CN107312749B - 一种卵母细胞激活的物理方法及其受精潜能的评价指标 - Google Patents
一种卵母细胞激活的物理方法及其受精潜能的评价指标 Download PDFInfo
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Abstract
本发明涉及一种卵母细胞激活的物理方法及其受精潜能的评价指标,采用电脉冲序列激活卵母细胞,用共聚焦显微镜下检测到的胞浆内钙离子浓度变化所形成的钙振荡特征作为卵母细胞受精潜能的评价指标。
Description
技术领域:
本发明涉及胚胎工程领域,特别涉及辅助生殖领域中一种卵母细胞激活的物理方法,以及一种评价精子或人工辅助方法激活卵母细胞时其受精潜能的评价指标。
技术背景
如今,全球约有10%-16%的夫妇面临着不孕不育的问题。尽管辅助生殖技术的诞生已经帮助了许多不孕不育的患者,但是受精失败的现象依然普遍存在。在辅助生殖领域中,卵母细胞单精子显微注射技术(intracytoplasmic sperm injection,ICSI)的应用最为广泛,它借助显微镜操作系统,人工地将单一的精子注射到卵母细胞内使其受精,可以使受精率明显提高到70%。但是任然有相当一部分患者在进行ICSI后无法受精,研究发现由于精子自身存在缺陷而导致的卵母细胞激活不足是ICSI受精失败的主要原因。
所谓的卵母细胞激活,就是指在成熟促进因子(M-Phase promoting factor,MPF)保持稳定的作用下,哺乳动物成熟的卵母细胞会发育并停留在减数第二次分裂中期,既MII期,只有在精子或人工方式的刺激下,卵母细胞才能恢复并完成减数分裂,这个过程被称为卵母细胞的激活。
精子刺激卵母细胞后,会引起卵母细胞发生一系列形态上和生理上的反应,主要包括细胞内钙浓度短暂性的升高以及随后发生的钙振荡、第二极体释放、形成雌、雄双原核等等。其中,细胞内钙离子浓度的升高和钙振荡的形成,不仅被认为是诱导激活后一系列反应的必要因素,更是接下来胚胎发育的重要环节。并且钙振荡的频率、振幅、持续时间等,都会影响着细胞周期的进程以及早期胚胎发育过程中蛋白质的表达
为了解决ICSI受精失败的问题,现在通常会采用人工激活的方式来帮助精子刺激卵母细胞,从而诱导卵母细胞内Ca2+浓度脉冲式的升高或者Ca2+振荡来实现激活。其中,卵母细胞激活率、囊胚率和能否诱导钙离子振荡是人们主要关注的问题。目前人工激活卵母细胞的方法主要有:化学激活法、电学激活法、机械激活法,在化学法中最常用的激活试剂包括乙醇、钙离子载体A23187、离子霉素、锶离子等等。
这些方法能在一定程度上提高卵母细胞的激活效率,但其中化学法和机械法的操作复杂、重复性差、且激活效率和胚胎发育潜能还有待提高。而传统的电学激活方法能量过大,容易对细胞产生损伤。
发明内容
本发明的目的是提供一种操作简便、高效可控的卵母细胞激活方法,可提高卵母细胞的激活效率和胚胎发育潜能,以及提供一种评价精子或人工方式刺激卵母细胞后其受精潜能的评价指标,为辅助生殖中胚胎移植提供理论依据。
为实现以上技术效果,本发明采用以下的技术方案:
本发明是基于纳秒级脉宽的脉冲电场能诱导卵母细胞出现钙离子振荡的现象,用胞浆内钙离子浓度变化所形成的钙振荡的特征作为卵母细胞受精潜能的评价指标。
上述的卵母细胞激活方法,首先将卵母细胞及卵母细胞培养液HTF置于电极杯后施加电脉冲序列进行处理,将处理后的卵母细胞经培养液洗涤2-4次,移入36-37℃、5-10%CO2的培养箱中连续培养7-8天。
优选地,先用电场强度为35kV/cm-50kV/cm,脉冲频率为0.5Hz-1.5Hz,脉冲宽度为30ns-300ns的脉冲作用3-10秒,紧接着用电场强度为5kV/cm-35kV/cm,脉冲频率为0.5Hz-1.5Hz,脉冲宽度为10ns-30ns的脉冲作用3-5秒。
上述的卵母细胞受精潜能的评价指标,其特征在于:用卵母细胞胞浆内钙离子浓度变化所形成钙振荡的特征作为评价卵母细胞受精潜能的指标。
上述的卵母细胞受精潜能的评价指标,其特征在于:将经过前处理激活后的卵母细胞用钙离子探针染色,置于共聚焦显微镜下,检测胞浆内钙离子浓度随时间的变化。
