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。     

先天性白内障的遗传咨询

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。     

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.     

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。     

哺乳动物精子质量的评价方法

精子的各种功能状态反应了精子的受精能力。检测精子质膜完整性的荧光探针有SYBR－14,SYTO－17,CFDA、CDMFDA、CAM、PI、Hoechst33258、Hoechst33342,其中以SYBR－14结合PI使用效果最好,检测线粒体活性的荧光探针有JC－1、MITO、Rh123,JC-1比MITO和Rh123更适用于检测精子线粒体功能,检测顶体状态的荧光探针有PNA－FITC、PSA－FITC、LYSO－G及CTC等。检测获能状态的荧光探针有CTC。此外,还可以通过检测精子与透明带的结合能力、精子穿入去透明带卵子的能力以及使卵子受精的能力和其后胚胎的发育能力等方面来评价精子的功能状态。     

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。     

应用高压液相层析（HPLC）评价乙肝疫苗纯度方法的建立和应用

应用高压液相层析（ＨＰＬＣ）方法，对血源乙肝疫苗、ＣＨＯ基因工程乙肝疫苗及酵母基因工程乙肝疫苗进行了检测，其纯度分别为８８～９４％，８７～９６％和９８～１００％。     

玉米生活精子的分离及扫描电镜观察(简报)

生殖细胞的分离和纯化是植物生殖生物学及生殖细胞工程的发展基础之一。70年代,Cass(1973)首次从大麦花粉管及花粉粒中分离精子;到80年代后期,分离精子已成为国际上实验生殖生物学中的热点。由于大量分离和纯化的技术进一步完善,已从一些植物的     

Experimental demonstration that mammalian oocytes are not selective towards X- or Y-bearing sperm

Mammalian oocytes are thought to be neutral as for X- or Y-bearing sperm selection is concerned, and penetration of an oocyte by an X- or a Y-bearing sperm is considered a random event. This assumption is mainly based on a posteriori evidences of a nearly equal sex ratio at birth, but it has never been experimentally demonstrated. We have designed a simple experiment, which allowed the penetration of an oocyte by more than one sperm and the further sexing by PCR of each single pronucleus present within the ooplasm. For the first time, we provide experimental evidence that mammalian oocytes do not play a selecting role since a single oocyte may be simultaneously fertilised by both X- and Y-bearing sperm.     

Simultaneous detection of X- and Y-bearing human sperm by double fluorescence in situ hybridization

Double fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in decondensed human sperm nuclei. Biotinylated X chromosome specific (TRX) and digoxigenin-labeled Y chromosome specific (HRY) probes were simultaneously hybridized to sperm preparations from 12 normal healthy donors. After the hybridization, the probes were detected immuno-cytochemically, using two different and independent affinity systems. Ninety-six percent of the 12,636 sperm showed fluorescent labeling, of which 47.4% were haploid X and 46.8% were haploid Y. A frequency of 0.46% of XX-bearing sperm (0.28% disomic, 0.18% diploid) and 0.38% YY-bearing sperm (0.21% disomic, 0.17% diploid) was found. The overall proportions of X- and Y-bearing sperm in the ejaculates were 47.9% and 47.2%, respectively, which was not significantly different from the expected 50:50 ratio. In addition 0.21% of cells appeared to be haploid XY-bearing sperm, 0.62% were diploid XY-bearing cells, and 0.05% of cells were considered to be tetraploid cells. The application of double FISH to human sperm using X-chro-mosome and Y-chromosome probes has allowed a more accurate assessment of the sex chromosal complements in sperm than single FISH method and quinacrine staining for Y-bodies. © 1993 Wiley-Liss, Inc.     

