猪生发泡期卵母细胞的玻璃化冷冻保存
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摘要
对卵母细胞进行冷冻保存,可以使得卵源不再受到时间和空间的限制,为体外受精、核移植和转基因等胚胎工程技术的研究提供充足的原材料。猪卵母细胞中富含脂滴,对低温十分敏感,以致猪卵母细胞冷冻保存的研究进展相对较慢。开展对猪卵母细胞冷冻保存的研究为猪和其他低温敏感性物种的卵母细胞冷冻保存提供方法。玻璃化冷冻卵母细胞不需要昂贵的仪器,操作方便、快速和高效。本试验以生发泡期(germinal vesicle,GV期)的猪卵母细胞为研究对象,研究了预平衡时间、颗粒细胞和冷冻载体对卵母细胞玻璃化冷冻效果的影响。
为了选择最佳的猪GV期卵母细胞在玻璃化冷冻液中的平衡时间,将卵母细胞分别用10%乙二醇(ethylene glycol,EG)、20%EG平衡1min、1min(总平衡时间2min)和3min、2min(总平衡时间5min),再通过30%EG的玻璃化冷冻液处理30s,直接装入半麦管,投入液氮中冷冻保存。解冻后,体外成熟培养44h,用透明质酸酶(hyaluronidase,HE)脱除卵丘,台盼蓝染色检查存活情况,并在体视镜下观察第一极体排出情况,统计存活率和成熟率。结果发现,平衡2min和5min分别得到51.33%、45.16%的存活率,两者差异不显著(P＞0.05);体外成熟后分别得到了3.54%和10.48%成熟率,差异显著(P＜0.05);说明平衡5min的冷冻效果优于平衡2min;同时表明,冷冻保存猪GV期卵母细胞是可行的。
为了研究颗粒细胞的存在对猪卵母细胞冷冻效果的影响,将包裹有3层以上或2层卵丘的猪卵母细胞在10%、20%和30%的EG分别处理3min、2min和30s后,冷冻解冻后体外成熟培养44h,统计存活率和成熟率;对照组卵母细胞不经冷冻直接体外成熟培养。结果发现,包裹有3层以上颗粒细胞的卵母细胞和包裹2层颗粒细胞的卵冷冻解冻后分别得到18.18%、42.11%的存活率,两者差异极显著(P＜0.01),均与对照组差异极显著(P＜0.01),对照组之间差异不显著(p＞0.05);体外成熟后,包裹有3层以上颗粒细胞的卵母细胞和2层颗粒细胞包裹的卵分别得到了4.55%、9.77%的成熟率,两者差异不显著(P＞0.05),但与对照组差异均极显著(P＜0.01),对照组之间差异不显著。说明脱除部分卵丘可以降低冷冻对卵母细胞造成的损伤。
为研究冷冻载体对猪卵母细胞冷冻效果的,将猪卵母细胞在10%、20%和30%EG中平衡3min、2min和30s后,分别装入开口式拉长麦管(open pullstraw,OPS)管和半麦管,冷冻解冻后体外成熟培养44h,检查存活和成熟情况;对照组不经冷冻直接体外成熟。结果表明,用OPS法、半麦管法冷冻猪GV期卵母细胞存活率分别为51.47%、55.41%和成熟率分别为8.82%、10.81%,两者之间差异不显著(P＞0.05),但均与对照组存活率和成熟率94.12%、69.12%差异极显著(P＜0.01)。结果说明半麦管法和OPS法冷冻效果相同,同样适合于猪GV期卵母细胞的冷冻保存。
综合本试验的结果表明,猪GV期卵母细胞玻璃化冷冻保存时,脱除包裹在卵母细胞外面的部分颗粒细胞,采用半麦管作为载体,在10%、20%EG平衡液中分别处理3min、2min后,再移入30%EG中处理30s,可以获得较好的发育能力。
The cryopreserved mammalian oocytes have been increasingly used in embryo biotechnologies,such as in vitro fertilization,nuclear transfer and transgene.The cryopreservation of oocytes can facilitate the transportation and,therefore,the research work.Nowadays,it has been a widely used basic biology.Porcine oocyte cryoperservation is of the great importance in both husbandary and human clinics.On the other hand,porcine oocyte cryopreservation has got less progress than that of other mammalian oocytes.Porcine oocytes are highly sensitive to low temperature stress due to the rich lipid droplets in their ooplasm.It is necessary to carry out more researches in order to optimize porcine oocytes cryopreservation.The vitrification is a recently developed method for oocyte cryopreservation.It has quite a few advantages. For instance,it is simple,handy,fast and effective without expensive instruments.The present study was to investigate the vitrification of porcine oocytes at germinal vesicle (GV) stage,including the effects of equilibration time in vitrification solution before cryopreservation,and the effects of cumulus cell existence carriers of oocytes on the survival rate and further developmental potential of thawed oocytes.
