获能处理对山羊精子结合外源DNA能力的影响  

Effect of goat sperm capacitation induced by heparin or calcium ionophore A23187 in vitro on the transfection exogenous DNA into spermatozoa

作　　者：赵永聚[1,2] 王炜[1] 王剑[1] 孙新明[2,3] 张家骅[1] 

机构地区：[1]西南大学动物科技学院,重庆400716 [2]第三军医大学基础部,重庆400038 [3]井冈山学院医学院,江西井冈山343000

出　　处：《中国兽医学报》2009年第4期528-532,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30600430);西南大学博士启动基金资助项目

摘　　要：鲜精离心洗涤除去精浆,然后用0.4μmol/L钙离子载体(IA组)作用10 min或用10 mg/L肝素(HE组)作用90 min获能后,与DNA孵育,再用地高锌(DIG)末端标记的线形外源DNA进行转染,用免疫组化的方法分别检测它们的转染效率;用透射电镜观察精子获能前后的超微结构变化,并用碘化丙锭和羟化荧光素双探针检测获能对精子质膜完整性的影响,探讨精子获能影响精子结合及内化外源DNA变化的原因。结果显示,获能处理降低了精子结合和内化外源DNA能力,IA组和HE组结合率分别为(17.41±4.69)%和(15.00±4.36)%,都极显著低于未经获能处理的精子(32.97±4.58)%(P<0.01)。IA组、HE组和对照组(未获能)内化率分别为(12.09±2.50)%、(8.93±1.79)%和(25.52±2.57)%,IA组、HE组均极差异小于对照组(P<0.01)。超微结构观察和双荧光探针检测都发现精子获能后质膜完整性降低。用IA或HE获能后的精子质膜完整率是分别是(33.16±6.46)%和(33.90±4.14)%,与对照组差异极显著(P<0.01)。结果表明,精子结合及内化外源DNA不是纯粹的分子渗透现象,而是受严格的分子调控的。The effect of goat spermatozoa capacitation induced by heparin or calcium ionophore A23187 in vitro on the transfection exogenous DNA into spermatozoa was evaluated in our study. Seminal ejaculates collected artificially from healthy adult bucks were pooled together and the washed sperm were incubated in vitro for 10 mins in the capacitation medium with 0.4/μmol/L calcium ionophore A23187（IA） or 90 rains in the medium with 10 mg/L hepa rin（HE）, of modified Tyrode＇s solution at 38. 5°C with sperm concentrations of 10 20 million/mL. The activated sperm motility and acrosome reaction were assessed. The induced sperm were incubated with exogenous DIG-labeled linear DNA for 60 mins. The transfection rate of sperm was observed by the in situ hybridization methods. The ultra-structure of activated spermatozoa were tested by transmission electron microscope （TEM）, and the integrity of sperm plasma membrane was evaluated with a combination of fluorescent probes-carboxifluorescein diacetate and propidium iodide （PI）. The results showed that sperm capacitation significantly reduced the efficiency of spermatozoa in picking up exogenous DNA （P〈0.01）. The efficiency （15.00±4. 36）% and （32. 97±4. 58）%,respectivelyo of IA, HE and untreated sperm were （ 17.41 ± 4.69） %, The efficiency of subsequently internalize the DNA into sperm nuclei were （ 12.09 ± 2.50） %, （8.9 3 ± 1.79） % and （25.5 2 ± 2.5 7） %, respectively. And the spermatozoa capacitation significantly reduced the integrity of sperm plasma membrane （P〈0.01）. It would suggest that DNA up- take by the mammalian spermatozoa is a very specific and high regulated phenomenon.

关 键 词：山羊 精子 体外获能 外源DNA 质膜 

分 类 号：S827[农业科学—畜牧学] S814[农业科学—畜牧兽医]