Name: using an extracellular flux analyzer to measure changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation
Uploaded: 2020-01-22T15:00:00
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作者希望感谢洛克菲勒高通量和光谱资源中心的拉沃西尔·拉莫斯-埃斯皮里图博士的支持。

精子是从8-16周大的CD-1雄性小鼠身上采集的。动物实验得到了威尔康奈尔医学机构动物护理和使用委员会（IACUC）的批准。
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传感器盒和细胞外通量分析仪校准的制备
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<li>要使传感器盒滋润，请从 XFe96 细胞外通量检测套件中取出传感器盒，并将传感器盒倒置在实用程序板旁边。</li>
		<li>使用多通道移液器将 25 mL 的双蒸馏 H2O 填充溶...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+assessment+of+bioenergetic+differences+caused+by+the+common+European+mitochondrial+DNA+haplogroups+H+and+T.">Experimental assessment of bioenergetic differences caused by the common European mitochondrial DNA haplogroups H and T.</a> Gene. 411 (1-2), 69-76 (2008).</li><li>Choi, S. W., Gerencser, A. A., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+analysis+of+isolated+cerebrocortical+nerve+terminals+on+a+microgram+scale:+spare+respiratory+capacity+and+stochastic+mitochondrial+failure.">Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure.</a> Journal of Neurochemistry. 109 (4), 1179-1191 (2009).</li><li>de Groof, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+OXPHOS+activity+precedes+rise+in+glycolytic+rate+in+H-RasV12/E1A+transformed+fibroblasts+that+develop+a+Warburg+phenotype.">Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype.</a> Molecular Cancer. 8, 54 (2009).</li><li>Chao, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+and+altered+systemic+glucose+metabolism+in+mice+lacking+Nur77.">Insulin resistance and altered systemic glucose metabolism in mice lacking Nur77.</a> Diabetes. 58 (12), 2788-2796 (2009).</li><li>Chang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilizing+capacity+of+spermatozoa+deposited+into+the+fallopian+tubes.">Fertilizing capacity of spermatozoa deposited into the fallopian tubes.</a> Nature. 168 (4277), 697-698 (1951).</li><li>Austin, C. R., Bishop, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+rodent+acrosome+and+perforatorium+in+fertilization.">Role of the rodent acrosome and perforatorium in fertilization.</a> Proceedings of the Royal Society of London. Series B, Biological Sciences. 149 (935), 241-248 (1958).</li><li>Ishijima, S., Baba, S. A., Mohri, H., Suarez, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+flagellar+movement+in+hyperactivated+and+acrosome-reacted+golden+hamster+spermatozoa.">Quantitative analysis of flagellar movement in hyperactivated and acrosome-reacted golden hamster spermatozoa.</a> Molecular Reproduction and Development. 61 (3), 376-384 (2002).</li><li>Suarez, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+hyperactivation+in+sperm.">Control of hyperactivation in sperm.</a> Human Reproduction Update. 14 (6), 647-657 (2008).</li><li>Goodson, S. G., Qiu, Y., Sutton, K. A., Xie, G., Jia, W., O'Brien, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+substrates+exhibit+differential+effects+on+functional+parameters+of+mouse+sperm+capacitation.">Metabolic substrates exhibit differential effects on functional parameters of mouse sperm capacitation.</a> Biology of Reproduction. 87 (3), 75 (2012).</li><li>Travis, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+relationships+between+capacitation-dependent+cell+signaling+and+compartmentalized+metabolic+pathways+in+murine+spermatozoa.">Functional relationships between capacitation-dependent cell signaling and compartmentalized metabolic pathways in murine spermatozoa.</a> Journal of Biological Chemistry. 276 (10), 7630-7636 (2001).</li><li>Urner, F., Leppens-Luisier, G., Sakkas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+tyrosine+phosphorylation+in+sperm+during+gamete+interaction+in+the+mouse:+the+influence+of+glucose.">Protein tyrosine phosphorylation in sperm during gamete interaction in the mouse: the influence of glucose.</a> Biology of Reproduction. 64 (5), 1350-1357 (2001).</li><li>Danshina, P. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoglycerate+kinase+2+(PGK2)+is+essential+for+sperm+function+and+male+fertility+in+mice.">Phosphoglycerate kinase 2 (PGK2) is essential for sperm function and male fertility in mice.</a> Biology of Reproduction. 82 (1), 136-145 (2010).