Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM, Cuernavaca, Mor., México.
Laboratorio de Imágenes y Visión por Computadora, Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, UNAM, Cuernavaca, Mor., México.
Biol Reprod. 2018 Nov 1;99(5):1010-1021. doi: 10.1093/biolre/ioy132.
Sperm motility is essential for fertilization. The asymmetry of flagellar beat in spermatozoa is finely regulated by intracellular calcium concentration ([Ca2+]i). Recently, we demonstrated that the application of high concentrations (10-20 μM) of the Ca2+ ionophore A23187 promotes sperm immobilization after 10 min, and its removal thereafter allows motility recovery, hyperactivation, and fertilization. In addition, the same ionophore treatment overcomes infertility observed in sperm from Catsper1-/-, Slo3-/-, and Adcy10-/-, but not PMCA4-/-, which strongly suggest that regulation of [Ca2+]i is mandatory for sperm motility and hyperactivation. In this study, we found that prior to inducing sperm immobilization, high A23187 concentrations (10 μM) increase flagellar beat. While 5-10 μM A23187 substantially elevates [Ca2+]i and rapidly immobilizes sperm in a few minutes, smaller concentrations (0.5 and 1 μM) provoke smaller [Ca2+]i increases and sperm hyperactivation, confirming that [Ca2+]i increases act as a motility switch. Until now, the [Ca2+]i thresholds that switch motility on and off were not fully understood. To study the relationship between [Ca2+]i and flagellar beating, we developed an automatic tool that allows the simultaneous measurement of these two parameters. Individual spermatozoa were treated with A23187, which is then washed to evaluate [Ca2+]i and flagellar beat recovery using the implemented method. We observe that [Ca2+]i must decrease below a threshold concentration range to facilitate subsequent flagellar beat recovery and sperm motility.
精子的运动能力对于受精至关重要。鞭毛的不对称拍动是由细胞内钙离子浓度（[Ca2+]i）精细调节的。最近，我们证明了应用高浓度（10-20 μM）的钙离子载体 A23187 可在 10 分钟后促进精子固定，随后去除 A23187 则允许运动恢复、超激活和受精。此外，相同的离子载体处理克服了 Catsper1-/-、Slo3-/-和 Adcy10-/-精子中观察到的不育，但 PMCA4-/-则不行，这强烈表明[Ca2+]i 的调节对于精子运动和超激活是必需的。在这项研究中，我们发现，在诱导精子固定之前，高浓度的 A23187（10 μM）会增加鞭毛的拍动。虽然 5-10 μM 的 A23187 会显著升高[Ca2+]i 并在几分钟内迅速使精子固定，但较小的浓度（0.5 和 1 μM）会引起较小的[Ca2+]i 升高和精子超激活，这证实了[Ca2+]i 的升高是运动的开关。到目前为止，尚不完全清楚作为运动开关的[Ca2+]i 阈值。为了研究[Ca2+]i 与鞭毛拍打之间的关系，我们开发了一种自动工具，允许同时测量这两个参数。用 A23187 处理单个精子，然后用实施的方法进行洗涤以评估[Ca2+]i 和鞭毛拍动的恢复。我们观察到[Ca2+]i 必须降低到一个阈值浓度范围以下，以促进随后的鞭毛拍动恢复和精子运动。
