人卵母细胞和胚胎在低温保存过程中细胞内外冰晶形成规律的研究
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摘要
目的研究人卵母细胞和胚胎在不同降温速率下细胞内外冰晶形成的规律,进一步解析其低温生物学特性,为开发合适的低温保存方案提供依据。方法将临床废弃卵母细胞和胚胎在包含1mol/L的乙二醇和植冰剂Snomax(10mg/L)PBS溶液中培养15min后,放置在低温显微镜下观察。通过设定不同的降温程序,实时记录细胞内外冰晶形成的过程。结果细胞外冰晶形成的温度为-(7.4±0.3)℃。当降温速率为-0.5℃/min时,人卵母细胞和胚胎细胞内均未有细胞内冰晶的形成;当降温速率分别为-2℃/min、-4℃/min、-8℃/min、-20℃/min时,人卵母细胞和胚胎的细胞内冰晶形成的温度分别为-(26.9±12.3)℃、-(26.2±12.0)℃;-(25.0±8.6)℃、-(20.4±6.3)℃以及-(23.0±6.5)℃、-(19.2±6.3)℃;-(25.4±8.4)℃、-(19.1±5.5)℃。桑椹胚细胞内冰晶形成有两种特点:卵裂球同时且均匀的形成冰晶或者单个卵裂球、一组卵裂球依次形成冰晶。结论卵母细胞细胞内冰晶形成的温度(-25.0℃)更低于胚胎(-20.4℃),且人卵母细胞细胞内冰晶形成的温度域宽且分散,而胚胎细胞内冰晶形成的温度域窄且集中。
Objective:To study the formation of intra-and extra-cellular ice crystals during cryopreservation of human oocytes and embryos at different cooling rates,and analyze its low-temperature biological characteristics for providing an objective basis for the development of suitable cryogenic storage solutions.Methods:The clinical abandoned oocytes and embryos were cultured in a solution containing ethylene glycol(1 mol/L)and Snomax(10 mg/L)PBS for 15 min,and place them under a cryogenic microscope to observe.The formation process of intracellular and extracellular ice crystals at the different cooling procedures was recorded in real time.Results:The temperature of extracellular ice crystal formation was-(7.4±0.3)℃.When the cooling rate is-0.5℃/min,no intracellular ice crystal was formed in human oocytes and embryonic cells.When the cooling rate is-2℃/min,-4℃/min,-8℃/min,-20℃/min respectively,the temperature of intracellular ice crystal formation was-(26.9±12.3)℃ and-(26.2±12.0)℃;-(25.0±8.6)℃ and-(20.4±6.3)℃in human oocytes,and-(23.0±6.5)℃ and-(19.2±6.3)℃;-(25.4±8.4)℃ and-(19.1±5.5)℃in human embryos respectively.There are two characteristics of ice crystal formation in the human Day 3 embryonic cells:the blastomeres simultaneously and uniformly formed ice crystals;or single blastomeres or a group of blastomeres sequentially formed ice crystals.Conclusions:The temperature of ice crystal formation in the oocyte(-25.0℃)is lower than that of the embryo(-20.4℃).The temperature range of ice crystal formation in oocyte is wide and dispersed,while the temperature range of ice crystal formation in embryo is narrow and concentrated.
Objective:To study the formation of intra-and extra-cellular ice crystals during cryopreservation of human oocytes and embryos at different cooling rates,and analyze its low-temperature biological characteristics for providing an objective basis for the development of suitable cryogenic storage solutions.Methods:The clinical abandoned oocytes and embryos were cultured in a solution containing ethylene glycol(1 mol/L)and Snomax(10 mg/L)PBS for 15 min,and place them under a cryogenic microscope to observe.The formation process of intracellular and extracellular ice crystals at the different cooling procedures was recorded in real time.Results:The temperature of extracellular ice crystal formation was-(7.4±0.3)℃.When the cooling rate is-0.5℃/min,no intracellular ice crystal was formed in human oocytes and embryonic cells.When the cooling rate is-2℃/min,-4℃/min,-8℃/min,-20℃/min respectively,the temperature of intracellular ice crystal formation was-(26.9±12.3)℃ and-(26.2±12.0)℃;-(25.0±8.6)℃ and-(20.4±6.3)℃in human oocytes,and-(23.0±6.5)℃ and-(19.2±6.3)℃;-(25.4±8.4)℃ and-(19.1±5.5)℃in human embryos respectively.There are two characteristics of ice crystal formation in the human Day 3 embryonic cells:the blastomeres simultaneously and uniformly formed ice crystals;or single blastomeres or a group of blastomeres sequentially formed ice crystals.Conclusions:The temperature of ice crystal formation in the oocyte(-25.0℃)is lower than that of the embryo(-20.4℃).The temperature range of ice crystal formation in oocyte is wide and dispersed,while the temperature range of ice crystal formation in embryo is narrow and concentrated.
