Name: the examination of peroxidase-positive leukocytes in semen
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作者感谢中山大学第三附属医院生殖医学中心男科实验室为本研究提供设施。本文的编写没有涉及任何资金。

本研究已通过中山大学附属第三医院医学伦理委员会评审批准。
1.工作溶液的制备
<ol><li>收集以下制备工作溶液所必需的试剂 - 邻甲苯胺底物溶液、饱和氯化铵 （NH4Cl） 溶液、148 mmol/L 乙二胺四乙酸二钠盐 （Na2EDTA） 溶液和 6% （v/v） 过氧化氢溶液。将它们与必要的工具一起放在室温 （RT） 实验室工作台上，例如移液器（1000 μL、200 μL、10 μL）和试管架。 注意：在处理上述试剂之前，请阅读安全数据表 （SDS）。</li>
	<li>准备一个不透明的棕色试剂瓶，以储存和保护制备的工作溶液免受光线照射。</li>
	<li>按照以下指定的比例将试剂转移到不透明的棕色试剂瓶中：<ol><li>使用 200 μL 移液器，转动旋钮将刻度调节至 100 μL。然后，将 100 μL NH4Cl 溶液转移到不透明的棕色试剂瓶中。</li>
		<li>更换移液器吸头，并使用上述200 μL移液管将100 μL 148 mmol/L Na2EDTA溶液转移到不透明的棕色试剂瓶中。然后，用移液轻轻吹气，使溶液彻底均质化。</li>
		<li>使用 1000 μL 移液管，转动旋钮将刻度调节至 900 μL。然后，将900μL邻甲苯胺底物溶液转移到不透明的棕色试剂瓶中，用移液轻轻吹气，并使溶液彻底匀浆。</li>
		<li>使用 10 μL 移液器，转动旋钮将刻度调节至 5 μL。然后，将 5 μL 的 6% （v/v） 过氧化氢溶液转移到不透明的棕色试剂瓶中。</li>
	</ol></li>
	<li>使用 1000 μL 移液管，转动旋钮将刻度调节至 1000 μL。更换移液器吸头，用移液器轻轻吹气，并在不透明的棕色试剂瓶中彻底均质化溶液。</li>
	<li>将即用型工作溶液储存在不透明的棕色试剂瓶中，在室温下避光。工作溶液反应24小时。</li>
	<li>每个精液样品需要 900 μL 的工作溶液。确保每天制备的工作溶液的数量与每天检查的样品数量相对应。</li>
</ol>2. 精液样本的制备
<ol><li>在采集精液样本之前，请确保患者已按照世界卫生组织 （WHO） 检查和处理人类精液的实验室手册（第 5 版）的要求戒射精 2 至 7 天。8.手淫是获取精液样本的首选方法。为了提高检查的准确性，指导患者收集所有射出的精液。</li>
	<li>打开恒温支架，提前加热至37°C，以便精液样品的后续液化能够顺利进行。</li>
	<li>收到患者的精液样本后，在聚合物基容器的外壁上仔细记录详细信息，包括识别码、采集时间、禁欲持续时间和其他相关信息。</li>
	<li>将装有精液样品的聚合物基容器放在预热至37°C的恒温支架上，这有利于精液液化。</li>
	<li>将精液样品吸入聚合物基移液管中，每5分钟观察一次液化状态，直到精液样品完全液化。</li>
	<li>如果精液样本在30分钟内未完全液化，则不要进行检查;继续等待 30 分钟。如果精液在60分钟后仍未完全液化，则加入等体积的磷酸盐缓冲溶液以1：1稀释并轻轻搅拌以促进液化。</li>
