BASIC STUDIES ON X- AND Y-BEARING SPERM

为了更有效和准确地鉴定人类携带X和Y染色体的精子，研究了荧光原位杂交（FISH）在精子细胞中的应用。用二硫苏糖醇处理洗涤后的精子以使头部去致密，用Carnoy溶液固定在载玻片上，并在-80 ° C下储存直至使用。在65 ℃硬化、变性和脱水后进行杂交。用DXZ 1探针检测x染色体，DYZ 1和DYZ 3探针检测y染色体。在荧光显微镜下观察标本。生物素标记的探针用亲和素-FITC染色，地高辛标记的探针用抗地高辛罗丹明染色。细胞核用碘化丙啶（PI）和DAPI染色。当生物素标记的DXZ 1被检查时，约50%的精子被识别为携带X染色体的精子，而地高辛标记的DXZ 1和生物素标记的DYZ 3的双重染色导致携带X染色体和Y染色体的精子的比例为1：1。该技术所需时间不到奎纳克林芥末染色的十分之一。遗憾的是，由于电泳装置的问题，FISH在自由流电泳的精子中的应用还没有完成，但是用Percoll密度梯度后的富含X的部分获得了有利的结果。此外，还对海胆卵尝试了一种由压力和电位组成的卵显微注射新技术。这将有助于将分离的带有x或y染色体的精子注入卵子，因为通过电泳分离的精子数量很少

In order to identify human x- and y-bearing sperm more efficiently and accurately, an application of fluorescence insitu hybridization (FISH) to the sperm cells was examined. Washed sperm were treated with dithiothreitol to decondense the heads, fixed with Carnoy solution on glass-slide, and stored at -80ﾟC until use. Hybridization was made after hardening at 65ﾟC,denaturation and dehydration. Probes used were DXZ1 for x chromosome and DYZ1 and DYZ3 for y chromosome. Specimens were observed under a flourescence microscope. Biotin-labeled probe was stained with avidin-FITC,while digoxigenin-labeled one was stained with anti-digoxigenin rhodamine. Nucleus was stained with propidium iodide (PI) and DAPI.When biotin-labeled DXZ1 was examined about 50% of sperm were recognized as x-bearing sperm, whereas double staining with digoxigenin-labeled DXZ1 and biotin-labeled DYZ3 resulted in a ratio of x- and y-bearing sperm, 1 : 1. The technique needs the time less than one tenth that for quinacrine mustard staining. Unfortunately, the application of FISH to the sperm subjected to free-flow electrophoresis has not yet been done because of the troble with the electrophoresis apparatus, but favorable results were obtained with the x-bearing rich fraction after Percoll density gradient. In addition, a new technique of microinjection into the egg, which consists of pressure and electric potential was tried with sea urchin eggs. This would be useful to inject isolated x- or y-bearing sperm into the egg, because the number of sperm isolated by the electrophoresis used to be small