Harrison R A, Ashworth P J, Miller N G
Department of Signalling, Babraham Institute, Cambridge, UK.
Mol Reprod Dev. 1996 Nov;45(3):378-91. doi: 10.1002/(SICI)1098-2795(199611)45:3<378::AID-MRD16>3.0.CO;2-V.
Bicarbonate/CO2 is believed to be the key in vitro effector of sperm capacitation, a process which induces major changes in the sperm plasma membrane in preparation for fertilization. In a flow cytometric study, we examined the effect of bicarbonate on boar spermatozoa using merocyanine, an impermeant lipophilic probe which binds to plasma membranes with increasing affinity as their lipid components become more disordered. We found that bicarbonate causes a rapid increase in the ability of live boar spermatozoa to bind merocyanine. First detected about 100 sec after exposure to bicarbonate and largely complete by 300 sec, this increase appears to result from individual cells within the sperm population switching from a low merocyanine-binding state to a high binding state. The majority of live spermatozoa are capable of responding in this way, and do so in proportion to bicarbonate concentration, half-maximal response being induced by about 3 mM bicarbonate; however, overall population response varies greatly between ejaculates. Increased merocyanine stainability is observed over the whole surface area of the cell, and is reversible both with respect to temperature (it is only manifested above 30 degrees C) and with respect to presence of bicarbonate. A similar effect can be induced by phosphodiesterase inhibitors such as isobutylmethylxanthine, and enhanced by a permeant cyclic nucleotide analogue. We conclude that bicarbonate causes a major alteration in sperm plasma membrane lipid architecture, apparently by perturbing enzymic control processes. This novel action of bicarbonate may represent an initial permissive event in the capacitation sequence.
碳酸氢根/二氧化碳被认为是精子获能的关键体外效应物,精子获能是一个诱导精子质膜发生重大变化以准备受精的过程。在一项流式细胞术研究中,我们使用部花青(一种非渗透性亲脂性探针,随着质膜脂质成分变得更加无序,其与质膜的结合亲和力增加)检测了碳酸氢根对猪精子的影响。我们发现,碳酸氢根可使活猪精子结合部花青的能力迅速增强。这种增强在接触碳酸氢根后约100秒首次检测到,到300秒时基本完成,似乎是由于精子群体中的单个细胞从低部花青结合状态转变为高结合状态所致。大多数活精子都有能力以这种方式做出反应,且反应程度与碳酸氢根浓度成正比,约3 mM碳酸氢根可诱导半数最大反应;然而,不同射精样本的总体反应差异很大。在细胞的整个表面积上都观察到部花青染色性增加,并且在温度方面(仅在30摄氏度以上才表现出来)和碳酸氢根的存在方面都是可逆的。磷酸二酯酶抑制剂如异丁基甲基黄嘌呤可诱导类似的效应,而渗透性环核苷酸类似物可增强这种效应。我们得出结论,碳酸氢根会导致精子质膜脂质结构发生重大改变,显然是通过干扰酶促控制过程实现的。碳酸氢根的这种新作用可能代表了获能序列中的一个初始允许事件。