	<li>在彻底搅拌和一致均质化后，保留完全液化的精液样本以备后续检查程序使用。</li>
</ol>3. 对过氧化物酶阳性白细胞进行染色并分析原始精液样本
<ol><li>在取样之前，使用基于聚合物的移液器通过移液反复吹气，并彻底均质化被检查的精液样品。</li>
	<li>使用 200 μL 移液器，转动旋钮将刻度调节至 100 μL。更换移液器吸头，然后将 100 μL 精液样品转移到离心管中。</li>
	<li>使用 1000 μL 移液管，转动旋钮将刻度调节至 900 μL。更换移液器吸头，然后将 900 μL 上述工作溶液转移到离心管中。</li>
	<li>使用1000μL移液管反复吹气并彻底均质化反应物，该反应物由精液样品和工作溶液组成。</li>
	<li>将离心管放置在二维振荡器上的试管架中，并在室温下以200rpm连续搅拌30分钟。</li>
	<li>在等待染色反应的同时，利用CASA系统分析精液样品，以确定聚合物基容器中原始精液样品中精子的浓度。<ol><li>打开CASA系统的电源开关，双击SCA SCOPE图标打开应用软件。</li>
		<li>取样前，用移液器反复吹气，并用聚合物基移液管再次将原始精液样品彻底均质化。</li>
		<li>使用 10 μL 移液器，转动旋钮将刻度调整至 10 μL。更换移液器吸头，移取 10 μL 原始精液样品，并将其放入 SCA 计数室。等待 60 秒，让精液样本停止漂移。</li>
		<li>将SCA计数室放置在CASA系统的样品架上，并在应用软件的对话框中设置患者信息，为正式检查做准备。</li>
		<li>通过单击 “开始 ”按钮，使用CASA系统检查SCA计数室中的原始精液样本，并记录必要的数据，例如精子的浓度。</li>
	</ol></li>
</ol>4.观察过氧化物酶阳性白细胞和精子
<ol><li>使用 1000 μL 移液管，转动旋钮将刻度调节至 1000 μL。更换移液器吸头，然后通过移液反复吹气，并在30分钟反应过程结束时再次在离心管中使反应物（由精液样品和工作溶液组成）彻底均质化。</li>
	<li>使用 10 μL 移液器，转动旋钮将刻度调整至 10 μL。更换移液器吸头，移取10μL上述反应物，并将其放入精子计数室。最后，用专用的玻璃盖玻片盖住精子计数室。</li>
	<li>将精子计数室放在显微载物台上，并用两个10倍目镜打开光学显微镜的电源开关。然后，等待 60 秒，让反应物样品停止漂移。</li>
	<li>在等待时间内将显微转换器切换到 10 倍物镜。然后依次调整粗调旋钮和微调旋钮。最后，在10×10的放大倍率下观察精子和圆形细胞。</li>
	<li>确定要分析的视场后，将显微转换器切换到40倍物镜，在10×40倍的放大倍率下观察精子和棕色过氧化物酶阳性白细胞。</li>
	<li>利用电子计数器系统计数相应字段内至少 200 个精子和棕色过氧化物酶阳性白细胞的数量。</li>
</ol>5.过氧化物酶阳性白细胞浓度的计算
<ol><li>使用以下公式计算过氧化物酶阳性白细胞的浓度： <img alt="Equation 1" src="/files/ftp_upload/66211/66211eq01v2.jpg" style="margin: auto;"></li>
</ol>

