重组KP52蛋白在小鼠GV期卵母细胞体外培养成熟的作用及相关机制

Effects and relevant mechanism of recombinant KP52 protein on the maturation of mouse GV oocytes in vitro culture

目的探索kisspeptin蛋白对生发泡(GV)期卵母细胞体外成熟(IVM)的影响及其机制。方法通过体外培养小鼠卵丘-卵母细胞复合体(COCs)过程中添加KP52蛋白,观察卵母细胞第一极体排出情况,评估卵母细胞成熟率,利用皮质颗粒染色观察卵母细胞胞质成熟情况,利用荧光定量的方法检测小鼠卵母细胞内cAMP水平。结果与IVM中的传统促成熟因子FSH相比,KP52蛋白组卵母细胞成熟率较低(F=240.556,P<0.01),但囊胚形成率显著升高(F=66.204,P<0.01),MⅡ期卵母细胞胞质中央的皮质颗粒向卵母细胞膜下扩散的更明显,MⅡ期卵母细胞胞质中皮质颗粒密度明显降低(F=48.602,P<0.01)。此外,体内成熟的卵母细胞和添加KP52蛋白进行体外培养的卵母细胞中cAMP水平在2h均有明显的增高,而添加FSH进行体外培养的卵母细胞内cAMP水平呈现下降趋势。结论KP52蛋白可以通过直接与COCs上的KISS1R结合,提高GV期卵母细胞内cAMP水平从而促进卵母细胞胞质胞核成熟同步化,改善IVM结局。

Objective To evaluate the effects of kisspeptin protein on in vitro maturation(IVM)of germinal vesicle(GV)oocyte and the underlying mechanisms.Methods The KP52 protein was added in the process of culturing mouse cumulus-oocyte complexes(COCs)in vitro to observe the extrusion of first polar body,evaluate the maturation rate of oocytes,and observe the oocyte cytoplasmic maturation by cortical granule staining.The level of cyclic adenosine monophosphate(cAMP)in mouse oocytes was detected by fluorescence quantitative method.Results Compared with the traditional maturation promoting factors follicle stimulating hormone(FSH)in IVM,the oocytes maturation rate in KP52 protein group was lower(F=240.556,P<0.01),but the blastocyst formation rate was significantly higher(F=66.204,P<0.01),and the diffusion of cortical granules in the central cytoplasm of MⅡoocytes to the oocyte membrane was more obvious,and the density of cortical granules in MⅡoocytes decreased significantly(F=48.602,P<0.01).In addition,the level of cAMP in mature oocytes in vivo and oocytes cultured in vitro with KP52 protein increased significantly at 2 h,while the level of cAMP in oocytes cultured in vitro with FSH protein decreased.Conclusion KP52 protein can directly bind to kisspeptin receptor(KISS1 R)on COCs to increase the level of cAMP in oocytes at GV stage,thereby promoting the synchronization of oocyte cytoplasmic and nuclear maturation,and improving the outcome of IVM.