In order to choose the appropriate equilibration time of oocytes in vitrification solution before cryopreservation,fresh porcine GV oocytes were treated with 10% and then 20%ethylene glycol(EG) for 1 min and 1 min(totally 2 min) respectively, or 3 min and 2 min(totally 5 min) respectively.And then all the oocytes were treated with 30%EG solution for 30 sec.Afterwards,the treated oocytes were moved into the Hemi-straws,and were cryopreserved in liquid nitrogen directly.The survival rate and further developmental potential of the thawed oocytes were examined after thawing and then in vitro maturation for 44 h.The oocytes were treated with hyaluronidase (HE) to remove the cumulus cells around oocytes,and then were stained by trypan blue to evaluate the survival of the oocytes.The oocytes with first polar body(PB1) were regarded as mature oocytes.The results showed that the survival rates of thawed porcine oocytes equiliberated in 10%and 20%EG for 2 min and 5 min were 51.33% and 45.16%respectively,which had no significant difference(P＞0.05).The maturation rate of oocytes equilibrated in EG for 5 min was significantly higher than that for 2 min after in vitro maturation(10.48%vs 3.54%,P＜0.05).
To test the effects of the number of cumulus cell layer around oocyte on the survival and maturation of frozen-thawed oocytes,the fresh GV oocytes with different layer of cumulus cells(multiple layers and two layers) were treated with 10%,20% and 30%EG for 3 min,2 min and 30s respectively followed by cryopreservation. Then their survival and maturation of the thawed oocytes were evaluated after staining and in vitro maturation.The fresh porcine GV oocytes were directly matured in vitro without cryoperservation as control group.The results showed that the oocytes enclosed with two layers of cumulus cells had a significantly higher survival rate that those enclossed with two layers(42.11%vs 18.18%,P＜0.01).In contrast,the maturation rates didn't differ significantly(9.77%vs 4.55%,P＞0.05).It indicated that the removal some cumulus cells around oocyte before vitrified cryopreservation may reduce the damage of vitrification.The survival rates in control group were 98.10% and 94.33%,respectively while the maturation rates in control group were 69.43% and 68.79%,respectively.Both the survival and maturation rates in control group were most significant higher than those of the couterparts(P＜0.01),which indicated the cryopreservation injury against porcine GV oocytes.
The third experiment was undertaken to investigate the effects of cryopreserved oocyte carriers on the survival and further developmental potential of the thawed oocytes.Two kinds of carriers of porcine oocytes,namely,open pulled straw(OPS) and Hemi-straw,were used with the same procedures of equiliberation, cryopreservation,thawing and maturation culture.The results showed that the survival rate of the oocytes in OPS and Hemi-straw were 51.47%and 55.41%, respectively after in vitro maturation,and they had no significant difference (P＞0.05).The maturation rates didn't have NO statistical difference was found for maturation rate either(8.82%vs 10.81%,P＞0.05).It was considered that both of OPS and Hemi-straw be used as the carriers of vitrified cryopreservation of porcine oocyte at stage of GV and Hemi-straw be a little better than OPS.
