Ferreira Juan J, Lybaert Pascale, Puga-Molina Lis C, Santi Celia M
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO, United States.
Department of Neuroscience, Washington University School of Medicine, St. Louis, MO, United States.
Front Cell Dev Biol. 2021 Sep 28;9:733653. doi: 10.3389/fcell.2021.733653. eCollection 2021.
To fertilize an egg, mammalian sperm must undergo capacitation in the female genital tract. A key contributor to capacitation is the calcium (Ca) channel CatSper, which is activated by membrane depolarization and intracellular alkalinization. In mouse epididymal sperm, membrane depolarization by exposure to high KCl triggers Ca entry through CatSper only in alkaline conditions (pH 8.6) or after incubation with bicarbonate (HCO ) and bovine serum albumin (capacitating conditions). However, in ejaculated human sperm, membrane depolarization triggers Ca entry through CatSper in non-capacitating conditions and at lower pH (< pH 7.4) than is required in mouse sperm. Here, we aimed to determine the mechanism(s) by which CatSper is activated in mouse and human sperm. We exposed ejaculated mouse and human sperm to high KCl to depolarize the membrane and found that intracellular Ca concentration increased at pH 7.4 in sperm from both species. Conversely, intracellular Ca concentration did not increase under these conditions in mouse epididymal or human epididymal sperm. Furthermore, pre-incubation with HCO triggered an intracellular Ca concentration increase in response to KCl in human epididymal sperm. Treatment with protein kinase A (PKA) inhibitors during exposure to HCO inhibited Ca concentration increases in mouse epididymal sperm and in both mouse and human ejaculated sperm. Finally, we show that soluble adenylyl cyclase and increased intracellular pH are required for the intracellular Ca concentration increase in both human and mouse sperm. In summary, our results suggest that a conserved mechanism of activation of CatSper channels is present in both human and mouse sperm. In this mechanism, HCO in semen activates the soluble adenylyl cyclase/protein kinase A pathway, which leads to increased intracellular pH and sensitizes CatSper channels to respond to membrane depolarization to allow Ca influx. This indirect mechanism of CatSper sensitization might be an early event capacitation that occurs as soon as the sperm contact the semen.
为使卵子受精，哺乳动物的精子必须在雌性生殖道中经历获能过程。获能的一个关键因素是钙（Ca）通道CatSper，它由膜去极化和细胞内碱化激活。在小鼠附睾精子中，暴露于高浓度氯化钾引起的膜去极化仅在碱性条件下（pH 8.6）或与碳酸氢盐（HCO）和牛血清白蛋白孵育后（获能条件）才会触发通过CatSper的钙内流。然而，在射出的人类精子中，膜去极化在非获能条件下且在比小鼠精子所需pH更低（<pH 7.4）时就能触发通过CatSper的钙内流。在此，我们旨在确定CatSper在小鼠和人类精子中被激活的机制。我们将射出的小鼠和人类精子暴露于高浓度氯化钾以使膜去极化，发现两种物种的精子在pH 7.4时细胞内钙浓度均增加。相反，在这些条件下，小鼠附睾精子或人类附睾精子的细胞内钙浓度并未增加。此外，预先用HCO孵育可触发人类附睾精子对氯化钾的反应，使其细胞内钙浓度增加。在暴露于HCO期间用蛋白激酶A（PKA）抑制剂处理可抑制小鼠附睾精子以及小鼠和人类射出精子中的钙浓度增加。最后，我们表明可溶性腺苷酸环化酶和细胞内pH升高是人类和小鼠精子细胞内钙浓度增加所必需的。总之，我们的结果表明，人类和小鼠精子中存在一种保守的CatSper通道激活机制。在这种机制中，精液中的HCO激活可溶性腺苷酸环化酶/蛋白激酶A途径，导致细胞内pH升高并使CatSper通道对膜去极化反应敏感，从而允许钙内流。这种CatSper致敏的间接机制可能是精子一接触精液就发生的早期获能事件。
