CN117143809B - 一种提高猪卵母细胞体外成熟质量的方法 - Google Patents
一种提高猪卵母细胞体外成熟质量的方法 Download PDFInfo
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Abstract
本发明涉及动物繁殖技术领域，具体为一种提高猪卵母细胞体外成熟质量的方法，在猪卵母细胞体外成熟培养液中添加PPARα激动剂，所述PPARα激动剂为非诺贝特酸，所述非诺贝特酸在猪卵母细胞体外成熟液中添加终浓度为50~300μM。本发明首次确定了PPARα激动剂可以作为脂代谢调节剂优化卵母细胞体外成熟体系，在猪卵母细胞体外成熟培养液中添加200μM非诺贝特酸，相比于已优化浓度的1μM吡格列酮和1μM GW501516，更能提高卵母细胞的成熟质量。
Description
技术领域
本发明涉及动物繁殖技术领域，具体涉及一种提高猪卵母细胞体外成熟质量的方法。
背景技术
猪作为重要的家畜，在异种器官移植和人类疾病模型构建中有潜在的应用价值。猪卵母细胞体外成熟是一项重要的家畜胚胎工程技术，指将卵泡内未成熟卵母细胞在体外适宜环境中培养，使之发育至第二次减数分裂中期。卵母细胞体外成熟是胚胎体外生产的关键步骤。由于猪卵母细胞体外成熟受众多因素影响，其质量仍远不如体内成熟卵母细胞，致使体外胚胎生产效率较低，限制了现代生物育种和生物医学的发展。因此，需要不断改善猪卵母细胞体外成熟培养体系，从而获得高质量的卵母细胞。
哺乳动物尤其是猪的卵母细胞内脂肪滴含量非常丰富，它们是由脂质形成的一种特殊细胞器，在储存能量、调节脂质动态平衡、膜结构组成及胞内信号传导方面具有重要作用。研究证实，脂肪酸氧化是卵母细胞成熟及早期胚胎发育必需的生物过程。卵母细胞在成熟过程中需要脂肪酸代谢供能，因而脂滴会发生明显下降，但仍然高于体内卵母细胞，说明体外环境下卵母细胞脂质代谢存在异常。因此，从调控脂肪酸代谢角度优化卵母细胞体外成熟体系是一条重要思路。现有研究已经有采用脂代谢调节剂优化卵母细胞体外成熟体系，例如，脂代谢调节剂GW501516、L-165041可以促进体外早期胚胎的发育能力；吡格列酮、罗格列酮能够提高卵母细胞的体外发育潜力，但是，在评价卵母细胞体外成熟质量的孤雌胚胎发育实验中，现有技术的脂代谢调节剂只能将囊胚率从53%左右提高到60%左右，然后就遭遇瓶颈，无法进一步提升囊胚率，说明现有的脂代谢调节剂对提高猪卵母细胞体外成熟质量的潜力已经用尽，想要获得进一步提高需要另辟蹊径。
现有技术文献主要包括：
1.牛体外受精胚胎培养过程中添加PPARδ激动剂GW501516，囊胚发育率提高了5.6个百分点（The PPARδ Agonist GW501516 Improves Lipolytic/Lipogenic Balance through CPT1 and PEPCK during the Development of Pre-Implantation Bovine Embryos, International Journal of Molecular Sciences 2019;20: 6066.）。
2.猪卵母细胞体外成熟过程中添加PPARγ激动剂吡格列酮，孤雌激活胚胎的囊胚发育率提高了4个百分点（《吡格列酮对猪卵母细胞体外成熟及孤雌胚胎发育的影响》, 湖北农业科学 2020;59: 93–96.）。
3. 猪卵母细胞体外成熟过程中添加PPARγ激动剂罗格列酮，其囊胚发育率提高了约6.5个百分点（
PPARγ is regulated by miR-27b-3p negatively and plays an important
role in porcine oocyte maturation
. Biochem Biophys Res Commun 2016;479: 224–30.）。
