山羊卵母细胞玻璃化冷冻保存液配方筛选研究
摘要
卵母细胞作为胚胎工程、核移植、转基因等技术研究的试验材料被广泛应用。卵母细胞冷冻保存不但可以使卵母细胞的供给不受时间和空间的限制，而且对保护品种资源、促进体外胚胎生产技术的商业化具有重要意义。为获得保存山羊体外成熟卵母细胞的优化玻璃化液配方，本试验开展了玻璃化液配方筛选研究。玻璃化液配方1、2、3、4、5和6所含渗透性抗冻保护剂浓度分别为30％EG、27.5％EG+2.5％DMSO、25％EG+5％DMSO、20％EG+10％DMSO、15％EG+15％DMSO和40％EG。将体外成熟的山羊卵母细胞分为3组：(1)对照组，卵母细胞不进行处理；(2)毒性组，卵母细胞在玻璃化液中进行暴露但不投入液氮中冷冻；(3)冷冻组，卵母细胞利用OPS法进行玻璃化冷冻。毒性组和冷冻组卵母细胞在浓度递减的蔗糖溶液中脱除抗冻保护剂。各处理对卵母细胞存活率、孤雌激活率、体外受精能力及胚胎发育能力的影响结果如下：
不同配方的玻璃化液对卵母细胞存活率影响的研究结果：卵母细胞经玻璃化液处理后，玻璃化液1-6组卵母细胞的形态正常率(97.1％、97.0％、97.0％、96.3％、98.1％、96.7％)与对照组无显著性差异(P＞0.05)；但在发育液培养15-17h后，玻璃化液6(EFS40)组的卵母细胞死亡率达54.1％，与对照组(0.0％)差异显著(P＜0.05)，玻璃化液1-5组卵母细胞与对照组无显著性差异(P＞0.05)。根据上述研究结果，对玻璃化液6予以淘汰。
不同配方的玻璃化液对卵母细胞孤雌激活作用的研究结果：玻璃化液4、5对卵母细胞的孤雌激活率分别为7.5％和9.0％，与对照组(0.0％)差异显著(P＜0.05)；玻璃化液1、2、3组的孤雌激活率分别为1.0％、2.7％、3.5％，与对照组无显著性差异(P＞0.05)。因此，对玻璃化液4、5予以淘汰。
玻璃化液1、2、3冷冻保存对卵母细胞受精能力的影响研究结果：卵母细胞在玻璃化液2中暴露后的正常受精率(31.8％)与对照组(51.2％)无显著性差异(P＞0.05)，在玻璃化液1、3中暴露的正常受精率(29.2％、29.3％)均较对照组(51.2％)显著降低(P＜0.05)；玻璃化液2冷冻保存卵母细胞的正常受精率(28.0％)较对照组(51.2％)显著降低(P＜0.05)，而玻璃化液1、3冷冻保存卵母细胞的正常受精率(22.73％、22.45％)则较对照组极显著降低(P＜0.01)。以24h卵裂数／48h卵裂数为卵裂速度参数，玻璃化液1、2的毒性组卵裂速度(51.9％和50.0％)与对照组(77.1％)之间差异显著(P＜0.05)；玻璃化液1、2、3毒性组和冷冻组卵母细胞的酶溶解透明带时间(424.0s～434.9s)较对照组(380.1s)显著增加(P＜0.05)。
玻璃化液1、2、3对卵母细胞胚胎发育能力的影响研究的结果：在体外受精胚胎的发育能力方面，玻璃化液1、2、3的毒性组桑椹胚率(37.0％、50.0％、37.5％)与对照组的桑椹胚率(58.3％)无显著性差异(P＞0.05)，玻璃化液1、2、3的各冷冻组桑椹胚率(25.0％、33.3％、32.1％)则显著低于对照组(P＜0.05)；体外受精胚胎囊胚发育率与对照组(27.1％)比较，玻璃化液2、3的毒性组(9.4％、9.4％)无显著性差异(P＞0.05)，其它组囊胚发育率显著降低(P＜0.05)。在孤雌激活胚胎发育能力方面，玻璃化液2的毒性组卵裂率和桑椹胚发育率(69.2％和53.3％)与对照组(80.0％、54.1％)无显著性差异(P＞0.05)，但冷冻组的卵裂率和桑椹胚率(61.4％、32.6％)显著低于对照组(P＜0.05)；孤雌激活胚胎的囊胚发育率，毒性组(6.7％)和冷冻组(2.3％)分别显著(P＜0.05)和极显著(P＜0.01)低于对照组20.8％。
综合上述研究结果，玻璃化液2为冷冻保存山羊体外成熟卵母细胞的最佳玻璃化液配方。
The goat oocyte took one kind of research experimental material, which waswidely applied in embryo project, the nucleus transplant and the genetransfer.Preservation of oocytes by vitrification, which were not only offer themassive oocytes for the laboratory and be overcome with time and the spatial limit,but also there was a vital significance on protecting the variety resources and IVPcommercialization.This study was conducted to get to the better OPS vitrifcationsolution, enhances the freezing effect of the oocyte. There are six differentvitrifcation solution in this study, which were named vitrifcation solutionONE(30%EG), vitrifcation solution TWO(27.5%EG+2.5%DMSO), vitrifcationsolution THREE (25%EG+5DMSO%), vitrifcation solution FOUR(20%EG+10%DMSO), vitrifcation solution FIVE(15%EG+15%DMSO),vitrifcationsolution SIX(40%EG).The goat IVM oocytes were divided into three groupsaccording to whether they were: (1) left untreated (control); (2) exposed tocryoprotectant agents (the toxicity group); or (3) cryopreserved by theopenpulled-straw (OPS) vitrification method(the vitrification group).oocytes in CPAsand vitrification group were thawed in 0.5mol/Lsucrose and 0.25mol/Lsucrose. In thisstudy,the experiment which we did about the effect of exposure to differentvitrification solution or cryopreservation on the survival of oocyte or thepartheno-activation of oocyte or the fertilization of oocytes and the development ofembryos.