上述的卵母细胞受精潜能度的评价指标,其特征在于:将钙离子浓度峰值30%处上升到峰值所需要的时间与从峰值下降到峰值30%处所需时间之比作为卵母细胞受精潜能的指标;钙振荡衰减因子如下,用于反映有效囊胚率:
其中:n为超过阈值的钙峰个数,阈值为最大钙离子浓度的70%,T为共聚焦显微镜的扫描时间;定义钙峰个数大于等于10时,有效囊胚率为100%。
虽然本发明已以较佳实施例披露如上,然而并非用以限定本发明。任何熟悉本领域的技术人员,在不脱离本发明技术方案范围情况下,都可利用上述揭示的方法和技术内容对本发明技术方案作出许多可能的变动和修饰,或修改为等同变化的等效实施例。因此,凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化及修饰,均仍属于本发明技术方案保护的范围内。
附图说明:
图1为兔卵母细胞激活的操作流程图
图2为电脉冲序列刺激后,小鼠卵母细胞内钙离子振荡图
图3为钙离子载体A23187刺激小鼠卵母细胞后引起的钙离子浓度变化图
图4为7%乙醇刺激小鼠卵母细胞后引起的钙离子浓度变化图
图5为正常精子刺激小鼠卵母细胞后引起的钙离子浓度变化图
具体实施方法
以下通过实施例对本发明进行进一步说明,当然本发明的实施范围并不仅限于下述的范围。
实施例一:激活兔卵母细胞
1.实验动物
6-15月龄,体重为2.5-3.5kg的性成熟期雌性日本大耳兔,购于北京维通利华实验动物技术有限公司。
2.实验试剂
孕马血清(PMSG Sigma)
人绒毛膜促性腺激素(hCG Sigma)
血浆替代品(SSS Irvine Scientific)
透明质酸酶(Sigma)
改良的人类输卵管液(mHTF Irvine Scientific)
人类输卵管液(HTF Irvine Scientific)
3.卵母细胞准备和培养
先用碘酒棉球消毒母兔颈部皮肤,于颈部皮下一次注射PMSG(150IU/只)。48h后,用酒精棉球消毒耳朵边缘,从耳静脉注射hCG(75IU/只)进行超促排卵。注射hCG 24h后,采用耳静脉注射10ml空气处死母兔。
将兔子背部朝下固定在手术台上,刮掉腹部中线兔毛并消毒后,用手术刀在腹部中线处皮肤切开2-3cm长的术口,打开腹腔后沿着子宫轻轻引出输卵管及卵巢。在卵巢附近找到输卵管伞,从伞部插入导卵管并固定,导卵管的另一端接培养皿。用10ml的注射器吸入含5%SSS的mHTF培养液作为冲卵液,注射器针头伸入输卵管后慢慢推动注射器,将冲卵液注入输卵管内,并由伞部的导卵管收集到培养皿中。
在显微镜下观察冲卵液中的卵母细胞,并采用80IU/ml透明质酸霉消化卵母细胞周围的颗粒细胞,将卵母细胞分散后再经含10%SSS的HTF培养液洗涤5次,最后移入HTF培养液中培养(37℃,饱和湿度,5%CO2),以待激活处理。
4.卵母细胞激活
将卵母细胞及适量的HTF培养液移入电极杯中,电极杯置于电脉冲序列发生器中。打开发生器的电源,此时脉冲电场会对电极杯内的卵母细胞施加电脉冲刺激。
在本实施例中,先用电场强度为50kV/cm,脉冲频率为1.5Hz,脉冲宽度为300ns的脉冲作用3秒,再用电场强度为30kV/cm,脉冲频率为1.0Hz,脉冲宽度为10ns的脉冲作用3秒。电极杯中两电极的间距为4mm。
将电场处理过的卵母细胞及HTF培养液从电极杯中吸出,转移至培养皿中,放置于培养箱中培养(37℃,饱和湿度,5%CO2)。15-24h后统计卵母细胞激活率,之后连续7-8天记录胚胎发育状况
在本实施例中,兔卵母细胞激活率可达到80%,囊胚率可达到37.5%。
实施例二:诱导小鼠卵母细胞内钙离子振荡
1.实验动物
6-8周龄的性成熟期雌性昆明小鼠,购于北京维通利华实验动物技术有限公司。
2.实验试剂
孕马血清(PMSG Sigma)
人绒毛膜促性腺激素(hCG Sigma)
血浆替代品(SSS Irvine Scientific)
透明质酸酶(Sigma)
改良的人类输卵管液(mHTF Irvine Scientific)
人类输卵管液(HTF Irvine Scientific)
Fluo-4AM/钙离子荧光探针(Dojindo Laboratories)
