甘氨酸对水貂GV期卵母细胞冷冻保存效率的影响
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摘要
试验旨在探究玻璃化冷冻及培养过程中添加甘氨酸(glycine,Gly)对水貂GV期卵母细胞冷冻解冻后存活率、核发育、线粒体和皮质颗粒分布的影响。试验分为3组:对照组(没有进行冷冻处理)、冷冻组和Gly添加处理组(1 mmol/L Gly)。对玻璃化冷冻解冻后的水貂GV期卵母细胞分别进行平衡恢复3 h和体外成熟培养,采用免疫荧光标记法检测各组GV期卵母细胞线粒体分布的差异及MⅡ期皮质颗粒分布的变化。结果显示,Gly添加处理组卵母细胞在解冻后3 h的存活率与冷冻组相比差异不显著(P>0.05),但显著低于对照组(P<0.05);Gly添加处理组卵母细胞的减数分裂恢复率显著高于冷冻组(P<0.05),但与对照组相比差异不显著(P>0.05)。免疫荧光结果显示,Gly添加处理组的GV期卵母细胞线粒体正常分布率显著高于冷冻组(P<0.05),但Gly添加处理组和冷冻组的GV期卵母细胞线粒体正常分布率均显著低于对照组(P<0.05)。皮质颗粒分布结果显示,水貂GV期卵母细胞在冷冻后体外成熟培养至MⅡ期时,Gly添加处理组皮质颗粒的正常皮质区分布比例显著高于冷冻组(P<0.05),但Gly添加处理组与冷冻组的正常皮质区分布比例均显著低于对照组(P<0.05)。结果表明,添加Gly可以提高冻融后水貂卵母细胞的减数分裂恢复率,降低冷冻对其线粒体及皮质颗粒的损失。
The purpose of this study was to investigate the effects of glycine(Gly) supplement on the survival rate,nuclear development,mitochondrial distribution and cortical granules distribution in mink oocytes during vitrification and in vitro maturation at germinal vesicle(GV) stage.The experiment was divided into three groups:Control group without vitrification,vitrification group and Gly supplement(1 mmol/L) vitrification group.Mink GV oocytes were cultured for 3 h after vitrification and in vitro maturation,respectively.Immunofluorescence labeling was used to detect the differences of mitochondrial distribution in GV oocytes and the changes of cortical granules distribution in MⅡ oocytes.The results showed that there was no significant difference in the survival rate of oocyte in Gly supplement vitrification group at 3 h after vitrification compared with vitrification group(P>0.05),but it was significantly lower than control group(P<0.05).The meiotic recovery rate of oocytes in Gly supplement vitrification group was significantly higher than vitrification group(P<0.05),but there was no significant difference with control group(P>0.05).The results of immunofluorescence showed that the normal distribution rate of mitochondria in GV oocytes of Gly supplement vitrification group was significantly higher than vitrification group(P<0.05),but the normal mitochondria distribution rate in GV oocytes of Gly supplement vitrification and vitrification groups were significantly lower than control group(P<0.05).The results of cortical granules distribution showed that when mink GV oocytes matured in vitro after vitrification to MⅡ stage,the proportion of cortical granules in Gly supplement vitrification group was significantly higher than vitrification group(P<0.05),but the proportion of cortical granules in Gly supplement vitrification and vitrification grous were significantly lower than control group(P<0.05).The results showed that the Gly supplement in mink oocytes could increase the meiotic recovery rate,and reduce the loss of mitochondria and cortical granules after vitrification.
The purpose of this study was to investigate the effects of glycine(Gly) supplement on the survival rate,nuclear development,mitochondrial distribution and cortical granules distribution in mink oocytes during vitrification and in vitro maturation at germinal vesicle(GV) stage.The experiment was divided into three groups:Control group without vitrification,vitrification group and Gly supplement(1 mmol/L) vitrification group.Mink GV oocytes were cultured for 3 h after vitrification and in vitro maturation,respectively.Immunofluorescence labeling was used to detect the differences of mitochondrial distribution in GV oocytes and the changes of cortical granules distribution in MⅡ oocytes.The results showed that there was no significant difference in the survival rate of oocyte in Gly supplement vitrification group at 3 h after vitrification compared with vitrification group(P>0.05),but it was significantly lower than control group(P<0.05).The meiotic recovery rate of oocytes in Gly supplement vitrification group was significantly higher than vitrification group(P<0.05),but there was no significant difference with control group(P>0.05).The results of immunofluorescence showed that the normal distribution rate of mitochondria in GV oocytes of Gly supplement vitrification group was significantly higher than vitrification group(P<0.05),but the normal mitochondria distribution rate in GV oocytes of Gly supplement vitrification and vitrification groups were significantly lower than control group(P<0.05).The results of cortical granules distribution showed that when mink GV oocytes matured in vitro after vitrification to MⅡ stage,the proportion of cortical granules in Gly supplement vitrification group was significantly higher than vitrification group(P<0.05),but the proportion of cortical granules in Gly supplement vitrification and vitrification grous were significantly lower than control group(P<0.05).The results showed that the Gly supplement in mink oocytes could increase the meiotic recovery rate,and reduce the loss of mitochondria and cortical granules after vitrification.
