CN112858242A - 一种高通量通过检测钙信号判断人精子质量的方法 - Google Patents
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Abstract
本发明公开了一种高通量通过检测钙信号判断人精子质量的方法，属于医学技术领域。本发明基于群体精子样本生理环境下，对同一样本进行多种生理因素刺激下[Ca2+]i变化高通量酶标仪检测，以大数据临床样本量对于各生理因子对[Ca2+]i调节情况作为研究对象，通过选取精子在配子受精之前功能调控各个过程中相应的生理刺激，观察各个精子样本为准备受精过程中各个方面正常与否情况。
Description
技术领域
本发明属于医学技术领域，具体涉及一种高通量通过检测钙信号判断人精子质量的方法。
背景技术
正常人成熟精子是一个形似蝌蚪状的长形细胞，分为头部、颈部、中段、主段和末段。由于哺乳动物成熟精子具有一个高度浓缩的细胞核，因而被认为是转录和翻译沉默的细胞，因此，精子生理功能的调控主要依赖于翻译后的蛋白修饰调节。基于胞内 Ca2+信号作为一种重要的第二信使，在该修饰调节过程中的关键作用，Ca2+在精子获能中扮演核心作用，在精子成熟、超活化运动、顶体反应、精卵结合等一系列精子生理功能的调控中也发挥着极其关键的作用。在体外实验研究细胞外Ca2+浓度对精子功能的影响时发现：获能液中含有0.22mM Ca2+就足够诱发人精子产生蛋白磷酸化反应与超活化运动，但要使精子发生顶体反应及精卵结合，获能液中的Ca2+浓度则必须达到0.58 mM。其中通过检测精子胞内Ca2+([Ca2+]i)含量时也发现，获能精子[Ca2+]i(100-300nM)明显高于未获能的精子(10-40nM)。获能精子[Ca2+]i升高后，胞内大量的Ca2+将与膜磷脂以及许多的功能酶结合，从而修饰细胞膜生理特性、改变酶活性，从而改变精子功能，使精子超活化，进而发生顶体反应、促进精卵结合，完成受精过程，因此，其对成熟精子各种功能调控过程尤为重要。而目前关于 Ca2+库如何调控胞内Ca2+信号进而影响精子功能仍知之甚少。因此，建立一种合适的方法检测成熟精子胞内 Ca2+信号调控情况将有助于全面认识受精过程中的精子功能调节机制。
目前测定钙信号响应的筛选方法有：利用流式细胞仪测定人精子胞内钙浓度、单细胞钙成像技术等。但这些方法步骤繁琐，耗时较长，不能高效、高通量的筛选多种生理指标下对应的生理因子刺激的钙信号变化情况。
发明内容
本发明的目的是提供一种高通量通过检测钙信号判断人精子质量的方法，通过模拟生理环境下，对同一样本进行体外群体精子测钙，实现对不同生理因子刺激下的钙信号[Ca2+]i变化荧光信号检测，以反映同一精液样本对于各生理因子对[Ca2+]i调节情况，通过选取精子在配子受精之前功能调控各个过程相应生理刺激，反应精子为准备受精过程中各个方面正常与否情况。
为了实现上述发明目的，本发明采用以下技术方案：
一种高通量通过检测钙信号判断人精子质量的方法，包括以下步骤：
步骤1，人精液样本的处理：将收集到的精液样本水浴液化；
步骤2，清洗纯化人精子：将步骤1的精液样本用HS清洗，以去除精浆；
步骤3，Fluo-4 AM精子孵育染色：采用钙荧光探针Fluo-4 AM 和Pluronic F-127对纯化后的精子进行染色；
步骤4，向染色的精子中加入刺激物，然后采用多功能酶标仪检测加入刺激物前后精子胞内钙信号[Ca2+]i的荧光值，根据精子胞内钙信号[Ca2+]i的荧光值变化，确定各刺激因子对人精子钙信号[Ca2+]i的影响，分析人精子样本对各生理过程响应是否正常，从而判断人精子质量。
进一步地，步骤1中水浴液化的条件是37℃水浴液化 60 min。
进一步地，步骤2中清洗具体是：向1 mL精液中加入0.5 mL 预热的HS溶液，混匀后，2000 rpm离心5 min，弃去上清，再加入1.0 mL HS溶液重悬，2000 rpm离心5 min，弃去上清，然后加入预热的HS溶液重悬并调整精子浓度为1×107个/mL以上。
进一步地，步骤3中染色具体是：向清洗纯化后的精子中加入5 μM 钙荧光探针Fluo-4 AM 和0.05% Pluronic F-127 的进行重悬混匀孵育，避光置于37℃、5 % CO2条件下染色30 min，1800 rpm 离心 6 min，吸去上清液以去除精子细胞外残留的染料，加入 HS溶液重悬精子沉淀，2000 rpm 离心 6 min，去除上清，重复一次，即得染色后的精子溶液。