3.卵母细胞准备和培养
对雌性小鼠腹腔注射PMSG(10IU/只小鼠),48h后腹腔注射hCG(10IU/只)进行超促排卵。注射hCG15-19h后,采用颈椎脱臼法处死小鼠,在无菌条件下暴露、取出输卵管。在含5%SSS的mHTF培养液中划破输卵管壶腹部膨大部位,将卵丘-卵母细胞复合体轻轻挤出。采用80IU/ml透明质酸霉消化卵母细胞周围的颗粒细胞,将卵母细胞分散后再经含10%SSS的HTF培养液洗涤5次,移入HTF培养液中。再按1:1的比例加入Fluo4-AM钙离子探针后,培养15分钟(37℃,饱和湿度,5%CO2),以待激活处理
4.钙离子振荡检测
将卵母细胞及适量的HTF与Fluo4-AM混合液(1:1)移入电极杯中,电极杯置于电脉冲序列发生器中。打开发生器的电源,此时脉冲电场会对电极杯内的卵母细胞施加电脉冲刺激。
在本实施例中,先用电场强度为40kV/cm,脉冲频率为1.0Hz,脉冲宽度为80ns的脉冲作用10秒,再用电场强度为15kV/cm,脉冲频率为1.0Hz,脉冲宽度为10ns的脉冲作用5秒。电极杯中两电极的间距为4mm。
将电场处理过的卵母细胞及与Fluo4-AM混合液从电极杯中吸出,转移至共聚焦培养皿中,放置于共聚焦显微镜(Nikon A1)下连续扫描2h,检测钙离子浓度在细胞内的变化。
实施例三:评价A23187激活小鼠卵母细胞的受精潜能
1.实验动物
6-8周龄的性成熟期雌性昆明小鼠,购于北京维通利华实验动物技术有限公司。
2.实验试剂
孕马血清(PMSG Sigma)
人绒毛膜促性腺激素(hCG Sigma)
血浆替代品(SSS Irvine Scientific)
透明质酸酶(Sigma)
改良的人类输卵管液(mHTF Irvine Scientific)
人类输卵管液(HTF Irvine Scientific)
Fluo-4AM/钙离子荧光探针(Dojindo Laboratories)
钙离子载体A23187(A23187Sigma)
3.卵母细胞准备
对雌性小鼠腹腔注射PMSG(10IU/只小鼠),48h后腹腔注射hCG(10IU/只)进行超促排卵。注射hCG15-19h后,采用颈椎脱臼法处死小鼠,在无菌条件下暴露、取出输卵管。在含5%SSS的mHTF培养液中划破输卵管壶腹部膨大部位,将卵丘-卵母细胞复合体轻轻挤出。采用80IU/ml透明质酸霉消化卵母细胞周围的颗粒细胞,将卵母细胞分散后再经含10%SSS的HTF培养液洗涤5次,移入HTF培养液中。
4.卵母细胞激活
将卵母细胞置于含10μM A23187的mHTF液中,37℃避光处理5min,经mHTF洗涤5次后,再置于含10%SSS的HTF液中继续培养4h。
5.检测钙离子浓度变化
按1:1的比例在含有卵母细胞的10%SSS的HTF液中加入Fluo4-AM钙离子探针,培养15分钟(37℃,饱和湿度,5%CO2)。之后将卵母细胞转移至共聚焦培养皿中,放置于共聚焦显微镜(Nikon A1)下连续扫描2h,检测钙离子浓度在细胞内的变化。
当钙离子浓度峰值30%处上升到峰值所需要的时间与从峰值下降到峰值30%处所需时间之比作大于0.50时,认为激活效果良好,且上升时间与下降时间之比越接近于1则激活效果越好。
在本实施例中,上升时间与下降时间之比约为0.54,有效囊胚率为0%。因此A23187激活卵母细胞效果良好,但激活后的卵母细胞很大程度上无法发育成囊胚。
实施例四:评价乙醇激活小鼠卵母细胞的受精潜能
1.实验动物
6-8周龄的性成熟期雌性昆明小鼠,购于北京维通利华实验动物技术有限公司。
2.实验试剂
孕马血清(PMSG Sigma)
人绒毛膜促性腺激素(hCG Sigma)
血浆替代品(SSS Irvine Scientific)
透明质酸酶(Sigma)
改良的人类输卵管液(mHTF Irvine Scientific)
人类输卵管液(HTF Irvine Scientific)
Fluo-4AM/钙离子荧光探针(Dojindo Laboratories)
7%乙醇(用HTF培养液将无水乙醇稀释到7%)
3.卵母细胞准备
