捕食者应激模型的建立及其对小鼠卵母细胞发育能力影响的研究
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摘要
众所周知,生活中应激无处不在,不可避免。近年来,随着社会生活节奏的不断加快以及自然灾害(比如地震、火山等)的频繁发生,人们面临的压力(应激)越来越大,有研究表明身兼多种角色于一身的女性,更容易受到应激相关疾病的骚扰,其生殖健康方面,甚至会引发不可逆转的不孕不育。随着人们对应激和应激性疾病机制研究的深入,大量的动物应激模型应运而生,但是作为单纯的心理应激模型,各种现有的模型(比如电击足部、束缚应激等)都存在不同的缺陷,引发不少研究者的争议。此外,人们对心理应激对雌性配子发育能力的研究涉及的也较少。因此,建立理想的心理应激动物模型研究心理应激对雌性卵母细胞发育能力的影响势在必行。
本研究基于以往所建立的捕食者应激模型,排除了躯体应激等其它应激因素干扰的影响,添加了小鼠自由饮水和采食装置,建立了一种单纯的心理应激模型。实验主要集中于小鼠注射促性腺激素24h后,再应激24h的研究,为保证较好的应激持续性,采取了四次换猫制(即每隔6h换一次猫)。应激过程中,利用高架十字迷宫(elevated plus maze, EPM)立即测定小鼠捕食者应激15min和注射促性腺激素24h后再应激17h(即第三次换猫后5h)后,5min内在EMP中的焦虑样行为;并测定注射促性腺激素后应激不同时间的小鼠血清皮质醇水平。利用行为学和生理学两方面相结合来确定所建立模型的有效性。同时实验通过观察捕食者应激24h的小鼠卵母细胞体外成熟培养、孤雌激活、体外受精以及胚胎体内外发育情况,研究单纯心理应激对小鼠卵母细胞发育能力的影响。结果发现:
(1)捕食者(猫)应激能够使小鼠产生焦虑样行为,这种焦虑样行为在应激过程中能够较好的维持。
(2)捕食者应激能够引起小鼠非条件性的生理激素皮质醇分泌增加,应激1h后,皮质醇浓度接近对照组的4倍(93.32±9.75 vs 25.76±1.53 ng/ml);应激12h时,实验组与对照组差距进一步拉大,为对照组的4.5倍(70.16±9.10 vs16.57±3.03 ng/ml);此后随应激时间的延长,应激小鼠皮质醇水平呈下降趋势,到应激24h时,应激组皮质醇水平仍然明显高于对照组,但是应激24h后撤销应激再恢复1h时,应激组皮质醇水平反而显著低于对照组。
(3)捕食者心理应激对注射促性腺激素小鼠卵母细胞体外成熟以及孤雌激活/受精能力均没有影响,但是显著降低囊胚发育率并严重影响了囊胚质量。
(4)捕食者心理应激虽然没有能够影响胚胎体内发育质量,但是却显著降低了胚胎移植后受体小鼠的产仔数或胎仔重。
As everyone knows, stress is found everywhere in our life, inevitable. In recent years, with the development of the society, the accelerating pace of life and the happening of natural disaster, people face more and more pressure(stress). Previous studies showed that women in social life are often several, and who is also the role of all in one, not only work well, but also take care of family, so career women are more vulnerable than other people to stressors. In worst-case scenarios, even this can lead to infertility. As further research on stress and mechanism of stress disorder, more and more animal models(e.g. foot shock, restraint stress ) have emerged, but as an ideal psychological stress model, they all have some disfigurement in some degrees and cause many controversies. In addition, there are a few researchs on effect of psychological stress on female gametes development ability. Consequently, Establishing an ideal model to study the effects of psychological stress on female oocytes is imperative.
In this study, a predator stress (psychological stress) system that not only avoids the physical stress and other stress factors but also allows mice free intake of feed and water was first established. Experiment mainly focused on predator stress for 24h following gonadotropin administration. To ensure good stress continuity, took four times for every cat system (i.e. change one cat every 6h). Elevated plus maze( EPM) was used to measure mouse anxiety-like behavior immediately following treatment for 15min or 17h and the mouse plasma cortisol was also measured at different time periods during predator stress, to further demonstrate the validity of our animal model as an ideal psychological stress model. At the same time , the effect of predator stress for 24h following gonadotropin stimulation on oocyte maturation, activation/fertilization, and embryo development in vitro and in vivo were then observed(investigated). The results are summarized as follows:
(1) Anxiety-like behavior is produced by brief exposure of mouse (Kun Ming) to a cat, and it can be preferably maintained through all the stress process.
(2) Cat-exposed to mice showed a pronounce rise in cortisol levels, and that peaked at 12h from the beginning of the predator exposure, nearly reached 4.5 times (70.16±9.10 vs 16.57±3.03 ng/ml)to contrast. With the extension of time, the cortisol levels presented downtrend, and exposure to cat for 24h, the difference was also significant. But after recovery 1h following exposure to cat for 24h, cortisol level is significantly lower than the control
(3) Stimulated mice were exposed to cat for 24 h starting from 24 h after eCG injection, and the maturation, activation/fertilization were unaffected by acute predator stress ,but blastocyst development were significantly reduced.
