卵母细胞玻璃化冷冻保存中凋亡的机制及缓解策略

doi:10.16431/j.cnki.1671-7236.2025.06.025

摘要/Abstract

摘要： 作为一种在超低温环境下长期保存卵母细胞的技术，卵母细胞玻璃化冷冻保存在优良种质资源保存方面具有重要意义。然而，玻璃化冷冻保存技术面临的核心挑战在于细胞凋亡信号的激活致使凋亡细胞比例升高，影响细胞功能、活力和发育潜能。细胞凋亡是指一种程序化的细胞死亡过程，通常被称为“细胞自杀”，这一机制在生物体内发挥着关键作用，有助于维持细胞的平衡和组织的稳态。然而，异常的细胞凋亡会导致细胞死亡等不利影响。因此，有效抑制或减缓细胞凋亡过程，成为提高冷冻卵母细胞存活率和发育潜能的关键所在。应用凋亡抑制剂和抗氧化剂可以减少玻璃化卵母细胞凋亡，提高其存活率和发育能力，从而改善解冻后的细胞质量。作者简述了玻璃化冷冻保存技术对卵母细胞的影响，以及冷冻引起的细胞内源性和外源性凋亡机制，并分析了相关凋亡抑制剂与抗氧化剂的应用，未来可深入探究新型抑制剂与抗氧化剂在玻璃化冷冻技术中的作用机理，以及探究细胞凋亡与坏死性凋亡等细胞死亡途径之间的交互影响，为卵母细胞玻璃化冷冻保存技术提供新的思路和方法。

关键词: 冷冻保存技术; 卵母细胞; 细胞凋亡; 凋亡抑制剂; 抗氧化剂

Abstract: Oocyte cryopreservation by vitrification,as a technique for long-term preservation of oocytes at ultra-low temperatures,is of great significance in the preservation of excellent germplasm resources.However,the key difficulty of cryopreservation is the activation of apoptotic signals,which leads to an increase in the proportion of apoptotic cells and affects cell function,viability and developmental potential.Apoptosis refers to a programmed cell death process,often referred to as "cell suicide",and this mechanism plays a key role in living organisms,helping to maintain cell balance and tissue homeostasis.However,abnormal apoptosis can lead to adverse effects such as cell death.Therefore,effectively inhibiting or slowing down the apoptotic process becomes the key to improve the survival and developmental potential of frozen oocytes.The application of apoptosis inhibitors and antioxidants can reduce the apoptosis of vitrification oocytes,improve their survival rate and development ability,and thus improve the cell quality after thawing.The authors briefly describe the effects of vitrification cryopreservation technology on oocytes,and the endogenous and exogenous apoptosis mechanisms induced by freezing,and analyzed the application of related apoptosis inhibitors and antioxidants.In the future,the mechanism of action of new inhibitors and antioxidants in vitrification cryopreservation technology can be further explored.And explore the interaction between apoptosis and necrotic apoptosis and other cell death pathways,to provide a new idea and method for oocyte vitrification cryopreservation technology.

Key words: cryopreservation techniques; oocyte; apoptotic; apoptosis inhibitor; antioxidant

中图分类号:

S814.8

陈丹丹, 齐雅天, 李俊杰. 卵母细胞玻璃化冷冻保存中凋亡的机制及缓解策略[J]. 中国畜牧兽医, 2025, 52(6): 2729-2735.

CHEN Dandan, QI Yatian, LI Junjie. Mechanisms of Apoptosis and Mitigation Strategies in Vitrification Cryopreservation of Oocytes[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2729-2735.