引文
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[17] 戴建军,吴彩凤,张廷宇,等.3种冷冻方法对猪MⅡ期卵母细胞线粒体分布和损伤的影响[J].畜牧兽医学报,2012,43(10):1525-1530.DAI J J,WU C F,ZHANG T Y,et al.Effects of three vitrification methods on the mitochondria distribution and damage of porcine MⅡ-stage oocytes[J].Acta Veterinaria et Zootechnica Sinica,2012,43(10):1525-1530.(in Chinese)
[18] 姜泽,玄彪,李作臣,等.LDL对玻璃化冷冻解冻培养MⅡ期猪卵母细胞线粒体膜电位和透明带泛素化水平的影响[J].中国畜牧兽医,2017,44(7):1961-1966.JIANG Z,XUAN B,LI Z C,et al.Effect of LDL on the mitochondrial transmembrane potential and ZP protein ubiquitination in vitrified-warmed MⅡ oocytes[J].China Animal Husbandry & Veterinary Medicine,2017,44(7):1961-1966.(in Chinese)
[19] BALTZ J M,ZHOU C.Cell volume regulation in mammalian oocytes and preimplantation embryos[J].Molecular Reproduction & Development,2012,79(12):821-831.
[20] ZHENG J,YIN X,GE W,et al.Post-ovulatory aging of mouse oocytes in vivo and in vitro:Effects of caffeine on exocytosis and translocation of cortical granules[J].Animal Science Journal,2016,87(11):1340-1346.
[21] CHEESEMAN L P,BOULANGER J,BOND L M,et al.Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes[J].Nature Communications,2016,7(1):13726.
[22] MIAO Y,ZHOU C,CUI Z,et al.Dynein promotes porcine oocyte meiotic progression by maintaining cytoskeletal structures and cortical granule arrangement[J].Cell Cycle,2017,16(21):2139-2145.
[23] TAN X,SONG E,LIU X,et al.Factors affecting the survival,fertilization,and embryonic development of mouse oocytes after vitrification using glass capillaries[J].In Vitro Cellular & Developmental Biology Animal,2009,45(8):420-429.
[24] MARCOJIMENEZ F,CASARESCRESPO L,VICENTE J S.Effect of cytochalasin B pre-treatment of in vitro matured porcine oocytes before vitrification[J].CryoLetters,2012,33(1):24-30.
[25] CHASOMBAT J,NAGAI T,PARNPAI R,et al.Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification:Effects on cytoskeleton integrity and developmental ability after warming[J].Cryobiology,2015,71(2):216-223.
[26] TIAN S J,YAN C L,YANG H X,et al.Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration[J].Animal Reproduction Science,2007,101(3):365-371.
[2] CAI Z,VILLUMSEN T M,ASP T,et al.SNP markers associated with body size and pelt length in American mink (Neovison vison)[J].BMC Genetics,2018,19(1):103.
[3] PERERA K D,GALASITI KANKANAMALAGE A C,RATHNAVAKE A D,et al.Protease inhibitors broadly effective against feline,ferret and mink coronaviruses[J].Antiviral Research,2018,160(1):79-86.
[4] EDGAR D H,GOOK D A.A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos[J].Human Reproduction Update,2012,18(5):536-554.
[5] RICHARDS T,WANG F,LIU L,et al.Rescue of postcompaction-stage mouse embryo development from hypertonicity by amino acid transporter substrates that may function as organic osmolytes[J].Biology of Reproduction,2010,82(4):769-777.
[6] VALOIERDI M R,SALEHNIA M.Developmental potential and ultrastructural injuries of metaphase Ⅱ (MⅡ) mouse oocytes after slow freezing or vitrification[J].Journal of Assisted Reproduction and Genetics,2005,22(3):119-127.
[7] STEEVES C L,HAMMER M A,WALKER G B,et al.The glycine neurotransmitter transporter GLYT1 is an organic osmolyte transporter regulating cell volume in cleavage-stage embryos[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100(24):13982-13987.
[8] CAO X Y,ROSE J,WANG S Y,et al.Glycine increases preimplantation development of mouse oocytes following vitrification at the germinal vesicle stage[J].Scientific Reports,2016,6(1):37262.
