Andrews J C, Nolan J P, Hammerstedt R H, Bavister B D
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802-4504.
Biol Reprod. 1994 Dec;51(6):1238-47. doi: 10.1095/biolreprod51.6.1238.
Zinc stabilizes somatic cell membranes and DNA, inhibits respiration, is present in high concentrations in the male reproductive tract, and may stabilize sperm during storage and ejaculation. Zinc removal from sperm may be necessary to prepare sperm for fertilization (capacitation). Incubation with Zn2+ chelators, e.g., D-penicillamine, can capacitate hamster sperm (Andrews and Bavister, Gamete Res 1989; 23:159-70). In the present study, the Zn(2+)-specific fluorochrome N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) and the vital stain propidium iodide were used to assess the zinc content of live hamster sperm with flow cytometry before and after capacitation. Capacitation was monitored with a salt-stored zona pellucida penetration assay or the occurrence of spontaneous or induced (with lysophosphatidylcholine) acrosome reactions. The effect of added zinc on sperm capacitation was also evaluated. Image Analysis was used to determine the subcellular location of zinc (TSQ fluorescence) and atomic absorption to determine whether the total zinc content of sperm changes during capacitation. Sperm incubated under non-capacitating conditions had high TSQ fluorescence and could not penetrate zonae pellucidae. Sperm incubated under capacitating conditions (plus BSA or D-penicillamine) were zinc-depleted (low fluorescence) and penetrated 90% or 78% of zonae, respectively. Image analysis showed a significant reduction in zinc in the acrosomal region during capacitation with BSA, but this did not correlate with the occurrence of spontaneous acrosome reactions. The atomic absorption data showed that the total zinc content of sperm was reduced by 44% or 40% when sperm were incubated under capacitating conditions (BSA or D-penicillamine, respectively). Zona pellucida penetration was completely inhibited when zinc was present throughout the capacitation period but not when it was added at the end of incubation. These data indicate that removal of zinc from hamster sperm is correlated with capacitation and may play a key regulatory role in this process.
锌可稳定体细胞的细胞膜和DNA，抑制呼吸作用，在雄性生殖道中含量很高，并且可能在精子储存和射精过程中使其保持稳定。为使精子具备受精能力（获能），可能需要去除精子中的锌。用锌离子螯合剂（如D-青霉胺）孵育可使仓鼠精子获能（安德鲁斯和巴维斯特，《配子研究》1989年；23:159 - 70）。在本研究中，锌特异性荧光染料N-(6-甲氧基-8-喹啉基)-对甲苯磺酰胺（TSQ）和活体染色剂碘化丙啶被用于通过流式细胞术评估获能前后活仓鼠精子的锌含量。通过盐储存的透明带穿透试验或自发或诱导（用溶血磷脂酰胆碱）顶体反应的发生来监测获能情况。还评估了添加锌对精子获能的影响。图像分析用于确定锌（TSQ荧光）的亚细胞定位，原子吸收法用于确定精子在获能过程中总锌含量是否发生变化。在非获能条件下孵育的精子具有高TSQ荧光，且不能穿透透明带。在获能条件下（加牛血清白蛋白或D-青霉胺）孵育的精子锌含量减少（低荧光），分别有90%或78%的精子穿透透明带。图像分析显示，在加牛血清白蛋白获能过程中，顶体区域的锌显著减少，但这与自发顶体反应的发生无关。原子吸收数据表明，当精子在获能条件下孵育时（分别加牛血清白蛋白或D-青霉胺），精子的总锌含量降低了44%或40%。在整个获能期都存在锌时，透明带穿透被完全抑制，但在孵育结束时添加锌则不会。这些数据表明，仓鼠精子中锌的去除与获能相关，并且可能在这一过程中起关键调节作用。
