Wozniak Katherine L, Mayfield Brianna L, Duray Alexis M, Tembo Maiwase, Beleny David O, Napolitano Marc A, Sauer Monica L, Wisner Bennett W, Carlson Anne E
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
PLoS One. 2017 Jan 23;12(1):e0170405. doi: 10.1371/journal.pone.0170405. eCollection 2017.
The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy.
METHODOLOGY/PRINCIPAL FINDING: Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+.
CONCLUSIONS/SIGNIFICANCE: Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu.
在非洲爪蟾（非洲爪蟾）中，细胞外钙离子对于受精和早期胚胎发育的必要性存在争议。据报道，顶体反应需要钙离子进入非洲爪蟾精子，但已有文献记载，在高浓度的钙离子螯合剂BAPTA中受精和胚胎发育仍会发生。在此，我们试图解决这一争议。
方法/主要发现：以卵裂沟的出现作为胚胎发育的指标，我们发现，在缺乏添加二价阳离子的溶液中受精的非洲爪蟾卵能够正常发育。相比之下，在毫摩尔浓度的BAPTA或EGTA中受精的卵无法发育。在添加精子后将胚胎转移至不同溶液中，我们发现，细胞外钙离子对于添加精子后最初30分钟内发生的事件是特异性必需的，但之后则不是。我们发现，荧光染色的精子无法穿透在高浓度BAPTA中受精的卵的包膜，而在没有钙离子螯合剂、或含有BAPTA和饱和氯化钙的情况下受精的卵，有几个精子穿透了卵黄膜。这些结果共同表明，在高浓度的钙离子螯合剂中不会发生受精。最后，我们发现卵胶膜中含有>5 mM易于扩散的钙离子。
结论/意义：综上所述，这些数据与受精需要细胞外钙离子一致。基于我们的发现，我们推测，围绕卵的卵胶膜作为易于获得的钙离子储备，在不断变化的细胞外环境中促进受精。