过氧化物酶阳性白细胞精液分析

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作者没有什么可透露的。

邻甲苯胺和过氧化氢具有光敏性，暴露在光线下会分解。为确保测试试剂的有效性，建议将制备的工作液存放在黑暗的地方。 
精液样本自然表现出异质性特征。在精液样品的制备过程中，正确判断精液的液化状态，并在每次取样前对精液样品进行彻底均质化，对于提高检测结果的可靠性至关重要。精液样品在15分钟内完全液化。如果它们在60分钟后仍未完全液化，则应考虑机械均质或酶消化。通过在液化过程中使用二维振荡器8连续轻轻摇动样品容器，可以获得更均匀的精液样品。
本文提出的检查方法具有简单、快速、经济等优点。然而，在某些情况下它可能没有好处：过氧化物酶储存在嗜天青颗粒中，这些颗粒在激活时将通过脱颗粒或细胞质分泌到细胞间隙中21,22。因此，中性粒细胞可能无法染色且无法识别。在这种情况下，有必要重复染色过程，甚至要求患者提供新的精液样本进行重新检查。为了尽量减少这种情况，建议尽可能使用新鲜精液样品，并尽量减少放置时间和温和的均质化过程。这种方法只能识别活动性疾病或感染，而不能识别后期的疾病，如慢性感染16。此外，过氧化物酶含量较低的其他类型的白细胞，如淋巴细胞、巨噬细胞和单核细胞，使用这种方法进行染色也存在困难;因此，通过采用白细胞抗原（例如 CD45）免疫组织化学染色可以获得可靠的结果。
根据 WHO 手册第 5 版的要求，传统的阳性髓过氧化物酶染色（Endtz 试验）需要在血细胞计数板中对至少 200 个过氧化物酶阳性白细胞进行显微镜计数，以计算其浓度8。与传统的手动Endtz测试相比，该协议引入了通过CASA检测获得的精子浓度数据，结合了手动和自动方法。为了计算过氧化物酶阳性白细胞的浓度，在显微镜下计数相应视场中的至少200个精子和白细胞。由于精液中精子的浓度远高于白细胞，因此与计数 200 个过氧化物酶阳性白细胞相比，计数 200 个精子所需的时间更少。
重度少精子症、隐精子症和无精子症患者在计数足够数量的精子时经常面临挑战。在这种情况下，过氧化物酶阳性白细胞的浓度可以使用血细胞计数板直接计算，如该板手册中所述。此外，对于不需要 CASA 或没有 CASA 设备的实验室的患者，还可以使用血细胞计数板来确定过氧化物酶阳性白细胞的浓度。

不孕不育已成为一个全球公共卫生问题，影响着大约15%的育龄夫妇。男性因素约占总不孕症病例的50%，近20%至30%可仅归因于男性因素1,2,3。男性生殖道感染 （MGTI） 是男性不育的重要原因之一，约占病例的 15% 4,5。
大多数人的精液中有白细胞，占非精子细胞的 13%，中性粒细胞占 12%，巨噬细胞占 0.9%，淋巴细胞占 0.1%6,7。根据世界卫生组织 （WHO） 人类精液检查和处理实验室手册（第 5 版），白细胞精子症通常定义为精液中存在 >1 × 106 个细胞/mL白细胞 8。这种情况可能由许多因素引起，例如环境毒素、药物滥用、精索静脉曲张、MGTI 等，所有这些都可能导致精液中白细胞浓度异常升高9.白细胞可提高精液中的活性氧 （ROS） 水平，导致脂质过氧化和蛋白质和 DNA 的氧化损伤，从而导致精子活力降低、精子形态异常增加、精子 DNA 片段化指数升高、精子顶体功能受损，甚至对胚胎发育产生负面影响10,11。
目前，男科医生在识别生殖道炎症时，通常选择检查精液中的圆形细胞或精浆中的弹性蛋白酶。然而，区分脱落的生精细胞和生殖道上皮细胞通常具有挑战性，它们无助于减少抗生素的不分青红皂白和不必要的使用6。后一种检查方法相对复杂、耗时长，结果报告速度慢，不利于MGTI等疾病的早期诊断和治疗。美国泌尿外科协会 （AUA） 建议，当精液分析中的圆形细胞浓度大于 1 ×10 6 个细胞/mL12 时，应进一步区分白细胞和生殖道脱落细胞。一些男科实验室利用流式细胞术13 或白细胞抗原（例如 CD45）免疫细胞化学14 来检查白细胞。虽然这些方法很精确，但它们既昂贵又耗时，使大规模临床实施变得困难，尤其是在发展中国家。
过氧化物酶广泛分布于各种类型的细胞中，在抵抗氧化应激损伤方面起着至关重要的作用。髓过氧化物酶 （MPO） 是过氧化物酶亚家族的成员，通常在免疫细胞中表达。它在中性粒细胞的嗜天青颗粒15,16 中表达最高，也在淋巴细胞17,18、单核细胞和巨噬细胞19 中表达。精液中白细胞（尤其是中性粒细胞）的浓度可以通过检查过氧化物酶阳性的圆形细胞来获得 7,20。为了使精液中白细胞的检查过程经济、方便、高效，我们重新设计了检查方法。在计算机辅助精液分析（CASA）系统的辅助下，可以在60分钟内获得白细胞的浓度。这种新方法降低了患者的检查成本和等待结果的时间，减轻了实验室技术人员的工作量，缩短了医生的诊疗等待时间。

