卵细胞体外成熟(IVM)在小鼠生物净化中的应用
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摘要
目的在小鼠生物净化中,从超排后未排卵的卵巢,取卵泡内未成熟卵采用卵母细胞体外成熟(in vitro maturation,IVM)使之在体外成熟并具备受精能力,以提高卵子利用率和作为常规促超排失败的一种补救措施。方法在小鼠生物净化中,取注射PMSG和HCG后未排卵卵巢,在实体显微镜下划破卵泡挑选卵丘卵母细胞复合物(COCs),置于成熟液滴中在体外发育成熟。同时以注射PMSG和HCG后正常超排卵、只注射PMSG后取未成熟卵体外成熟和注射PMSG和HCG后取疑似成熟卵和裸卵体外成熟作为对照。经体外受精、体外胚胎发育后,将2-细胞胚移植到受体输卵管,使其在受体内发育成为成熟的个体。结果注射PMSG和HCG后未排卵组(A组),卵细胞体外成熟率为87.0%±3.2%,二胞率为55.1%±12.3%,囊胚率为23.1%,移植41枚二细胞胚至2只受体鼠,出生5只幼崽,产仔率12.2%。只注射PMSG,48 h后取未成熟卵组(B组),体外成熟率为83.9%±3.9%,二胞率为51.8%±9.3%,囊胚率为38.5%±13.9%。注射PMSG和HCG正常超排组(C组),其二胞率为78.9%±0.6%,囊胚率为78.0%±3.8%。注射PMSG和HCG未排卵卵巢,取裸卵和疑似成熟卵(D组),体外成熟培养0 h,6 h和16～18 h,其成熟率、二胞率和囊胚率与其它三组相比均较低且有极显著性差异。A组和B组与正常对照C组相比,二胞率均有显著性差异,囊胚率均有极显著性差异。结论卵母细胞体外成熟(IVM)可以作为小鼠生物净化中促超排失败的一种补救措施,并且可以提高珍稀品系小鼠的卵子利用率。
Objective In mice biological purification,ovaries that did not ovulate after ovarian stimulation,were dissected and immature oocytes were isolated from follicles,then subjected to in vitro maturation for the ability of fertilization.By IVM,our aim was to improve oocytes utility ratios and as a remedial measure when conventional hyperstimulation fails. Method In mice biological purification,COCs were isolated by puncturing of ovarian follicles with a needle, that did not ovulate after PMSG and HCG stimulation, then subjected immediately to maturation in IVM culture.At the same time, the normal superovulation after PMSG and HCG injection, the immature oocytes in vitro maturation after PMSG injection, and suspected matured oocytes and nude oocytes in vitro maturation after PMSG and HCG injection were used as controls. After IVM and IVF, the 2-cell embryos were introduced to oviducts of the pseudo-pregnant foster mothers, and developed matured individuals in vivo. Result Ovaries that did not ovulate after PMSG and HCG injection(group A),the mature rate of immature oocytes was 87.0%±3.2%,2-cell rate was 55.1%±12.3%,blastocyst rate was 23.1%,41 2-cell embryos were introduced to oviducts of two pseudo-pregnant foster mothers, and 5 cubs were born, the birth rate was 12.2%.PMSG injection only, immature oocytes were obtained 48 h later(group B),in vitro maturation rate was 83.9%±3.9%, 2-cell rate was 51.8%±9.3%,blastocyst rate was 38.5%±13.9%.The normal superovulation after PMSG and HCG injection(group C),2-cell rate was 78.9%±0.6%,blastocyst rate was 78.0%±3.8%.The suspected matured oocytes and nude oocytes were obtained from ovaries that did not ovulate after PMSG and HCG injection(group D),matured in vitro 0 h,6 h and 16～18 h,the mature rate,2-cell rate and blastocyst rate were all lower than the other three groups and had significant differences.The 2-cell rate of group A and group B had significant differences,and blastocyst rate had extremely significant differences compared with group C. Conclusion Oocytes IVM can be used as a remedial measure when conventional hyperstimulation fails, and also can be used to improve rare strains of mice oocytes utility ratios.