</li><li>Miki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glyceraldehyde+3-phosphate+dehydrogenase-S,+a+sperm-specific+glycolytic+enzyme,+is+required+for+sperm+motility+and+male+fertility.">Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility.</a> Proceedings of the National Academy of Sciences. 101 (47), 16501-16506 (2004).</li><li>Mori, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+spermatogenic+cell-specific+type+1+hexokinase+(mHk1-s)+transcripts+are+expressed+by+alternative+splicing+from+the+mHk1+gene+and+the+HK1-S+protein+is+localized+mainly+in+the+sperm+tail.">Mouse spermatogenic cell-specific type 1 hexokinase (mHk1-s) transcripts are expressed by alternative splicing from the mHk1 gene and the HK1-S protein is localized mainly in the sperm tail.</a> Molecular Reproduction and Development. 49 (4), 374-385 (1998).</li><li>Westhoff, D., Kamp, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glyceraldehyde+3-phosphate+dehydrogenase+is+bound+to+the+fibrous+sheath+of+mammalian+spermatozoa.">Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa.</a> Journal of Cell Science. 110 (15), 1821-1829 (1997).</li><li>Nevo, A. C., Rikmenspoel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+ATP+in+sperm+flagella.">Diffusion of ATP in sperm flagella.</a> Journal of Theoretical Biology. 26 (1), 11-18 (1970).</li><li>Adam, D. E., Wei, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+transport+of+ATP+within+the+motile+sperm.">Mass transport of ATP within the motile sperm.</a> Journal of Theoretical Biology. 49 (1), 125-145 (1975).</li><li>Tombes, R. M., Shapiro, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+termini+of+a+phosphocreatine+shuttle.+Purification+and+characterization+of+two+creatine+kinase+isozymes+from+sea+urchin+sperm.">Enzyme termini of a phosphocreatine shuttle. Purification and characterization of two creatine kinase isozymes from sea urchin sperm.</a> Journal of Biological Chemistry. 262 (33), 16011-16019 (1987).</li><li>Gomendio, M., Tourmente, M., Roldan, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+mammalian+lineages+respond+differently+to+sexual+selection:+metabolic+rate+constrains+the+evolution+of+sperm+size.">Why mammalian lineages respond differently to sexual selection: metabolic rate constrains the evolution of sperm size.</a> Proceedings of the Royal Society of Biological Sciences. 278 (1721), 3135-3141 (2011).</li><li>Tourmente, M., Villar-Moya, P., Rial, E., Roldan, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+ATP+generation+via+glycolysis+and+oxidative+phosphorylation+and+relationships+with+sperm+motility+in+mouse+species.">Differences in ATP generation via glycolysis and oxidative phosphorylation and relationships with sperm motility in mouse species.</a> Journal of Biological Chemistry. 290 (33), 20613-20626 (2015).</li><li>Visconti, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacitation+of+mouse+spermatozoa.+II.+Protein+tyrosine+phosphorylation+and+capacitation+are+regulated+by+a+cAMP-dependent+pathway.">Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway.</a> Development. 121 (4), 1139-1150 (1995).</li><li>Buck, J., Sinclair, M. L., Schapal, L., Cann, M. J., Levin, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytosolic+adenylyl+cyclase+defines+a+unique+signaling+molecule+in+mammals.">Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals.</a> PNAS. 96 (1), 79-84 (1999).</li><li>Visconti, P. E., Bailey, J. L., Moore, G. D., Pan, D., Olds-Clarke, P., Kopf, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacitation+of+mouse+spermatozoa.+I.+Correlation+between+the+capacitation+state+and+protein+tyrosine+phosphorylation.">Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation.</a> Development. 121 (4), 1129-1137 (1995).</li><li>Morgan, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+PKA+inhibition+using+a+chemical+genetic+approach+and+its+application+to+studies+on+sperm+capacitation.">Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation.</a> PNAS. 105 (52), 20740-20745 (2008).</li><li>Lybaert, P., Danguy, A., Leleux, F., Meuris, S., Lebrun, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methodology+for+the+detection+and+quantification+of+the+acrosome+reaction+in+mouse+spermatozoa.">Improved methodology for the detection and quantification of the acrosome reaction in mouse spermatozoa.</a> Histology and Histopathology. 24 (8), 999-1007 (2009).</li></ol>