发明内容
为解决上述问题，本发明提出一种提高猪卵母细胞体外成熟质量的方法。目的在于有效提高体外培养猪卵母细胞的成熟质量，进而提高早期胚胎的发育潜力。
本发明提供的提高猪卵母细胞体外成熟质量的方法，在猪卵母细胞体外成熟培养液中添加PPARα激动剂。
进一步，所述PPARα激动剂为非诺贝特酸。
进一步，所述非诺贝特酸在猪卵母细胞体外成熟液中添加终浓度为50~300μM。
优选地，所述非诺贝特酸在猪卵母细胞体外成熟液中添加终浓度为200μM。
本发明的有益效果为：
1.首次确定了PPARα激动剂可以作为脂代谢调节剂优化卵母细胞体外成熟体系。现有技术中的GW501516、L-165041为PPARδ激动剂，吡格列酮、罗格列酮为PPARγ激动剂，而过氧化物酶体增殖物激活受体（PPARs）属于配体激活的II型核受体超家族成员，有PPARα、PPARγ和PPARδ三种主要亚型。现有技术常用的脂代谢调节剂属于PPARδ和PPARγ激动剂，有研究报道，体外成熟过程中添加1μM吡格列酮能够提高猪卵母细胞的早期胚胎发育，1μMGW501516对体外成熟牛卵母细胞也有相似效果，因而，它们可作为评价非诺贝特酸功效的参照组。而本发明首次采用PPARα激动剂作为脂代谢调节剂优化卵母细胞体外成熟体系，获得囊胚率提高到70%左右的技术效果，显著优于PPARδ和PPARγ激动剂。
2.本发明采用的PPARα激动剂是非诺贝特酸，能够增强脂肪酸分解代谢，降低低密度脂蛋白胆固醇和甘油三脂。目前，尚无在家畜胚胎工程领域应用非诺贝特酸的报道。因此，研究非诺贝特酸是否能够有效调控猪卵母细胞体外成熟过程中脂肪酸代谢，进而提高卵母细胞成熟质量及其早期胚胎体外发育潜力，具有重要意义。
3.本发明提供的猪卵母细胞体外成熟培养液，通过添加200μM非诺贝特酸，相比于已优化浓度的1μM吡格列酮和1μM GW501516，更能提高卵母细胞的成熟质量。孤雌激活胚胎体外培养试验发现，采用本发明所述技术方案获得的成熟卵母细胞，其胚胎第7天囊胚发育率指标超过常规方案15个百分点，超过吡格列酮或GW501516方案约8个百分点。本发明提供了非诺贝特酸的一种新用途，即用于进一步开发新型卵母细胞体外培养液，从而提高体外成熟卵母细胞质量，为体外受精、体细胞核移植等胚胎生物技术提供优质卵母细胞，
4.由本发明提供的体外成熟培养液获得的猪成熟卵母细胞，其胞内脂肪酸含量减少、氧化应激水平降低。采用代谢组和脂质组分析发现，差异代谢物有101个，差异脂类代谢物有82个。差异代谢物富集在卵母细胞减数分裂、类固醇激素生物合成、色氨酸代谢、苯丙氨酸代谢、硫代谢等通路；差异脂类代谢物富集在甘油磷脂代谢、α-亚麻酸代谢、花生四烯酸代谢、亚油酸代谢、鞘脂代谢等通路。这些结果进一步证实本发明提供的非诺贝特酸在猪卵母细胞体外成熟过程中能够调节脂代谢能力，弥补了卵母细胞体外成熟期间脂肪酸氧化能力较差的问题，从而有效地提高卵母细胞的体外成熟质量。
附图说明
图1为实施例4对照组和非诺贝特酸组猪成熟卵母细胞的脂肪酸荧光标记图片（图1中A）和相对荧光强度值（图1中B）；
图2为实施例5对照组和非诺贝特酸组猪成熟卵母细胞的活性氧和谷胱甘肽荧光标记图片（图2中A）和相对荧光强度值（图2中B、图2中C）；
图3为实施例6对照组和非诺贝特酸组猪成熟卵母细胞的差异代谢物火山图；
图4为实施例6对照组和非诺贝特酸组猪成熟卵母细胞中差异代谢物的KEGG通路富集气泡图；
图5为实施例6对照组和非诺贝特酸组猪成熟卵母细胞的差异脂类代谢物火山图；
图6为实施例6对照组和非诺贝特酸组猪成熟卵母细胞中差异脂类代谢物的KEGG通路富气泡集图。
具体实施方式
以下通过具体实施例进一步说明本发明的技术方案。
若未特别指明，实施例中所用技术手段为本领域技术人员所熟知的常规手段。
实施例1：非诺贝特酸提高猪卵母细胞体外成熟质量的方法：
（一）试剂来源和溶液配制
