小鼠休眠胚胎的冷冻研究
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摘要
本研究对小鼠休眠胚胎与正常孵化期胚胎进行冷冻试验,目的是检验小鼠休眠胚胎抗冻能力及经过冻融后的体内外发育潜力,并观察其特殊的细胞结构和细胞器分布,为胚胎休眠技术应用于实际生产奠定基础。
本研究内容分三部分。第一部分研究目的是提高小鼠休眠胚胎的制备效率,尝试用超数排卵方法来获取小鼠休眠胚胎。结果表明:见栓后注射抗PMSG组的平均出胚数(9.4枚/只)最高,显著高于其他时间注射抗PMSG实验组,常规超排处理结合注射抗PMSG血清法能有效提高小鼠休眠胚胎的回收率。
试验第二部分研究目的是检验休眠胚胎冷冻后质量变化以及冻融后体内外的发育潜力。运用常规冷冻方法对正常孵化期胚胎和休眠胚胎以及冷冻效果较好的囊胚期胚胎进行冷冻,并在解冻后分别进行体外复苏培养试验和胚胎移植试验,验证胚胎经过冻融后的体内外发育潜力,同时运用双重荧光染色的方法,进行胚胎细胞计数,观察经过冷冻过程胚胎内细胞团细胞数和滋养层细胞数的变化。利用透射电子显微镜,观察小鼠休眠胚胎与正常孵化期胚胎在细胞连接和各细胞器形态与分布上的差异,以及在冻融培养后的变化。结果表明:囊胚期胚胎的冷冻解冻回收率(80.8%)极显著高于其他两组。休眠胚胎的冷冻解冻回收率(72.1%)极显著高于孵化期胚胎(50.2%)、休眠胚胎(94.2%)和囊胚期胚胎的发育率(90.9%)差异不显著,但是都极显著(p<0.01)高于孵化期胚胎(42.1%)。囊胚期胚胎的移植妊娠率(49.7%)显著高于休眠胚胎(40.8%)。休眠胚胎的移植妊娠率(40.8%)显著高于孵化期胚胎(30.1%)。休眠胚胎的内细胞团细胞数(27.83)显著高于孵化期胚胎(19.53),两种胚胎的滋养层细胞数无差异。冻融,培养后休眠胚胎的内细胞团数(25.18)显著高于孵化期胚胎(14.68),滋养层细胞数(114.09)也显著高于孵化期胚胎(73.88)。冻融,培养前后休眠胚胎内细胞团数和滋养层细胞数差异均不显著,培养后孵化期胚胎的内细胞团和滋养层细胞数都显著低于冷冻前的。利用透射电子显微镜观察各组胚胎超微结构。结果显示:两种胚胎在细胞连接,细胞核的形状,脂滴的数量与体积,内质网的形态与数量等方面都有明显差异,冻融培养后观察发现,休眠胚胎在细胞连接,细胞器形态等方面与冻融前变化明显,其在培养过程中有一个快速发育恢复的过程。
试验的第三部分目的是揭示休眠胚胎冷冻效果较好的分子机理。通过RT PCR对小鼠孵化囊胚和休眠胚中的Cx43、Cx31的转录水平进行了检测,意在检验休眠胚与孵化胚缝隙连接的差异。实验收集两种类型胚胎各100枚,提取总RNA,对其进行反转录然后PCR,测定其相对于b-actin的表达量。结果显示:孵化囊胚中Cx43、Cx31的相对值低于休眠胚,但差异不显著。表明休眠胚具有和孵化胚相同的Cx43、Cx31转录水平。
本实验结论:1.常规超排处理结合注射抗PMSG血清能有效提高小鼠休眠胚胎的回收率。2.小鼠休眠胚胎冻融后体内外发育潜力均优于小鼠正常孵化期胚胎。3.小鼠休眠胚胎在滋养层细胞连接、细胞核中异染色质分布、胚胎细胞中脂滴和核糖体分布上都与小鼠正常孵化期胚胎有较大差异。4.休眠胚具有与正常孵化囊胚相同的Cx31、Cx43转录水平。
In order to investigate the potential endurance of delayed mouse embryos to resist the freezing and thawing treatment, the delayed and normal hatched murine blastocysts were frozen by cryopreservation and rapid thawing process. In addition, the special cellular structure and disposition of those dormant embryos, were also revealed as well by transmission electron microscope observation.
There are total three experiments in it. Experiment I: In order to improve the collecting efficiency of mouse dormant embryos, the normal superovulation combined with anti-PMSG serum (A-PMSG) was applied to it. According to the different doses of A-PMSG injected into the mouse after injection of PMSG, the disposal animals were divided into five groups: 1) injecting A-PMSG before mating, 2) injecting A-PMSG after finding a vaginal plug, 3) injecting A-PMSG after ovariectomy in the morning of day 4 (day 1= vaginalplug), 4) superovulating without A-PMSG, and 5) the control one. It was shown that the average recovery number of delayed embryos in the group injected with the A-PMSG after finding a vaginal plug, is significantly higher than the others (P<0.01).