参考文献

[1] DIEZ C,MUNOZ M,CAAMANO J N,et al.Cryopreservation of the bovine oocyte:Current status and perspectives[J].Reproduction in Domestic Animals,2012,3:76-83.
[2] FU Y,DANG W,HE X,et al.Biomolecular pathways of cryoinjuries in low-temperature storage for mammalian specimens[J].Bioengineering Basel,2022,9(10):545.
[3] WU C,RUI R,DAI J,et al.Effects of cryopreservation on the developmental competence,ultrastructure and cytoskeletal structure of porcine oocytes[J].Molecular Reproduction and Development,2006,73(11):1454-1462.
[4] QUAN G,WU G,HONG Q.Oocyte cryopreservation based in sheep:The current status and future perspective[J].Biopreservation and Biobanking,2017,15(6):535-547.
[5] WHALEY D,DAMYAR K,WITEK R P,et al.Cryopreservation:An overview of principles and cell-specific considerations[J].Cell Transplantation,2021,30:963689721999617.
[6] TAMURA A N,HUANG T T,MARIKAWA Y.Impact of vitrification on the meiotic spindle and components of the microtubule-organizing center in mouse mature oocytes[J].Biology of Reproduction,2013,89(5):112.
[7] CHANG C C,SHAPIRO D B,NAGY Z P.The effects of vitrification on oocyte quality[J].Biology of Reproduction,2022,106(2):316-327.
[8] MANDAWALA A A,HARVEY S C,ROY T K,et al.Cryopreservation of animal oocytes and embryos:Current progress and future prospects[J].Theriogenology,2016,86(7):1637-1644.
[9] SUN H,GUO Y,YU R,et al.Ru360 protects against vitrification-induced oocyte meiotic defects by restoring mitochondrial function[J].Theriogenology,2023,204:40-49.
[10] ABEDPOUR N,RAJAEI F.Vitrification by cryotop and the maturation,fertilization,and developmental rates of mouse oocytes[J].Iranian Red Crescent Medical Journal,2015,17(10):e18172.
[11] 张迪,房晓欢,李俊杰,等.羊GV期卵母细胞玻璃化冷冻保存研究进展[J].家畜生态学报,2024,45(6):9-13.ZHANG D,FANG X H,LI J J,et al.Advances in vitrification cryopreservation of ovine GV oocytes[J].Journal of Domestic Animal Ecology,2024,45(6)：9-13.(in Chinese)
[12] YODRUG T,PARNPAI R,HIRAO Y,et al.Effect of vitrification at different meiotic stages on epigenetic characteristics of bovine oocytes and subsequently developing embryos[J].Animal Science Journal,2021,92(1):e13596.
[13] RAJAEI F,ABOUREDP N,SALEHNIA M,et al.The effect of vitrification on mouse oocyte apoptosis by cryotop method[J].Iranian Biomedical Journal,2013,17(4):200-205.
[14] AHMADI E,SHIRAZI A,SHAMS-ESFANDABADI N,et al.Antioxidants and glycine can improve the developmental competence of vitrified/warmed ovine immature oocytes[J].Reproduction in Domestic Animals,2019,54(3):595-603.
[15] MEN H,MONSON R L,PARRISH J J,et al.Degeneration of cryopreserved bovine oocytes via apoptosis during subsequent culture[J].Cryobiology,2003,47(1):73-81.
[16] VALLORANI C,SPINACI M,BUCCI D,et al.Pig oocyte vitrification by Cryotop method and the activation of the apoptotic cascade[J].Animal Reproduction Science,2012,135(1-4):68-74.
[17] ZHAO J,LI Y.Adenosine triphosphate content in human unfertilized oocytes,undivided zygotes and embryos unsuitable for transfer or cryopreservation[J].Journal of International Medical Research,2012,40(2):734-739.
[18] STOJKOVIC M,MACHADO S A,STOJKOVIC P,et al.Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation:Correlation with morphological criteria and developmental capacity after in vitro fertilization and culture[J].Biology of Reproduction,2001,64(3):904-909.
[19] LAN T,ZHANG K,LIN F,et al.Effects of MICU1-mediated mitochondrial calcium uptake on energy metabolism and quality of vitrified-thawed mouse metaphase Ⅱ oocytes[J].International Journal of Molecular Sciences,2022,23(15):8629.
[20] LEIBO S P,SONGSASEN N.Cryopreservation of gametes and embryos of non-domestic species[J].Theriogenology,2002,57(1):303-326.