(4) Stimulated mice were exposed to cat for 24 h starting from 24 h after eCG injection, and the embryo development in vivo was not affected, but reduced the litter size or the birth weight of young.
本研究基于以往所建立的捕食者应激模型,排除了躯体应激等其它应激因素干扰的影响,添加了小鼠自由饮水和采食装置,建立了一种单纯的心理应激模型。实验主要集中于小鼠注射促性腺激素24h后,再应激24h的研究,为保证较好的应激持续性,采取了四次换猫制(即每隔6h换一次猫)。应激过程中,利用高架十字迷宫(elevated plus maze, EPM)立即测定小鼠捕食者应激15min和注射促性腺激素24h后再应激17h(即第三次换猫后5h)后,5min内在EMP中的焦虑样行为;并测定注射促性腺激素后应激不同时间的小鼠血清皮质醇水平。利用行为学和生理学两方面相结合来确定所建立模型的有效性。同时实验通过观察捕食者应激24h的小鼠卵母细胞体外成熟培养、孤雌激活、体外受精以及胚胎体内外发育情况,研究单纯心理应激对小鼠卵母细胞发育能力的影响。结果发现:
(1)捕食者(猫)应激能够使小鼠产生焦虑样行为,这种焦虑样行为在应激过程中能够较好的维持。
(2)捕食者应激能够引起小鼠非条件性的生理激素皮质醇分泌增加,应激1h后,皮质醇浓度接近对照组的4倍(93.32±9.75 vs 25.76±1.53 ng/ml);应激12h时,实验组与对照组差距进一步拉大,为对照组的4.5倍(70.16±9.10 vs16.57±3.03 ng/ml);此后随应激时间的延长,应激小鼠皮质醇水平呈下降趋势,到应激24h时,应激组皮质醇水平仍然明显高于对照组,但是应激24h后撤销应激再恢复1h时,应激组皮质醇水平反而显著低于对照组。
(3)捕食者心理应激对注射促性腺激素小鼠卵母细胞体外成熟以及孤雌激活/受精能力均没有影响,但是显著降低囊胚发育率并严重影响了囊胚质量。
(4)捕食者心理应激虽然没有能够影响胚胎体内发育质量,但是却显著降低了胚胎移植后受体小鼠的产仔数或胎仔重。
As everyone knows, stress is found everywhere in our life, inevitable. In recent years, with the development of the society, the accelerating pace of life and the happening of natural disaster, people face more and more pressure(stress). Previous studies showed that women in social life are often several, and who is also the role of all in one, not only work well, but also take care of family, so career women are more vulnerable than other people to stressors. In worst-case scenarios, even this can lead to infertility. As further research on stress and mechanism of stress disorder, more and more animal models(e.g. foot shock, restraint stress ) have emerged, but as an ideal psychological stress model, they all have some disfigurement in some degrees and cause many controversies. In addition, there are a few researchs on effect of psychological stress on female gametes development ability. Consequently, Establishing an ideal model to study the effects of psychological stress on female oocytes is imperative.
In this study, a predator stress (psychological stress) system that not only avoids the physical stress and other stress factors but also allows mice free intake of feed and water was first established. Experiment mainly focused on predator stress for 24h following gonadotropin administration. To ensure good stress continuity, took four times for every cat system (i.e. change one cat every 6h). Elevated plus maze( EPM) was used to measure mouse anxiety-like behavior immediately following treatment for 15min or 17h and the mouse plasma cortisol was also measured at different time periods during predator stress, to further demonstrate the validity of our animal model as an ideal psychological stress model. At the same time , the effect of predator stress for 24h following gonadotropin stimulation on oocyte maturation, activation/fertilization, and embryo development in vitro and in vivo were then observed(investigated). The results are summarized as follows:
(1) Anxiety-like behavior is produced by brief exposure of mouse (Kun Ming) to a cat, and it can be preferably maintained through all the stress process.
(2) Cat-exposed to mice showed a pronounce rise in cortisol levels, and that peaked at 12h from the beginning of the predator exposure, nearly reached 4.5 times (70.16±9.10 vs 16.57±3.03 ng/ml)to contrast. With the extension of time, the cortisol levels presented downtrend, and exposure to cat for 24h, the difference was also significant. But after recovery 1h following exposure to cat for 24h, cortisol level is significantly lower than the control
(3) Stimulated mice were exposed to cat for 24 h starting from 24 h after eCG injection, and the maturation, activation/fertilization were unaffected by acute predator stress ,but blastocyst development were significantly reduced.
(4) Stimulated mice were exposed to cat for 24 h starting from 24 h after eCG injection, and the embryo development in vivo was not affected, but reduced the litter size or the birth weight of young.
引文
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