在缺乏某些代谢基质或关键代谢酶的情况下，精子的丧失表明能量代谢是支持成功受精的关键因素。细胞激活过程中的代谢开关是其他细胞类型的一个既定概念，然而，我们才刚刚开始了解精子如何适应细胞加速过程中日益增长的能源需求。我们使用细胞外通量分析仪开发了一种易于应用的工具，用于实时监测精子增生过程中糖解和氧化磷酸化的变化。在细胞外H+和O2的变化检测?...

质谱学的应用彻底改变了代谢学的研究。靶向代谢特征分析和代谢追踪可以精确监测能量代谢的变化。然而，成功实施代谢组学需要大量的培训、经验丰富的工作人员和昂贵、高度敏感的质谱仪，而每个实验室都不容易获得。近年来，使用细胞外通量分析仪，如海马XFe96，作为测量各种细胞类型1、2、3、4、5能量代谢变化的替代方法，已经越来越流行。
精子是高度专业化的活细胞;其任务是把父系基因组传送给卵母细胞。射精后离开男性生殖道的精子在功能上仍然不成熟，不能受精卵母细胞，因为它们不能穿透卵母细胞的背心。精子在成熟过程中通过女性生殖道，称为"生育能力6，7"，获得受精能力。从 cauda 表皮解剖的新鲜射精精子或精子可在体外孵育，在定义的电容培养基中孵育，其中含有 Ca2+、碳酸氢盐 （HCO3-） 或细胞渗透 cAMP 模拟（例如，二丁基林-cAMP）、胆固醇接受器（例如，牛血清白蛋白、BSA）和能量源（如葡萄糖）。在细胞发育过程中，精子将运动模式改变为不对称的鞭状节拍，代表一种称为超活化8、9的游泳模式，它们能够进行acrosome反应7，其中蛋白水解酶被释放，消化卵母细胞的背心。这些过程需要能量，并且类似于体细胞，精子通过糖解以及线粒体TCA周期和氧化磷酸化（牛磷酸化）10生成ATP和其他高能化合物。虽然多项研究表明糖解是必要的，足以支持精子的上限11，12，13，14，但牛磷的贡献则不那么明显。与其他细胞类型不同，糖解在物理上与TCA周期结合，精子高度分裂，被认为能将这些过程保持在单独的鞭节内：中间部分浓缩线粒体机械，而糖解的关键酶似乎仅限于主要部分15，16。这种分割导致一场持续的争论，关于糖解在主要部分产生的丙酮酸盐是否可以支持线粒体牛磷在中间部分，以及ATP产生的牛磷在中间是否能够足够迅速地扩散沿旗杆的长度，以支持能量需求在主要部分17，18，19。也有支持牛磷在精子能力中的作用。牛磷不仅比糖解更有利，产生的ATP比糖解多16倍，而且中片体积和线粒体含量与哺乳动物物种的生殖适应性直接相关，而哺乳动物之间对配偶20的竞争程度更大。解决这些问题需要检查糖解和牛磷在精子发作期间的相对贡献的方法。
Tourmente等人应用了24井细胞外通量分析仪，比较了与精子性能参数明显不同的小鼠物种的能量代谢21。在这里，我们使用96井细胞外通量分析仪来实时监测小鼠精子吸收过程中能量代谢的变化，而不是报告非电容精子的基础ECAR和OCR值。我们开发了一种方法，通过测量氧的通量（O2）和质子（H+）（图1A），在多达12个不同的实验条件下，通过实时监测精子中的糖解和牛磷，在多达12个不同的实验条件下击败鞭状藻。由于在糖解过程中乳酸分解为乳酸，并通过TCA循环生产CO2，非电容和容积的精子挤出H-进入检测介质，由细胞外通量分析仪通过H+敏感荧光管固定到传感器盒的探针尖端检测到。同时，通过氧化磷酸化检测O2消耗，通过O2-敏感荧光道固定到同一探针尖端（图1B）。有效检测释放的H+和消耗的O2需要经过修饰的精子缓冲液，缓冲能力低，不含碳酸氢盐或苯酚红。因此，为了在没有碳酸氢盐的情况下诱导电容，我们采用了一种可渗透的cAMP模拟注射与广泛PDE抑制剂IBMX22一起。三个额外的独立注射端口允许注射药理活化剂和/或抑制剂，这有助于实时检测细胞呼吸和糖解率因实验操作而发生的变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Deoxy-D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D8375</td>
			<td>2-DG</td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I7018</td>
			<td>IBMX; prepare a 500 mM stock solution in DMSO (111.1 mg/ml) and store in small aliquots</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td>AntA; prepare a 5 mM stock solution in DMSO (2.7 mg/ml) and store in small aliquots</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A1470</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>CaCl2</td>
		</tr>
		<tr>
			<td>Concanacalin A, Lectin from Arachis hypogaea (peanut)</td>
			<td>Sigma-Aldrich</td>
			<td>L7381</td>
			<td>ConA</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>Isothesia</td>
			<td>Henry Schein Animal Health</td>
			<td>1169567761</td>
			<td>Isoflurane</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td>MgSO4</td>
		</tr>
		<tr>
			<td>N6,2&#39;-O-Dibutyryladenosine 3&#39;,5&#39;-cyclic monophosphate sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>D0627</td>
			<td>db-cAMP</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>KH2PO4</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Cayman Chemical Company</td>
			<td>13995</td>
			<td>Rot; prepare a 5 mM stock solution in DMSO (2mg/ml) and store in small aliquots</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>NaHCO3-</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Equipment and materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12 channel pipette 10-100 &#956;L</td>
			<td>eppendorf</td>
			<td>ES-12-100</td>
			<td></td>
		</tr>
		<tr>
			<td>12 channel pipette 50-300 &#956;L</td>
			<td>vwr</td>
			<td>613-5257</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C, non-CO2 incubator</td>
			<td>vwr</td>
			<td>1545</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL cetrifuge tubes</td>
			<td>eppendorf</td>
			<td>30119380</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>vwr</td>
			<td>76211-286</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge with plate adapter</td>
			<td>Thermo Scientific</td>
			<td>IEC FL40R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection kit</td>
			<td>World Precision Instruments</td>
			<td>MOUSEKIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted phase contrast microscope with 40X objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OctaPool Solution Reservoirs, 25 ml, divided</td>
			<td>Thomas Scientific</td>
			<td>1159X93</td>
			<td></td>
		</tr>
		<tr>
			<td>OctaPool Solution Reservoirs, 25 mL, divided</td>
			<td>Thomas Scientific</td>
			<td>1159X95</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td>Agilent</td>
			<td>102416-100</td>
			<td>Also sold as XFe96 FluxPak mini (102601-100) with 6 instead of 18 cartidges.</td>
		</tr>
	</tbody>
</table>