对雌性小鼠腹腔注射PMSG(10IU/只小鼠),48h后腹腔注射hCG(10IU/只)进行超促排卵。注射hCG15-19h后,采用颈椎脱臼法处死小鼠,在无菌条件下暴露、取出输卵管。在含5%SSS的mHTF培养液中划破输卵管壶腹部膨大部位,将卵丘-卵母细胞复合体轻轻挤出。采用80IU/ml透明质酸霉消化卵母细胞周围的颗粒细胞,将卵母细胞分散后再经含10%SSS的HTF培养液洗涤5次,移入HTF培养液中。
4.卵母细胞激活
将卵母细胞置于含7%乙醇的mHTF液,37℃处理5min,经mHTF洗涤5次后,再置于含10%SSS的HTF液中继续培养4h
5.检测钙离子浓度变化
按1:1的比例在含有卵母细胞的10%SSS的HTF液中加入Fluo4-AM钙离子探针,培养15分钟(37℃,饱和湿度,5%CO2)。之后将卵母细胞转移至共聚焦培养皿中,放置于共聚焦显微镜(Nikon A1)下连续扫描2h,检测钙离子浓度在细胞内的变化。
当钙离子浓度峰值30%处上升到峰值所需要的时间与从峰值下降到峰值30%处所需时间之比作大于0.50时,认为激活效果良好,且上升时间与下降时间之比越接近于1则激活效果越好。
在本实施例中,上升时间与下降时间之比约为0.11,有效囊胚率为0%。因此7%乙醇虽然可以激活卵母细胞,但激活效果不好,且卵母细胞激活后无法发育成囊胚。
实施例五:评价正常精子激活小鼠卵母细胞的受精潜能
1.实验动物
6-8周龄的性成熟期雌性与雄性昆明小鼠,购于北京维通利华实验动物技术有限公司。
2.实验试剂
孕马血清(PMSG Sigma)
人绒毛膜促性腺激素(hCG Sigma)
血浆替代品(SSS Irvine Scientific)
透明质酸酶(Sigma)
改良的人类输卵管液(mHTF Irvine Scientific)
人类输卵管液(HTF Irvine Scientific)
Fluo-4AM/钙离子荧光探针(Dojindo Laboratories)
3.卵母细胞准备
对雌性小鼠腹腔注射PMSG(10IU/只小鼠),48h后腹腔注射hCG(10IU/只)进行超促排卵。注射hCG15-19h后,采用颈椎脱臼法处死小鼠,在无菌条件下暴露、取出输卵管。在含5%SSS的mHTF培养液中划破输卵管壶腹部膨大部位,将卵丘-卵母细胞复合体轻轻挤出。采用80IU/ml透明质酸霉消化卵母细胞周围的颗粒细胞,将卵母细胞分散后再经含10%SSS的HTF培养液洗涤5次,移入HTF培养液中。
4.精子准备
采用颈椎脱臼法处死雄性小鼠,在无菌条件下暴露、取出输精管。在含5%SSS的mHTF培养液中划破输精管,将精子轻轻挤出。经含10%SSS的HTF培养液洗涤5次,移入含有卵母细胞的HTF培养液中5.检测钙离子浓度变化
按1:1的比例在含有卵母细胞和精子的10%SSS的HTF液中加入Fluo4-AM钙离子探针,培养15分钟(37℃,饱和湿度,5%CO2)。之后将卵母细胞转移至共聚焦培养皿中,放置于共聚焦显微镜(Nikon A1)下连续扫描2h,检测钙离子浓度在细胞内的变化。
当钙离子浓度峰值30%处上升到峰值所需要的时间与从峰值下降到峰值30%处所需时间之比作大于0.50时,认为激活效果良好,且上升时间与下降时间之比越接近于1则激活效果越好。
在本实施例中,上升时间与下降时间之比约为0.92,有效囊胚率为66.7%。因此认为正常精子激活卵母细胞的效果很好,且卵母细胞激活后很大程度上可以发育成囊胚。
Claims (2)
1.一种卵母细胞激活的物理方法,其特征在于:将卵母细胞及卵母细胞培养液置于电极杯后施加电脉冲序列刺激卵母细胞;将电脉冲激活后的卵母细胞经培养液洗涤2-4次,移入36-37 ℃ 、5-10%CO2 的培养箱中连续培养7-8天;所述的电脉冲序列进行处理的具体步骤是先用电场强度为35kV/cm-50kV/cm,脉冲频率为0.5Hz-1.5Hz,脉冲宽度为30ns-300ns的脉冲作用3-10秒,再用电场强度为5kV/cm-35kV/cm,脉冲频率为0.5Hz-1.5Hz,脉冲宽度为10ns-30ns的脉冲作用3-5秒,其中卵母细胞为小鼠和大耳兔的卵母细胞。
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