进一步地，步骤4中所述刺激物为NaHCO3溶液、孕酮溶液、Bourgeonal溶液、前列腺素1-前列腺素2的混合溶液。
进一步地，步骤4中多功能酶标仪的检测条件为：激发波长502 nm，发散波长525nm，温度37℃。
本发明基于群体精子样本生理环境下，对同一样本进行多种生理因素刺激下[Ca2 +]i变化高通量酶标仪检测，以大数据临床样本量对于各生理因子对[Ca2+]i调节情况作为研究对象，通过选取精子在配子受精之前功能调控各个过程中相应的生理刺激，观察各个精子样本为准备受精过程中各个方面正常与否情况。在自动加样、避免加样时间差的同时快速高效的检测同一样本对各功能调控过程指标刺激[Ca2+]i的检测，进一步判断精子样本的生理功能正常与否，实现对临床生殖中心检验中监测精子是否因生理功能响应缺陷引起的不育不孕作出诊断，为临床男性不育的诊断提供新的方法和思考；另一方面可为实验室科研中对于影响人精子[Ca2+]i调节的其他生理因子、蛋白或合成药物的筛选，进而验证其他生理因子、蛋白或合成各种药物的调控机制，为科学研究提供前期的筛选方法。
本发明充分利用了多功能酶标仪（Flexstation 3）自动加药以及高通量检测的特点，避免因加样时间差而无法检测瞬时钙信号变化，可快速、稳定、成批次的大量检测人精子样本充分满足大样本量的数据检测；并且利用人精子在受精前需要发生的各个生理功能调控过程的相关生理因子，对响应各生理功能调控的钙信号检测，有效的反应出人精子各个功能调控过程生理指标的正常情况，为临床生殖中心等检测人精子是否正常作参考，高效筛选人精子样本。利用该方法还可为实验室科研中影响人精子[Ca2+]i调节的其他生理因子、蛋白或合成药物的筛选，进而验证其他生理因子、蛋白或合成各种药物的调控机制，为科学研究提供前期的筛选方法。钙荧光探针（Fluo-4 AM）稳定性高、灵敏度高，可得到稳定的精子[Ca2+]i数据情况。从图1可知正常人精子样本均反应出各生理刺激稳定的[Ca2+]i数据表现，提示该方法可简便、快捷、节省大量的时间、准确的监测人精子样本响应各生理刺激的[Ca2+]i情况。
附图说明
图1为对正常精子样本各种生理刺激下胞内钙信号的测量结果。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
本发明中所采用的HS 溶液配方为：135 mM NaCl，5 mM KCl，1 mM MgSO4，2 mMCaCl2，20 mM HEPES，5 mM 蔗糖，10 mM 乳酸，1 mM 丙酮酸钠，使用NaOH调节 pH至7.4。
实施例1
1. 人精液样本的处理
将当天上午收集到的精液样本37℃水浴液化 60 min。轻吹混匀，取10 µL精液，进行伟力计算机辅助精液分析仪（CASA）精子活力检测，记录人精子各项参数，对比《世界卫生组织人类精液检查与处理实验室手册》第5版(WHO5)精液标准参数（精子活力≥40%、前向运动≥32%、精子浓度≥15%），初筛精液常规参数正常的精子样本，结果如表1所示。
表1. 计算机辅助精液分析仪（CASA）分析精子样本各项功能参数
| 精子浓度 | 总活力 | 前向运动 | |
| 标准 | ≥ 15*10<sup>6</sup>/mL | ≥ 40% | ≥ 32% |
| 正常样本1 | 52.71*10<sup>6</sup>/mL | 55.94% | 43.71% |
2. 清洗纯化人精子
人精液样本利用HS清洗两遍，去除精浆。取1.5mL离心管，加入1 mL精液，在加入0.5 mL HS溶液(提前置37℃ 预热30 min备用)。轻混匀，2000 rpm离心5 min。弃去上清，再加入1.0 mL HS溶液重悬，2000 rpm离心5 min，弃去上清，重新加入适量温育后的HS溶液重悬并调整精子浓度为1×107个/mL以上。取10 µL置于精液分析板中，于计算机辅助精液分析仪（CASA）中检测精子样本的各项参数。清洗纯化后精子活力参数正常。
3. Fluo-4 AM精子孵育染色
在清洗纯化后的精子中，加入5 μM 钙荧光探针Fluo-4 AM 和0.05% Pluronic F-127 的进行重悬混匀孵育，避光置于37°C，5 % CO2培养箱染色30 min，1800 rpm 离心 6min，吸去上清液以去除精子细胞外残留的染料，加入 HS溶液重悬精子沉淀，2000 rpm 离心 6 min，去除上清，重复一次，避光备用既得染色后的精子溶液。
4. 人精子[Ca2+]i高通量测定
