Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca Morelos 62210, Mexico.
Science Department, School of Pure and Applied Sciences, Florida SouthWestern State College, Fort Myers, FL 33919, USA.
Int J Mol Sci. 2020 Dec 24;22(1):93. doi: 10.3390/ijms22010093.
Plasma membrane (PM) hyperpolarization, increased intracellular pH (pH), and changes in intracellular calcium concentration ([Ca]) are physiological events that occur during human sperm capacitation. These parameters are potential predictors of successful outcomes for men undergoing artificial reproduction techniques (ARTs), but methods currently available for their determination pose various technical challenges and limitations. Here, we developed a novel strategy employing time-lapse flow cytometry (TLFC) to determine capacitation-related membrane potential () and pH changes, and progesterone-induced [Ca] increases. Our results show that TLFC is a robust method to measure absolute and pH values and to qualitatively evaluate [Ca] changes. To support the usefulness of our methodology, we used sperm from two types of normozoospermic donors: (subjects with self-reported paternity) and (subjects without self-reported paternity and no known fertility problems). We found relevant differences between them. The incidences of membrane hyperpolarization, pH alkalinization, and increased [Ca] were consistently high among samples (100%, 100%, and 86%, respectively), while they varied widely among samples (44%, 17%, and 45%, respectively). Our results indicate that TLFC is a powerful tool to analyze key physiological parameters of human sperm, which pending clinical validation, could potentially be employed as fertility predictors.
质膜（PM）超极化、细胞内 pH 值升高（pH）和细胞内钙浓度变化([Ca])是人类精子获能过程中的生理事件。这些参数是接受人工生殖技术（ART）的男性成功的潜在预测指标，但目前用于确定这些参数的方法存在各种技术挑战和限制。在这里，我们开发了一种使用时间分辨流动 cytometry（TLFC）来确定与获能相关的膜电位 () 和 pH 值变化以及孕酮诱导的 [Ca] 增加的新策略。我们的结果表明，TLFC 是一种强大的方法，可以测量绝对 和 pH 值，并定性评估 [Ca] 的变化。为了支持我们的方法的有用性，我们使用了来自两种类型的正常精子供体的精子： (有自我报告生育能力的个体) 和 (没有自我报告生育能力且没有已知生育问题的个体)。我们发现它们之间存在相关差异。 在 样本中，膜超极化、pH 值碱化和 [Ca] 增加的发生率始终很高（分别为 100%、100%和 86%），而在 样本中差异很大（分别为 44%、17%和 45%）。我们的结果表明，TLFC 是分析人类精子关键生理参数的有力工具，在经过临床验证后，它可能被用作生育能力的预测指标。
