Pommer Angela C, Rutllant Josep, Meyers Stuart A
School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology, University of California, Davis, California 95616, USA.
Biol Reprod. 2003 Apr;68(4):1208-14. doi: 10.1095/biolreprod.102.011106. Epub 2002 Oct 31.
Phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism regulating sperm function that may be a meaningful indicator of capacitation. There is substantial evidence that cryopreservation promotes the capacitation of sperm and this cryocapacitation is frequently cited as one factor associated with the reduced longevity of cryopreserved sperm in the female reproductive tract. This study was designed to determine whether stallion sperm express different levels of tyrosine phosphorylation after in vitro capacitation and whether thawed sperm display similar phosphorylation characteristics in comparison with freshly ejaculated sperm. Experiments were performed to facilitate comparisons of tyrosine phosphorylation, motility, and viability of sperm prior to and following in vitro capacitation in fresh and frozen-thawed sperm. We hypothesized that equine spermatozoa undergo tyrosine phosphorylation during capacitation and that this phosphorylation is modified when sperm have been cryopreserved. We also hypothesized that tyrosine phosphorylation could be enhanced by the use of the activators dibutyryl cAMP (db cAMP) and caffeine, as well as methyl beta-cyclodextrin-which causes cholesterol efflux from the spermatozoa-and inhibited by the protein kinase A (PK-A) inhibitor H-89. Our results indicate that equine sperm capacitation is mediated by a signaling pathway that involves cAMP-dependent PK-A and tyrosine kinases and that cryopreserved sperm may be more sensitive to inducers of capacitation, which could explain their limited life span when compared with fresh sperm.
精子蛋白酪氨酸残基的磷酸化是调节精子功能的一种重要细胞内机制，可能是获能的一个有意义指标。有大量证据表明冷冻保存可促进精子获能，这种冷冻获能常被认为是与冷冻保存精子在雌性生殖道中寿命缩短相关的一个因素。本研究旨在确定马精子在体外获能后酪氨酸磷酸化水平是否不同，以及解冻精子与新鲜射出精子相比是否表现出相似的磷酸化特征。进行实验以方便比较新鲜精子和冻融精子在体外获能前后的酪氨酸磷酸化、活力和生存力。我们假设马精子在获能过程中发生酪氨酸磷酸化，并且当精子被冷冻保存时这种磷酸化会发生改变。我们还假设使用激活剂二丁酰环磷腺苷（db cAMP）和咖啡因，以及导致精子胆固醇外流的甲基-β-环糊精可增强酪氨酸磷酸化，而蛋白激酶A（PK-A）抑制剂H-89可抑制酪氨酸磷酸化。我们的结果表明马精子获能是由一条涉及cAMP依赖性PK-A和酪氨酸激酶的信号通路介导的，并且冷冻保存的精子可能对获能诱导剂更敏感，这可以解释与新鲜精子相比它们有限的寿命。