（1）TCM-199培养液为ThermoFisher Scientific公司产品，非诺贝特酸和吡格列酮购自Selleck公司，GW501516购自MedChemExpress公司，其他未说明的均为Sigma-Aldrich公司。
（2）TL-Hepes-PVA猪卵母细胞体外操作液：6.662g/L氯化钠、0.2386g/L氯化钾、0.168g/L碳酸氢钠、0.046g/L磷酸二氢钾、2.383g/L HEPES、0.022g/L丙酮酸钠、0.856mL乳酸钠、2.186g/L山梨醇、0.102g/L六水合氯化镁、0.294g/L二水合氯化钙、0.065g/L青霉素、0.05g/L链霉素、3g/L牛血清白蛋白，充分溶解于超纯水中，定容至1L，调节pH值为7.2~7.4，0.22μm滤器过滤分装，4℃冰箱储存备用。
（3）猪卵母细胞体外成熟培养液：
对照组（不含非诺贝特酸的体外成熟培养液）：90%体积分数TCM-199、10%体积分数猪卵泡液、0.1g/L丙酮酸钠、0.07g/L半胱氨酸、0.55g/L葡萄糖、10ng/mL表皮生长因子、0.5μg/mL促卵泡素、0.5μg/mL促黄体生成素。
试验组（含非诺贝特酸的体外成熟培养液）：90%体积分数TCM-199、10%体积分数猪卵泡液、0.1g/L丙酮酸钠、0.07g/L半胱氨酸、0.55g/L葡萄糖、10ng/mL表皮生长因子、0.5μg/mL促卵泡素、0.5μg/mL促黄体生成素和不同浓度（50、100、200、400μM）的非诺贝特酸。
（4）电激活液：0.3mol/L甘露醇、0.1mmol/L二水合氯化钙、0.1mmol/L七水合硫酸镁、0.1g/L聚乙烯醇，0.22μm滤器过滤分装，-20℃冰箱储存备用。
（5）PZM-3猪胚胎体外培养液：6.312g/L氯化钠、2.106g/L碳酸氢钠、0.746g/L氯化钾、0.048g/L磷酸二氢钾、0.022g/L丙酮酸钠、0.048g/L硫酸镁、0.616g/L L-乳酸半钙盐、0.292 g/L L-谷氨酰胺、0.05 g/L庆大霉素、0.546g/L亚牛磺酸钠、4g/L BSA、20mL非必需氨基酸溶液、40mL必需氨基酸溶液，定容至1L，调节pH值为7.2~7.4，渗透压为272~288mOsm，0.22μm滤器过滤分装，4℃冰箱储存备用。
（二）猪卵母细胞采集和体外成熟培养：从当地屠宰场收集猪卵巢，置于37℃含有青霉素和链霉素的生理盐水中，4h内运回实验室。卵巢清洗后，使用带有18号针头的20mL一次性注射器抽取卵巢表面3~8mm直径卵泡内卵泡液，然后放入50mL离心管中使卵丘-卵母细胞复合体（COCs）自然沉降。沉淀在TL-Hepes-PVA液中清洗2次后转入培养皿中，在体视显微镜下挑选卵母细胞胞质均匀、颗粒细胞包裹3层以上的COCs。
对照组：将不含非诺贝特酸的体外成熟培养液加入24孔板中，每个孔为500μＬ，覆盖石蜡油后在39℃、5% CO2和100%湿度的培养箱中预热4h以上。将挑出的COCs在体外成熟培养液中清洗3次，然后以50枚左右为一组放入24孔板每个中，置于39℃、5% CO2和100%湿度的培养箱中培养42~44h。
试验组：将含不同浓度非诺贝特酸的体外成熟培养液加入24孔板中，每个孔为500μＬ，覆盖石蜡油后在39℃、5% CO2和100%湿度的培养箱中预热4h以上。将挑出的COCs在含不同浓度非诺贝特酸的体外成熟培养液中清洗3次，然后以50枚左右为一组放入24孔板每个中，置于39℃、5% CO2和100%湿度的培养箱中培养42~44h。
（三）猪成熟卵母细胞收集：体外成熟培养完成后，取0.5mL离心管加入100μL的0.1%（w/v）透明质酸酶溶液，使用200μL移液器将COCs转移到离心管中，轻轻吹打50次左右以除去卵母细胞周围的卵丘颗粒细胞，然后用TL-Hepes-PVA液清洗3次，在体视显微镜下挑选胞质均匀且排出第一极体的卵母细胞并统计数量。以上方法在对照组和试验组中均适用。
