Inhibition of sperm capacitation and fertilizing capacity by adjudin is mediated by chloride and its channels in humans
Inhibition of sperm capacitation and fertilizing capacity by adjudin is mediated by chloride and its channels in humans
复制标题
调节蛋白对精子获能和受精能力的抑制是由氯及其通道介导的
DOI：
10.1093/humrep/des384
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发表时间：
2013-01-01
影响因子：
6.1
通讯作者：
Shi, Qi-Xian
中科院分区：
文献类型：
--
作者：
Li, Kun;Ni, Ya;Shi, Qi-Xian
研究问题：adjudin 是否会破坏人类精子中的氯离子 (Cl-) 离子转运功能，从而阻碍精子体外获能和受精能力？总结答案：本研究结果表明，adjudin 是 Cl- 通道的有效阻断剂：破坏 Cl- 离子转运功能会导致人类体外精子获能和受精能力下降。 已知信息： 尽管我们之前的研究已证明 adjudin Adjudin 通过破坏支持细胞粘附连接来发挥作用，最显着的是通过靶向测试蛋白和肌动蛋白丝束来破坏支持细胞中基于肌动蛋白的细胞骨架，从而实现顶端外质特化，但目前尚不清楚 adjudin 是否会阻碍人类精子中的 Cl- 离子转运功能。 研究设计、大小和持续时间：精液样本取自 45 名有生育能力的男性（年龄 25-32 岁）。通过不连续 Percoll 梯度离心，从人管液 (HTF) 培养基中的精液中分离精子，并在获能条件下与 10 nM-10 muM 的 adjudin 和/或其他试剂一起孵育 0-5 小时。 参与者/材料、设置、方法：我们评估了在 37 度下孵育的 adjudin 和不同试剂对精子功能的影响C. 通过计算机辅助精子分析 (CASA) 系统分析精子活力和过度激活。通过金霉素荧光染色评估精子获能和顶体反应。通过精子穿透无带仓鼠卵试验评估精子受精能力，并通过 EIA 试剂盒定量精子中的细胞 cAMP 水平。通过精子免疫检测分析了在存在或不存在adjudin的情况下蛋白质酪氨酸、丝氨酸和苏氨酸的磷酸化，特别是比较了adjudin在完全HTF培养基中与Cl-缺乏的HTF培养基以及各种Cl-通道阻滞剂中对精子过度活化和获能的影响。 主要结果和机会的作用：Adjudin显着抑制精子过度活化，但不抑制精子。动力。 Adjudin 诱导的精子获能抑制是可逆的，并且发现它以剂量依赖性方式阻断 rhuZP(3)β 和孕酮诱导的顶体反应。 Adjudin 还阻止精子穿透无透明带的仓鼠卵，并显着抑制毛喉素激活的跨膜腺苷酸环化酶和可溶性腺苷酸环化酶活性，导致人类精子中细胞 cAMP 水平显着下降。 Adjudin 未能降低精子蛋白酪氨酸磷酸化，但它确实阻止精子丝氨酸和苏氨酸蛋白磷酸化。有趣的是，发现调节蛋白仅在完全 Cl--HTF 培养基中发挥其对精子获能和获能相关事件的抑制作用，而在缺乏 Cl- 的培养基中则不然，这说明 Cl- 可能参与其中。 Adjudin 通过破坏氯离子及其运输功能来抑制人类精子的生育能力。 局限性、注意原因：本研究仅在体外检验了 Adjudin 对人类精子获能和受精能力的影响，因此具有一些局限性。需要进一步的体内研究来证实 adjudin 是一种有效的男性避孕药。 研究结果的更广泛意义：我们的研究表明，adjudin 对获能的抑制是可逆的，并且毒性较低，为检查 adjudin 作为男性生育控制介质打开了大门。 Adjudin可能是一种安全、有效、可逆的男性抗生育剂，适用于Adjudin作为男性抗生育剂的人体初步临床试验。
STUDY QUESTION: Does adjudin disrupt chloride ion (Cl-) ion transport function in human sperm and impede sperm capacitation and fertilizing ability in vitro?SUMMARY ANSWER: In this study the results indicate that adjudin is a potent blocker of Cl- channels: disrupting Cl- ion transport function results in a decline in sperm capacitation and fertilizing ability in humans in vitro.WHAT IS KNOWN ALREADY: Although our previous studies have demonstrated that adjudin exerts its effect by disrupting sertoligerm cell adhesion junctions, most notably apical ectoplasmic specialization by targeting testin and actin filament bundles that disrupts the actin-based cytoskeleton in sertoli cells, it remains unclear whether adjudin impedes Cl- ion transport function in the human sperm.STUDY DESIGIN, SIZE AND DURATION: Semen samples were obtained from 45 fertile men (aged 25-32). Spermatozoa were isolated from the semen in the human tube fluid (HTF) medium by centrifugation through a discontinuous Percoll gradient, and incubated with adjudin at 10 nM-10 mu M and/or other reagents under capacitating conditions for 0-5 h.PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated the effect of adjudin and different reagents on sperm functions with which they were incubated at 37 degrees C. Sperm motility and hyperactivation were analyzed by a computer-assisted sperm analysis (CASA) system. Sperm capacitation and the acrosome reaction were assessed by chlortetracycline fluorescence staining. Sperm fertilizing ability was evaluated by sperm penetration of zona-free hamster egg assay, and cellular cAMP levels in spermatozoa were quantified by the EIA kit. The proteins tyrosine, serine and threonine phosphorylation in the presence or absence of adjudin were analyzed by means of a immunodetection of spermatozoa, especially, compared the effect of adjudin on sperm hyperactivation and capacitation in the complete HTF medium with the Cl--deficient HTF medium as well as the various Cl- channel blockers.MAIN RESULTS AND THE ROLE OF CHANCE: Adjudin significantly inhibited sperm hyperactivation but not sperm motility. Adjudin-induced inhibition of sperm capacitation was reversible, and it was found to block the rhuZP(3)beta- and progesterone-induced acrosome reaction in a dose-dependent manner. Adjudin also blocked sperm penetration of zona-free hamster eggs, and significantly inhibited both forskolin-activated transmembrane adenylyl cyclase and soluble adenylyl cyclase activities leading to a significant decline in the cellular cAMP levels in human spermatozoa. Adjudin failed to reduce sperm protein tyrosine phosphorylation but it did prevent sperm serine and threonine protein phosphorylation. Interestingly, adjudin was found to exert its inhibitory effects on sperm capacitation and capacitation-associated events only in the complete Cl--HTF medium but not Cl--deficient medium, illustrating the likely involvement of Cl-. Adjudin inhibits the fertility capacity of human sperm is mediated by disrupting chloride ion and its transport function.LIMITATIONS, REASONS FOR CAUTION: This study has examined the effect of adjudin only on human sperm capacitation and fertilizing ability in vitro and thus has some limitations. Further investigations in vivo are needed to confirm adjudin is a potent male contraceptive.WIDER IMPLICATIONS OF THE FINDINGS: Our studies demonstrated that adjudin inhibition of capacitation is reversible and its toxicity is low, opening the door for the examination of adjudin as a mediator of male fertility control. Adjudin may be a safe, efficient and reversible male antifertility agent and applicable to initial clinical trials of adjudin as a male antifertility agent in humans.