Difference in sperm head volume as a theoretical basis for sorting X- and Y-bearing spermatozoa: potentials and limitations

Volume-based sorting of X- and Y-chromosome-bearing sperm cells could be an interesting alternative to the existing technique based on DNA content. Advantages would be that DNA staining and ultraviolet excitation, used in the existing technique, could be avoided. To assess the possibilities and limitations of sperm-head volume as sorting criterion, achievable purity and yield are determined for bull sperm. Two important parameters in this respect are the magnitude of the volume difference and the biological variation within each (X or Y) population. Earlier, we established a difference in volume matching the difference in DNA content (3.8%) between X- and Y-bearing bull sperm heads by comparing thicknesses and areas of high numbers of pre-sorted X- and Y-bearing bull sperm heads by interference microscopy and subsequent image analysis. Unfortunately, despite the high number of measurements, a direct determination of biological variations was not possible due to an unknown contribution of instrumental variations. In this paper, we determine the contribution of instrumental errors by measuring a single sperm head, varying parameters such as location in the image, orientation angle, focusing etc., simulating the behavior of the measuring system. After correction, both for the instrumental variation, and for the fact that the original samples were not pure, biological variations in volume of 5.9 +/- 0.8% were found. Our results indicate that when 10% of the bull sperm are sorted on basis of their head volume, a theoretical enrichment of 80% could be achieved. Expected purity and yield are lower than what is standard for the existing technique. At the moment, a technique to physically separate X- and Y-bearing sperm cells based on volume is not available. However, for applications for which the potential hazards of DNA staining and UV excitation are problematic, the development of such technique should be considered.     

On the selective elimination of Y-bearing sperm

The potential use of antibodies that selectively recognize either X-bearing or Y-bearing sperm is self-evident. Thus our attention was directed to the fact that under optimal conditions, H-Y antibody lyses 50% of mouse spermatozoa. Accordingly, we asked whether expression of H-Y antigen is haploid in spermatozoa from XY male mice heterozygous for the autosomal dominantSxr gene, for if H-Y expression were haploid, H-Y antibody would be expected to kill 75% of spermatozoa derived from these XY,Sxr/- males. However, maximal lysis remained at the 50% level, which indicates that haploid expression of H-Y antigen and the potential immunoselection of Y-(or X-) bearing spermatozoa are unlikely.     

Differences in motility of human X- and Y-bearing spermatozoa.

Human Y-bearing spermatozoa, as identified by the quinacrine staining technique, were found to be significantly more motile than X-bearing spermatozoa under laboratory conditions. This difference is consistent with current estimates of the difference in mean head DNA content.     

CAG repeat lengths in X- and Y-bearing sperm indicate that gender bias during transmission of Huntington's disease gene is determined in the embryo

The size of the CAG tract at the Huntington's disease (HD) locus upon transmission depends on the gender of the parent. However, the basis for the parent-of-origin effect is unknown. To test whether expansion and contraction in HD are "imprinted" in the germ cells, we isolated the X- and Y-bearing sperm of HD transgenic mice. Here we show that CAG repeat distributions in the X- and Y-bearing spermatozoa of founding fathers do not differ. These data show that gender-dependent changes in CAG repeat length arise in the embryo.     

Binding of anti-H-Y monoclonal antibodies to separated X and Y chromosome bearing porcine and bovine sperm

Studies designed to answer the question whether or not H-Y antigen is preferentially expressed on Y chromosome bearing sperm have resulted in conflicting results. This is probably due to the absence of reliable methods for estimating the percentage of X and Y chromosome bearing sperm in fractions, enriched or depleted for H-Y antigen positive sperm. In recent years a reliable method for separating X and Y chromosome bearing sperm has been published. With this method, separation is achieved by using a flow cytometer/cell sorter, which detects differences in DNA content. This technique provided the first opportunity for testing anti-H-Y antibody binding to fractions enriched for X and Y chromosme bearing sperm, directly. A total of 7 anti-H-Y monoclonal antibodies were tested using sorted porcine sperm and in one experiment also sorted bovine sperm. All monoclonal antibodies bound only a fraction of the sperm (20 to 50%). However, no difference in binding to the X and Y sperm enriched fractions was found. Therefore, the present experiments do not yield evidence that H-Y antigen is preferentially expressed in Y chromosome bearing sperm. © 1993 Wiley-Liss, Inc.