Instituto de Biología y Medicina Experimental (IByME) - CONICET, Buenos Aires, Argentina.
Mol Hum Reprod. 2012 Jan;18(1):22-32. doi: 10.1093/molehr/gar058. Epub 2011 Sep 20.
Calcium (Ca(2+)) is an absolute requirement for a decisive sperm function event: the acrosome reaction (AR). Physiologically, sperm capacitation is a prerequisite for this specialized exocytosis and both events are intimately related. In an effort to separate capacitation from AR, we have been using a modified sperm incubation medium where Ca(2+) is replaced by Strontium (Sr(2+)). The aim of this report is to analyze with more detail the difference between sperm incubated with Ca(2+) or Sr(2+) in several events. We found that sperm undergo the capacitation-related changes in the chlortetracycline (CTC) pattern and tyrosine phosphorylation, and also bind to the zona pellucida (ZP) when using Sr(2+)-instead of Ca(2+)-containing media. However, the spontaneous AR typical of hamster sperm does not take place in Sr(2+)-medium, even if sperm are previously capacitated with Ca(2+). Nevertheless, Sr(2+) was able to sustain AR when cells were treated with thapsigargin or depolarized with K(+) in Na(+)-depleted medium. Considering that the absence of Na(+) increased spontaneous AR in Sr(2+)-medium, we tested whether Na(+)-transport systems could be involved in the inability of Sr(2+)-incubated sperm to undergo AR. We found that when sperm incubated in Sr(2+)-medium are treated with amiloride to inhibit epithelial Na(+) channel (ENaC), they are able to undergo spontaneous AR. The same result was obtained when analyzing AR on the ZP. On the contrary, addition of ouabain (a Na(+)/K(+)-ATPase inhibitor) or DIDS (a Na(+)/HCO3(-) co-transporter inhibitor) showed no effect. These results suggest that, differing from what happens in Ca(2+)-incubated sperm, cells incubated in Sr(2+)-modified medium would have an active ENaC.
钙离子（Ca(2+)）是决定精子功能事件的绝对要求：顶体反应（AR）。在生理上，精子获能是这种特殊胞吐作用的前提，这两个事件密切相关。为了将获能与 AR 分离，我们一直在使用一种改良的精子孵育培养基，其中 Ca(2+)被锶（Sr(2+)）取代。本报告的目的是更详细地分析在几种事件中用 Ca(2+)或 Sr(2+)孵育的精子之间的差异。我们发现，当使用不含 Ca(2+)的 Sr(2+)孵育时，精子会经历氯四环素（CTC）模式和酪氨酸磷酸化的获能相关变化，并且与透明带（ZP）结合。然而，仓鼠精子的自发 AR 并不发生在 Sr(2+)-介质中，即使精子先前用 Ca(2+)获能。然而，当细胞用 thapsigargin 处理或在 Na(+)-耗尽培养基中用 K(+)去极化时，Sr(2+)能够维持 AR。考虑到 Na(+)的缺失增加了 Sr(2+)-介质中的自发 AR，我们测试了 Sr(2+)-孵育的精子是否无法发生 AR 是否与 Na(+)转运系统有关。我们发现，当在 Sr(2+)-培养基中孵育的精子用阿米洛利处理以抑制上皮 Na(+)通道（ENaC）时，它们能够发生自发 AR。在 ZP 上分析 AR 时也得到了相同的结果。相反，添加哇巴因（一种 Na(+)/K(+)-ATP 酶抑制剂）或 DIDS（一种 Na(+)/HCO3(-)共转运体抑制剂）没有效果。这些结果表明，与在 Ca(2+)-孵育的精子不同，在 Sr(2+)-修饰培养基中孵育的细胞会有活性的 ENaC。