Experiment II: To test the developmental potentiality of mouse dormant embryos after freezing-thawing treatment, the delayed embryos, blastocysts and normal hatched blastocysts, were frozen and thawed separately, then, they were cultured in vitro and transferred into suitable recipients after thawing. Meanwhile, the cell number of inner cell mass (ICM) and trophectoderm of both dormant and activated embryos, were counted by double label fluorescent staining. Then the specific cellular structure and disposition of cell organelle of those delayed embryos was revealed by transmission electron microscope. It was shown that the freeze-thawing recovery rate of hatched blastocyst (80.8%) was significantly higher than the others’(p<0.01). The freeze-thawing recovery rate of dormant embryos(72.1%)was extremely significantly higher than normal blastocysts(50.2%). There was no significantly difference on developmental rate between dormant ones(94.2%)and normal blastocysts (90.9%), but they were all extremely significantly higher than that of the hatched blastocyst group (42.1%). The pregnancy rate in group of blastocysts (49.7%), was significantly higher than that in the dormant one (40.8%). The average cell number of ICM from the delayed embryos was 27.83, which was significantly higher than that in hatched ones (19.53). The cell number of trophectcderm kept stablely among all the groups. After thawing and in vitro culture, the cell number of ICM for delayed embryos was 25.18, which was significantly higher than that for hatched ones (14.68). Their average cell number of trophectcderm (114.9) was also significantly higher than that of hatched ones (73.88) The ultrastructure of embryos in different groups, was also observed through transmission electron microscope respectively. The result implied that there were some significant differences of the shape of nucleus, number of lipid droplet and the morphogenesis of endoplasmic reticulum , especially of the significant change of cell-cell connection and cell organelle compared with the pre-freezing one. It indicated that the delayed embryos might restore their developmental potentiality during in vitro culture.
Experiment III: To investigate the potential transcriptional level of cx31 and cx43 between the dormant embryos and mormal hatched blastocyst, the RT- PCR method was applied to evaluate the semi-quantity of those two genes. The results showed that there is no significant difference of mRNA level of cx43 and cx31 between the dormant embryos and the hatched blastocysts.
In general, it can be concluded as following: 1) The collecting efficiency of the dormant mouse embryos can be improved by superovulation with anti-PMSG serum. 2) The different morphological change and the disposition of cell organelle in delayed mouse embryos from that in normal ones, might sustain the dormant embryos to defeat the freezing shock . 3) . The dormant mouse embryos might initiate another way to play a better role on anti-freezing shock than those hatched blastocysts , but not depending on the signal of cx43 and cx31.
本研究内容分三部分。第一部分研究目的是提高小鼠休眠胚胎的制备效率,尝试用超数排卵方法来获取小鼠休眠胚胎。结果表明:见栓后注射抗PMSG组的平均出胚数(9.4枚/只)最高,显著高于其他时间注射抗PMSG实验组,常规超排处理结合注射抗PMSG血清法能有效提高小鼠休眠胚胎的回收率。
试验第二部分研究目的是检验休眠胚胎冷冻后质量变化以及冻融后体内外的发育潜力。运用常规冷冻方法对正常孵化期胚胎和休眠胚胎以及冷冻效果较好的囊胚期胚胎进行冷冻,并在解冻后分别进行体外复苏培养试验和胚胎移植试验,验证胚胎经过冻融后的体内外发育潜力,同时运用双重荧光染色的方法,进行胚胎细胞计数,观察经过冷冻过程胚胎内细胞团细胞数和滋养层细胞数的变化。利用透射电子显微镜,观察小鼠休眠胚胎与正常孵化期胚胎在细胞连接和各细胞器形态与分布上的差异,以及在冻融培养后的变化。结果表明:囊胚期胚胎的冷冻解冻回收率(80.8%)极显著高于其他两组。休眠胚胎的冷冻解冻回收率(72.1%)极显著高于孵化期胚胎(50.2%)、休眠胚胎(94.2%)和囊胚期胚胎的发育率(90.9%)差异不显著,但是都极显著(p<0.01)高于孵化期胚胎(42.1%)。囊胚期胚胎的移植妊娠率(49.7%)显著高于休眠胚胎(40.8%)。休眠胚胎的移植妊娠率(40.8%)显著高于孵化期胚胎(30.1%)。休眠胚胎的内细胞团细胞数(27.83)显著高于孵化期胚胎(19.53),两种胚胎的滋养层细胞数无差异。冻融,培养后休眠胚胎的内细胞团数(25.18)显著高于孵化期胚胎(14.68),滋养层细胞数(114.09)也显著高于孵化期胚胎(73.88)。冻融,培养前后休眠胚胎内细胞团数和滋养层细胞数差异均不显著,培养后孵化期胚胎的内细胞团和滋养层细胞数都显著低于冷冻前的。利用透射电子显微镜观察各组胚胎超微结构。结果显示:两种胚胎在细胞连接,细胞核的形状,脂滴的数量与体积,内质网的形态与数量等方面都有明显差异,冻融培养后观察发现,休眠胚胎在细胞连接,细胞器形态等方面与冻融前变化明显,其在培养过程中有一个快速发育恢复的过程。
试验的第三部分目的是揭示休眠胚胎冷冻效果较好的分子机理。通过RT PCR对小鼠孵化囊胚和休眠胚中的Cx43、Cx31的转录水平进行了检测,意在检验休眠胚与孵化胚缝隙连接的差异。实验收集两种类型胚胎各100枚,提取总RNA,对其进行反转录然后PCR,测定其相对于b-actin的表达量。结果显示:孵化囊胚中Cx43、Cx31的相对值低于休眠胚,但差异不显著。表明休眠胚具有和孵化胚相同的Cx43、Cx31转录水平。
本实验结论:1.常规超排处理结合注射抗PMSG血清能有效提高小鼠休眠胚胎的回收率。2.小鼠休眠胚胎冻融后体内外发育潜力均优于小鼠正常孵化期胚胎。3.小鼠休眠胚胎在滋养层细胞连接、细胞核中异染色质分布、胚胎细胞中脂滴和核糖体分布上都与小鼠正常孵化期胚胎有较大差异。4.休眠胚具有与正常孵化囊胚相同的Cx31、Cx43转录水平。