It was concluded that the procedure of cryopreservation of porcine GV oocytes be as follows:to remove part of cumulus cells around fresh oocytes,treat oocytes with 10%,20%and 30%EG for 3 min,2 min and 30 s respectively,use Hemi-straw as cryopreservation carrier and cryopreserve oocytes in liquid nitrogen.
为了选择最佳的猪GV期卵母细胞在玻璃化冷冻液中的平衡时间,将卵母细胞分别用10%乙二醇(ethylene glycol,EG)、20%EG平衡1min、1min(总平衡时间2min)和3min、2min(总平衡时间5min),再通过30%EG的玻璃化冷冻液处理30s,直接装入半麦管,投入液氮中冷冻保存。解冻后,体外成熟培养44h,用透明质酸酶(hyaluronidase,HE)脱除卵丘,台盼蓝染色检查存活情况,并在体视镜下观察第一极体排出情况,统计存活率和成熟率。结果发现,平衡2min和5min分别得到51.33%、45.16%的存活率,两者差异不显著(P＞0.05);体外成熟后分别得到了3.54%和10.48%成熟率,差异显著(P＜0.05);说明平衡5min的冷冻效果优于平衡2min;同时表明,冷冻保存猪GV期卵母细胞是可行的。
为了研究颗粒细胞的存在对猪卵母细胞冷冻效果的影响,将包裹有3层以上或2层卵丘的猪卵母细胞在10%、20%和30%的EG分别处理3min、2min和30s后,冷冻解冻后体外成熟培养44h,统计存活率和成熟率;对照组卵母细胞不经冷冻直接体外成熟培养。结果发现,包裹有3层以上颗粒细胞的卵母细胞和包裹2层颗粒细胞的卵冷冻解冻后分别得到18.18%、42.11%的存活率,两者差异极显著(P＜0.01),均与对照组差异极显著(P＜0.01),对照组之间差异不显著(p＞0.05);体外成熟后,包裹有3层以上颗粒细胞的卵母细胞和2层颗粒细胞包裹的卵分别得到了4.55%、9.77%的成熟率,两者差异不显著(P＞0.05),但与对照组差异均极显著(P＜0.01),对照组之间差异不显著。说明脱除部分卵丘可以降低冷冻对卵母细胞造成的损伤。
为研究冷冻载体对猪卵母细胞冷冻效果的,将猪卵母细胞在10%、20%和30%EG中平衡3min、2min和30s后,分别装入开口式拉长麦管(open pullstraw,OPS)管和半麦管,冷冻解冻后体外成熟培养44h,检查存活和成熟情况;对照组不经冷冻直接体外成熟。结果表明,用OPS法、半麦管法冷冻猪GV期卵母细胞存活率分别为51.47%、55.41%和成熟率分别为8.82%、10.81%,两者之间差异不显著(P＞0.05),但均与对照组存活率和成熟率94.12%、69.12%差异极显著(P＜0.01)。结果说明半麦管法和OPS法冷冻效果相同,同样适合于猪GV期卵母细胞的冷冻保存。
综合本试验的结果表明,猪GV期卵母细胞玻璃化冷冻保存时,脱除包裹在卵母细胞外面的部分颗粒细胞,采用半麦管作为载体,在10%、20%EG平衡液中分别处理3min、2min后,再移入30%EG中处理30s,可以获得较好的发育能力。
The cryopreserved mammalian oocytes have been increasingly used in embryo biotechnologies,such as in vitro fertilization,nuclear transfer and transgene.The cryopreservation of oocytes can facilitate the transportation and,therefore,the research work.Nowadays,it has been a widely used basic biology.Porcine oocyte cryoperservation is of the great importance in both husbandary and human clinics.On the other hand,porcine oocyte cryopreservation has got less progress than that of other mammalian oocytes.Porcine oocytes are highly sensitive to low temperature stress due to the rich lipid droplets in their ooplasm.It is necessary to carry out more researches in order to optimize porcine oocytes cryopreservation.The vitrification is a recently developed method for oocyte cryopreservation.It has quite a few advantages. For instance,it is simple,handy,fast and effective without expensive instruments.The present study was to investigate the vitrification of porcine oocytes at germinal vesicle (GV) stage,including the effects of equilibration time in vitrification solution before cryopreservation,and the effects of cumulus cell existence carriers of oocytes on the survival rate and further developmental potential of thawed oocytes.
