Ca2＋、HCO －3和 BSA 对塔里木马鹿冻融精子体外获能及蛋白酪氨酸磷酸化的影响

摘要

The frozen thawing sperm of Cervus elaphus yarkandensis was used as experimental materi-als,sperm capacitation status were assessed by chlorotetracycline (CTC)staining,the expression levels of tyrosine phosphorylated protein were detected with Western blot analysis of sperm membrane protein sep-arated by SDS-PAGE,following the sperm were suspended in spTALP liquids of cattle-haemocyanin where there were,Ca2 + ,HCO -3 and BSA deficiency or containing,and cultured for 0,2,4 h.The results showed that the sperm motility and capacitation rate decreased significantly (P <0.05),and the levels of sperm protein tyrosine phosphorylation were relatively lower (P < 0.05 );when Cervus elaphus yarkandensis sperm in vitro capacitation system simultaneously or separately lack BSA,HCO -3 and Ca2 + ,the sperm mo-tility and capacitation rate decreased significantly (P <0.05),On the contrary,when BSA,HCO -3 and Ca2 +existed at the same time in the vitro capacitation system,sperm motility and sperm capacitation rate signif-icantly increased (P <0.05).Moreover,the level of sperm protein tyrosine phosphorylation expressed when there were Ca2 + and BSA deficiency in capacilation system,while contained HCO -3 ,was basically close to that expressed when there were HCO -3 and BSA deficiency but Ca2 + contained.But when there were HCO -3 and Ca2 + deficiency at the same time,level of the sperm protein tyrosine phosphorylation significantly de-creased (P <0.05).It was suggested that HCO -3 and Ca2 + played an important regulatory role in Cervus elaphus yarkandensis sperm capacitation and protein tyrosine phosphorylation.%以塔里木马鹿冻融精子为材料，将精子分别悬浮于缺或含 Ca2＋、HCO －3和牛血清白蛋白(BSA)的spTALP液中，在培养0、2、4 h 时，采用金霉素(CTC)染色法评价精子获能状态，采用 SDS-PAGE 分离精子膜蛋白，进行免疫印迹分析，检测酪氨酸磷酸化蛋白的表达水平.结果表明，塔里木马鹿精子体外获能体系中同时或单独缺少 BSA、HCO －3和 Ca2＋时，精子活力及获能率明显下降(P ＜0．05)，精子蛋白酪氨酸磷酸化水平也相对较低(P＜0．05)；相反获能体系中这三种成分同时存在时，精子活力及获能率明显提高(P ＜0．05)，精子蛋白酪氨酸磷酸化水平也相对较高(P ＜0．05)；另外，培养体系中缺 Ca2＋和 BSA 但存在 HCO －3时的精子蛋白酪氨酸磷酸化水平，与缺 HCO －3和 BSA 但存在 Ca2＋时的基本相似；同时缺少 HCO －3和 Ca2＋时，精子蛋白酪氨酸磷酸化水平显著降低(P ＜0．05).提示 HCO －3和 Ca2＋在塔里木马鹿精子获能及其蛋白酪氨酸磷酸化过程中具有重要调控作用，而BSA 协助增强精子获能及其蛋白酪氨酸磷酸化过程.