In the study on the effect of exposure to different vitrification solution onmortality of oocytes:it was showed that oocytes exposure to different vitrificationsolution, there was no significant differences(P＞0.05)in the rate of complete shapebetween the CPAs control (97.1%,97.0%,97.0%,96.3%,98.1%,96.7%)andcontrol(100.0%); the oocytes which was exposured to in different vitrificationsolution was cultured in SOF for 15-17h,the mortality rate of the vitrification solutionsix (54.1%)was significantly higher(P＜0.05)than the control(0.0%), but there was nosignificant differences (P＞0.05)between other group and the control.so thevitrification solution SIX was eliminated.
In the study on the effect of partheno-activation on oocytes exposure to differetvitrification solution:the partheno-activation rate of oocytes which was exposured tovitrification solution FOUR (10%DMSO and 20%EG) or FIVE(EDFS30)wassignificantly higher(P＜0.05)than the controls(0.0%), the partheno-activation rate ofoocytes which was exposured to vitrification solution ONE, TWO, THREE(1.0%,2.7%,3.5%)were not significantly differences(P＞0.05)than the controls(0.0%),so the vitrification solution FOUR, FIVE were eliminated.
In the study on the effect of vitrification by different OPS vitrification solutionon the fertilization of oocytes:the fertilization rate (31.8%)of oocytes which were exposed to vitrification solution TWO was not significantly differences(P＞0.05) thanthe controls(51.2%), the fertilization rate(29.2%,29.3%) of oocytes which wereexposed to vitrification solution ONE, THREE were significantly differences(P＜0.05)than the controls(51.2%), the fertilization rate of oocytes which werecryopreserved to vitrification solution TWO was significantly lower(P＜0.05)than thecontrols(51.2%), the fertilization rate of oocytes which were cryopreserved tovitrification solution ONE, THREE were remarkably significantly lower (P＜0.01)thanthe controls (51.2%);by the post-24h cleavage rate/the post-48h cleavage rate forparameter, the cleavage speed (51.9%,50.0%)of oocytes which were cryopreserved tovitrification solution ONE, TWO were significantly slower(P＜0.05)than thecontrols(77.1%),the cleavage speed of oocytes in other groups were remarkablysignificantly slower(P＜0.01)than the controls(77.1%); The results was ZP hardeningthat exposure or vitrification in vitrification solutions on oocytes, dissolutiontime(424.0s～434.9s) of zona pellucida were significantly longer (P＜0.05)for oocytesexposed to vitrification solutions or vitrified in OPS, compared to controls(380.1s).
In the study on the effect of vitrification by different OPS vitrification solutionONE, TWO, THREE on the development of IVF embryos: for the IVF embryos, Themorulae rates(37.0%,50.0%,37.5%) of oocytes which were exposed to vitrificationsolution ONE, TWO,THREE were not significantly differences(P＞0.05) than thecontrols(58.3%), while The morulae rates(37.0%,50.0%,37.5%) of oocytes whichwere cryopreserved to vitrification solution ONE, TWO,THREE were significantlydifferences(P＞0.05) than the controls(58.3%), The blastocyst rate of the oocyteswhich was exposed to vitrification solution TWO, THREE (9.4%,9.4%) were notsignificantly differences(P＞0.05) than the controls(27.1%),but The blastocyst rate ofother oocytes were significantly lower (P＜0.05) than the controls(27.1%), Theoocytes were exposed or vitrified in vitrification solutions TWO, it was showed thatafter parthenogenetic activation the cleavage rate(69.2%)and the morulaerates(53.3%) of oocytes in toxicity group were no significantly differences(P＞0.05)than the controls (80.0% and 54.2%),but the cleavage rate (61.4%)and themorulae rates(32.6%)of oocytes in vitrification group were significantlydifferences(P＜0.05) than the controls. The blastocyst rates of oocytes in toxicitygroup (6.7%) showed significant differences (P＜0.05) compared to controls (20.8%),while a remarkably significantly (P＜0.01) reduced percentage of blastocyst (2.3%) byOPS vitrification.
