EGF和IGF-1对水牛卵母细胞体外成熟培养的影响

作者研究了在培养液中添加不同浓度的EGF、IGF-1以及EGF联合IGF-1对水牛卵母细胞体外成熟的影响。结果表明：①添加各种浓度的EGF都可以提高水牛卵母细胞的成熟率，其中50 ng/ml EGF有显著影响(P＜0.05)。②10、20 ng/ml的IGF 1对水牛卵母细胞体外成熟无显著影响(P＞0.05)； 30 ng/ml的IGF-1能显著提高水牛卵母细胞体外成熟率(P＜0.05)。③添加20 ng/ml EGF+30 ng/ml IGF-1组卵母细胞体外成熟率高于添加30 ng/ml的IGF-1组，显著高于添加20 ng/ml EGF组和对照组(P＜0.05)。可见，EGF和IGF-1对水牛卵母细胞体外成熟有协同作用。     

表皮生长因子和胰岛素样生长因子对水牛卵母细胞胞质成熟的影响

以皮层颗粒（CG）单层分布于质膜下做为卵母细胞胞质成熟的标志,利用CG荧光染色法,研究了不同浓度表皮生长因子（EGF）和胰岛素样生长因子（IGF-1）及其组合对水牛卵母细胞胞质成熟的影响。结果发现：（1）添加不同浓度的EGF（10、20、30、50ng／mL）都可以提高胞质的成熟率,但是组间差异不显著（P〉0．05）;皮质颗粒的分布随着EGF浓度的升高逐步由中间分布向皮层分布转变;（2）在成熟液中添加IGF-130ng／mL时效果较好,能显著提高卵母细胞胞质的成熟率;皮质颗粒的分布随着IGF-1浓度的升高逐步由中间分布向皮层分布转变,在添加IGF一130ng／mL时,在皮层分布最好,随着IGF-1量的进一步增加,皮质颗粒又向中间分布转变;（3）添加20ng／mL EGF＋30ng／mL IGF-1组卵母细胞体外成熟率高于添加30ng／mL的IGF-1组,显著高于添加20ng／m LEGF组和对照组（P〈0.05）。由此表明,EGF和IGF-1对水牛卵母细胞体外成熟有协同作用。     

兔卵母细胞体外成熟培养影响因素的研究

试验旨在探讨卵丘细胞包裹层数、表皮生长因子(epidermal growth factor,EGF)和谷胱甘肽(glutathione,GSH)对兔卵母细胞体外成熟培养的影响。分别采用切割法和针刺挤压法取母兔卵巢表面的卵丘卵母细胞复合体(cumulus oocyte complexes,COCS),将获取的COCS在基础培养液中添加不同浓度表皮生长因子(0ng/mL、10ng/mL、20ng/mL、30ng/mL)及不同浓度的谷胱甘肽(0μmol/mL、0.5μmol/mL、1.0μmol/mL、1.5μmol/mL、2.0μmol/mL)进行体外成熟培养并观察第一极体排出率。结果表明:①手术刀切割法和针刺挤压法平均回收卵母细胞率分别为(22.83、39.29)枚,针刺挤压法显著高于手术刀切割法(P<0.05)。②3层以上卵丘细胞包裹的卵母细胞体外成熟率为81.27%,显著高于1~3层卵丘细胞包裹的体外成熟率63.75%和裸卵细胞体外成熟率23.45%(P<0.05)。③添加20ng/mL的EGF对兔卵母细胞体外成熟培养后成熟率为82.14%,效果最好,且显著高于对照组10ng/mL和30ng/mL EGF组(P<0.05)。④添加不同浓度的谷胱甘肽可以促进和提高兔卵母细胞体外成熟率,其中添加1.0μmol/mL的谷胱甘肽时,卵母细胞成熟率为71.44%,与添加1.5μmol/mL组的相比差异不显著(P>0.05),但显著高于其他组(P<0.05)。结论为添加20ng/mL的EGF和1.0μmol/mL谷胱甘肽,对用针刺挤压法获得3层以上卵丘细胞包裹的兔卵母细胞体外培养效果最佳。     

