Baumber Julie, Meyers Stuart A
Sperm Biology Laboratory, Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, 95616, USA.
J Androl. 2006 May-Jun;27(3):459-68. doi: 10.2164/jandrol.05107. Epub 2005 Dec 8.
Macaque spermatozoa can be capacitated according to a defined protocol and exhibit hyperactivated motility similar to that described in other species. The aim of this study was to create a method for defining hyperactivation that could be routinely used in the laboratory alongside our existing sperm motility analysis protocol. Percoll-separated macaque spermatozoa were incubated for 2 hours (37 degrees C; 5% CO(2) in air) at a concentration of 20 x 10(6)/mL in bicarbonate (36 mmol)-buffered Biggers, Whitten and Whittingham medium (BWW) containing 30 mg/mL bovine serum albumin (BSA), followed by an additional 30 minutes with (capacitated) or without (incubated) caffeine (1 mmol) and dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP; 1.2 mmol). One hundred and fifty progressive and hyperactivated tracks were selected from each of three monkeys. Thresholds for hyperactivation were based on the 10th (amplitude of lateral head displacement, ALH) and 90th (linearity, LIN) percentiles of the hyperactivated kinematic data set and were LIN less than or equal to 69% and ALH greater than or equal to 7.5 microM; a threshold of greater than or equal to 130 microM/s was also included for curvilinear velocity (VCL). These thresholds were 91% effective at identifying hyperactivated tracks. Capacitation of macaque spermatozoa, by the addition of caffeine and dbcAMP, resulted in a significant increase in ALH, VCL, and beat cross frequency and a significant decrease in total and progressive motility, straight line velocity, straightness, and LIN when compared to incubated spermatozoa, suggesting the expression of hyperactivated motility. Utilizing the above thresholds, hyperactivation was expressed by 5% +/- 0.8% of the incubated sperm population vs 53 +/- 3.7% of the capacitated sperm population (P < .0001). Hyperactivation was not observed when dbcAMP and caffeine were added separately and was significantly (P < .005) reduced by the addition of H-89. The results of this paper demonstrate that hyperactivation can be reliably estimated for rhesus macaque spermatozoa.
猕猴精子可根据既定方案进行获能，并表现出与其他物种中所描述的类似的超激活运动。本研究的目的是创建一种定义超激活的方法，该方法可在实验室中与我们现有的精子运动分析方案一起常规使用。将经Percoll分离的猕猴精子以20×10⁶/mL的浓度在含有30mg/mL牛血清白蛋白（BSA）的碳酸氢盐（36mmol）缓冲的比格斯、惠滕和惠廷厄姆培养基（BWW）中于37℃、5%二氧化碳（空气中）孵育2小时，然后再分别在有（获能）或无（孵育）咖啡因（1mmol）和二丁酰腺苷3',5'-环磷酸（dbcAMP；1.2mmol）的条件下孵育30分钟。从三只猴子的精子中各选取150条进行性运动和超激活轨迹。超激活的阈值基于超激活运动学数据集的第10百分位数（头部横向位移幅度，ALH）和第90百分位数（线性度，LIN），即LIN小于或等于69%且ALH大于或等于7.5微米；曲线速度（VCL）还包括大于或等于130微米/秒的阈值。这些阈值在识别超激活轨迹方面的有效性为91%。与孵育的精子相比，添加咖啡因和dbcAMP使猕猴精子获能后，ALH、VCL和摆动交叉频率显著增加，总运动性和进行性运动性、直线速度、直线度和LIN显著降低，表明超激活运动得以表达。利用上述阈值，孵育的精子群体中有5%±0.8%表现出超激活，而获能的精子群体中有53±3.7%表现出超激活（P<.0001）。单独添加dbcAMP和咖啡因时未观察到超激活，添加H-89后超激活显著（P<.005）降低。本文结果表明，恒河猴精子的超激活可以可靠地评估。
