Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction.
Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction.
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体温过演的能量代谢状态使用修订的方案进行了有效的ATP提取方案。
DOI：
10.1242/bio.017954
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发表时间：
2016-11-15
期刊：
影响因子：
2.4
通讯作者：
Henning H
中科院分区：
文献类型：
--
作者：
Nguyen QT;Wallner U;Schmicke M;Waberski D;Henning H
哺乳动物精子利用三磷酸腺苷作为能量来源，在受精过程中发挥关键作用。ATP及其前体核苷酸ADP和AMP在精子生理学研究中经常被研究，主要是通过生物发光分析。检测结果差异很大，主要是因为核苷酸提取和防止其酶降解的效率不同。在这里，我们描述了一种经过修订和验证的方案，用于有效地抑制磷酸酶和腺嘌呤核苷酸提取，从而导致持续高的ATP浓度，超过先前报道的猪精子的值高达20倍。修订后的分析方法适用于测定新鲜和冷冻样本提取液中的ATP浓度和腺苷能荷，从而可以同时评估长期储存实验的精液样本。经验证后，将该方法应用于猪精子在17℃和5℃下保存24和72 h，冷冻至5℃，但不影响保存时间的情况下，精子中的三磷酸腺苷浓度降低(P<；0.05)，并伴随着腺苷二磷酸和腺苷二磷酸的出现。ATP和能荷与膜完整精子的比例高度相关，支持了核苷酸在冷休克细胞中通过破裂的膜泄漏的观点。本试验允许对精子能量代谢进行高度标准化的研究。摘要：提出了一种从猪精子中高效提取ATP的修订方案，该方案可从新鲜和冷冻的样品中持续获得高的ATP含量和能量电荷值。
Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa. Summary: A revised protocol for efficient extraction of ATP from boar spermatozoa is presented that consistently yields high ATP contents and energy charge values from fresh and frozen samples.
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DOI：
10.1128/aem.30.5.713-721.1975
发表时间：
1975-01-01
期刊：
APPLIED MICROBIOLOGY
影响因子：
--
作者：
LUNDIN, A;THORE, A
通讯作者：
THORE, A
影响因子：
9.7
作者：
Wen, G;Voroney, RP;Chikushi, J
通讯作者：
Chikushi, J
影响因子：
4.5
作者：
Henning, H.;Ngo, T. T.;Waberski, D.
通讯作者：
Waberski, D.
影响因子：
2.9
作者：
du Plessis SS;Agarwal A;Mohanty G;van der Linde M
通讯作者：
van der Linde M
影响因子：
0.8
作者：
Dziekonska, A.;Strzezek, J.
通讯作者：
Strzezek, J.