Name: evaluation of intracellular location of reactive oxygen species in solea senegalensis spermatozoa
Uploaded: 2018-03-11T13:00:00
Description: Watch this Scientific Journal Video about Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa at JoVE.com

我们感谢 AQUAGAMETE 1205 的成本行动。这项工作得到了 AGL201568330-C2-1-R 项目 (MINECO/菲德) 的财政支持。大卫 g. Valcarce 由 EDU1084/2012 和 Fondo 社会 Europeo 资助。作者承认安娜 Riaza 博士和 Stolt 海洋农场, Paulino 博士, 拉巴斯博士蒙特罗和何塞拉蒙 Guiérrez。我们还感谢宝拉. 费尔南德斯. Colado 为录像。

注: Fluorochrome 孵化和共焦分析将采取至少2-3 小时的控制和治疗样品。在这个时间计算中不包括数据处理。所需材料可在材料表中找到。本协议可应用于新鲜或冷冻保存的精子。Solea senegalensis是一种在冷水中产生的鱼类, 通常在寒冷的环境下工作 (4-7 摄氏度)。有关协议的一般视图, 请参见图 1 。
1. 实验前的准备工作
<ol><li>在分析级二?...

<ol>
	<li>Amaral, S., 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=Mitochondrial+functionality+and+chemical+compound+action+on+sperm+function.">Mitochondrial functionality and chemical compound action on sperm function.</a> Curr Med Chem. , (2016).</li><li>Morielli, T., O'Flaherty, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+impairs+function+and+increases+redox+protein+modifications+in+human+spermatozoa.">Oxidative stress impairs function and increases redox protein modifications in human spermatozoa.</a> Reproduction. 149 (1), 113-123 (2015).</li><li>G&#252;rler, H., 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=Effects+of+cryopreservation+on+sperm+viability,+synthesis+of+reactive+oxygen+species,+and+DNA+damage+of+bovine+sperm.">Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.</a> Theriogenology. , (2016).</li><li>Zhu, Z., 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=Vitamin+E+Analogue+Improves+Rabbit+Sperm+Quality+during+the+Process+of+Cryopreservation+through+Its+Antioxidative+Action.">Vitamin E Analogue Improves Rabbit Sperm Quality during the Process of Cryopreservation through Its Antioxidative Action.</a> PLoS One. 10 (12), e0145383 (2015).</li><li>Fang, L., 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=Inhibition+of+ROS+production+through+mitochondria-targeted+antioxidant+and+mitochondrial+uncoupling+increases+post-thaw+sperm+viability+in+yellow+catfish.">Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish.</a> Cryobiology. 69 (3), (2014).</li><li>Thomson, L. K., Fleming, S. D., Aitken, R. J., De Iuliis, G. N., Zieschang, J. A., Clark, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation-induced+human+sperm+DNA+damage+is+predominantly+mediated+by+oxidative+stress+rather+than+apoptosis.">Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis.</a> Hum Reprod. 24 (9), 2061-2070 (2009).</li><li>Kim, S. H., Yu, D. H., Kim, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+phosphatidylserine+translocation,+intracellular+hydrogen+peroxide,+and+DNA+integrity+in+canine+sperm.">Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm.</a> Theriogenology. 73 (3), (2010).</li><li>Guthrie, H. D., Welch, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reactive+oxygen+species+on+sperm+function.">Effects of reactive oxygen species on sperm function.</a> Theriogenology. 78 (8), 1700-1708 (2012).</li><li>Aitken, R. J., Jones, K. T., Robertson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+and+sperm+function--in+sickness+and+in+health.">Reactive oxygen species and sperm function--in sickness and in health.</a> J Androl. 33 (6), (2012).</li><li>Valcarce, D. G., Robles, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+captivity+and+cryopreservation+on+ROS+production+in+Solea+senegalensis+spermatozoa.">Effect of captivity and cryopreservation on ROS production in Solea senegalensis spermatozoa.</a> Reproduction. 152 (5), (2016).</li><li>Gibb, Z., Lambourne, S. R., Aitken, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+paradoxical+relationship+between+stallion+fertility+and+oxidative+stress.">The paradoxical relationship between stallion fertility and oxidative stress.</a> Biol Reprod. 91 (3), (2014).</li><li>Cabrita, 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=Factors+enhancing+fish+sperm+quality+and+emerging+tools+for+sperm+analysis.">Factors enhancing fish sperm quality and emerging tools for sperm analysis.</a> Aquaculture. 432, 389-401 (2014).</li></ol>

众所周知, 线粒体是精子运动和功能的关键细胞器。这些细胞器同时直接参与 ROS 的生产。有趣的是, 适当的精子功能需要控制的活性氧含量为1。在哺乳动物中, 生育率和氧化应激之间的正相关关系已显示在11中, 但过度的水平影响精子质量12。一个关键的因素, 可能是决定性的积极或消极的影响, 不仅是 ros 水平, 而且 ros 细胞内定位。ros 的有害?...

