小檗碱对玻璃化冷冻猪卵母细胞脂滴含量及胚胎发育的影响

Effects of berberine on lipid droplets content and embryo development in vitrified frozen porcine oocytes

【目的】探讨小檗碱对玻璃化冷冻猪卵母细胞脂滴含量及体外受精胚胎发育效果的影响。【方法】体外成熟液和胚胎培养液中添加小檗碱为Ber组,未添加小檗碱为对照组,MII期卵母细胞进行冷冻液处理为冷冻液处理对照组和冷冻液处理+Ber组;采用开放式拉长塑料细管法对卵母细胞玻璃化冷冻与解冻,试验各组别为GV期冷冻组、GV期冷冻+Ber组、MII期冷冻组、MII期冷冻+Ber组。观察卵母细胞体外成熟效果及其体外受精胚胎体外发育效果;观察卵母细胞冷冻化学损伤存活率及其体外受精胚胎发育率;采用尼罗红染色法观察卵母细胞中脂滴含量,观察卵母细胞玻璃化冷冻解冻后存活率及其体外受精胚胎发育率;利用差异染色法和TUNEL法计数囊胚内细胞团和细胞总数以及细胞凋亡数。【结果】Ber组能够极显著促进猪卵母细胞体外成熟,显著降低猪卵母细胞玻璃化冷冻的化学损伤,促进猪卵母细胞冷冻解冻后的细胞存活率,而猪MII期卵母细胞相对于GV期卵母细胞具有很好的冷冻解冻后的存活率;小檗碱能降低猪卵母细胞玻璃化冷冻后的脂滴含量,MⅡ期卵母细胞冷冻前后脂滴含量均低于GV期冷冻前后;小檗碱能够促进猪GV期卵母细胞玻璃化冷冻解冻后的体外成熟,且有利于猪卵母细胞玻璃化冷冻解冻后早期胚胎的发育,相对于冷冻MII期卵母细胞,冷冻GV期卵母细胞具有更好的体外发育效果;小檗碱能够提高猪卵母细胞玻璃化冷冻解冻后体外发育囊胚内细胞团数与细胞总数,降低其细胞凋亡,有利于提高猪卵母细胞玻璃化冷冻解冻后体外受精胚胎发育至囊胚的质量。而GV期相对MII期则冷冻效果更佳。【结论】小檗碱能够通过降低玻璃化冷冻猪卵母细胞脂滴含量,从而提高其冷冻解冻后体外成熟率和体外受精胚胎发育质量;GV期玻璃化冷冻卵母细胞相对于MII期,在冷冻解冻后其体外受精胚胎发育质量有更好提高。相关机制还有待今后进一步研究。

【Objective】To investigate the effects of berberine on lipid content and in vitro fertilization embryo development in vitrified porcine oocytes.【Methods】Ber was added to the maturation medium and embryo culture medium as Ber group,and Ber was not added as control group,the effect of chemical damage on oocytes was treated with cryopreservation solution of MII oocytes,and the groups were frozen solution treatment control group and frozen solution treatment+Ber group.The vitrification and thawing of oocytes by Open pulled straw method in GV vitrification group,GV vitrification+Ber group,MII vitrification group and MII vitrification+Ber group.The effect of oocytes in vitro maturation and in vitro fertilization embryos development were observed.The survival rate of cryopreservation chemical damage of oocytes and the development rate of IVF embryos were observed.Lipid droplets content in oocytes by Nile Red staining was observed,the survival rate and IVF embryo development rate of oocytes after vitrification and thawing were observed.Differential staining and TUNEL method were used to number of inner cell masses and total cells counts and apoptosis counts in blastocysts.【Results】Ber significantly promoted the IVM of porcine oocytes,significantly reduced the chemical damage of porcine oocytes vitrification,and promoted the survival rate of porcine oocytes after vitrification and thawing,compared with GV stage oocytes,porcine MII stage oocytes had a good survival rate after vitrification and thawing;Ber reduced the lipid droplets content in porcine oocytes after vitrification,and the lipid droplets content in MII oocytes before and after vitrification were lower than those before and after GV vitrification.Ber promoted the porcine GV stage oocytes IVM after vitrification and thawing,and was beneficial to the development of early embryos after vitrification and thawing of porcine oocytes,compared with vitrification MII stage oocytes,vitrification GV stage oocytes had better in vitro development effect.Ber increased the number of blastocyst inner cell masses and the total number of cells after vitrification and thawing of pig oocytes,and reduced the apoptosis,which was beneficial to improve the quality of IVF embryo development to blastocyst after vitrification and thawing of porcine oocytes.The vitrification effect of GV stage was better than that of MII stage.【Conclusion】Ber improves the IVM rate and the quality of IVF embryo development after vitrification and thawing by reducing the content of lipid droplets in vitrified porcine oocytes.Compared with the MII stage,the GV stage vitrified frozen oocytes shows a better improvement in the quality of IVF embryo development after freezing and thawing.The relevant mechanism is to be further studied in the future.