Objective In mice biological purification,ovaries that did not ovulate after ovarian stimulation,were dissected and immature oocytes were isolated from follicles,then subjected to in vitro maturation for the ability of fertilization.By IVM,our aim was to improve oocytes utility ratios and as a remedial measure when conventional hyperstimulation fails. Method In mice biological purification,COCs were isolated by puncturing of ovarian follicles with a needle, that did not ovulate after PMSG and HCG stimulation, then subjected immediately to maturation in IVM culture.At the same time, the normal superovulation after PMSG and HCG injection, the immature oocytes in vitro maturation after PMSG injection, and suspected matured oocytes and nude oocytes in vitro maturation after PMSG and HCG injection were used as controls. After IVM and IVF, the 2-cell embryos were introduced to oviducts of the pseudo-pregnant foster mothers, and developed matured individuals in vivo. Result Ovaries that did not ovulate after PMSG and HCG injection(group A),the mature rate of immature oocytes was 87.0%±3.2%,2-cell rate was 55.1%±12.3%,blastocyst rate was 23.1%,41 2-cell embryos were introduced to oviducts of two pseudo-pregnant foster mothers, and 5 cubs were born, the birth rate was 12.2%.PMSG injection only, immature oocytes were obtained 48 h later(group B),in vitro maturation rate was 83.9%±3.9%, 2-cell rate was 51.8%±9.3%,blastocyst rate was 38.5%±13.9%.The normal superovulation after PMSG and HCG injection(group C),2-cell rate was 78.9%±0.6%,blastocyst rate was 78.0%±3.8%.The suspected matured oocytes and nude oocytes were obtained from ovaries that did not ovulate after PMSG and HCG injection(group D),matured in vitro 0 h,6 h and 16～18 h,the mature rate,2-cell rate and blastocyst rate were all lower than the other three groups and had significant differences.The 2-cell rate of group A and group B had significant differences,and blastocyst rate had extremely significant differences compared with group C. Conclusion Oocytes IVM can be used as a remedial measure when conventional hyperstimulation fails, and also can be used to improve rare strains of mice oocytes utility ratios.
引文
[1] 杜江涛,宋晓明,戴方伟,等.小鼠腺病毒荧光定量PCR方法的建立及初步应用[J].实验动物科学,2017,34(3):49-54.
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[8] Sugiura K,Pendola F L,Eppig J J.Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells:energy metabolism[J].Dev Biol,2005,279(1):20-30.
[9] Richani D,Wang X,Zeng H T,et al.Pre-maturation with cAMP modulators in conjunction with EGF-like peptides during in vitro maturation enhances mouse oocyte developmental competence[J].Mol Reprod Dev,2014,81(5):422-435.
[10] Gilchrist R B,De Vos M,Smitz J,et al.IVM media are designed specifically to support immature cumulus-oocyte complexes not denuded oocytes that have failed to respond to hyperstimulation[J].Fertil Steril,2011,96(2):e141.
[11] Frank L A,Sutton-McDowall M L,Russell D L,et al.Effect of varying glucose and glucosamine concentration in vitro on mouse oocyte maturation and developmental competence[J].Reprod Fertil Dev,2013,25(8):1095-1104.
[12] Albuz F K,Sasseville M,Lane M,et al.Simulated physiological oocyte maturation (SPOM):a novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes[J].Hum Reprod,2010,25(12):2999-3011.
[13] Eppig J J,O′Brien M J,Wigglesworth,K,et al.Effect of in vitro maturation of mouse oocytes on the health and lifespan of adult offspring[J].Hum Reprod,2009,24(4):922-928.
[2] 李娜,杨秋龙,李思璧,等.模型动物生物净化方法的探讨[J].上海交通大学学报(农业科学版),2014,32(5):84-88.
[3] Zhang Y,Shao L,Xu Y,et al.Effect of anti-Mullerian hormone in culture medium on quality of mouse oocytes matured in vitro[J].PLoS One,2014,9(6):e99393.
[4] 刘丽均,郁丽丽,朱权凤,等.生物工程技术在实验小鼠生物净化上的应用[J].中国比较医学杂志,2009,19(11):63-66.
[5] 宋绍征,王怡,王宝珠,等.激素剂量、小鼠品系及周龄对超数排卵的影响[J].实验动物科学,2011,28(4):5-8.
[6] 徐平.不同日龄和品系小鼠超排卵、体外受精及受孕率的比较研究[J].中国实验动物学杂志,2001,11(2):78-81.
[7] Zeng H T,Richani D,Sutton-McDowall M L,et al.Prematuration with cyclic adenosine monophosphate modulators alters cumulus cell and oocyte metabolism and enhances developmental competence of in vitro-matured mouse oocytes[J].Biol Reprod,2014,91(2):47.
[8] Sugiura K,Pendola F L,Eppig J J.Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells:energy metabolism[J].Dev Biol,2005,279(1):20-30.
[9] Richani D,Wang X,Zeng H T,et al.Pre-maturation with cAMP modulators in conjunction with EGF-like peptides during in vitro maturation enhances mouse oocyte developmental competence[J].Mol Reprod Dev,2014,81(5):422-435.
[10] Gilchrist R B,De Vos M,Smitz J,et al.IVM media are designed specifically to support immature cumulus-oocyte complexes not denuded oocytes that have failed to respond to hyperstimulation[J].Fertil Steril,2011,96(2):e141.
[11] Frank L A,Sutton-McDowall M L,Russell D L,et al.Effect of varying glucose and glucosamine concentration in vitro on mouse oocyte maturation and developmental competence[J].Reprod Fertil Dev,2013,25(8):1095-1104.
[12] Albuz F K,Sasseville M,Lane M,et al.Simulated physiological oocyte maturation (SPOM):a novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes[J].Hum Reprod,2010,25(12):2999-3011.
[13] Eppig J J,O′Brien M J,Wigglesworth,K,et al.Effect of in vitro maturation of mouse oocytes on the health and lifespan of adult offspring[J].Hum Reprod,2009,24(4):922-928.