Zhang H, Zheng R L
Department of Biology, Lanzhou University, China.
Free Radic Res. 1996 Apr;24(4):261-8. doi: 10.3109/10715769609088023.
Capacitation of spermatozoa is an essential procedure for fertilization. Capacitated spermatozoa have an increase in the intracellular cAMP and acrosome reaction (AR) occurs immediately. The effect of exogenous superoxide anion (O2-) on the level of intracellular cAMP and the percentages of both spontaneous AR and lysophosphatidylcholine-induced AR (LPC-AR) were studied using semen samples collected from 10 healthy and fertile volunteers working or studying in Lanzhou Medical College. Spermatozoa were separated by Percoll and incubated at 37 degrees C in Ham's F-10 medium with O2- generation system: xanthine+xanthine oxidase +catalase+diethylenetriaminepentaacetic acid+ sodium formate. The intracellular cAMP was determined by (3H)-cAMP radioimmunoassay at 3 h of incubation, and the percentages of AR and LPC-AR were evaluated by the triple-stain technique at 3.5 h of incubation. The effects of SOD with different concentration were also determined. The results showed:the level of intracellular cAMP (pmol/10(8) spermatozoa) of spermatozoa increased from 14.0 +/- 1.3 to 23.2 +/- 2.5 (P < 0.01), and the percentages of AR and LPC-AR increased from 4.5 +/- 1.1% and 14 +/- 1.9% to 16 +/- 2.0% and 32.5 +/- 1.7%, respectively (P < 0.01 in both comparisons). SOD inhibited these processes concentration dependently. To investigate the source of O2- during in vivo sperm capacitation, female genital tract fluids collected from 6 healthy nonpregnant donors of reproductive age, and seminal plasma, capacitated and noncapacitated spermatozoa from 10 fertile volunteers were investigated by spin trapping method. The results showed: A typical electron paramagnetic resonance spectrum for O2- spin adduct was exhibited only in capacitated spermatozoa but not in vaginal or cervical secretions, uterine and fallopian tubal fluids, nor in seminal plasma and noncapacitated spermatozoa. These results suggested that only capacitated spermatozoa themselves are able to generate O2- which stimulated their capacitation in turn. Furthermore, on the basis of these data, we propose that it may be possible to utilize the inhibitory effect of SOD on sperm capacitation so as to regulate fertilization.
精子获能是受精的一个必要过程。获能的精子细胞内cAMP增加，并且顶体反应（AR）随即发生。使用从在兰州医学院工作或学习的10名健康且可育志愿者采集的精液样本，研究了外源性超氧阴离子（O2-）对细胞内cAMP水平以及自发AR和溶血磷脂酰胆碱诱导的AR（LPC-AR）百分比的影响。精子通过Percoll分离，并在含有O2-生成系统（黄嘌呤+黄嘌呤氧化酶+过氧化氢酶+二亚乙基三胺五乙酸+甲酸钠）的Ham's F-10培养基中于37℃孵育。孵育3小时时通过（3H）-cAMP放射免疫测定法测定细胞内cAMP，孵育3.5小时时通过三重染色技术评估AR和LPC-AR的百分比。还测定了不同浓度超氧化物歧化酶（SOD）的作用。结果显示：精子细胞内cAMP水平（pmol/10（8）个精子）从14.0±1.3增加到23.2±2.5（P<0.01），AR和LPC-AR的百分比分别从4.5±1.1%和14±1.9%增加到16±2.0%和32.5±1.7%（两项比较均P<0.01）。SOD浓度依赖性地抑制这些过程。为了研究体内精子获能过程中O2-的来源，通过自旋捕获法研究了从6名健康育龄未孕供体采集的女性生殖道液，以及来自10名可育志愿者的精浆、获能和未获能精子。结果显示：仅在获能精子中呈现出O2-自旋加合物的典型电子顺磁共振谱，而在阴道或宫颈分泌物、子宫和输卵管液中未呈现，在精浆和未获能精子中也未呈现。这些结果表明，只有获能的精子自身能够产生O2-，而O2-又反过来刺激它们的获能。此外，基于这些数据，我们提出有可能利用SOD对精子获能的抑制作用来调节受精。