不同配方的玻璃化液对卵母细胞存活率影响的研究结果：卵母细胞经玻璃化液处理后，玻璃化液1-6组卵母细胞的形态正常率(97.1％、97.0％、97.0％、96.3％、98.1％、96.7％)与对照组无显著性差异(P＞0.05)；但在发育液培养15-17h后，玻璃化液6(EFS40)组的卵母细胞死亡率达54.1％，与对照组(0.0％)差异显著(P＜0.05)，玻璃化液1-5组卵母细胞与对照组无显著性差异(P＞0.05)。根据上述研究结果，对玻璃化液6予以淘汰。
不同配方的玻璃化液对卵母细胞孤雌激活作用的研究结果：玻璃化液4、5对卵母细胞的孤雌激活率分别为7.5％和9.0％，与对照组(0.0％)差异显著(P＜0.05)；玻璃化液1、2、3组的孤雌激活率分别为1.0％、2.7％、3.5％，与对照组无显著性差异(P＞0.05)。因此，对玻璃化液4、5予以淘汰。
玻璃化液1、2、3冷冻保存对卵母细胞受精能力的影响研究结果：卵母细胞在玻璃化液2中暴露后的正常受精率(31.8％)与对照组(51.2％)无显著性差异(P＞0.05)，在玻璃化液1、3中暴露的正常受精率(29.2％、29.3％)均较对照组(51.2％)显著降低(P＜0.05)；玻璃化液2冷冻保存卵母细胞的正常受精率(28.0％)较对照组(51.2％)显著降低(P＜0.05)，而玻璃化液1、3冷冻保存卵母细胞的正常受精率(22.73％、22.45％)则较对照组极显著降低(P＜0.01)。以24h卵裂数／48h卵裂数为卵裂速度参数，玻璃化液1、2的毒性组卵裂速度(51.9％和50.0％)与对照组(77.1％)之间差异显著(P＜0.05)；玻璃化液1、2、3毒性组和冷冻组卵母细胞的酶溶解透明带时间(424.0s～434.9s)较对照组(380.1s)显著增加(P＜0.05)。
玻璃化液1、2、3对卵母细胞胚胎发育能力的影响研究的结果：在体外受精胚胎的发育能力方面，玻璃化液1、2、3的毒性组桑椹胚率(37.0％、50.0％、37.5％)与对照组的桑椹胚率(58.3％)无显著性差异(P＞0.05)，玻璃化液1、2、3的各冷冻组桑椹胚率(25.0％、33.3％、32.1％)则显著低于对照组(P＜0.05)；体外受精胚胎囊胚发育率与对照组(27.1％)比较，玻璃化液2、3的毒性组(9.4％、9.4％)无显著性差异(P＞0.05)，其它组囊胚发育率显著降低(P＜0.05)。在孤雌激活胚胎发育能力方面，玻璃化液2的毒性组卵裂率和桑椹胚发育率(69.2％和53.3％)与对照组(80.0％、54.1％)无显著性差异(P＞0.05)，但冷冻组的卵裂率和桑椹胚率(61.4％、32.6％)显著低于对照组(P＜0.05)；孤雌激活胚胎的囊胚发育率，毒性组(6.7％)和冷冻组(2.3％)分别显著(P＜0.05)和极显著(P＜0.01)低于对照组20.8％。
综合上述研究结果，玻璃化液2为冷冻保存山羊体外成熟卵母细胞的最佳玻璃化液配方。
The goat oocyte took one kind of research experimental material, which waswidely applied in embryo project, the nucleus transplant and the genetransfer.Preservation of oocytes by vitrification, which were not only offer themassive oocytes for the laboratory and be overcome with time and the spatial limit,but also there was a vital significance on protecting the variety resources and IVPcommercialization.This study was conducted to get to the better OPS vitrifcationsolution, enhances the freezing effect of the oocyte. There are six differentvitrifcation solution in this study, which were named vitrifcation solutionONE(30%EG), vitrifcation solution TWO(27.5%EG+2.5%DMSO), vitrifcationsolution THREE (25%EG+5DMSO%), vitrifcation solution FOUR(20%EG+10%DMSO), vitrifcation solution FIVE(15%EG+15%DMSO),vitrifcationsolution SIX(40%EG).The goat IVM oocytes were divided into three groupsaccording to whether they were: (1) left untreated (control); (2) exposed tocryoprotectant agents (the toxicity group); or (3) cryopreserved by theopenpulled-straw (OPS) vitrification method(the vitrification group).oocytes in CPAsand vitrification group were thawed in 0.5mol/Lsucrose and 0.25mol/Lsucrose. In thisstudy,the experiment which we did about the effect of exposure to differentvitrification solution or cryopreservation on the survival of oocyte or thepartheno-activation of oocyte or the fertilization of oocytes and the development ofembryos.
