STUDY QUESTION What role do sphingolipids have in mediating human sperm capacitation? SUMMARY ANSWER Sphingosine 1-phosphate (S1P) mediates the acquisition of fertilizing competency in human spermatozoa by engaging with its Gi-coupled receptor S1PR1 and promoting production of reactive oxygen species such as nitric oxide and superoxide anion. WHAT IS KNOWN ALREADY Bioactive sphingolipids, such as S1P, are fundamental for regulating numerous physiological domains and processes, such as cell membranes and signalling, cell death and proliferation, cell migration and invasiveness, inflammation, and central nervous system development. STUDY DESIGN, SIZE, DURATION Semen samples were obtained from a cohort of 10 healthy non-smoking volunteers (18-30 years old) to investigate the role of S1P in sperm. PARTICIPANTS/MATERIALS, SETTING, METHODS Percoll-selected human spermatozoa were incubated at 37°C for 3.5 h in BWW media with or without foetal cord serum ultrafiltrate (FCSu), sphingosine (Sph), or ceramide (Cer). Spermatozoa were also incubated with or without pharmacological inhibitors of sphingolipid metabolism. Protein tyrosine phosphorylation was determined by immunoblotting. The acrosome reaction was determined by PSA-FTIC labelling of the acrosome and analysed using fluorescence microscopy. Intracellular nitric oxide (NO•) production was determined using a DAF-2DA probe. Immunocytochemistry was performed to localize and assess the functional relationship of key components of lipid signalling in spermatozoa. Sperm viability and motility of the samples were evaluated by the hypo-osmotic swelling (HOS) test and computer-aided sperm analysis (CASA). Statistical differences between groups were determined using ANOVA and Tukey's test. Normal distribution of the data and variance homogeneity were assessed using Shapiro-Wilk and Levene's test, respectively. A difference was considered significant when the P-value was ≤0.05. MAIN RESULTS AND THE ROLE OF CHANCE S1P mediates the acquisition of fertilizing competency in human spermatozoa by engaging with its Gi-coupled receptor S1PR1. We found that S1PR1 redistributes to the post-acrosomal region upon induction of capacitation. S1P signalling promotes the activation of the PI3K-AKT pathway, leading to NO• production during sperm capacitation. L-NAME, an nitric oxide synthase inhibitor, impaired the Sph- and Cer-dependent capacitation. Additionally, Sph and Cer promote superoxide anion (O2•-) production, and the extracellular addition of superoxide dismutase (SOD) prevented Sph- and Cer-dependent capacitation, suggesting that Sph and Cer stimulate O2•- production during sperm capacitation. Protein kinase type R (PKR), ceramide kinase (CERK), and protein kinase C (PKC) are responsible for translocating and activating sphingosine kinase 1 (SphK1), which is necessary to promote S1P production for sperm capacitation. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION The utilization and actions of sphingolipids may differ in spermatozoa of different species. WIDER IMPLICATIONS OF THE FINDINGS Sphingolipid metabolites such as Sph, Cer, S1P, and ceramide 1-phosphate (C1P) play a crucial role in inducing human sperm capacitation. Our research has provided new insights into fundamental sphingolipid processes in human sperm, including the importance of C1P in translocating and activating SphK1 as well as the S1P signalling to regulate the PI3K/AKT/NOS pathway to generate NO• for sperm capacitation. We are the first to identify the presence of PKR in human spermatozoa and its role in the phosphorylation activities of SphK1 with the subsequent activation of S1P signalling. Furthermore, our study has identified that S1PR1 and S1PR3 are involved in capacitation and the acrosome reaction, respectively. These findings shed light on a novel mechanism by which sphingolipids drive capacitation in human sperm and pave the way for further exploration of the role of bioactive sphingolipid metabolites in this process. Lastly, our studies lay the foundation for examining the lipid profile of infertile males, as potential discrepancies can affect the functional capacity of spermatozoa to reach fertilizing potential. STUDY FUNDING/COMPETING INTEREST(S) This research was funded by the Canadian Institutes of Health Research (CIHR), grant number PJT-165962 to C.O.F. S.S. was awarded a Research Institute-MUHC Desjardins Studentship. There are no competing interests to report.

