多种激素对小鼠卵母细胞体外成熟的影响
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摘要
为了探讨促卵泡素(FSH)、人绒毛膜促性膜激素(HCG)、雌二醇(E_2)三种激素和丙酮酸钠在卵母细胞体外成熟培养液中的最佳添加浓度,深入研究小鼠卵母细胞体外成熟的规律,进而完善小鼠卵母细胞体外成熟培养体系,试验对小鼠卵母细胞进行体外培养,在完全培养基中单独或组合添加FSH、HCG、E_2三种激素,并在此基础上添加不同浓度的丙酮酸钠,分别测定第一极体(PB1)排出率和自然裸卵细胞(NO)死亡率。结果表明:小鼠卵母细胞经体外培养14 h后,在完全培养基中只添加FSH,1 IU/mL FSH试验组的卵母细胞体外成熟率最高,PB1排出率为52.50%, NO死亡率为15.00%;在只添加HCG以及1 IU/mL FSH和HCG组合添加试验中, 1 IU/mL HCG试验组卵母细胞体外成熟率最高,PB1排出率分别为35.70%和53.83%,NO死亡率分别为67.50%和25.00%;在只添加E_2试验中,1μg/mL E_2试验组卵母细胞体外成熟率最高,PB1排出率为47.00%,NO死亡率为0;在添加1 IU/mL FSH、1 IU/mL HCG和1μg/mL E_2的条件下添加不同浓度的丙酮酸钠,0.04 mg/mL丙酮酸钠试验组卵母细胞体外成熟率最高,PB1排出率为75.80%,NO死亡率为11.37%。说明在完全培养基中添加1 IU/mL FSH、1 IU/mL HCG、1μg/mL E_2和0.04 mg/mL丙酮酸钠,对小鼠卵母细胞PB1排出的促进作用最好,对NO体外成熟培养效果最好。
In order to discuss the optimal concentrations of three hormones FSH,HCG,E_2,and sodium pyruvate in oocyte in vitro maturation medium,and to deeply understand the rules of maturation of mouse oocytes in vitro,the in vitro maturation culture system of mouse oocytes was further improved. In vitro culture of mouse oocytes was conducted in this experiment. Three hormones,FSH,HCG and E2,were added alone or in combination in the complete culture medium,followed by adding different concentrations of sodium pyruvate on this basis,for the determination of the first-polar excretion rate(PB1%) and the mortality rate of natural naked oocytes(NO). The results showed that after 14 h of in vitro culture of mouse oocytes,it was found that only FSH was added in the complete culture medium,and the in vitro maturation rate of oocytes in the 1 IU/mL FSH group was the highest;the first-polar expulsion rate PB1% was 52.50% and the mortality rate of NO was 15.00%. In the addition of HCG alone and the combination of 1 IU/mL FSH and HCG,the in vitro maturation rate of oocytes in the 1 IU/mL HCG group was the highest,with PB1% of 35.73% and 53.83%,and NO mortality rate of 67.50% and 25.00%,respectively. In the E_2 test alone,the in vitro maturation rate of oocytes in the 1 μg/mL E_2 group was the highest,PB1% was 47%,and NO mortality rate was 0. When adding 1 IU/mL FSH,1 IU/mL HCG and 1 μg/mL E_2 with different concentrations of sodium pyruvate,it was found that the in vitro maturation rate of oocytes in the 0.04 mg/mL sodium pyruvate group was the highest,PB1% was 75.80%,and NO mortality rate was 11.37%. The results suggested that adding 1 IU/mL FSH,1 IU/mL HCG,1 μg/mL E_2 and 0.04 mg/mL sodium pyruvate to complete culture medium had the best effect on promoting the excretion of PB1 of mouse oocytes and NO in vitro maturation culture.
In order to discuss the optimal concentrations of three hormones FSH,HCG,E_2,and sodium pyruvate in oocyte in vitro maturation medium,and to deeply understand the rules of maturation of mouse oocytes in vitro,the in vitro maturation culture system of mouse oocytes was further improved. In vitro culture of mouse oocytes was conducted in this experiment. Three hormones,FSH,HCG and E2,were added alone or in combination in the complete culture medium,followed by adding different concentrations of sodium pyruvate on this basis,for the determination of the first-polar excretion rate(PB1%) and the mortality rate of natural naked oocytes(NO). The results showed that after 14 h of in vitro culture of mouse oocytes,it was found that only FSH was added in the complete culture medium,and the in vitro maturation rate of oocytes in the 1 IU/mL FSH group was the highest;the first-polar expulsion rate PB1% was 52.50% and the mortality rate of NO was 15.00%. In the addition of HCG alone and the combination of 1 IU/mL FSH and HCG,the in vitro maturation rate of oocytes in the 1 IU/mL HCG group was the highest,with PB1% of 35.73% and 53.83%,and NO mortality rate of 67.50% and 25.00%,respectively. In the E_2 test alone,the in vitro maturation rate of oocytes in the 1 μg/mL E_2 group was the highest,PB1% was 47%,and NO mortality rate was 0. When adding 1 IU/mL FSH,1 IU/mL HCG and 1 μg/mL E_2 with different concentrations of sodium pyruvate,it was found that the in vitro maturation rate of oocytes in the 0.04 mg/mL sodium pyruvate group was the highest,PB1% was 75.80%,and NO mortality rate was 11.37%. The results suggested that adding 1 IU/mL FSH,1 IU/mL HCG,1 μg/mL E_2 and 0.04 mg/mL sodium pyruvate to complete culture medium had the best effect on promoting the excretion of PB1 of mouse oocytes and NO in vitro maturation culture.
