Chamberland A, Fournier V, Tardif S, Sirard M A, Sullivan R, Bailey J L
Département des sciences animales, Centre de recherche en Biologie de la Reproduction, Université Laval, Québec, Canada.
Theriogenology. 2001 Feb 1;55(3):823-35. doi: 10.1016/s0093-691x(01)00446-0.
It is generally accepted that incubation with heparin is required to induce capacitation of ejaculated bovine spermatozoa in vitro. The capacitation process implicates many biochemical events, and is correlated with modified sperm motility and the phosphorylation of specific proteins on tyrosine residues. To better understand the molecular basis of heparin-induced capacitation, bovine spermatozoa were incorporated with a radioactive substrate of protein kinases [gamma32P]-ATP, to observe protein phosphorylation dynamics over time. Sperm motion parameters including the percentage of motile spermatozoa, amplitude of lateral head displacement (ALH) and flagellar beat cross frequency (BCF) were assessed to determine whether the protein phosphorylation patterns induced by heparin also promote changes in motility. Capacitation was confirmed using the chlortetracycline fluorescence assay and the appearance of 'pattern B' stained spermatozoa. Evaluation of the different motility parameters during capacitation reveal that heparin has a marked negative effect, over time, on the percentage of motile spermatozoa, consistent with hyperactivation. Indeed, the presence of heparin greatly increases the BCF of bull spermatozoa and induces a significant increase in the ALH compared to spermatozoa incubated without heparin. We detected several sperm proteins that are phosphorylated over time. A 45 kDa protein is the most intensely phosphorylated of the sperm proteins. However, it is visible regardless of the presence of heparin. Interestingly, a second phosphorylated protein of approximately 50 kDa undergoes more intense phosphorylation with heparin than without. In summary, the present study demonstrated that heparin induces physiological changes in several sperm motility parameters including ALH, BCF and the percentage of motile spermatozoa. Heparin also increases the intensity of phosphorylation of a 50 kDa sperm protein. These results suggest that capacitation of bovine spermatozoa and capacitation-associated motility changes may be regulated by a mechanism that includes protein phosphorylation, and that a presently unknown protein kinase is involved.
一般认为，体外诱导射出的牛精子获能需要用肝素孵育。获能过程涉及许多生化事件，并与精子活力的改变以及酪氨酸残基上特定蛋白质的磷酸化相关。为了更好地理解肝素诱导获能的分子基础，将牛精子与蛋白激酶的放射性底物[γ32P]-ATP结合，以观察蛋白质磷酸化随时间的动态变化。评估精子运动参数，包括活动精子的百分比、头部侧向位移幅度（ALH）和鞭毛摆动交叉频率（BCF），以确定肝素诱导的蛋白质磷酸化模式是否也促进活力变化。使用金霉素荧光测定法和“B型”染色精子的出现来确认获能。对获能过程中不同运动参数的评估表明，随着时间的推移，肝素对活动精子的百分比有显著的负面影响，这与超活化一致。事实上，与未用肝素孵育的精子相比，肝素的存在极大地增加了公牛精子的BCF，并导致ALH显著增加。我们检测到几种随时间磷酸化的精子蛋白。一种45 kDa的蛋白是精子蛋白中磷酸化最强烈的。然而，无论是否存在肝素，它都可见。有趣的是，第二种约50 kDa的磷酸化蛋白在有肝素的情况下比没有肝素时磷酸化更强烈。总之，本研究表明，肝素可诱导包括ALH、BCF和活动精子百分比在内的几种精子运动参数发生生理变化。肝素还增加了一种50 kDa精子蛋白的磷酸化强度。这些结果表明，牛精子的获能和与获能相关的活力变化可能受一种包括蛋白质磷酸化的机制调节，并且涉及一种目前未知的蛋白激酶。
