AMP-activated kinase, AMPK, is involved in the maintenance of plasma membrane organization in boar spermatozoa
AMP-activated kinase, AMPK, is involved in the maintenance of plasma membrane organization in boar spermatozoa
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DOI：
10.1016/j.bbamem.2013.05.026
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发表时间：
2013-09-01
影响因子：
3.4
通讯作者：
Julia Bragado, M.
中科院分区：
文献类型：
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作者：
Hurtado de Llera, Ana;Martin-Hidalgo, David;Julia Bragado, M.
精子经历能量和代谢依赖性过程才能成功使卵母细胞受精。 AMP 激活蛋白 Idnase，AMPK，是细胞能量的传感器。我们最近证明 AMPK 控制精子活力。我们的目标是 i) 通过免疫荧光研究 AMPK 在公猪精子中的细胞内定位，ii) 研究 AMPK 是否在精子的其他相关过程中发挥作用：线粒体膜电位 (Delta Psi m)、质膜脂质解体、向外磷脂酰丝氨酸 (PS) 暴露、顶体完整性和通过流式细胞术诱导顶体反应，以及 iii) 通过蛋白质印迹研究细胞内 AMPK 通路。在存在或不存在化合物C(CC，30μM)、AMPK抑制剂和/或cAMP类似物8Br-cAMP的情况下，在不同条件下孵育精子。 AMPK α 蛋白在整个顶体和精子鞭毛中段表达，而磷酸-Thr(172)-AMPK 特异性定位于顶体顶端部分和鞭毛中段。 CC 处理迅速赋予精子头对头聚集促进特性，对在 TCM 中孵育的精子进行长期 AMPK 抑制可显着降低高 Delta Psi m。此外，AMPK 抑制显着诱导质膜脂质解体，同时以时间依赖性方式减少质膜向外的 PS 易位。当 AMPK 受到抑制时，TCM 顶体完整性显着增强。然而，顶体反应和离子载体 A23187 诱导的膜脂解体均不受 CC 影响。精子中 PKA 激活后，AMPK 磷酸化会受到强烈刺激。这项工作表明，位于 PKA 下游的 AMPK 在不同水平上调节哺乳动物精子膜功能。 (C) 2013 Elsevier B.V. 保留所有权利。
Spermatozoa undergo energy- and metabolism-dependent processes to successfully fertilize the oocyte. AMP-activated protein Idnase, AMPK, is a sensor of cell energy. We recently showed that AMPK controls spermatozoa motility. Our aims are i) to investigate the intracellular localization of AMPK in boar spermatozoa by immunofluorescence, ii) to study whether AMPK plays a role in other relevant processes of spermatozoa: mitochondrial membrane potential (Delta Psi m), plasma membrane lipid disorganization, outward phosphatidylserine (PS) exposure, acrosome integrity and induced-acrosome reaction by flow cytometry and iii) to investigate intracellular AMPK pathways by western blot. Spermatozoa were incubated under different conditions in the presence or absence of compound C (CC, 30 mu M), an AMPK inhibitor and/or cAMP analog 8Br-cAMP. AMPK alpha protein is expressed at the entire acrosome and at the midpiece of spermatozoa flagellum, whereas phospho-Thr(172)-AMPK is specifically localized at the apical part of acrosome and at flagellum midpiece. CC treatment rapidly confers head-to-head aggregation-promoting property to spermatozoa Long term AMPK inhibition in spermatozoa incubated in TCM significantly reduces high Delta Psi m. Moreover, AMPK inhibition significantly induces plasma membrane lipid disorganization and simultaneously reduces outward PS translocation at plasma membrane in a time-dependent manner. Acrosomal integrity in TCM is significantly enhanced when AMPK is inhibited. However, neither acrosome reaction nor membrane lipid disorganization induced by ionophore A23187 are affected by CC. AMPK phosphorylation is potently stimulated upon PKA activation in spermatozoa This work suggests that AMPK, lying downstream of PKA, regulates at different levels mammalian spermatozoa membrane function. (C) 2013 Elsevier B.V. All rights reserved.