Ghrelin对绵羊卵母细胞体外成熟的影响

Effects of Ghrelin on in Vitro Maturation of Ovine Oocytes

该研究旨在探讨外源Ghrelin对绵羊卵母细胞体外成熟的调控作用。在成熟培养液中分别添加浓度为0、100、200和300ng/m L的Ghrelin,进行绵羊卵母细胞体外培养,成熟培养24h,观察统计处于MII期的细胞数目。选择最佳添加浓度,在体外成熟培养0、6、8、14、16、24h,通过Hoechst33342染色持续检测卵细胞中细胞核成熟随时间变化,并对不同培养时期细胞进行实时荧光定量PCR检测,以证明细胞质量相关基因的相对表达量。成熟培养24h时,添加0、100、200、300 ng/m LGhrelin组卵母细胞成熟率分别为:63.7%(220/345)、69.4%(230/332)、85.6%(270/316)、75.9%(231/305)。200ng/m L Ghrelin组显著高于对照组(P<0.01),其余组并未显著高于对照组(P<0.05);染色证明:200ng/m L Ghrelin组于成熟培养0h、6h、14h、16h到达GV、GVBD、MI、MII期(P<0.05);实时荧光定量PCR证明:对卵母细胞成熟GV、GVBD、MI、MII期,Ghrelin受体基因GH-SR-1a和卵母细胞质量相关基因OCT-4、GLUT1和IFNT的相对表达时间提前,但并不显著影响这些基因的表达水平。外源Ghrelin参与了绵羊卵母细胞的体外成熟调控,可在没有改变其成熟过程的前提下加速卵母细胞的体外成熟过程,但对绵羊卵母细胞的质量和进一步发育相关的基因的表达没有明显影响。

To study the regulation effect of exogenous Ghrelin in vitro maturation of sheep oocytes,0,100,200 and 300 ng/mL Ghrelin were added in mature culture medium,in vitro culture of sheep oocytes and 24h in mature cul-ture,and the observation statistics were in the number of MII period.To choose the best dosage and mature in vitro-culture 0,6,8,14,16,24 h,continuous detection of egg in the nucleus by dyeing Hoechst33342 mature and change over time,and the real-time fluorescent quantitative PCR to detect cell culture period,to show that cell quality rela-tive expression of related genes.When the mature culture was incubated for 24h,the maturation rate of the oocytes of 0,100,200,300ng/mLGhrelin group was 63.7%(220/345),69.4%(230/332),85.6%(270/316)and 75.9%(231/305).The 200ng/mL Ghrelin group was significantly higher than the control group(P<0.01),while the remaining group was not significantly higher than the control group(P<0.05).The results showed that 200 ng/mL Ghrelin group was established in mature culture 0h,6h,14h,16h to GV,GVBD,MI,MII(P<0.05).Real-time fluorescent quantitative PCR to prove:the GV oocytes mature,GVBD,MI,MII,Ghrelin receptor gene GHSR-1a and oocyte quality related gene OCT-4,the relative expression of GLUT1 and IFNT ahead of time,but not dramatically affect the gene expression level.exogenous Ghrelin participated in the sheep oocyte in vitro maturation regulation,under the premise of no change in the mature process can be accelerated in vitro maturation of oocytes,but the quality of sheep oocytes and related gene expression did not significantly affect the further development.