[21] ZHAO X M,DU W H,WANG D,et al.Recovery of mitochondrial function and endogenous antioxidant systems in vitrified bovine oocytes during extended in vitro culture[J].Molecular Reproduction and Development,2011,78(12):942-950.
[22] GUITERREZ CASTILLO E,DIAZ F A,TALBOT S A,et al.Recovery of spindle morphology and mitochondrial function through extended culture after vitrification-warming of bovine oocytes[J].Theriogenology,2022,189:192-198.
[23] DENG D,XIE J,TIAN Y,et al.Effects of meiotic stage-specific oocyte vitrification on mouse oocyte quality and developmental competence[J].Frontiers in Endocrinology,2023,14:1200051.
[24] AMSTISLAVSKY S,MOKROUSOVE V,BRUSENTSEV E,et al.Influence of cellular lipids on cryopreservation of mammalian oocytes and preimplantation embryos:A review[J].Biopreservation and Biobanking,2019,17(1):76-83.
[25] SHIRZEYLI M H,EINI F,SHIRZEYLI F H,et al.Assessment of mitochondrial function and developmental potential of mouse oocytes after mitoquinone supplementation during vitrification[J].Journal of the American Association for Laboratory Animal Science,2021,60(4):388-395.
[26] NAKAGAWA S,YONEDA A,HAYAKAWA K,et al.Improvement in the in vitro maturation rate of porcine oocytes vitrified at the germinal vesicle stage by treatment with a mitochondrial permeability transition inhibitor[J].Cryobiology,2008,57(3):269-275.
[27] REDZA DUTORDOIR M,AVERLL BATES D A.Activation of apoptosis signalling pathways by reactive oxygen species[J].Biochimica et Biophysica Acta,2016,1863(12):2977-2992.
[28] QIN J,GUO S,YANG J,et al.Melatonin promotes in vitro development of vitrified-warmed mouse GV oocytes,potentially by modulating phosphorylation of Drp1[J].Frontiers in Veterinary Science,2021,8:752001.
[29] ZHAO X M,HAO H S,DU W H,et al.Melatonin inhibits apoptosis and improves the developmental potential of vitrified bovine oocytes[J].Journal of Pineal Research,2016,60(2):132-141.
[30] ELMORE S.Apoptosis:A review of programmed cell death[J].Toxicologic Pathology,2007,35(4):495-516.
[31] IGNEYE F H,KRAMMER P H.Death and anti-death:Tumour resistance to apoptosis[J].Nature Reviews Cancer,2002,2(4):277-288.
[32] XU J,SUN L,WU C,et al.Involvement of PINK1/Parkin-mediated mitophagy in mitochondrial functional disruption under oxidative stress in vitrified porcine oocytes[J].Theriogenology,2021,174:160-168.
[33] DARCY M S.Cell death:A review of the major forms of apoptosis,necrosis and autophagy[J].Cell Biology International,2019,43(6):582-592.
[34] GAO H H,LI J T,LIU J J,et al.Autophagy inhibition of immature oocytes during vitrification-34warming and in vitro mature activates apoptosis via Caspase-9 and -12 pathway[J].European Journal of Obstetrics & Gynecology and Reproductive Biology,2017,217:89-93.
[35] DAI J,WU C,MUNERI C W,et al.Changes in mitochondrial function in porcine vitrified MⅡ-stage oocytes and their impacts on apoptosis and developmental ability[J]. Cryobiology,2015,71(2):291-298.
[36] NIU Y,DAI J,WU C,et al.The application of apoptotic inhibitor in apoptotic pathways of MⅡ stage porcine oocytes after vitrification[J]. Reproduction in Domestic Animals,2016,51(6):953-959.
[37] KAUR S,KUROAWA M.Regulation of oocyte apoptosis:A view from gene knockout mice[J].International Journal of Molecular Sciences,2023,24(2):1345.
[38] CHEN Y N,DAI J J,WU C F,et al.Apoptosis and developmental capacity of vitrified parthenogenetic pig blastocysts[J].Animal Reproduction Science,2018,8:137-144.
[39] NIU Y,DAI J,CHEN Y,et al.Positive effect of apoptotic inhibitor Z-VAD-FMK on vitrified-thawed porcine MⅡ stage oocytes[J].Cryoletters,2016,37(3):188-195.
[40] SHEPEL E,DOSENKO V,VOZNESENSKAYA T,et al.Changes in expression of TNF alpha and its receptors mRNAs in oocytes and granulosa cells in mice with experimental immune ovarian failure[J].Journal of Health Sciences,2014,4(8):081-090.