在孵育染色精子时，将多功能酶标仪（Flexstation 3 , Molecular Devices）提前开机预热 30 min。同时配制10×刺激物： pH7.4 HS溶液（对照）、250 mM NaHCO3、10 μM孕酮(Progesterone)、pH8.5 HS溶液（1M NaOH调节pH）、800 μM Bourgeonal、10 μM 前列腺素1+10 μM 前列素2(PEG1+PEG2)。
取180 μL孵育后的样本置于康宁黑色底板透明的96孔板中放置在仪器下层，添加200 μL 各刺激物至试剂透明96孔板，设置仪器的激发波长：502 nm，发散波长：525 nm，温度37°C，在第120 s加样，自动吸取20 μL刺激物，至刺激物浓度稀释10倍（加样体积为180+20 μL）开启程序读取数据10 min。确定各刺激因子对人精子[Ca2+]i的影响，进一步分析人精子样本对各生理过程响应是否正常。其中，加样前检测的120 s钙信号的平均荧光基底值为F0，检测的所有钙信号荧光值为F，计算精子样本对响应各刺激因子精子胞内钙信号[Ca2 +]i的影响情况。公式如下：钙信号荧光信号变化百分数（%）=ΔF/F0×100 %（F0, 刺激前平均荧光；ΔF，每个测量点荧光值减去F0）。
从图1可知正常人精子样本均反应出各生理刺激稳定的[Ca2+]i数据表现，说明该方法可简便、快捷、节省大量的时间、准确的监测人精子样本响应各生理刺激的[Ca2+]i情况。
本发明模拟生理环境下，对同一样本进行体外群体精子测钙，实现对不同生理因子刺激下的钙信号[Ca2+]i变化荧光信号检测，以反映同一精液样本对于各生理因子对[Ca2 +]i调节情况，通过选取精子在配子受精之前功能调控各个过程相应生理刺激，反应精子为准备受精过程中各个方面正常与否情况。
本发明一方面可为实验室科研对于影响人精子[Ca2+]i调节的其他生理因子、蛋白或合成药物的筛选，进而验证其他生理因子、蛋白或合成各种药物的调控机制；另一方面趋向于高通量临床精液样本各生理指标响应是否异常的判断，实现以建成临床生殖中心检验中监测精子是否因生理指标响应缺陷引起不育不孕的诊断方法，为男性不育的诊断与治疗提供新的方法和思考。
Claims (6)
1.一种高通量通过检测钙信号判断人精子质量的方法，其特征在于：包括以下步骤：
步骤1，人精液样本的处理：将收集到的精液样本水浴液化；
步骤2，清洗纯化人精子：将步骤1的精液样本用HS清洗，以去除精浆；
步骤3，Fluo-4 AM精子孵育染色：采用钙荧光探针Fluo-4 AM 和Pluronic F-127对纯化后的精子进行染色；
步骤4，向染色的精子中加入刺激物，然后采用多功能酶标仪检测加入刺激物前后精子胞内钙信号[Ca2+]i的荧光值，根据精子胞内钙信号[Ca2+]i的荧光值变化，确定各刺激因子对人精子钙信号[Ca2+]i的影响，分析人精子样本对各生理过程响应是否正常，从而判断人精子质量。
2.根据权利要求1所述的方法，其特征在于：步骤1中水浴液化的条件是37℃水浴液化60 min。
3.根据权利要求1所述的方法，其特征在于：步骤2中清洗具体是：向1 mL精液中加入0.5 mL 预热的HS溶液，混匀后，2000 rpm离心5 min，弃去上清，再加入1.0 mL HS溶液重悬，2000 rpm离心5 min，弃去上清，然后加入预热的HS溶液重悬并调整精子浓度为1×107个/mL以上。
4.根据权利要求1所述的方法，其特征在于：步骤3中染色具体是：向清洗纯化后的精子中加入5 μM 钙荧光探针Fluo-4 AM 和0.05% Pluronic F-127 的进行重悬混匀孵育，避光置于37℃、5 % CO2条件下染色30 min，1800 rpm 离心 6 min，吸去上清液以去除精子细胞外残留的染料，加入 HS溶液重悬精子沉淀，2000 rpm 离心 6 min，去除上清，重复一次，即得染色后的精子溶液。
5.根据权利要求1所述的方法，其特征在于：步骤4中所述刺激物为NaHCO3溶液、孕酮溶液、Bourgeonal溶液、前列腺素1-前列腺素2的混合溶液。
6.根据权利要求1所述的方法，其特征在于：步骤4中多功能酶标仪的检测条件为：激发波长502 nm，发散波长525 nm，温度37℃。
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| CN110873706A (zh) * | 2019-11-11 | 2020-03-10 | 南通大学 | 一种孕酮响应缺陷不育人精子样本的高通量筛选方法 |
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