In order to choose the appropriate equilibration time of oocytes in vitrification solution before cryopreservation,fresh porcine GV oocytes were treated with 10% and then 20%ethylene glycol(EG) for 1 min and 1 min(totally 2 min) respectively, or 3 min and 2 min(totally 5 min) respectively.And then all the oocytes were treated with 30%EG solution for 30 sec.Afterwards,the treated oocytes were moved into the Hemi-straws,and were cryopreserved in liquid nitrogen directly.The survival rate and further developmental potential of the thawed oocytes were examined after thawing and then in vitro maturation for 44 h.The oocytes were treated with hyaluronidase (HE) to remove the cumulus cells around oocytes,and then were stained by trypan blue to evaluate the survival of the oocytes.The oocytes with first polar body(PB1) were regarded as mature oocytes.The results showed that the survival rates of thawed porcine oocytes equiliberated in 10%and 20%EG for 2 min and 5 min were 51.33% and 45.16%respectively,which had no significant difference(P＞0.05).The maturation rate of oocytes equilibrated in EG for 5 min was significantly higher than that for 2 min after in vitro maturation(10.48%vs 3.54%,P＜0.05).
To test the effects of the number of cumulus cell layer around oocyte on the survival and maturation of frozen-thawed oocytes,the fresh GV oocytes with different layer of cumulus cells(multiple layers and two layers) were treated with 10%,20% and 30%EG for 3 min,2 min and 30s respectively followed by cryopreservation. Then their survival and maturation of the thawed oocytes were evaluated after staining and in vitro maturation.The fresh porcine GV oocytes were directly matured in vitro without cryoperservation as control group.The results showed that the oocytes enclosed with two layers of cumulus cells had a significantly higher survival rate that those enclossed with two layers(42.11%vs 18.18%,P＜0.01).In contrast,the maturation rates didn't differ significantly(9.77%vs 4.55%,P＞0.05).It indicated that the removal some cumulus cells around oocyte before vitrified cryopreservation may reduce the damage of vitrification.The survival rates in control group were 98.10% and 94.33%,respectively while the maturation rates in control group were 69.43% and 68.79%,respectively.Both the survival and maturation rates in control group were most significant higher than those of the couterparts(P＜0.01),which indicated the cryopreservation injury against porcine GV oocytes.
The third experiment was undertaken to investigate the effects of cryopreserved oocyte carriers on the survival and further developmental potential of the thawed oocytes.Two kinds of carriers of porcine oocytes,namely,open pulled straw(OPS) and Hemi-straw,were used with the same procedures of equiliberation, cryopreservation,thawing and maturation culture.The results showed that the survival rate of the oocytes in OPS and Hemi-straw were 51.47%and 55.41%, respectively after in vitro maturation,and they had no significant difference (P＞0.05).The maturation rates didn't have NO statistical difference was found for maturation rate either(8.82%vs 10.81%,P＞0.05).It was considered that both of OPS and Hemi-straw be used as the carriers of vitrified cryopreservation of porcine oocyte at stage of GV and Hemi-straw be a little better than OPS.
It was concluded that the procedure of cryopreservation of porcine GV oocytes be as follows:to remove part of cumulus cells around fresh oocytes,treat oocytes with 10%,20%and 30%EG for 3 min,2 min and 30 s respectively,use Hemi-straw as cryopreservation carrier and cryopreserve oocytes in liquid nitrogen.
引文
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