[9] LEVISETTI P E,PATRIZIO P,SCARAVELLI G.Evolution of human oocyte cryopreservation:Slow freezing versus vitrification[J].Current Opinion in Endocrinology Diabetes & Obesity,2016,23(6):445-450.
[10] SETTI P E L,PORCU E,PATRIZIO P,et al.Human oocyte cryopreservation with slow freezing versus vitrification.Results from the national Italian Registry data,2007-2011[J].Fertility & Sterility,2014,102(1):90-95.
[11] MOAWAD A R,XU B,TAN S L,et al.L-carnitine supplementation during vitrification of mouse germinal vesicle stage-oocytes and their subsequent in vitro maturation improves meiotic spindle configuration and mitochondrial distribution in metaphase Ⅱ oocytes[J].Human Reproduction,2014,29(10):2256-2268.
[12] ZHOU C,BALTZ J M.JAK2 mediates the acute response to decreased cell volume in mouse preimplantation embryos by activating NHE1[J].Journal of Cellular Physiology,2012,228(2):428-438.
[13] TARTIA A P,RUDRARAJU N,RICHARDS T,et al.Cell volume regulation is initiated in mouse oocytes after ovulation[J].Development,2009,136(13):2247-2254.
[14] ZANDERFOX D,CASHMAN K S,LANE M.The presence of 1 mM glycine in vitrification solutions protects oocyte mitochondrial homeostasis and improves blastocyst development[J].Journal of Assisted Reproduction & Genetics,2013,30(1):107-116.
[15] KOTIL T,KERVANCLOGLU M,KANTEN G,et al.Mitochondrial activity and cytoskeleton organization in three pronuclei oocytes after intracytoplasmic sperm injection[J].Zygote,2018,26(4):319-325.
[16] COSTANZO M,CISTERNA B,VELLA A,et al.Low ozone concentrations stimulate cytoskeletal organization,mitochondrial activity and nuclear transcription[J].European Journal of Histochemistry,2015,59(2):2515.
[17] 戴建军,吴彩凤,张廷宇,等.3种冷冻方法对猪MⅡ期卵母细胞线粒体分布和损伤的影响[J].畜牧兽医学报,2012,43(10):1525-1530.DAI J J,WU C F,ZHANG T Y,et al.Effects of three vitrification methods on the mitochondria distribution and damage of porcine MⅡ-stage oocytes[J].Acta Veterinaria et Zootechnica Sinica,2012,43(10):1525-1530.(in Chinese)
[18] 姜泽,玄彪,李作臣,等.LDL对玻璃化冷冻解冻培养MⅡ期猪卵母细胞线粒体膜电位和透明带泛素化水平的影响[J].中国畜牧兽医,2017,44(7):1961-1966.JIANG Z,XUAN B,LI Z C,et al.Effect of LDL on the mitochondrial transmembrane potential and ZP protein ubiquitination in vitrified-warmed MⅡ oocytes[J].China Animal Husbandry & Veterinary Medicine,2017,44(7):1961-1966.(in Chinese)
[19] BALTZ J M,ZHOU C.Cell volume regulation in mammalian oocytes and preimplantation embryos[J].Molecular Reproduction & Development,2012,79(12):821-831.
[20] ZHENG J,YIN X,GE W,et al.Post-ovulatory aging of mouse oocytes in vivo and in vitro:Effects of caffeine on exocytosis and translocation of cortical granules[J].Animal Science Journal,2016,87(11):1340-1346.
[21] CHEESEMAN L P,BOULANGER J,BOND L M,et al.Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes[J].Nature Communications,2016,7(1):13726.
[22] MIAO Y,ZHOU C,CUI Z,et al.Dynein promotes porcine oocyte meiotic progression by maintaining cytoskeletal structures and cortical granule arrangement[J].Cell Cycle,2017,16(21):2139-2145.
[23] TAN X,SONG E,LIU X,et al.Factors affecting the survival,fertilization,and embryonic development of mouse oocytes after vitrification using glass capillaries[J].In Vitro Cellular & Developmental Biology Animal,2009,45(8):420-429.
[24] MARCOJIMENEZ F,CASARESCRESPO L,VICENTE J S.Effect of cytochalasin B pre-treatment of in vitro matured porcine oocytes before vitrification[J].CryoLetters,2012,33(1):24-30.
[25] CHASOMBAT J,NAGAI T,PARNPAI R,et al.Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification:Effects on cytoskeleton integrity and developmental ability after warming[J].Cryobiology,2015,71(2):216-223.
[26] TIAN S J,YAN C L,YANG H X,et al.Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration[J].Animal Reproduction Science,2007,101(3):365-371.