<table><tbody><tr><td>148 mmol/L ethylenediamine tetraacetic acid disodium (Na&lt;sub&gt;2&lt;/sub&gt;EDTA) solution</td><td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td><td>20230301</td><td>Agentia B</td></tr><tr><td>6% (v/v) hydrogen peroxide solution</td><td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td><td>20230301</td><td>Agentia D</td></tr><tr><td>Automatic Sperm Class Analyzer &amp;ndash; SCA SCOPE&amp;nbsp;</td><td>Microptic S.L.</td><td>1222996; Model type: SCA-SCOPE-H</td><td>Computer-Assisted Semen Analysis (CASA) equipment</td></tr><tr><td>Disposable centrifugal tube (1.5 mL)</td><td>Zhejiang Gongdong Medical Equipment Co., LTD</td><td>2210009</td><td>Centrifugal tube for Staining process</td></tr><tr><td>Disposable transfer pipette (3 mL)</td><td>Jiangsu Kangjian Medical Supplies Co., LTD</td><td>20221101</td><td>Polymer-based pipette</td></tr><tr><td>Electronic counter</td><td>None</td><td>None</td><td>A multichannel counter</td></tr><tr><td>Electronic scales</td><td>Shanghai Liangping Instrument Co., LTD</td><td>D9008084</td><td>MAX = 100 g, e = 10 d, d = 0.01 g</td></tr><tr><td>Makler counting chamber</td><td>Makler</td><td>MQ30004</td><td>0.01 sq.mm, 10 &amp;mu;m deep</td></tr><tr><td>Optical microscope</td><td>Olympus</td><td>3G41067201307</td><td>CX31, 10&amp;times;40</td></tr><tr><td>Ortho-toluidine substrate solution</td><td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td><td>20230301</td><td>Agentia C</td></tr><tr><td>Pipet tips(1 mL)</td><td>Coming Life Sciences (Wujiang) Co.,Ltd</td><td>02923205</td><td>1000 tips/unit, 5 units/case</td></tr><tr><td>Pipet tips(10 &amp;micro;L)</td><td>Coming Life Sciences (Wujiang) Co.,Ltd</td><td>00323961</td><td>1000 tips/unit, 20 units/case</td></tr><tr><td>Pipet tips(200 &amp;micro;L)</td><td>Coming Life Sciences (Wujiang) Co.,Ltd</td><td>32822810</td><td>1000 tips/unit, 20 units/case</td></tr><tr><td>Saturated ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl) solution</td><td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td><td>20230301</td><td>Agentia A</td></tr><tr><td>SCA counting chamber</td><td>Microptic S.L.</td><td>102230608</td><td>10 &amp;micro;m, 2 Chambers</td></tr><tr><td>The container for semen sample</td><td>Jiangsu Kangjian Medical Supplies Co., LTD</td><td>20230701</td><td>Polymer-based receptacle for semen sample</td></tr><tr><td>Thermostatic table</td><td>Shenzhen MIAOQUAN Instrument Co., LTD</td><td>MQ30004</td><td>MQ-300</td></tr><tr><td>Transfer pipette (10 &amp;micro;L)</td><td>Eppendoff</td><td>3121000.015</td><td></td></tr><tr><td>Transfer pipette (1000 &amp;micro;L)</td><td>Eppendoff</td><td>3121000.12</td><td></td></tr><tr><td>Transfer pipette (200 &amp;micro;L)</td><td>Eppendoff</td><td>3121000.082</td><td></td></tr><tr><td>Two-dimensional shaker</td><td>DragonLab</td><td>822000010000</td><td>VC5A002205</td></tr></tbody></table>

