness. If these mutations also affect sperm competition ability or sperm production, then females will benefit from polyandry as it incites sperm competition and, consequently, minimizes the mutational load in the offspring. We tested this hypothesis in the guppy (Poecilia reticulata), a species characterized by polyandry and intense sperm competition, by investigating whether age affects post‐copulatory male traits and sperm competition success. Females did not discriminate between old and young males in a mate choice experiment. While old males produced longer and slower sperm with larger reserves of strippable sperm, compared to young males, artificial insemination did not reveal any effect of age on sperm competition success. Altogether, these results do not support the hypothesis that polyandry evolved in response to costs associated with mating with old males in the guppy.