using an extracellular flux analyzer to measure changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation

哺乳动物精子在女性生殖道中获得受精能力，这个过程称为卵子。与电容相关的过程需要能量。关于产生ATP的来源，促进精子渐进性、充能性、超活化和杂技反应的来源，仍有持续的争论。在这里，我们描述了细胞外通量分析仪的应用，作为分析小鼠精子细胞内能量代谢变化的工具。使用H+- 和O2-敏感荧光草，该方法允许在非电容和电容精子中实时监测糖解和氧化磷酸化。在存在不同能量基质和/或药理活化剂和/或抑制剂的情况下使用此测定，可以深入了解不同代谢途径的贡献以及精子加速期间信号级联和代谢之间的交集。

该方法使用细胞外通量分析仪监测小鼠精子增生期间糖解和牛磷速率的实时变化。图4显示了一个示范性实验，其中精子在葡萄糖作为唯一能量基质的情况下被电位，而抗霉素和罗酮作为药理调节剂。细胞外通量分析仪TYH缓冲液和药理调制器的能量基板可根据实验目标自由选择。BSA/TYH中的未电容小鼠精子通过其头部附着在康A涂层瞬态室的底部。在此?...

Watch this Scientific Journal Video about Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation at JoVE.com

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

我们描述了细胞外通量分析仪在小鼠精子增生期间实时监测糖解和氧化磷酸化变化的应用。

使用细胞外通量分析仪测量小鼠精子发作期间糖解和氧化磷酸化的变化

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

该协议可用于应用细胞外通量分析仪来监测精子电容过程中能量代谢的变化。我们开发了此方案，以比较非电容和电容小鼠精子之间糖解和氧化磷酸化的变化。在测定前一天，从细胞外通量检测套件中取出传感器盒，将墨盒倒置放在公用电源板旁边。向溶液储液罐中加满 25 毫升的双蒸馏水，并将 200 微升水添加到公用板的每个井中，然后将传感器盒放回公用板中。将板放入37摄氏度的非二氧化碳培养箱中。接下来，将25毫升的细胞外通量分析仪卡兰特放入50毫升锥形管中，将管放入37摄氏度的非二氧化碳培养箱中。要准备 ConA 涂层微板，在五毫升双蒸馏水中溶解 2.5 毫克 ConA，并使用多通道移液器将 96 孔外通量分析仪的每口井用 50 微升的 ConA 溶液填充，然后将盖子打开，让板干燥过夜。为了准备精子缓冲液，将250毫升的细胞外通量分析仪TYH缓冲液加热到37摄氏度，将pH调整为7.4。在测定当天，将公用板每井中每井200微升的XF卡兰特水更换，将该水板放回非二氧化碳培养箱至少一小时。要生成波模板，请打开细胞外通量分析仪。当温度稳定到37摄氏度时，打开波软件并打开一个空白模板。在组定义选项卡下，将检测介质定义为TYH，将细胞类型定义为小鼠精子，使注射策略和预处理空白。为每个测定条件创建不同的组，并打开板图，将每个组分配给特定的井。在协议选项卡下，取消高光平衡，添加四个不同的测量周期，选择相应的端口，并编辑测量细节，以便突出显示注入后测量，然后保存测定模板，填写项目摘要并保存结果。在收获精子之前，在37摄氏度下预热50毫升TYH缓冲液。从成年雄性小鼠身上采集出秋千后，将每只卡达放在24孔板的个别井中，将预加热TYH缓冲液的500微升。使用钳子将每根井底部的每个阴唇单独固定，并使用羽毛剪刀在每个组织中快速进行五到七个小切口。