引文
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[11]Valdimarsson G,Kidder GM.Temporal control of gap junction assembly in preimplantation mouse embryos[J].JCell Sci,1995,108:1715-1722.
[12]Davidson JS,Baumgarten IM,Harley EH.Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid[J].Biochem Biophys Res Commun,1986,134:29-36.
[13]Donnez J,Dolmans MM.Fertility preservation in women[J].Nat Rev Endocrinol,2013,9:735-749.
[14]Shufaro Y,Schenker JG.Cryopreservation of human genetic material[J].Ann N Y Acad Sci,2010,1205:220-224.
[15]Guenther JF,Seki S,Kleinhans FW,et al.Extra-and intracellular ice formation in Stage I and II Xenopus laevis oocytes[J].Cryobiology,2006,52:401-416.
[16]Acker JP,Mcgann LE.Cell-cell contact affects membrane integrity after intracellular freezing[J].Cryobiology,2000,40:54-63.
[2]Mazur P.Freezing of living cells:mechanisms and implications[J].Am J Physiol,1984,247:C125-142.
[3]Fabbri R,Pasquinelli G,Bracone G,et al.Cryopreservation of human ovarian tissue[J].Cell Tissue Bank,2006,7,123-133.
[4]K9rber C,Englich S,Rau G,et al.Intracellular ice formation:cryomicroscopical observation and calorimetric measurement[J].J Microsc,1991,61:313-325.
[5]Jin B,Seki S,Paredes E,et al.Intracellular ice formation in mouse zygotes and early morulae vs.cooling rate and temperature-experimental vs.theory[J].Cryobiology,2016,73:181-186.
[6]Berger WK,Uhrík B.Freeze-induced shrinkage of individual cells and cell-to-cell propagation of intracellular ice in cell chains from salivary glands[J].Experientia,1996,52:843-850.
[7]Acker JP,Larese A,Yang H,et al.Intracellular ice formation is affected by cell interactions[J].Cryobiology,1999,38:363-371.
[8]Acker JP,Elliott JA,Mcgann LE,et al.Intercellular ice propagation:experimental evidence for ice growth through membrane pores[J].Biophys J,2001,81:1389-1397.
[9]Kleinhans FW,Guenther JF,Roberts DM,et al.Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry[J].Cryobiology,2006,52:128-138.
[10]Seki S,Mazur P.The temperature and type of intracellular ice formation in preimplantation mouse embryos as a function of the developmental stage[J].Biol Reprod,2010,82:1198-1205.
[11]Valdimarsson G,Kidder GM.Temporal control of gap junction assembly in preimplantation mouse embryos[J].JCell Sci,1995,108:1715-1722.
[12]Davidson JS,Baumgarten IM,Harley EH.Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid[J].Biochem Biophys Res Commun,1986,134:29-36.
[13]Donnez J,Dolmans MM.Fertility preservation in women[J].Nat Rev Endocrinol,2013,9:735-749.
[14]Shufaro Y,Schenker JG.Cryopreservation of human genetic material[J].Ann N Y Acad Sci,2010,1205:220-224.
[15]Guenther JF,Seki S,Kleinhans FW,et al.Extra-and intracellular ice formation in Stage I and II Xenopus laevis oocytes[J].Cryobiology,2006,52:401-416.
[16]Acker JP,Mcgann LE.Cell-cell contact affects membrane integrity after intracellular freezing[J].Cryobiology,2000,40:54-63.