Rajamanickam Gayathri D, Kastelic John P, Thundathil Jacob C
Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Heritage Medical Research Building RM 400, Calgary, AB, T2N4N1, Canada.
Cell Tissue Res. 2017 Apr;368(1):187-200. doi: 10.1007/s00441-016-2514-7. Epub 2016 Nov 2.
Capacitation comprises a series of structural and functional modifications of sperm that confer fertilizing ability. We previously reported that the testis-specific isoform of Na/K-ATPase (ATP1A4) regulated bovine sperm capacitation through signaling mechanisms involving kinases. During subsequent investigations to elucidate mechanisms by which ATP1A4 regulates sperm capacitation, we observed that ATP1A4 was localised in both raft and non-raft fractions of the sperm plasma membrane and that its total content was increased in both membrane fractions during capacitation. The objective of the present study was to investigate mechanism(s) of capacitation-associated increase in the content of ATP1A4. Despite the widely accepted dogma of transcriptional/translational quiescence, incubation of sperm with either ouabain (specific ligand for ATP1A4) or heparin increased ATP1A4 content in raft and non-raft sperm membrane fractions, total sperm protein extracts (immunoblotting) and fixed sperm (flow cytometry), with a concurrent increase in Na/K-ATPase enzyme activity. This capacitation-associated increase in ATP1A4 content was partially decreased by chloramphenicol (mitochondrial translation inhibitor) but not affected by actinomycin D (transcription inhibitor). To demonstrate de novo ATP1A4 synthesis, we evaluated incorporation of bodipy conjugated lysine in this protein during capacitation. A partial decrease in bodipy-lysine incorporation occurred in ATP1A4 from sperm capacitated in the presence of chloramphenicol. Therefore, increased ATP1A4 content during capacitation was attributed to mitochondrial translation of ATP1A4 mRNA present in ejaculated sperm, rather than gene transcription. To our knowledge, this is the first report demonstrating ATP1A4 synthesis during bovine sperm capacitation.
精子获能包括精子的一系列结构和功能修饰，这些修饰赋予精子受精能力。我们之前报道过，钠钾ATP酶（ATP1A4）的睾丸特异性同工型通过涉及激酶的信号机制调节牛精子的获能。在随后阐明ATP1A4调节精子获能机制的研究中，我们观察到ATP1A4定位于精子质膜的脂筏和非脂筏部分，并且在获能过程中其在两个膜部分的总含量均增加。本研究的目的是探究获能相关的ATP1A4含量增加的机制。尽管转录/翻译静止这一观点已被广泛接受，但用哇巴因（ATP1A4的特异性配体）或肝素孵育精子，会增加脂筏和非脂筏精子膜部分、精子总蛋白提取物（免疫印迹法）以及固定精子（流式细胞术）中的ATP1A4含量，同时钠钾ATP酶活性也会增加。这种与获能相关的ATP1A4含量增加部分被氯霉素（线粒体翻译抑制剂）降低，但不受放线菌素D（转录抑制剂）影响。为了证明ATP1A4的从头合成，我们评估了在获能过程中硼二吡咯共轭赖氨酸在该蛋白中的掺入情况。在氯霉素存在下获能的精子中，ATP1A4的硼二吡咯 - 赖氨酸掺入量出现部分下降。因此，获能过程中ATP1A4含量的增加归因于射精精子中存在的ATP1A4 mRNA的线粒体翻译，而非基因转录。据我们所知，这是首次报道在牛精子获能过程中ATP1A4的合成。
