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。     

用分离精子进行性别控制研究的现状

哺乳动物后代性别控制的方法有两种,即植入前胚胎性别的选择和受精前精子的分离,后者是动物性别控制最有效的途径。目前最有重复性、科学性和有效性的分离精子的方法,是根据精子DNA含量存在差异的原理,利用荧光染料与DNA相结合,并通过流动细胞检索分离仪进行精子的分离。     

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。     

X,Y精子分离技术的进展

自本世纪伊始,就有许多有关分离X精子(具有X染色体的精子)和Y精子(具有Y染色体的精子)的研究报告.近年来,美国、日本等国相继报道分离人和牛的X、Y精子获得成功,然而其实验效果的重复性极低.本文仅就最近有关X、Y精子的分离研究进展情况作以扼要的介绍.     

铅对小鼠精子形态影响的观察

通过精子畸形试验,观察了醋酸铅对小鼠雄性生殖细胞的影响。结果表明,随着醋酸铅浓度的增大,精子畸形率增加,提示了铅对哺乳动物的雄性生殖细胞具有潜在的诱变能力。     

哺乳动物精子获能过程中信号通路研究进展

哺乳动物精子在雌性生殖道内及体外获能培养过程中伴随着胆固醇外流、质膜重组、离子通道调节及获能相关蛋白磷酸化状态改变等相关生理调节过程,其中信号通路及相应信号分子对精子获能及功能修饰起到重要调节作用,成为精子细胞超激活运动及完成受精作用的关键环节。根据近年来的研究报道,对哺乳动物精子获能过程中已知的信号通路、信号分子及调节因子、离子通道、存在的问题及未来研究主要方向进行综述,为精子体外培养及辅助生殖等提供理论参考。     

精子功能相关的蛋白质调控受精过程的研究进展

哺乳动物的受精过程涉及到精子一系列的功能活动,如精子在雌性生殖道的运行、精子的超活化与获能、顶体反应以及精卵融合等。在精子经历的这一系列过程中,精子功能相关的蛋白质发挥着不可或缺的作用,这些蛋白分子的正常与否与雄性个体的繁殖力高低密切相关,因此精子功能相关的蛋白质能够作为评定哺乳动物精液受精能力的生物标记。文章主要对哺乳动物精子功能相关的蛋白质进行了综述,以阐述相关蛋白分子对精子运动活力、精子获能、顶体反应、透明带穿入和精卵融合等方面的重要作用以及这些蛋白分子在家畜遗传改良上的潜在应用。     

哺乳动物受精过程中精子和透明带的初级及次级结合

在哺乳动物的受精过程中,获能的精子穿过卵丘细胞层到达卵子透明带后,精子头部便结合在透明带的表面上.精子和透明带的结合可以分为初级结合和次级结合两个阶段,初级结合是指二者在顶体反应之前的结合,次级结合是指二者在顶体反应之后的结合.精子和透明带的结合是精子顶体反应和精子入卵的先决条件,是哺乳动物受精过程中非常重要的环节,涉及到精子以及透明带的多种蛋白质分子之间的相互作用.这方面的研究工作主要是以小鼠为实验对象进行的,在其他哺乳动物中也有很多报道.本文将主要以小鼠为例,概述哺乳动物受精过程中精子和透明带结合的分子机制,并兼述在二者的结合过程中透明带糖蛋白ZP3对精子顶体反应的诱导作用.     

LDH-C：睾丸重要的特异表达基因

乳酸脱氢酶C(LDHC)是目前已知的最早在男性生精细胞中发现的睾丸特异同功酶。LDHC最早通过凝胶电泳技术在人精子及睾丸生精细胞中被发现。免疫组化结果显示LDHC最早出现在早期的粗线期初级精母细胞中,其数量随着减数分裂逐渐增加。成熟精子中LDHC主要定位在精子尾部的主段区域。研究显示,乳酸脱氢酶家族的同功酶在哺乳动物细胞中无所不在,他们受到发育的调控,具有组织细胞的特异性,且功能多样。本文就LDHC的发展史及他们在帮助精子完成受精过程中的作用作一综述。     

