School of Biosciences, University of Birmingham, Birmingham, UK.
School of Applied Sciences, Division of Health Sciences, Abertay University, Dundee, UK.
Mol Hum Reprod. 2023 May 31;29(6). doi: 10.1093/molehr/gaad015.
Exposure of human sperm to progesterone (P4) activates cation channel of sperm (CatSper) channels, inducing an intracellular Ca2+ concentration ([Ca2+]i) transient followed by repetitive [Ca2+]i activity (oscillations), which are believed to be functionally important. We investigated the potential significance of store-operated Ca2+-entry in these oscillations using the inhibitor SKF96365 (30 µM; SKF). Following pre-treatment of human sperm with 3 µM P4, exposure to SKF doubled the proportion of oscillating cells (P = 0.00004). In non-pre-treated cells, SKF had an effect similar to P4, inducing a [Ca2+]i transient in >80% of cells which was followed by oscillations in ≈50% of cells. The CatSper blocker RU1968 (11 µM) inhibited the SKF-induced [Ca2+]i increase and reversibly arrested [Ca2+]i oscillations. Using whole-cell patch clamp, we observed that SKF enhanced CatSper currents by 100% within 30 s, but amplitude then decayed to levels below control over the next minute. When cells were stimulated with P4, CatSper currents were stably increased (by 200%). Application of SKF then returned current amplitude to control level or less. When sperm were prepared in medium lacking bovine serum albumin (BSA), both P4 and SKF induced a [Ca2+]i transient in >95% of cells but the ability of SKF to induce oscillations was greatly reduced (P = 0.0009). We conclude that SKF, similar to a range of small organic molecules, activates CatSper channels, but that a secondary blocking action also occurs, which was detected only during patch-clamp recording. The failure of SKF to induce oscillations when cells were prepared without BSA emphasizes that the drug does not fully mimic the actions of P4.
人类精子暴露于孕激素（P4）会激活精子阳离子通道（CatSper），诱导细胞内钙离子浓度([Ca2+]i)短暂增加，随后是重复的[Ca2+]i 活性（振荡），这被认为具有重要的功能意义。我们使用抑制剂 SKF96365（30μM；SKF）研究了储存操作的 Ca2+-进入在这些振荡中的潜在意义。在人类精子用 3μM P4 预处理后，暴露于 SKF 使振荡细胞的比例增加了一倍（P=0.00004）。在未经预处理的细胞中，SKF 的作用类似于 P4，诱导超过 80%的细胞发生 [Ca2+]i 短暂增加，随后约 50%的细胞发生振荡。CatSper 阻滞剂 RU1968（11μM）抑制 SKF 诱导的 [Ca2+]i 增加，并可逆地阻止 [Ca2+]i 振荡。通过全细胞膜片钳，我们观察到 SKF 在 30 秒内使 CatSper 电流增加 100%，但在接下来的一分钟内，电流幅度衰减至低于对照水平。当细胞用 P4 刺激时，CatSper 电流稳定增加（增加 200%）。然后应用 SKF 将电流幅度恢复到对照水平或更低。当精子在缺乏牛血清白蛋白（BSA）的培养基中制备时，P4 和 SKF 均诱导超过 95%的细胞发生 [Ca2+]i 短暂增加，但 SKF 诱导振荡的能力大大降低（P=0.0009）。我们得出结论，SKF 与一系列小分子类似，激活 CatSper 通道，但也发生继发性阻断作用，仅在膜片钳记录中检测到。当细胞在没有 BSA 的情况下制备时，SKF 未能诱导振荡，这强调了该药物不能完全模拟 P4 的作用。