EGF和IGF-1对山羊卵母细胞体外成熟的影响

研究了在培养液中添加不同浓度的EGF、IGF-1以及EGF联合IGF-1对山羊有腔卵母细胞体外成熟的影响。结果表明: (1)10, 20, 30μg/LEGF对山羊卵母细胞体外成熟无显著影响(P>0.05)。(2) 10μg/L的IGF-1对山羊卵母细胞体外成熟无显著影响(P>0.05); 20, 30μg/L的IGF-1能显著提高山羊卵母细胞体外成熟率 (P<0.05)。(3)添加 10μg/LEGF+20μg/LIGF-1组卵母细胞体外成熟率显著高于添加 20μg/L的IGF-1组(P<0.05),极显著高于添加 10μg/LEGF组和对照组(P<0.01)。可见,EGF和IGF-1对山羊卵母细胞体外成熟有协同作用。     

表皮生长因子对水牛胚胎体外发育及凋亡的影响

为了探讨表皮生长因子(EGF)对水牛早期胚胎体外发育及凋亡的影响,通过收集屠宰场卵巢卵母细胞进行体外成熟和体外受精,将假定的受精卵置于含不同浓度EGF(0,25,50和100 ng/mL)的培养液中培养,检查分裂率和囊胚发育率,用细胞凋亡试剂盒(Annexin-V-FluosStaining kit)试剂染色,统计囊胚细胞凋亡率和坏死率。结果表明:50 ng/mL EGF组的孵化囊胚率显著高于对照组(P<0.05),该组细胞凋亡率和坏死率显著低于对照组(P<0.05)。100 ng/mL EGF的卵裂率、囊胚率、D7囊胚率和孵化囊胚率显著低于对照组和其他试验组(P<0.05)。细胞凋亡率和坏死率显著高于其他各组(P<0.05)。提示:一定浓度的EGF可提高囊胚孵化率,并可抑制胚胎细胞的凋亡。     

激素及生长因子对牛卵母细胞体外成熟的影响

在卵母细胞体外成熟液(含FSH)中分别添加10, 30, 50ng/mL表皮生长因子(EGF),24h检查其成熟率.结果表明:添加 EGF50ng/mL时牛卵母细胞第一极体排出率显著提高,达85.4%(P＜0.01);同时发现在含FSH和EGF的成熟液中添加HMG(尿促性素)并不能提高牛卵母细胞体外成熟效果(P>0.05);同时还发现单独添加HMG成熟效果显著好于FSH(P＜0.05).     

人尿促性腺激素、雌二醇及表皮生长因子对牛卵母细胞体外成熟质量的影响

试验旨在研究不同激素配比及表皮生长因子(EGF)浓度对牛卵母细胞体外成熟及卵母细胞质量的影响。将随机分组的卵丘-卵母细胞复合体于添加FSH+LH、HMG、FSH+LH+E₂、HMG+E₂ 4种不同激素组合配比的成熟基础液中培养,对比其体外成熟率,比较了EGF对牛卵母细胞体外成熟率和孤雌胚胎体外发育的影响,并采用TUNEL法检测添加不同浓度EGF的牛孤雌激活囊胚细胞凋亡情况。结果表明,添加HMG的成熟试验结果稳定,E₂对牛卵母细胞成熟有一定的促进作用,HMG+E₂联合使用可以得到高效稳定的成熟结果;在此基础上,在成熟液中添加30 ng/mL EGF对牛卵母细胞的成熟质量、胚胎发育及降低胚胎细胞凋亡都有明显的促进作用。因此,在体外成熟培养液中添加0.075 IU/mL HMG、1 μg/mL E₂和30 ng/mL EGF对牛卵母细胞的成熟和质量较为有益。     

表皮生长因子对水牛卵母细胞体外培养核质成熟的影响

为了探讨表皮生长因子（EGF）对水牛卵泡卵母细胞体外培养核质成熟的影响，在以TCM199为基础的成熟液中加入不同浓度的EGF（0、10、25、50、100 ng/ml），体外成熟培养24~26 h，观察第一极体（PB1）的排放；随后进行孤雌激活检测其分裂率、囊胚发育率、囊胚孵化率，并用Hoechst33342染色后计算囊胚的细胞数。结果发现添加EGF各组的卵母细胞第一极体排放率显著提高（P<0.05）；成熟液中添加25 ng/ml EGF时，卵裂率及囊胚发育率（分别为80.0%、44.8%）明显高于对照组（分别为69.3%、31.9%，P<0.05），但对囊胚的细胞数影响不大。EGF不仅促进水牛卵母细胞体外培养的核成熟，而且有利于卵母细胞的胞质成熟，其中EGF的最佳浓度为25 ng/ml。     