双酚A暴露对哺乳动物精子蛋白酪氨酸磷酸化的影响

双酚A(BPA)是一种人工合成的雌激素性化合物,广泛存在于环境中,对哺乳动物内分泌有干扰作用,影响雄性生殖系统功能。本研究以新鲜猪精子、17 ℃保存猪精子以及小鼠精子为对象,采用体外培养方法,利用蛋白免疫印迹(WB)和免疫荧光技术,分析不同浓度BPA(0、0.1、1、10、100 μmol·L^-1)暴露对哺乳动物精子蛋白酪氨酸磷酸化的影响及分子机制。结果表明: 低中浓度(0.1、1 μmol·L^-1)BPA暴露对新鲜猪获能精子蛋白酪氨酸磷酸化具有显著促进作用,但在高浓度(10、100 μmol·L^-1)BPA暴露下,猪获能精子蛋白酪氨酸磷酸化呈现降低趋势。BPA暴露下,小鼠获能精子蛋白酪氨酸磷酸化随BPA浓度的增加而增强,并且BPA影响猪和小鼠精子获能相关酪氨酸磷酸化修饰的蛋白种类不同。表明BPA暴露对哺乳动物精子的影响具有物种特异性。免疫荧光结果显示BPA对精子蛋白酪氨酸磷酸化的影响主要发生在鞭毛的中段和主段。     

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.     

Flow cytometric sexing of mammalian sperm

This review reexamines parameters needed for optimization of flow cytometric sexing mammalian sperm and updates the current status of sperm sexing for various species where this technology is currently being applied. Differences in DNA content have provided both a method to differentiate between these sex-determining gametes and a method to sort them that can be used for predetermining sex in mammals. Although the DNA content of all cells for each mammalian species is highly conserved, slight but measurable DNA content differences of sperm occur within species even among cattle breeds due to different sizes of Y-chromosomes. Most mammals produce flattened, oval-headed sperm that can be oriented within a sorter using hydrodynamic forces. Multiplying the percentage the difference in DNA content of the X- or Y-chromosome bearing sperm times the area of the flat profile of the sperm head gives a simple sorting index that suggests that bull and boar sperm are well suited for separation in a flow sorter. Successful sperm sexing of various species must take into account the relative susceptibilities of gametes to the stresses that occur during sexing. Sorting conditions must be optimized for each species to achieve acceptable sperm sexing efficiency, usually at 90% accuracy. In the commercial application of sperm sexing to cattle, fertility of sex-sorted bull sperm at 2 x 10(6)/dose remains at 70-80% of unsexed sperm at normal doses of 10 to 20 x 10(6) sperm. DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species, and normal-appearing offspring have been produced from sexed sperm of at least seven species.     

Attachment of the ascidian sperm surface egg receptor N-acetylglucosaminidase to the cell membrane

Ascidian sperm bind to vitelline coat N-acetylglucosamine groups of the egg bvia sperm surface N-acetylglucosaminidase. This sperm surface egg receptor remains anchored throughout penetration. Localization to the sperm surface was verified by biotinylation of intact sperm followed by solubilization in Triton X-100 and binding to streptavidin agarose. The enzyme was determined to be an integral membrane protein as judged by resistance to release by KI and high pH. Linkage of the enzyme to the sperm surface was probed through differential solubilization followed by measuring released enzymatic activity with a fluorogenic substrate. Nonionic detergents released 90% of the activity. Proteases released about 40%. No activity was released by a phosphatidyl–inositol specific phospholipase C. This finding, combined with the similarity of release level by all the detergents, including triton X-114 phase separation experiment. This observation, coupled with the finding of release by nonionic detergents, suggests that the protein is hydrophilic once released from the membrane. Thus, although clearly an integral membrane protein, the enzyme has limited bydrophobicity such as would be present in a single transmembrane sequence or extensive glycosylation. © 1994 Wiley-Liss, Inc.     

Sexual selection and the temporal separation of reproductive events: sperm storage data from reptiles, birds and mammals

