Lijun Shan, Linmei Gao, Yuhao Chai, Kun Li, Jianmin Yu, Fei Liang, Jiangfeng Qin, Ya Ni, Peibei Sun
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引用次数: 0
Abstract
Background: Sperm capacitation is a prerequisite for natural or in vitro fertilization. After capacitation, sperm become hyperactivated and undergo an acrosome reaction, which helps them penetrate the oocyte. Cordycepin, a bioactive compound first isolated from Cordyceps militaris, is an adenosine analog with numerous physiological activities. However, its effects on sperm capacitation remain unclear. This study aims to elucidate the effects and mechanisms of cordycepin on human sperm capacitation.
Methods: During in vitro capacitation culture, healthy human sperm were treated with cordycepin (20, 100, 500 µM). Sperm motility and hyperactivation were detected using a computer-assisted sperm analyzer. Sperm acrosome reaction was measured using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin. Sperm protein kinase A (PKA) activity was analyzed using an ELISA kit. The levels of sperm protein tyrosine phosphorylation were detected by western blotting. Sperm DNA damage was detected by a sperm chromatin dispersion assay. Reactive oxygen species (ROS) were measured using the fluorescence probe 2',7'-dichlorodihydrofluorescein diacetate. The expression and localization of adenosine receptors were analyzed by western blotting and immunofluorescence. The specific inhibitors of adenosine receptors were used to confirm their effects on cordycepin-induced sperm capacitation. Finally, molecular docking was performed to analyze the interaction between cordycepin and adenosine receptors.
Results: Cordycepin improved hyperactivated sperm motility, acrosome reaction, PKA activity, and protein tyrosine phosphorylation during capacitation while having no obvious effects on sperm ROS or DNA damage. Four adenosine receptor subtypes were expressed in human sperm, but their localizations differed. Inhibition of adenosine receptors significantly decreased cordycepin-induced sperm hyperactivation and the acrosome reaction. Molecular docking showed that cordycepin can bind to the four subtypes of adenosine receptors.
Conclusion: Cordycepin may promote human sperm capacitation through adenosine receptor-mediated signaling pathways. These findings may be useful for assisted reproductive technology and animal breeding.
背景:精子获能是自然受精或体外受精的先决条件。获能后,精子变得活性亢进并发生顶体反应,这有助于它们穿透卵母细胞。虫草素是一种生物活性化合物,最早从冬虫夏草中分离出来,是一种具有多种生理活性的腺苷类似物。然而,它对精子获能的影响仍不清楚。本研究旨在阐明虫草素对人类精子获能的影响和机制:方法:在体外获能培养过程中,用虫草素(20、100、500 µM)处理健康人类精子。使用计算机辅助精子分析仪检测精子活力和超活化。精子顶体反应是用异硫氰酸荧光素结合的豌豆凝集素来测量的。使用 ELISA 试剂盒分析精子蛋白激酶 A(PKA)的活性。精子蛋白酪氨酸磷酸化水平通过 Western 印迹法检测。精子染色质分散试验检测精子 DNA 损伤。使用荧光探针 2',7'-二氯二氢荧光素二乙酸酯测量活性氧(ROS)。腺苷受体的表达和定位是通过 Western 印迹法和免疫荧光法分析的。使用腺苷受体的特异性抑制剂证实了它们对虫草素诱导的精子获能的影响。最后,通过分子对接分析了虫草素与腺苷受体之间的相互作用:结果:虫草素改善了精子获能过程中的超活化精子活力、顶体反应、PKA活性和蛋白酪氨酸磷酸化,但对精子的ROS和DNA损伤没有明显影响。人类精子中表达了四种腺苷受体亚型,但它们的定位不同。抑制腺苷受体可显著降低虫草素诱导的精子过度活化和顶体反应。分子对接显示,虫草素能与四种亚型的腺苷受体结合:结论:虫草素可通过腺苷受体介导的信号通路促进人类精子获能。这些发现可能有助于辅助生殖技术和动物育种。
期刊介绍:
Reproductive Biology and Endocrinology publishes and disseminates high-quality results from excellent research in the reproductive sciences.
The journal publishes on topics covering gametogenesis, fertilization, early embryonic development, embryo-uterus interaction, reproductive development, pregnancy, uterine biology, endocrinology of reproduction, control of reproduction, reproductive immunology, neuroendocrinology, and veterinary and human reproductive medicine, including all vertebrate species.