肝素处理山羊精子体外获能的研究

Studies on in vitro Capacitation of Goat Spermatozoa by Heparin Treatment

系统研究了作用浓度、时间和温度以及输卵管上皮细胞和卵丘细胞对肝素处理山羊精子体外获能后的精子活力、质膜完整性、顶体完整率、获能比例及受精和卵裂的影响 ,为改善山羊精子体外获能效果和研究获能机理提供了必要的数据。主要实验结果如下 :1 在获能液中添加 5、10、2 5、50和 10 0 μg mL肝素处理 45min时 ,添加 50和10 0 μg mL肝素精子获能比率最高 (分别为 55%和 56% ) ,但添加 10 0 μg mL肝素处理后顶体完整率明显 (P <0 0 5)低于对照组。说明山羊精子获能的最佳肝素浓度为 50 μg mL。 2 随肝素作用时间 (0 ,10 ,2 0 ,3 0 ,45,60和 12 0min)的延长 ,获能精子比例逐渐提高。其中 ,肝素处理 45～ 12 0min各组的获能精子比例差异不显著 (P >0 0 5) ,处理12 0min组的精子活力和质膜完整率显著低于其它各组。说明 50 μg mL肝素处理精子获能的最佳时间是 45～ 60min。 3 在 42℃和 3 8 5℃下处理时 ,获能精子比例显著高于 15℃和 3 7℃ ,但 42℃处理后精子活力和顶体完整率显著低于其它温度。因此 ,3 8 5℃为山羊精子获能的最佳温度。 4 与输卵管上皮细胞共培养获能精子比例显著高于对照组和卵丘细胞组 ,但精子活力、质膜完整率和顶体完整率差异不显著。输卵管上皮组的受精率 (91 3 % )和卵?

Systematical studies are lacking on the influencing factors and mechanisms of th e heparin enhanced sperm capacitation, although many studies have shown that hep arin enhanced sperm capacitation. The effect of heparin concentration and exposu re time, incubation temperature and co_culture with oviductal epithelial cells o r cumulus cells on goat sperm capacitation were investigated in this study. The motility, membrane and acrosome integrity and capacitated percentage of goat spe rmatozoa were assessed after different heparin treatments, and rates of fertiliz ation and embryo cleavage were compared after in vitro insemination of oocyt es w ith spermatozoa capacitated by different heparin treatments. The major results a re summarized as follows: 1) When spermatozoa were capacitated with heparin at 5 , 10, 25, 50 and 100μg/mL for 45 min, 50 and 100μg/mL heparin treatments pro du ced the highest capacitated percentages of 55% and 56%, respectively, but th e pe rcentage of spermatozoa with intact acrosomes in the 100μg/mL heparin treatmen t decreased significantly (P<0 05) in comparison with that in the control gr oup, indicating that the optimal heparin concentration for goat sperm capacitation wo uld be 50μg/mL. 2) Capacitated percentage of spermatozoa increased with extensi on of treatment time when goat sperm were treated with 50μg/mL heparin for 0, 1 0, 20, 30, 45, 60 or 120 min. Although heparin treatments for 45 to 120 min did not differ significantly (P>0 05) in capacitated sperm percentages, sperm m otili ty and membrane integrity decreased significantly when treated with heparin for 120 min. This suggested that the optimal exposure time of heparin at 50μg/mL f o r goat sperm capacitation would be 45 to 60 min. 3) Significantly higher capacit ated percentages of spermatozoa were obtained when goat sperm were treated at 42 and 38 5 ℃ than at 15 and 37 ℃, but sperm motility and acrosome integrity we re significantly lower when spermatozoa were treated at 42 ℃ than they were tre ated at other temperatures. Temperature of 38 5 ℃ would, therefore, be the opt im a l temperature for goat sperm capacitation. 4) The capacitated percentage of sper matozoa was significantly higher when goat sperm were co_cultured with oviductal epithelial cells than when treated with heparin alone or co_cultured with cumul us cells, but sperm motility and membrane and acrosome integrity did not differ significantly among the three treatments. Rates of fertilization (91 3%) and cl e avage (72 2%) were significantly higher in the oviductal epithelial cell co_cul t ure group than those in the heparin alone group. This indicated that co_culture with oviductal epithelial cells significantly enhanced goat sperm capacitation b y heparin treatment.