In the study on the effect of exposure to different vitrification solution onmortality of oocytes:it was showed that oocytes exposure to different vitrificationsolution, there was no significant differences(P＞0.05)in the rate of complete shapebetween the CPAs control (97.1%,97.0%,97.0%,96.3%,98.1%,96.7%)andcontrol(100.0%); the oocytes which was exposured to in different vitrificationsolution was cultured in SOF for 15-17h,the mortality rate of the vitrification solutionsix (54.1%)was significantly higher(P＜0.05)than the control(0.0%), but there was nosignificant differences (P＞0.05)between other group and the control.so thevitrification solution SIX was eliminated.
In the study on the effect of partheno-activation on oocytes exposure to differetvitrification solution:the partheno-activation rate of oocytes which was exposured tovitrification solution FOUR (10%DMSO and 20%EG) or FIVE(EDFS30)wassignificantly higher(P＜0.05)than the controls(0.0%), the partheno-activation rate ofoocytes which was exposured to vitrification solution ONE, TWO, THREE(1.0%,2.7%,3.5%)were not significantly differences(P＞0.05)than the controls(0.0%),so the vitrification solution FOUR, FIVE were eliminated.
In the study on the effect of vitrification by different OPS vitrification solutionon the fertilization of oocytes:the fertilization rate (31.8%)of oocytes which were exposed to vitrification solution TWO was not significantly differences(P＞0.05) thanthe controls(51.2%), the fertilization rate(29.2%,29.3%) of oocytes which wereexposed to vitrification solution ONE, THREE were significantly differences(P＜0.05)than the controls(51.2%), the fertilization rate of oocytes which werecryopreserved to vitrification solution TWO was significantly lower(P＜0.05)than thecontrols(51.2%), the fertilization rate of oocytes which were cryopreserved tovitrification solution ONE, THREE were remarkably significantly lower (P＜0.01)thanthe controls (51.2%);by the post-24h cleavage rate/the post-48h cleavage rate forparameter, the cleavage speed (51.9%,50.0%)of oocytes which were cryopreserved tovitrification solution ONE, TWO were significantly slower(P＜0.05)than thecontrols(77.1%),the cleavage speed of oocytes in other groups were remarkablysignificantly slower(P＜0.01)than the controls(77.1%); The results was ZP hardeningthat exposure or vitrification in vitrification solutions on oocytes, dissolutiontime(424.0s～434.9s) of zona pellucida were significantly longer (P＜0.05)for oocytesexposed to vitrification solutions or vitrified in OPS, compared to controls(380.1s).
In the study on the effect of vitrification by different OPS vitrification solutionONE, TWO, THREE on the development of IVF embryos: for the IVF embryos, Themorulae rates(37.0%,50.0%,37.5%) of oocytes which were exposed to vitrificationsolution ONE, TWO,THREE were not significantly differences(P＞0.05) than thecontrols(58.3%), while The morulae rates(37.0%,50.0%,37.5%) of oocytes whichwere cryopreserved to vitrification solution ONE, TWO,THREE were significantlydifferences(P＞0.05) than the controls(58.3%), The blastocyst rate of the oocyteswhich was exposed to vitrification solution TWO, THREE (9.4%,9.4%) were notsignificantly differences(P＞0.05) than the controls(27.1%),but The blastocyst rate ofother oocytes were significantly lower (P＜0.05) than the controls(27.1%), Theoocytes were exposed or vitrified in vitrification solutions TWO, it was showed thatafter parthenogenetic activation the cleavage rate(69.2%)and the morulaerates(53.3%) of oocytes in toxicity group were no significantly differences(P＞0.05)than the controls (80.0% and 54.2%),but the cleavage rate (61.4%)and themorulae rates(32.6%)of oocytes in vitrification group were significantlydifferences(P＜0.05) than the controls. The blastocyst rates of oocytes in toxicitygroup (6.7%) showed significant differences (P＜0.05) compared to controls (20.8%),while a remarkably significantly (P＜0.01) reduced percentage of blastocyst (2.3%) byOPS vitrification.
引文
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