（四）孤雌激活和胚胎体外培养：卵母细胞在电激活液中平衡30s，迅速转入覆盖电激活液的0.5 mm融合槽电极之间，用BLS CF-150/B细胞融合仪激活，条件为电场强度1.3kV/cm、直流脉冲时程80μs、脉冲1次。电激活后卵母细胞在辅助激活液（PZM-3液添加5μg/mL细胞松弛素B和10μg/mL放线菌酮）中培养4h，然后转入PZM-3液中进行胚胎体外培养，条件为39℃、5% CO2和100%湿度。第2天观察卵裂，第7天观察囊胚发育（孤雌激活当天为第0天）。
试验结果：
数据采用SPSS 20软件进行统计分析，Duncan多重比较判断差异显著性，结果以平均值±标准误表示。体外成熟液中不添加非诺贝特酸为对照组，添加不同浓度非诺贝特酸为试验组，分别观察各组所得成熟卵母细胞的孤雌胚胎发育情况。由表1可知，与对照组相比，添加100~300μM非诺贝特酸的体外成熟液能显著促进猪卵母细胞的早期胚胎发育能力，其中200μM非诺贝特酸的促进效果最佳，囊胚率为69.5%，效率提升超过15%。
表1 不同浓度非诺贝特酸组中卵母细胞的孤雌胚胎发育情况
注：同列数据肩标不同字母代表差异显著(P<0.05)，无字母表示差异不显著(P>0.05)。
实施例2：采用非诺贝特酸提高猪卵母细胞体外成熟质量的方法，同实施例1相比，区别在于非诺贝特酸组的用量为200μM。此外，吡格列酮组的用量为1μM，用于对比非诺贝特酸的作用效果。其他操作同实施例1。
试验结果：
由表2可知，与对照组相比，非诺贝特酸组和吡格列酮组的囊胚率显著提高；此外，非诺贝特酸组的囊胚率明显高于吡格列酮组，超过8%左右。上述结果说明最佳浓度非诺贝特酸对猪卵母细胞的成熟效果优于最佳浓度吡格列酮。
表2 非诺贝特酸和吡格列酮组中卵母细胞的孤雌胚胎发育情况
注：同列数据肩标不同字母代表差异显著(P<0.05)，无字母表示差异不显著(P>0.05)。
实施例3：采用非诺贝特酸提高猪卵母细胞体外成熟质量的方法，同实施例1相比，区别在于非诺贝特酸组的用量为200μM。此外，GW501516组的用量为1μM，用于进一步对比非诺贝特酸的作用效果。其他操作同实施例1。
试验结果：
由表3可知，与对照组相比，非诺贝特酸组和GW501516组的囊胚率显著提高；此外，非诺贝特酸组的囊胚率也明显超过GW501516组9.5%左右。上述结果说明最佳浓度非诺贝特酸对猪卵母细胞的成熟效果优于最佳浓度GW501516。
表3 非诺贝特酸和GW501516组中卵母细胞的孤雌胚胎发育情况
注：同列数据肩标不同字母代表差异显著(P<0.05)，无字母表示差异不显著(P>0.05)。
实施例4：根据实施例1所采用非诺贝特酸提高猪卵母细胞体外成熟质量的方法，获得成熟卵母细胞后检测胞内脂肪酸水平，以验证非诺贝特酸对脂肪酸代谢的影响。
卵母细胞内脂肪酸水平检测：猪卵母细胞在4%多聚甲醛液中固定1h，DPBS液清洗3次后在5μM BODIPY-FA荧光探针液中避光孵育30min，再用DPBS液清洗3次，然后置于激光扫描共聚焦显微镜下观察（Nikon，Tokyo，Japan），激发光为535nm。拍照采集TIFF格式图片，用ImageJ软件分析胞内荧光强度值，即代表脂肪酸水平。
试验结果：
对照组中卵母细胞的荧光强度值设定为1，非诺贝特酸组中卵母细胞的荧光强度值为与对照组的相对值。由图1可知，与对照组相比，非诺贝特酸组中成熟卵母细胞的脂肪酸水平显著下降（P＜0.05），表明非诺贝特酸有助于促进卵母细胞体外成熟过程中的脂肪酸代谢。
实施例5：根据实施例1所采用非诺贝特酸提高猪卵母细胞体外成熟质量的方法，获得成熟卵母细胞后检测胞内活性氧和谷胱甘肽水平，以验证非诺贝特酸对氧化应激的影响。