In order to investigate the potential endurance of delayed mouse embryos to resist the freezing and thawing treatment, the delayed and normal hatched murine blastocysts were frozen by cryopreservation and rapid thawing process. In addition, the special cellular structure and disposition of those dormant embryos, were also revealed as well by transmission electron microscope observation.
There are total three experiments in it. Experiment I: In order to improve the collecting efficiency of mouse dormant embryos, the normal superovulation combined with anti-PMSG serum (A-PMSG) was applied to it. According to the different doses of A-PMSG injected into the mouse after injection of PMSG, the disposal animals were divided into five groups: 1) injecting A-PMSG before mating, 2) injecting A-PMSG after finding a vaginal plug, 3) injecting A-PMSG after ovariectomy in the morning of day 4 (day 1= vaginalplug), 4) superovulating without A-PMSG, and 5) the control one. It was shown that the average recovery number of delayed embryos in the group injected with the A-PMSG after finding a vaginal plug, is significantly higher than the others (P<0.01).
Experiment II: To test the developmental potentiality of mouse dormant embryos after freezing-thawing treatment, the delayed embryos, blastocysts and normal hatched blastocysts, were frozen and thawed separately, then, they were cultured in vitro and transferred into suitable recipients after thawing. Meanwhile, the cell number of inner cell mass (ICM) and trophectoderm of both dormant and activated embryos, were counted by double label fluorescent staining. Then the specific cellular structure and disposition of cell organelle of those delayed embryos was revealed by transmission electron microscope. It was shown that the freeze-thawing recovery rate of hatched blastocyst (80.8%) was significantly higher than the others’(p<0.01). The freeze-thawing recovery rate of dormant embryos(72.1%)was extremely significantly higher than normal blastocysts(50.2%). There was no significantly difference on developmental rate between dormant ones(94.2%)and normal blastocysts (90.9%), but they were all extremely significantly higher than that of the hatched blastocyst group (42.1%). The pregnancy rate in group of blastocysts (49.7%), was significantly higher than that in the dormant one (40.8%). The average cell number of ICM from the delayed embryos was 27.83, which was significantly higher than that in hatched ones (19.53). The cell number of trophectcderm kept stablely among all the groups. After thawing and in vitro culture, the cell number of ICM for delayed embryos was 25.18, which was significantly higher than that for hatched ones (14.68). Their average cell number of trophectcderm (114.9) was also significantly higher than that of hatched ones (73.88) The ultrastructure of embryos in different groups, was also observed through transmission electron microscope respectively. The result implied that there were some significant differences of the shape of nucleus, number of lipid droplet and the morphogenesis of endoplasmic reticulum , especially of the significant change of cell-cell connection and cell organelle compared with the pre-freezing one. It indicated that the delayed embryos might restore their developmental potentiality during in vitro culture.
Experiment III: To investigate the potential transcriptional level of cx31 and cx43 between the dormant embryos and mormal hatched blastocyst, the RT- PCR method was applied to evaluate the semi-quantity of those two genes. The results showed that there is no significant difference of mRNA level of cx43 and cx31 between the dormant embryos and the hatched blastocysts.
In general, it can be concluded as following: 1) The collecting efficiency of the dormant mouse embryos can be improved by superovulation with anti-PMSG serum. 2) The different morphological change and the disposition of cell organelle in delayed mouse embryos from that in normal ones, might sustain the dormant embryos to defeat the freezing shock . 3) . The dormant mouse embryos might initiate another way to play a better role on anti-freezing shock than those hatched blastocysts , but not depending on the signal of cx43 and cx31.
引文
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