当所有的阴极被切入时，立即将盘子放入非二氧化碳培养箱，让精子分散15分钟。要加载传感器墨盒，请从传感器墨盒中取出盖子，将相应端口加载导轨的字母与墨盒的左上角对齐。将端口装载导轨放置到位，将移液器尖端垂直插入端口 A 的港口装载导孔中，并将 20 微升 TYH 缓冲液注入每列。对于端口 B，将 22 微升 TYH 缓冲液注入第一至四列，将 22 微升 500 毫摩尔 2-脱氧葡萄糖注入列 5 至 8 中，将 5 微摩尔抗霉素 A rotenone 的 25 微升注入列 9 至 12 列。对于端口 C，将 25 微升 TYH 缓冲器注入每列，其中奇数和 25 微升 10 毫摩尔的二丁二醇-环环放大器 IBMX 到每个列中，具有偶数，然后将带校准板的传感器盒放入细胞外通量分析仪中，然后开始检测。校准需要 10 到 15 分钟。要准备精子板，在TYH缓冲液中加入三毫升三毫克三毫克的牛血清白蛋白，每毫升离心管。接下来，将两口精子汇集到三个1.5毫升离心管中。计数后，将精子稀释至新鲜TYH中每毫升浓度2倍10至7精子，并辅以牛血清白蛋白。通过离心沉淀精子，将精子颗粒重新悬浮在一毫升TYH缓冲液中。再次离心精子，取出超级精子，注意不要干扰颗粒。将每个精子悬浮液转移到准备好的五毫升离心管中，并将 180 微升TYH缓冲液加入 ConA 涂层板的每个角落井中。将180微升精子加入 ConA 涂层板的每个空井中，并在离心之间两次将板旋转 180 度，然后将校准板与精子板一起放置并继续测定。在这个具有代表性的实验中，精子在葡萄糖作为唯一能量基质的情况下进行电容，作为药理调节剂的2-脱氧葡萄糖和抗霉素和罗酮。箭头指示电容感应。在葡萄糖存在的情况下，在葡萄糖作用的同时，细胞外酸化率增加了七倍，通过用2-脱氧葡萄糖阻断糖解抑制了这种增酸率。电容精子的耗氧率比非电容精子增加了20倍，这表明小鼠精子可增强糖解和氧化磷酸化，以支持电容过程中不断增长的能量需求。精子容塞期间细胞外酸化率的上升不受氧化磷酸化抑制剂抗霉素甲和罗泰诺的影响，表明该速率的变化主要是由糖解氢释放所推动的。然而，抗霉素 A 和 rotenone 以及 2-脱氧葡萄糖的氧气消耗率的增加表明，精子封顶期间 OXPHOS 的增加取决于糖解活性。能量源和药理激活剂和抑制剂可以根据实验的目标而变化，最多可以同时测量多达12种不同的条件。该协议可以很容易地适应其他物种，如人类或牛，其中能量代谢的变化在风化期间同样神秘。

Research

JoVE Journal

Biochemistry

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

高度敏感和快速荧光检测用便携式分析仪FRET

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

科研

生物化学

Oligopeptide Competition Assay for Phosphorylation Site Determination

寡肽竞争测定磷酸化位点测定

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

在氧化应激期间细胞粘附和纤维蛋白扩散动力学

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

DetectSyn：一种快速、无偏倚的荧光方法，用于检测突触密度的变化

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

用于蛋白质磷酸化定量的优化单分子下拉测定

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...