The duration of sperm storage by females differs markedly between reptiles (maximum: 2555 d [7 years] and birds (maximum: 117d), with mammals showing both very short (< 1 d) and relatively long periods (maximum: 198 days). The females of many reptiles, probably all birds and some mammals have specialized structures for storing sperm, suggesting that selection for sperm storage has operated on females. Sperm storage, together with delayed implantation and delayed development, separates copulation from fertilization, and hence, copulation from the timing of birth. All three types of separation mechanism occur in mammals. Delayed implantation cannot occur in reptiles and birds because their eggs do not implant, but delayed development occurs in some reptiles. Comparisons among vertebrate classes suggest that sperm storage in the female tract may have an upper limit of a little over 100 days in endotherms: non-hibernating mammals have circumvented this limit by using delayed implantation as an uncoupling mechanism. A long reproductive cycle (up to four years) probably makes sperm storage obligatory for some reptiles. We consider existing hypotheses for prolonged sperm storage and the other reproductive delays, and propose a new hypothesis. Our hypothesis extends Sandell's (1990) hypothesis which states that in mammals delayed implantation has evolved to allow females to time both their copulation and birth seasons optimally. We propose that the other separation mechanisms, namely sperm storage and delayed development, have also evolved for this reason. Sandell suggested that the optimal time for females to copulate is when the opportunities to obtain the best quality male exist, and thus the dislocation of copulation and birth seasons has occurred through sexual selection. We propose that the other two separation mechanisms may also have evolved through sexual selection, in part at least. Of the three separation mechanisms, sperm storage has additional advantages for females in that it also allows them to modify their choice of male after copulation has occurred, through sperm competition. Thus we propose that: (1) all separation mechanisms have evolved when the optimal time for copulation is not compatible with either the optimal time for fertilization (birds) or the optimal time for birth, given a constant gestation period (reptiles, birds and mammals); (2) separation mechanisms have evolved through sexual selection enabling females to improve the quality of the male that fertilizes their eggs, either through pre-copulatory male-male competition (via any of the separation mechanisms) or through post-copulatory sperm competition (via sperm storage).     

Hoechst 33342: the dye that enabled differentiation of living X-and Y-chromosome bearing mammalian sperm

Hoechst 33342 is the fluorophore used routinely to measure DNA in X- and Y-chromosome-bearing mammalian sperm so they can be separated by flow sorting. A difference of <3% in DNA mass can be detected. This synthetic dye consists of two adjacent benzimidazole rings with N-methyl-piperazine and phenolic groups at the ends. The molecule permeates the cell membrane of living cells and binds selectively to A-T base pairs exposed in the minor-groove of double stranded DNA. Capability to distinguish and separate X- and Y-chromosome-bearing sperm has led to artificial insemination of somewhere around a million female mammals. Offspring with obvious abnormalities are no more frequent than after insemination of unsorted sperm into cows, horses, humans, pigs, sheep, rabbits, dolphins and other mammals. There is no apparent genotoxic effect from exposure of sperm to Hoechst 33342, although information on cellular toxicity or development of embryos resulting from Hoechst 33342-stained sperm is less reassuring. Little is known about the fate of sperm-delivered Hoechst dye in the female reproductive tract or on progeny of resultant offspring.     

Clinical experience with flow cytometric separation of human X- and Y-chromosome bearing sperm

Numerous methods to separate human X- and Y-bearing sperm have been reported with unconfirmed separation after DNA analysis and inconsistent birth results. Successful flow cytometric separation of sperm resulting in alteration of the sex ratio of young born has been demonstrated in several animal species. Flow cytometric separation of human X- and Y-bearing sperm (MicroSort) has been confirmed after DNA analysis by fluorescence in situ hybridization (FISH). Pregnancies and births have resulted from the use of MicroSort after intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).     

Verification of flow cytometorically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique

Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method for sexing the spermatozoa of mammals. The standard method for verifying the purity of sorted X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and accuracy of DNA reanalysis by fluorescence in situ hybridization (FISH) using chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromosome Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flow cytometric reanalysis; purity for the sorted Y-bearing spermatozoa was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH across the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa and 5,000 Y spermatozoa were reanalyzed for DNA content for each ejaculate. These results confirm the high purity of flow sorted porcine X and Y sperm cells and the validity of reanalysis of DNA in determining the proportions of X- and Y-sorted spermatozoa from viewing thousands of individual sperm chromosomes directly using FISH.     