不同培养液对牛卵母细胞体外成熟及孤雌胚胎发育的影响

为研究表皮生长因子(EGF)、β-巯基乙醇(β-ME)、亚硫磺酸(HTAU)对牛卵母细胞体外成熟(IVM)及孤雌激活胚胎体外发育(IVC)效果的影响,实验采集牛卵巢采用切割法收集卵丘-卵母细胞复合体(COCs)随机处于不同浓度EGF、β-ME培养液成熟培养24 h后孤雌激活,研究其在不同胚胎培养液中的后续发育,以期筛选出最好的牛卵母细胞IVM-IVC条件。结果表明:添加25、50、100 ng/mL EGF组的成熟率、卵裂率均高于对照组,其中50 ng/mL EGF组的成熟率、卵裂率与对照组差异显著(P<0.05);50、100、500μmol/L的β-ME对牛卵母细胞体外成熟没有促进作用;50 ng/mL EGF与50、100μmol/Lβ-ME组合并无协同作用;胚胎培养基中添加不同浓度EGF对早期胚胎的体外发育无显著影响;而添加100μmol/Lβ-ME组的囊胚率、囊胚细胞数均极显著高于对照组(P<0.01);在成熟液中添加50 ng/mL EGF和100μmol/Lβ-ME、胚胎培养液中添加0.5 mmol/L HTAU孤雌胚发育最好。     

卵丘细胞对猪卵母细胞体外成熟及孤雌胚胎体外发育的影响

为探讨表皮生长因子(epidermal growth factor,EGF)的添加浓度及脱卵丘细胞时间对猪卵母细胞体外成熟及孤雌胚胎体外发育的影响.试验通过在体外成熟液中添加不同浓度(0、10、15、20、30、40 ng/mL)的EGF来研究其对培养44 h的卵母细胞成熟率以及孤雌胚胎发育的影响;在培养开始后的不同时间(18、24、38、44 h)进行脱卵丘细胞处理来研究不同时间脱卵丘处理对培养44 h的卵母细胞成熟率以及孤雌胚胎发育的影响.结果表明,成熟培养基中添加10 ng/mL EGF能显著提高卵母细胞的卵裂率和囊胚率(P <0.05).共培组和独培组卵母细胞培养18 h后脱卵丘细胞成熟率均低于44 h,但差异不显著(P >0.05);共培组卵母细胞培养18 h后脱卵丘细胞的卵裂率和囊胚率显著高于培养44 h(P <0.05);独培组卵母细胞培养18 h后脱卵丘细胞的卵裂率与44 h无显著差异(P >0.05),但囊胚率显著高于培养44 h后脱卵丘细胞(P <0.05).添加10 ng/mL EGF对猪卵母细胞体外成熟及孤雌胚胎体外发育较好;卵母细胞培养18 h后脱卵丘细胞可提高孤雌胚胎早期发育能力.     

Effects of EGF and IGF-I on in vitro Maturation and Cleavage of Ovine Oocytes

The study investigated the effects of epidermal growth factor(EGF) and insulin-like growth factor 1(IGF-I),alone or together,on the in vitro maturation and cleavage of ovine oocytes,aimed to optimize the in vitro maturation conditions for ovine oocytes.The results showed that the maturation and cleavage rates were 71.2% and 45.5% respectively when the medium was supplemented with 50 ng/mL EGF alone,which was significantly higher than other EGF supplemented groups (0,10,20,30,and 40 ng/mL) (P<0.05).The highest maturation and cleavage rates were 72.9% and 45.7% when the EGF concentration reached 100 ng/mL.The maturation and cleavage rates were 70.7% and 58.5% with 40 ng/mL IGF-I supplemented,which were significantly higher than other treatments (0,10,20,60,80,and 100 ng/mL) (P<0.05).The lowest maturation and cleavage rates were 38.8% and 20.0% when the IGF-I concentration reached 100 ng/mL (P<0.05).When 50 ng/mL EGF and 40 ng/mL IGF-I were used concomitantly,the maturation and cleavage rates were 85.6% and 61.0% respectively,which were significantly higher than the treatments with EGF or IGF-I alone (P<0.05).     