[41] JUNG S E,RYU B Y.New strategies for germ cell cryopreservation:Cryoinjury modulation[J].Clinical and Experimental Reproductive Medicine-cerm,2023,4:213-222.
[42] ANCHAMPARUTHY V M,PEARSON R E,GWAZDAUSKAS F C.Expression pattern of apoptotic genes in vitrified-thawed bovine oocytes[J].Reproduction in Domestic Animals,2010,45(5):e83-90.
[43] HWANG I S,HARA H,CHUNG H J,et al.Rescue of vitrified-warmed bovine oocytes with rho-associated coiled-coil kinase inhibitor[J].Biology of Reproduction,2013,89(2):26.
[44] COLEMAN M L,OLSON M F.Rho GTPase signalling pathways in the morphological changes associated with apoptosis[J].Cell Death and Differentiation,2002,9(5):493-504.
[45] MINAMBRES R,GUASCH R M,PEREZ ARAGO A,et al.The RhoA/ROCK-I/MLC pathway is involved in the ethanol-induced apoptosis by anoikis in astrocytes[J].Journal of Cell Science,2006,119(2):271-282.
[46] TIWARI M,PRASAD S,TRIPATHI A,et al.Apoptosis in mammalian oocytes:A review[J].Apoptosis,2015,20(8):1019-1025.
[47] BISSOYI A,NAYAK B,PRAMANIK K,et al.Targeting cryopreservation-induced cell death:A review[J].Biopreservation and Biobanking,2014,12(1):23-34.
[48] 莫显红,李俊杰,郭成,等.家畜卵母细胞玻璃化冷冻损伤研究进展[J].中国畜牧兽医,2024,51(6):2524-2532.MO X H,LI J J,GUO C,et al..Research progress on vitrification injury of oocytes in livestock[J]. China Animal Husbandry & Veterinary Medicine,2024,51(6):2524-2532.(in Chinese)
[49] ZHANG A,ZHANG Z,LIU Y,et al.The role of caspase family in acute brain injury:The potential therapeutic tin the future[J].Current Neuropharmacology,2022,20(6):1194-1211.
[50] PERO M E,ZULLO G,ESPOSITO L,et al.Inhibition of apoptosis by caspase inhibitor Z-VAD-FMK improves cryotolerance of in vitro derived bovine embryos[J].Theriogenology,2018,108:127-135.
[51] COLOMBO M,ZAHMEL J,JANSCH S,et al.Inhibition of apoptotic pathways improves DNA integrity but not developmental competence of domestic cat immature vitrified oocytes[J].Frontiers in Veterinary Science,2020,7:588334.
[52] ARAYATHAM S,TIPTANAVATTANA N,THARASANIT T.Effects of vitrification and a Rho-associated coiled-coil containing protein kinase 1 inhibitor on the meiotic and developmental competence of feline oocytes[J].Journal of Reproduction and Development,2017,63(5):511-517.
[53] GUPTA M K,UHM S J,LEE H T.Effect of vitrification and beta-mercaptoethanol on reactive oxygen species activity and in vitro development of oocytes vitrified before or after in vitro fertilization[J].Fertility and Sterility,2010,93(8):2602-2607.
[54] ITO J,SHIRASUNA K,KUWAYAMA T,et al.Resveratrol treatment increases mitochondrial biogenesis and improves viability of porcine germinal-vesicle stage vitrified-warmed oocytes[J].Cryobiology,2020,93:37-43.
[55] GIARETTA E,SPINACI M,BUCCI D,et al.Effects of resveratrol on vitrified porcine oocytes[J].Oxidative Medicine and Cellular Longevity,2013,2013:920257.
[56] RUIZ CONCA M,GARDELA J,MOGAS T,et al.Apoptosis and glucocorticoid-related genes mRNA expression is modulated by coenzyme Q10 supplementation during in vitro maturation and vitrification of bovine oocytes and cumulus cells[J].Theriogenology,2022,192:62-72.
[57] HWANG I S,KWON D,KWAK T U,et al.Improved survival and developmental rates in vitrified-warmed pig oocytes after recovery culture with coenzyme Q10[J].Cryoletters,2016,37(1):59-67.
[58] YANG T,KONG B,GU J W,et al.Anti-apoptotic and anti-oxidative roles of quercetin after traumatic brain injury[J]. Cellular and Molecular Neurobiology,2014,34(6):797-804.
[59] SILVA A A A,SILVA M N P,FIGUEIREDO L B F,et al.Quercetin influences in vitro maturation,apoptosis and metabolically active mitochondria of goat oocytes[J].Zygote,2018,26(6):465-470.
[60] DAVOODIAN N,KADIVAR A,AHMADI E,et al.Quercetin effect on the efficiency of ovine oocyte vitrification at GV stage[J].Theriogenology,2021,174:53-59.