On the integrity of sperm DNA

Ejaculated rabbit spermatozoa washed with buffer prior to decondensation by Triton X-100 and dithiothreitol were good templates for DNA synthesis by Escherichia coli DNA polymerase. This result agrees with the observations of Zirkin and Chang [1977], and implies that the sperm DNA is nicked. Template activity, however, was reduced if spermatozoa were extensively washed before decondensation, and if DNase inhibitors EDTA or Na₂SO₄ were present during decondensation. Template activity was also low if decondensation was induced with DNase inhibitors thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate and dithiothreitol instead of with Triton X-100 and dithiothreitol. Calf thymus DNA was completely degraded when incubated with rabbit seminal plasma or buffer-washed spermatozoa, but much less degradation was observed if EDTA, Na₂SO₄, thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate were also present, or if spermatozoa were extensively washed with buffer. Centrifugation of spermatozoa through 2.05 M sucrose completely removed contaminating DNase, and such spermatozoa were inactive as DNA templates after decondensation. The DNA template activity of swollen rabbit sperm nuclei thus parallels the activity of a contaminating seminal plasma DNase. This suggest that the nicks in sperm DNA enabling it to act as a template for DNA synthesis were generated by the DNase during decondensation and thus are not a natural structural feature of the DNA. The presence of breaks in the DNA of decondensed buffer-washed spermatozoa (DNase contaminated) was confirmed by their incorporation of phosphate from [γ^?32 P] ATP in the presence of the enzyme polynucleotide kinase. These spermatozoa were found to contain as few as two breaks/mole of DNA, but sucrose-washed spermatozoa (DNase free) were free of breaks. The possible use of this enzymic procedure for the assessment of sperm genome damage and the evaluation of the quality of a sperm population are discussed.     

Preselection of sex of offspring in swine for production: current status of the process and its application

It is estimated that as many as 30,000 offspring, mostly cattle, have been produced in the past 5 years using AI or some other means of transport with spermatozoa sexed by flow cytometric sperm sorting and DNA as the marker of differentiation. It is well documented that the only marker in sperm that can be effectively used for the separation of X- and Y-chromosome bearing spermatozoa is DNA. The method, as it is currently used worldwide, is commonly known as the Beltsville Sperm Sexing Technology. The method is based on the separation of sperm using flow cytometric sorting to sort fluorescently (Hoechst 33342) labeled sperm based on their relative content of DNA within each population of X- and Y-spermatozoa. Currently, sperm can be produced routinely at a rate of 15 million X- and an equal number of Y-sperm per hour. The technology is being applied in livestock, laboratory animals, and zoo animals; and in humans with a success rate of 90-95% in shifting the sex ratio of offspring. Delivery of sexed sperm to the site of fertilization varies with species. Conventional AI, intrauterine insemination, intra-tubal insemination, IVF with embryo transfer and deep intrauterine insemination are effectively used to obtain pregnancies dependent on species. Although sperm of all species can be sorted with high purity, achieving pregnancies with the low numbers of sperm needed for commercial application remains particularly elusive in swine. Deep intrauterine insemination with 50-100 million sexed boar sperm per AI has given encouragement to the view that insemination with one-fiftieth of the standard insemination number will be sufficient to achieve pregnancies with sexed sperm when specialized catheters are used. Catheter design, volume of inseminate, number of sexed sperm are areas where further development is needed before routine inseminations with sexed sperm can be conducted in swine. Cryopreservation of sex-sorted sperm has been routinely applied in cattle. Although piglets have been born from frozen sex-sorted boar sperm, freezing and processing protocols in combination with sex-sorted sperm are not yet optimal for routine use. This review will discuss the most recent results and advances in sex-sorting swine sperm with emphasis on what developments must take place for the sexing technology to be applied in commercial practice.     

The eggstraordinary story of how life begins

More than 15 years have elapsed since the identification of phospholipase C ζ1 (PLCζ) from a genomic search for mouse testis/sperm‐specific PLCs. This molecule was proposed to represent the sperm factor responsible for the initiation of calcium (Ca²⁺) oscillations required for egg activation and embryo development in mammals. Supporting evidence for this role emerged from studies documenting its expression in all mammals and other vertebrate species, the physiological Ca²⁺ rises induced by injection of its messenger RNA into mammalian and nonmammalian eggs, and the lack of expression in infertile males that fail intracytoplasmic sperm injection. In the last year, genetic animal models have added support to its role as the long sought‐after sperm factor. In this review, we highlight the findings that demonstrated the role of Ca²⁺ as the universal signal of egg activation and the experimental buildup that culminated with the identification of PLCζ as the soluble sperm factor. We also discuss the structural–functional properties that make PLCζ especially suited to evoke oscillations in eggs. Lastly, we examine unresolved aspects of the function and regulation of PLCζ and whether or not it is the only sperm factor in mammalian sperm.