Regulation of in vitro growth of preantral follicles by growth factors in goats

Goat preantral follicles were cultured to investigate the effects of insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the in vitro growth and viability of oocytes. Preantral follicles were isolated mechanically and enzymatically (using collagenase and DNase) from prepuberal goat ovaries. The working medium was composed of Defined Eagle's Minimum Essential Medium (DMEM) supplemented with HEPES (20 mM), 10% fetal calf serum (FCS), hypoxanthine (2 mM), dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) (2 mM), penicillin (75 ng/ml) and streptomycin (50 ng/ml). The culture medium consisted of the working medium with follicle stimulating hormone (FSH) (100 ng/ml) and hydrocortisone (40 ng/ml) added. In the experiment, goat preantral follicles were cultured for 9 days in the culture medium and in the culture medium supplemented with either IGF-I (100 ng/ml), EGF (50 ng/ml), bFGF (50 ng/ml) or IGF-I (100 ng/ml)+EGF (50 ng/ml). The results indicated that IGF-I (100 ng/ml) effectively maintained the survival of oocytes and promoted their growth; EGF (50 ng/ml) enhanced the survival rate of oocytes but had a negative effect on oocyte growth; bFGF (50 ng/ml) stimulated oocyte survival but had no obvious effect on their growth while IGF-I (100 ng/ml) and EGF (50 ng/ml) in combination had a greater effect on both survival and growth rate of oocytes than IGF-I or EGF alone. The supplementation of IGF-1 and EGF to the culture medium is recommended in the culture of goat preantral follicles.     

培养时间和表皮生长因子对兔卵母细胞体外成熟的影响

本试验旨在探讨培养时间和表皮生长因子(EGF)对兔卵母细胞体外成熟及受精的影响。分别用切割法和针刺挤压法取经超排后母兔卵巢表面的卵母细胞颗粒细胞复合体(COCs),将COCs分别在不同时间(24、30、36及40h)和在基础培养液中添加不同浓度表皮生长因子(0、50、100ng/mL)进行体外成熟培养并观察其第一极体排出率。结果表明:①针刺挤压法获得COCs数明显高于切割法(P0.05);②体外成熟培养时间30(36)h的第一极体排出率高于24(40)h(P0.05);③添加50和100ng/mLEGF组卵母细胞第一极体排出率明显高于对照组(P0.05)。因此,①兔卵巢卵母细胞的获得方法以针刺挤压法较好;②兔卵母细胞成熟时间以30～36h为宜;③EGF对兔卵母细胞体外成熟有促进作用,在基础培养液中添加100ng/mLEGF为宜。     

谷氨酰胺和IGF-I对晋岚绒山羊卵母细胞体外成熟的影响

为了探讨在成熟培养液中添加谷氨酰胺和IGF-I对晋岚绒山羊母羔卵母细胞体外成熟的影响。随机选择12只6-8周龄晋岚绒山羊母羔为试验动物,用FSH进行超数排卵,活体采集卵母细胞,并对未成熟的卵母细胞进行体外成熟培养。在培养液中添加0、50、100和150μg/mL的谷氨酰胺及0、25、50和100 ng/mL的IGF-I。结果表明：培养液中谷氨酰胺添加量为100μg/mL时,能显著提高卵母细胞成熟率（P〈0.05）;在培养液中添加100μg/mL谷氨酰胺的基础上,再添加50 ng/mL的IGF-I可以进一步显著提高卵母细胞的成熟率（P〈0.01）。     

Effect of Human Menopausal Gonadotropin,17β-estradiol and Epidermal Growth Factor on the Quality of in vitro Maturation Bovine Oocytes

For optimizing in vitro maturation system of bovine oocytes,we firstly examined the influence of four different hormonal regimes(FSH+LH,HMG,FSH+LH+E₂ and HMG+E₂) on oocyte maturation rates.Then we studied the effects of epidermal growth factor (EGF) in the above defined medium on bovine oocyte maturation,in vitro development and quality of parthenogenetic embryos.The cell apoptotic index of parthenogenetic blastocysts was detected by TUNEL.No significant difference was observed in maturation rates in four groups supplemented with different hormones.However,human menopausal gonadotropin (HMG) provided steady maturation results in replicates.Maturation of oocytes was promoted by supplementation with 17β-estradiol (E₂).Combination of HMG and E₂ gave rise to steady and efficient mature results.The presence of EGF at 30 ng/mL concentration significantly increased maturation rate and blastocyst rate and reduced apoptotic cells in parthenogenetic blastocysts.Therefore,the optimal oocyte maturation solution could be supplemented with 0.075 IU/mL HMG,1 μg/mL E₂ and 30 ng/mL EGF.