卵母细胞内活性氧和谷胱甘肽水平检测：猪卵母细胞于DPBS液清洗3次，转入10μMDCFH-DA荧光探针液中39 ℃条件下避光孵育30 min，再用DPBS清洗3次，然后置于激光扫描共聚焦显微镜下观察，激发光为488nm。拍照采集TIFF格式图片，用ImageJ软件分析胞内荧光强度值，即代表活性氧水平。谷胱甘肽采用ThiolTrackerTM Violet荧光探针标记，染色浓度为10μM，操作程序与活性氧检测相同，激发光为405nm。
试验结果：
由图2可知，与对照组相比，非诺贝特酸组成熟卵母细胞的活性氧水平显著降低（P＜0.05）、谷胱甘肽水平显著升高（P＜0.05），表明非诺贝特酸具有增强卵母细胞抗氧化能力的作用。
实施例6：为研究非诺贝特酸处理提高猪卵母细胞体外成熟质量的具体机制，进一步分析卵母细胞的代谢组和脂质组变化特征。
代谢组学测序：参照实施例1，收集不添加或添加200μM非诺贝特酸的成熟猪卵母细胞，作为对照组和非诺贝特酸样品放入离心管中-80℃超低温冰箱保存备用。实验启动后，在对照组和非诺贝特酸样品中加入50%甲醇后震动混匀，常温静置10min，提取液放-20°C过夜，沉淀样品中的蛋白质，4000g离心20min后转移上清液到96孔板。非靶向脂质代谢物提取需利用稀释液（异丙醇:乙腈:水=2:1:1，体积比）稀释脂质提取液。数据采集的液相体系为超高压液相，柱温设置为35℃，流速为0.4ml/min，流动相A为水（1%甲酸），流动相B为乙腈（0.1%甲酸）。液相梯度设置为：5%B，0~0.5min；5%~100%B，0.5~7min；100%B，7~8min；100%~5%B，8~8.1min；5%B，8.1~10min。采集所用的高分辨率质谱仪为TripleTOF 5600plus飞行时间质谱，每个样本进行一次正离子模式采集和一次负离子模式采集。采集数据的模式为信息依赖性采集模式，每间隔10个样本进行一次质控样品扫描。质谱数据用MSConvert软件转化为mzXML格式，导入XCMS软件进行峰提取并进行质控，使用CAMERA进行加和离子注释，接着用metaX软件鉴定代谢物。二级质谱信息与in-house标准数据库进行匹配，利用HMDB数据库进行注释。代谢物进行定量、筛选差异代谢物，使用MSEA、MetPA等数据库和软件进行生物信息学分析。
试验结果：
由图3可知，与对照组相比，非诺贝特酸组成熟卵母细胞中有101个差异代谢产物，其中显著上调的98个、显著下调的13个。这些差异代谢产物主要富集在卵母细胞减数分裂、类固醇激素生物合成、色氨酸代谢、苯丙氨酸代谢、硫代谢、孕酮介导卵母细胞成熟、皮质醇及醛固酮合成和分泌等通路（图4）。结果表明，非诺贝特酸能够调节卵母细胞的代谢水平，进而影响体外培育成熟效果。由图5可知，与对照组相比，非诺贝特酸组成熟卵母细胞中有82个差异脂质代谢产物，其中显著上调的60个、显著下调的22个。差异脂质代谢产物富集在甘油磷脂代谢、α-亚麻酸代谢、花生四烯酸代谢、亚油酸代谢、鞘脂代谢等通路（图6）。可见，本发明提供的非诺贝特酸也可改变卵母细胞的脂肪酸代谢能力，因而更有利于提高卵母细胞的成熟质量。
以上对本发明所提供的一种提高猪卵母细胞体外成熟质量的方法进行了详细介绍。本文通过具体实施方式对本发明的原理和实施方式进行了阐述，以上说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
Claims (2)
1.一种提高猪卵母细胞体外成熟质量的方法，其特征在于，在猪卵母细胞体外成熟培养液中添加PPARα激动剂，所述PPARα激动剂为非诺贝特酸，所述非诺贝特酸在猪卵母细胞体外成熟液中添加终浓度为50~300μM。
2.根据权利要求1所述提高猪卵母细胞体外成熟质量的方法，其特征在于，所述非诺贝特酸在猪卵母细胞体外成熟液中添加终浓度为200μM。
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