Effects of disulfide bond reducing agents on sperm chromatin structural integrity and developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection in pigs
Effects of disulfide bond reducing agents on sperm chromatin structural integrity and developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection in pigs
复制标题
二硫键还原剂对猪胞浆内单精子注射后精子染色质结构完整性及体外成熟卵母细胞发育能力的影响
DOI：
10.1530/rep-08-0143
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发表时间：
2009-04-01
期刊：
影响因子：
3.8
通讯作者：
Tian, Jian-Hui
中科院分区：
文献类型：
--
作者：
Cheng, Wen-Min;An, Lei;Tian, Jian-Hui
我们最近报道了电激活后的二次化学激活大大提高了体外成熟猪卵母细胞通过胞浆内单精子注射（ICSI）受精的发育能力。我们假设用二硫键还原剂处理精子会提高这种ICSI程序产生的猪胚胎的发育能力。我们研究了谷胱甘肽（GSH）、二硫苏糖醇（DTT）、谷胱甘肽或DTT联合肝素对体外成熟猪卵母细胞ICSI后精子DNA结构、父系染色体完整性、原核形成和发育能力的影响。采用吖啶橙染色和基于流式细胞术的精子染色质结构测定，通过计算主要种群外的细胞（COMP α T）来确定精子DNA的完整性。gsh处理组和对照组之间COMP α T值无差异。gsh处理组COMP α T值显著低于dtt处理组。ICSI后，GSH治疗没有显著改变父系染色体完整性。DTT加或减肝素处理的精子的父系染色体完整性也在所有组中最低。gsh处理的精子正常受精率和囊胚形成率最高，显著高于对照组和dtt处理组。对照和谷胱甘肽处理的精子产生的囊胚多数为二倍体，而dtt处理的精子产生的囊胚为单倍体。总之，用谷胱甘肽处理精子增强了我们优化的ICSI程序产生的猪胚胎的发育能力。
We recently reported that electrical activation followed by secondary chemical activation greatly enhanced the developmental competence of in vitro matured porcine oocytes fertilized by intracytoplasmic sperm injection (ICSI). We hypothesized that sperm treatment with disulfide bond reducing agents will enhance the development competence of porcine embryos produced by this ICSI procedure. We examined the effects of glutathione (GSH), dithiothreitol (DTT), GSH or DTT in combination with heparin on sperm DNA structure, paternal chromosomal integrity, pronuclear formation, and developmental competence of in vitro matured porcine oocytes after ICSI. Acridine orange staining and flow cytometry based sperm chromatin structure assay were used to determine sperm DNA integrity by calculating the cells outside the main population (COMP alpha T). No differences were observed in COMP alpha T values among GSH-treated and control groups. COMP alpha T values in GSH-treated groups were significantly lower than that in DTT-treated groups. Following ICSI, GSH treatments did not significantly alter paternal chromosomal integrity. Paternal chromosomal integrity in sperm treated with DTT plus or minus heparin was also the lowest among all groups. GSH-treated sperm yielded the highest rates of normal fertilization and blastocyst formation, which were significantly higher than that of control and DTT-treated groups. The majority of blastocysts derived from control and GSH-treated spermatozoa were diploid, whereas blastocysts derived from DTT-treated spermatozoa were haploid. In conclusion, sperm treatment with GSH enhanced the developmental capacity of porcine embryos produced by our optimized ICSI procedure.