the examination of peroxidase-positive leukocytes in semen

白细胞精子症可导致精子活力降低、精子形态异常增加、精子DNA片段化指数升高、精子顶体功能受损，甚至影响胚胎发育。它是临床实践中常见的男科疾病，也是男性不育的重要原因之一。在确定是否存在男性生殖道炎症时，男科医生通常选择检查精液中的圆形细胞或精浆弹性蛋白酶作为临床诊断依据。然而，圆形细胞的检查很容易受到脱落的生精细胞和生殖道上皮细胞的影响，这无助于减少抗生素的不分青红皂白和不必要的使用。同时，弹性蛋白酶的检测过程相对复杂、耗时、报告结果缓慢，不利于男性生殖道感染（MGTIs）等疾病的早期诊断和治疗。我们创新地将计算机辅助精液分析（CASA）系统辅助的精液中过氧化物酶阳性白细胞的检测作为白细胞精子症的诊断标准，成功解决了这些问题。该检查只需将四种试剂组成的操作液加入标本中，室温下的总反应时间可控制在20-30min以内。通过随后的涂片和显微镜检查，可以在总共60分钟内获得精液中过氧化物酶阳性白细胞的浓度，可用于诊断男性生殖道是否存在炎症。

在实施上述程序时，离心管中的反应物经常表现出半透明的乳白色外观（图1）。检测到明显的棕色可显示白细胞的潜在脱颗粒，这可能导致液体染色。因此，染色过程可能会失败，因此需要重新染色或收集新的精液样本进行重新测试21。
过氧化物酶阳性白细胞呈棕色，与未染色的普通圆形细胞形成鲜明对比（图2，普通载玻片）。随后，在当前显微镜视野下对每个精子和棕色白细胞进行计数，然后转移到一个全新的视野进行连续观察和计数。为确保最佳准确性，有必要计数至少 200 个精子和相应视野中存在的白细胞。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66211/66211fig01.jpg"> 图1：反应物溶液。 完成反应过程的反应物是亚半透明乳白色液体。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66211/66211fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66211/66211fig02.jpg"> 图2：光学显微镜下的观察（普通载玻片）。 （A）过氧化物酶阳性白细胞和（B）在10×40放大倍率光学显微镜下观察到的普通圆形细胞。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66211/66211fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Simple and Cost-Effective Method for Leukocyte Analysis in Semen at JoVE.com

作者聚焦：一种简单且经济高效的精液白细胞分析方法 

本文提出了一种经济有效的治疗精液中过氧化物酶阳性白细胞的方法。在计算机辅助精液分析（CASA）系统的辅助下，可以在总共60分钟内获得精液中过氧化物酶阳性白细胞的浓度，有效提高了男科实验室和男科医生的效率。

精液中过氧化物酶阳性白细胞的检查

Leukocytospermia can lead to decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation ...

男性生殖医学是我的主要研究重点，我们的方案主要解决分析精液中过氧化物酶阳性白细胞的速度和成本问题。精液本质上是异质性的，这极大地影响了精液分析结果的稳定性。因此，在检查前必须确保精液液化并充分混合。我们的方案结合了手动和自动方法，利用精子浓度来计算白细胞的浓度，与即用计数的白细胞相比，这节省了时间并且更具成本效益。

the-examination-of-peroxidase-positive-leukocytes-in-semen

Universidad San Sebastian

Research

JoVE Journal

Medicine

The Examination of Peroxidase-Positive Leukocytes in Semen

3.7K 次观看.  The Third Affiliated Hospital of Sun Yat-sen University. 男性生殖医学是我的主要研究重点，我们的方案主要解决分析精液中过氧化物酶阳性白细胞的速度和成本问题。精液本质上是异质性的，这极大地影响了精液分析结果的稳定性。因此，在检查前必须确保精液液化并充分混合。我们的方案结合了手动和自动方法，利用精子浓度来计算白细胞的浓度，与即用计数的白细胞相比，这节省了时间并且更具成本效益。

视频: 作者聚焦：一种简单且经济高效的精液白细胞分析方法 

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

听力：IMAP测试电池的决策意识

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

科研

医学

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

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) in spermatozoa is of high importance in any species, but these methods are rarely used and even less in teleost. Cryopreservation is a useful technique in aquaculture for different purposes, including gene banking and guaranteed sperm availability throughout the year. Freezing/thawing procedures could cause ROS production and damage the sperm cells. Considering the prospective damage that an excess of ROS production could cause in spermatozoa depending on their localization, here a detailed methodology to detect H2O2 and to evaluate its intracellular localization by confocal microscopy is provided. For this purpose, a combination of 3 fluorochromes (2&#8242;,7&#8242;-Dichlorodihydrofluorescein diacetate (DCFH-DA), a live mitochondria stain and 4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)) are used to evaluate the co-localization of H2O2 with spermatozoa nuclei or mitochondria in Solea senegalesis sperm samples.