中文翻译：

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鞘脂在人类精子获能过程中调节氧化还原信号传导。


研究问题 鞘脂在介导人类精子获能方面有什么作用？摘要答案：1-磷酸鞘氨醇 （S1P） 通过与 Gi 偶联受体 S1PR1 结合并促进活性氧（如一氧化氮和超氧阴离子）的产生，介导人类精子获得受精能力。已知的生物活性鞘脂，如 S1P，是调节许多生理领域和过程的基础，例如细胞膜和信号传导、细胞死亡和增殖、细胞迁移和侵袭性、炎症和中枢神经系统发育。研究设计、大小、持续时间 精液样本是从 10 名健康不吸烟志愿者 （18-30 岁） 的队列中获得的，以研究 S1P 在精子中的作用。参与者/材料、设置、方法 将 Percoll 选择的人精子在 37°C 下在 BWW 培养基中孵育 3.5 小时，有或没有胎脐带血清超滤液 （FCSu）、鞘氨醇 （Sph） 或神经酰胺 （Cer）。精子也与或没有鞘脂代谢的药物抑制剂一起孵育。通过免疫印迹法测定蛋白酪氨酸磷酸化。通过顶体的 PSA-FTIC 标记测定顶体反应，并使用荧光显微镜进行分析。使用 DAF-2DA 探针测定细胞内一氧化氮 （NO•） 的产生。进行免疫细胞化学以定位和评估精子中脂质信号关键成分的功能关系。通过低渗肿胀 （HOS） 测试和计算机辅助精子分析 （CASA） 评估样本的精子活力和活力。使用 ANOVA 和 Tukey 检验确定组间的统计差异。 分别使用 Shapiro-Wilk 和 Levene 检验评估数据的正态分布和方差同质性。当 P 值为 ≤0.05 时，差异被认为是显著的。主要结果和机会 S1P 的作用通过与 Gi 偶联受体 S1PR1 结合介导人类精子受精能力的获得。我们发现 S1PR1 在诱导获能后重新分布到顶体后区域。S1P 信号传导促进 PI3K-AKT 通路的激活，导致精子获能期间产生 NO•。L-NAME 是一种一氧化氮合酶抑制剂，损害了 Sph 和 Cer 依赖性获能。此外，Sph 和 Cer 促进超氧阴离子 （O2•-） 的产生，超氧化物歧化酶 （SOD） 的细胞外添加阻止了 Sph 和 Cer 依赖性获能，表明 Sph 和 Cer 刺激精子获能期间 O2•- 的产生。R 型蛋白激酶 （PKR）、神经酰胺激酶 （CERK） 和蛋白激酶 C （PKC） 负责易位和激活鞘氨醇激酶 1 （SphK1），这是促进精子获能的 S1P 产生所必需的。大规模数据 N/A. 局限性，谨慎原因 鞘脂的利用和作用在不同物种的精子中可能有所不同。研究结果的更广泛意义 鞘脂代谢物如 Sph、Cer、S1P 和神经酰胺 1-磷酸 （C1P） 在诱导人类精子获能方面起着至关重要的作用。我们的研究为人类精子的基本鞘脂过程提供了新的见解，包括 C1P 在易位和激活 SphK1 中的重要性，以及 S1P 信号传导调节 PI3K/AKT/NOS 通路以产生 NO• 用于精子获能。 我们首次确定了人精子中 PKR 的存在及其在 SphK1 磷酸化活性中的作用以及随后的 S1P 信号激活。此外，我们的研究还发现 S1PR1 和 S1PR3 分别参与获能和顶体反应。这些发现揭示了鞘脂驱动人类精子获能的新机制，并为进一步探索生物活性鞘脂代谢物在此过程中的作用铺平了道路。最后，我们的研究为检查不育雄性的脂质谱奠定了基础，因为潜在的差异会影响精子达到受精潜力的功能能力。研究资金/竞争利益 这项研究由加拿大卫生研究院 （CIHR） 资助，授予 C.O.F. S.S. 的资助号 PJT-165962 被授予研究所-MUHC Desjardins 学生奖学金。没有需要报告的竞争利益。