STUDY QUESTION What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? SUMMARY ANSWER EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. WHAT IS KNOWN ALREADY EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. STUDY DESIGN, SIZE, DURATION EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. MAIN RESULTS AND THE ROLE OF CHANCE EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

中文翻译：

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人精浆细胞外囊泡中的蛋白质和非蛋白质成分通过 CatSper 介导的钙信号传导改善人类精子功能

研究问题人精浆细胞外囊泡（EV）在调节人类精子功能中的意义和机制是什么？摘要答案 EV 通过激活 CatSper 通道，通过细胞外 Ca2+ 流入增加细胞内 Ca2+ 浓度 [Ca2+]i，并随后调节人类精子活力，尤其是过度活跃的活力，这归因于 EV 中的蛋白质和非蛋白质成分。已知的信息 EV 是人类精子功能的功能调节剂，来自正常精浆和弱精子症精浆的 EV 货物是不同的。 EV与精子在酸性非生理性蔗糖缓冲溶液中预融合可以升高人类精子中的[Ca2+]i。 CatSper 是人类精子中的一个主要 Ca2+ 通道，当精子对不同的细胞外刺激做出反应时，负责 [Ca2+]i 调节。然而，CatSper 在 EV 诱发的钙信号传导中的作用及其潜在的生理意义仍不清楚。研究设计、大小、持续时间 从正常和弱精子精液的精浆中分离出的 EV 用于研究 EV 调节人类精子中钙信号的机制，包括 CatSper 和 EV 中负责货物的参与。此外，还评估了EV和EV蛋白衍生肽的临床应用潜力。这是一项持续了 5 年多的实验室研究，涉及 200 多个单独的实验。参与者/材料、地点、方法根据南通大学附属医院和江西省妇幼保健院人体受试者伦理委员会的规定招募精液捐献者。使用 Flow NanoAnalyzer、蛋白质印迹法和透射电子显微镜系统地表征精浆 EV。通过荧光测量检查精子 [Ca2+]i 反应。采用全细胞膜片钳技术记录 CatSper 电流。通过计算机辅助精子分析评估精子活力参数。还通过检查精子在粘性甲基纤维素介质中的渗透能力来评估精子过度活化。采用液相色谱-质谱法分析EV中的蛋白质和非蛋白质成分。通过商业试剂盒检测前列腺素、活性氧、丙二醛和DNA完整性的水平。主要结果和机会 EV 的作用 通过细胞外 Ca2+ 内流增加 [Ca2+]i，这可以被 CatSper 抑制剂抑制。此外，EV 还增强了人类精子中的 CatSper 电流。此外，在 CatSper 缺陷的精子中，EV-in [Ca2+]i 增加并且 CatSper 电流不存在，这证实了 CatSper 在 EV 诱导的人类精子 Ca2+ 信号传导中的关键作用。 EV的蛋白质和非蛋白质成分均有助于[Ca2+]i的增加，这对于EV对人类精子的影响非常重要。因此，EV 及其货物促进了精子过度活跃的运动。此外，精浆EV蛋白衍生肽，如NAT1衍生肽（NP）和THBS-1衍生肽（TP），可以激活精子钙信号并增强精子功能。有趣的是，与从正常精浆（N-EV）分离的EV相比，源自弱精子精液的EV引起的[Ca2+]i增加较低，并且N-EV显着改善了弱精子样本和冻融精子中的精子活力和功能。大规模数据不适用。局限性、谨慎原因这是一项体外研究，在推断与精子体内调节的生理相关性时必须谨慎。研究结果的更广泛意义我们的研究结果表明，CatSper 介导的 Ca2+ 信号传导在接近生理条件下参与 EV 调节精子功能，EV 及其衍生物是一种新型 CatSper 和精子功能调节剂，具有临床应用潜力。它们可以被开发来改善由低 [Ca2+]i 反应和/或冷冻和解冻引起的精子活力。研究经费/竞争利益本研究得到国家自然科学基金项目（32271167）、江苏省社会发展项目（BE2022765）、南通市社会民生科技计划（MS22022087）、南通市基础科学研究计划（JC22022086）、江苏省创新创业人才计划（JSSCRC2021543）。作者宣称没有利益冲突。作者宣称没有利益冲突。作者宣称没有利益冲突。