活性氧种类的生产已与精子质量最近的1。虽然线粒体中的 ros 生产可以被认为是正常的生理过程, 但氧化应激是由过量的 ros 产生的, 是导致精子在不同水平上损伤的明显原因。在人类中, 氧化应激与男性不育、改变运动和接受精子2的能力有关;在哺乳动物中, 冷冻精子样品中 DNA 完整性的变化也与 H2O23的合成有关。
冷冻保存是水产养殖中的一种常用的基因银行技术。这种技术在具有生殖问题 (如Solea senegalensis) 的物种中尤为重要。这个市场上有价值的物种显示, 由于缺乏求爱, 在圈养的个体中繁殖功能失调。这一事实使精子的超低温保存必须有精子可用性的人工受精。然而, 冷冻保存可能是氧化应激的来源, 可能是有害的精子4 , 因为研究报告了有益的效果, 补充抗氧化剂。据报道, 通过线粒体靶向抗氧化剂的活性氧抑制有助于黄鲶鱼的精子超低温保存5。
因此, 精子样本中 ROS 的含量是很重要的, 特别是在保存6、7之后, 因为这些分子被认为是精子生存和生育能力8的一个缺点。此外, 研究细胞中 ROS 的分布对于推断潜在损伤的程度是至关重要的。例如, 线粒体中的低活性氧能被认为是正常的, 与精子功能相容, 但细胞核内的高活性氧含量可能是精子 DNA 损伤的指标。H2O2是最相关的 ROS 之一, 可以从线粒体中释放出来, 穿透细胞核, 因为它是一个小而无电荷的分子9。Dichlorofluorescein 双乙酸 (DCFH) 可以明确揭示胞内过氧化物发光的绿色荧光。本文介绍了用共聚焦显微镜检测Solea senegalensis精子中 H2O2细胞内定位的详细协议。

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			<td height="21" style="height:21px;width:351px;">2&#8242;,7&#8242;-Dichlorodihydrofluorescein diacetate (DCFH-DA)&#160;</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6883</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">4&#8242;,6-diamidino-2-phenylindole (DAPI)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C1016&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscopy</td>
			<td>Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slips</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-541B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO, Analytical Grade</td>
			<td>Sigma-Aldrich</td>
			<td>W387520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol, Analytical Grade</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MitoTrackerDeep Red&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>M22426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge (refrigerated)</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neubauerchamber</td>
			<td>Sigma-Aldrich</td>
			<td>BR717810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>10143562BEF</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of intracellular location of reactive oxygen species in solea senegalensis spermatozoa

氧化应激是降低精子质量的重要因素之一。开发有效的检测精子中活性氧 (ROS) 的协议在任何物种中都是非常重要的, 但是这些方法在硬骨鱼类中很少使用, 甚至更少。冷冻保存是水产养殖的一种有用的技术, 为不同的目的, 包括基因银行和保证精子的可用性全年。冷冻/解冻程序可能导致 ROS 的产生和损伤精子细胞。考虑到过量的 ROS 生产可能导致精子的潜在损害取决于它们的定位, 这里有一个详细的方法来检测 H2O2和评估其细胞内定位的共焦显微镜提供。为此, 采用3荧光 (-2′-、7′-Dichlorodihydrofluorescein 双乙酸 (DCFH DA)、活线粒体染色和4′-、6-Diamidino-2-苯基吲哚二盐酸盐 (DAPI)) 来评价H2 O 2 的协同定位与精子细胞核或线粒体在Solea senegalesis精子样本。

共焦显微术是硬骨鱼类精子细胞内 ROS 评价的理想方法。这项研究 (图 1) 中提出的三荧光 (DAPI、线粒体染色和 DCFH) 的组合提供了许多有用的信息, 可应用于基础研究, 并可应用于改进工业水产养殖植物, 如冷冻保存协议。可以进行不同类型的分析, 以关联细胞内 ROS 的存在和其他参数: 运动, 生存能力, 不同模式之间的良好和不良育种, 活化运动或季节性?...

Watch this Scientific Journal Video about Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa at JoVE.com

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

此协议描述了一个用于检测Solea senegalensis精子中的 H2O2本地化的详细方法, 使用敏感的 fluorochrome DCFH-DA 为 ROS、线粒体的活线粒体染色和细胞核的 DAPI可视化, 分别。该协议的设计是在2小时内进行, 无论是新鲜或解冻精子。

Solea Senegalensis精子中活性氧的细胞内定位评价

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