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

This protocol describes a detailed methodology for detecting H2O2 localization within Solea senegalensis spermatozoa using a sensitive fluorochrome DCFH-DA for ROS, a live mitochondria stain for mitochondria, and DAPI for nuclei visualization, respectively. The protocol is designed to be performed within 2 h with either fresh or thawed spermatozoa.

Solea Senegalensis精子中活性氧的细胞内定位评价

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) ...

发育生物学

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male&#39;s cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 &#956;L capillary pipette was placed into the tube at a 180&#176; angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 &#956;L line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 &#956;L capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

雌性生殖道交配后精子的收集及对小鼠精子液化的测定

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

Fluorimetric Techniques for the Assessment of Sperm Membranes

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

精子膜评价的荧光技术

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

使用紫罗兰兴奋细胞渗透DNA结合染料分离穆林精子生成细胞

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

在富含白细胞的血液样本卤化过氧化物酶活性的快速，具体的评估

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

免疫与感染

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

啮齿动物附睾精子磷酸分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

生物学

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility.
With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 &#176;C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5&#8242;,6,6&#8242;-tetrachloro-1,1&#8242;,3,3&#8242;-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.

The present protocol provides a practical solution that allows for the measurement of apoptosis, mitochondrial membrane potential, and DNA damage in human sperm with a single cytometer.

用于检测人类精子功能缺陷的生物标志物的流式细胞术分析

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

密度梯度离心法测定微分品质的精子

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

U-Shaped Horizontal Swimming Technique for Preparing High-Quality Sperm with Low DNA Fragmentation Index

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and viscous seminal plasma. High-quality sperm refers to progressively motile spermatozoa with normal morphology, which often exhibit a lower DNA fragmentation index (DFI) and higher fertilization potential. Preparing high-quality sperm is a critical step in human-assisted reproductive technology. The traditional sperm preparation method, discontinuous density gradient centrifugation (DGC), is time-consuming and labor-intensive. Repeated centrifugation can damage sperm DNA, thereby affecting subsequent fertilization and embryo development. This study introduces a U-shaped horizontal swimming (UHS) method for preparing intracytoplasmic sperm injection (ICSI) sperm, which significantly eliminates the detrimental effects of&#160;centrifugation on sperm DNA.&#160;The UHS method involves creating a UHS lane using a fertilization medium within an ICSI operating dish. A 10 &#181;L fertilization medium microdroplet is placed at the starting point on the left side of the UHS lane to hold the semen. Two additional 10 &#181;L fertilization medium buffer droplets are positioned at intervals along the left middle section of the lane, with all droplets connected by fertilization medium. The dish is then covered with culture oil and incubated overnight at 37 &#176;C with 6% CO2 to equilibrate. Subsequently, 3 &#181;L of semen is added to the microdroplet at the starting point. High-quality spermatozoa swim to the right side of the UHS lane, facilitating their suction into the ICSI injection needle. Dead sperm, cell debris, and other viscous impurities largely remain at the initial point or in the buffer droplets. We simultaneously processed 21 semen samples using both UHS and DGC techniques and compared their DFI. The results demonstrated that the DFI in the DGC group was 5.5% &#177; 3.2%, whereas the DFI in the UHS group was 1.7% &#177; 1.1%. The difference between the two groups was statistically significant (P &#60; 0.05).

This protocol describes a method for screening high-quality sperm with a low DNA fragmentation index by utilizing sperm motility characteristics and thigmotaxis. The method employs a U-shaped horizontal swimming lane, enabling high-quality sperm to reach the opposite side through buffer droplets, while dead sperm, cell debris, and impurities are excluded.

U 型水平游泳技术制备低 DNA 片段化指数的高质量精子

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and ...