single-cell pulsed-field gel electrophoresis to detect the early stage of dna fragmentation in human sperm nuclei单细胞pulsed-field凝胶电泳检测早期人类精子核dna碎片.pdfVIP

Single-Cell Pulsed-Field Gel Electrophoresis to Detect the Early Stage of DNA Fragmentation in Human Sperm Nuclei 1 1 2 1 Satoru Kaneko *, Joji Yoshida , Hiromichi Ishikawa , Kiyoshi Takamatsu 1 Reproduction Center, Gynecology, Ichikawa General Hospital, Tokyo Dental College, Ichikawa, Chiba, Japan, 2 Reproduction Center, Urology, Ichikawa General Hospital, Tokyo Dental College, Ichikawa, Chiba, Japan Abstract Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 mg/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra- cytoplasmic sperm injectio