引文
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[11] ZENG J,JIANG M,WU X,et al.SIRT4 is essential for metabolic control and meiotic structure during mouse oocyte maturation[J].Aging Cell,2018,17(4):e12789.
[12] 陈媛,杨菁,李洁,等.促性腺激素及雌二醇对小鼠卵母细胞体外成熟的影响[J].武汉大学学报(医学版),2007,28(5):568-587.
[13] 常卫华,张勇,王燕玲,等.重离子束辐照激素FSH/LH及丙酮酸钠对绵羊卵母细胞体外成熟的影响[J].核农学报,2011,25(2):391-396.
[14] GESHI M,TAKENOUCHI N,YAMAUCHI N,et al.Effects of sodium pyruvate in nonserum maturation medium on maturation,fertilization,and subsequent development of bovine oocytes with or without cumulus cells[J].Biol Reprod,2000,63(6):1730-1734.
[2] APPELTANT R,SOMFAI T,KIKUCHI K,et al.Influence of co-culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus-enclosed porcine oocytes in a defined system[J].Anim Sci J,2016,87(4):503-510.
[3] HIRAO Y.Conditions affecting growth and developmental competence of mannalian oocytes in vitro[J].Anim Sci J,2011,82(2):187-197.
[4] TOORI M A,MOSAVI E,NIKSERESHT M,et al.Influence of insulin-like growth factor-i on maturation and fertilization rate of immature oocyte and embryo development in NMRI mouse with TCM199 and α-MEM medium[J].J Clin Diaqn Res,2014,8(12):AC05-08.
[5] CHENG Y,ZHONG Z,LATHAM K E.Strain-specific spontaneous activation during mouse oocyte maturation[J].Fertil Steril,2012,98(1):200-206.
[6] 许惠艳,王塑天,杨巧蓉,等.不同生理状态及激素量对小鼠超数排卵及后续胚胎发育的影响研究[J].西南农业学报,2013,26(4):1691-1694.
[7] BOQLITTI Y,CHITWOOD J L,FRESNO C,et al.Comparison of different fertilisation media for an in vitro maturation-fertilisation-culture system using flow-cytometrically sorted X chromosome-bearing spermatozoa for bovine embryo production[J].Reprod Fertil Dev,2015,28:1695-1703.
[8] STROWITZKI T.In vitro Maturation(IVM) of human oocytes[J].Arch Gynecol Obstet,2013,288(5):971-975.
[9] WRENZYCKI C,STINSOFF H.Maturation environment and impact on subsequent developmental competence of bovine oocytes[J].Reprod Dom Anom,2013,48(1):38-43.
[10] COTICCHIO G,DAL C M,MIQNINI R M,et al.Oocyte maturation:gamete-somatic cells interactions,meiotic resumption,cytoskeletal dynamics and cytoplasmic reorganization[J].Hum Reprod Uprod,2015,21(4):427-454.
[11] ZENG J,JIANG M,WU X,et al.SIRT4 is essential for metabolic control and meiotic structure during mouse oocyte maturation[J].Aging Cell,2018,17(4):e12789.
[12] 陈媛,杨菁,李洁,等.促性腺激素及雌二醇对小鼠卵母细胞体外成熟的影响[J].武汉大学学报(医学版),2007,28(5):568-587.
[13] 常卫华,张勇,王燕玲,等.重离子束辐照激素FSH/LH及丙酮酸钠对绵羊卵母细胞体外成熟的影响[J].核农学报,2011,25(2):391-396.
[14] GESHI M,TAKENOUCHI N,YAMAUCHI N,et al.Effects of sodium pyruvate in nonserum maturation medium on maturation,fertilization,and subsequent development of bovine oocytes with or without cumulus cells[J].Biol Reprod,2000,63(6):1730-1734.