摘要
旨在探讨玻璃化冷冻-解冻对牦牛未成熟卵母细胞发育能力及卵丘-卵母细胞复合体(COCs)转录组的影响,为完善牦牛COCs冷冻保存技术提供理论依据。本研究将未经成熟培养的牦牛COCs进行玻璃化冷冻-解冻后分为2组,A组:COCs体外成熟(IVM)后用普通牛精子进行体外受精(IVF),获得的受精卵在G-1胚胎培养液中培养72 h后转入G-2培养液培养96 h;B组:IVF后,受精卵在G-1培养液培养120 h后转入G-2培养液培养48 h;以未进行冷冻处理的新鲜COCs作为对照组(C组):IVF后,受精卵在G-1培养液培养72 h后转入G-2培养液培养96 h。对牦牛新鲜COCs(n=3)和玻璃化冷冻-解冻的COCs(n=3)进行扩增、建库和转录组测序(RNA-seq)分析。结果发现,B组的卵裂率、囊胚率显著高于A组(P<0.05),但A组和B组的卵裂率、囊胚率均显著低于C组(P<0.05)。以|log2(fold change)|≥2,Q<0.05为阈值,牦牛冻融COCs相对于新鲜COCs共筛选出851个差异表达基因(DEGs),其中上调846个,下调5个。GO分析表明,DEGs主要富集于生物过程、细胞组分和分子功能3大类;KEGG注释结果表明,DEGs富集到258条通路,其中16条通路显著富集(P<0.05)。研究表明,IVF后在G-1培养液中培养120 h可以提高牦牛玻璃化冷冻卵母细胞的后续发育能力;玻璃化冷冻影响牦牛COCs转录组,从而降低卵母细胞的发育潜力。该发现为完善牦牛COCs玻璃化冷冻技术提供了一定的理论基础。
The objective of this study was to investigate the effect of vitrification/thawing on developmental competence of immature oocytes and cumulus-oocyte-complexes(COCs) transcriptome of yaks(Bos grunniens), in order to provide theoretical foundation to improve vitrification techniques of yak COCs. Vitrified/thawed yak immature COCs were divided into two groups. Group A: COCs were in vitro matured(IVM) and in vitro fertilized(IVF) with cattle sperms, then in vitro cultured(IVC) in G-1 for 72 h followed by IVC in G-2 for 96 h;Group B: after IVF, zygotes were IVC in G-1 for 120 h followed by IVC in G-2 for 48 h. Fresh immature yak COCs were used as the control(Group C): after IVF, zygotes were IVC in G-1 for 72 h followed by IVC in G-2 for 96 h. Yak fresh immature COCs(n=3) and vitrified/thawed immature COCs(n=3) were used for amplification, library preparation and RNA-seq analysis. The results showed that cleavage rate and blastocyst rate in Group B were significantly higher than those in Group A(P<0.05), but cleavage rate and blastocyst rate in both Group A and B were significantly lower than those in Group C(P<0.05). When |log2(fold change)| ≥2 and Q-value <0.05 were set as thresholds for identifying deferentially expressed genes(DEGs), a total of 851 DEGs were detected, of which, 846 were up-regulated and 5 were down-regulated in virtrified/thawed COCs compared to fresh COCs. GO analysis showed DEGs were classified into 3 categories: biological processes, cellular components and molecular functions. KEGG annotation showed that there were 258 pathways, of which, 16 were significantly enriched(P<0.05). In conclusion, the results showed that IVC in G-1 for 120 h after IVF could increase subsequent developmental competence of vitrified yak oocytes. Vitrification affected transcriptome of yak COCs, which reduced developmental potential of yak vitrified oocytes. The result provided a theoretical basis for improving vitrification techniques of yak COCs.
作者
杨远潇
字向东
YANG Yuanxiao;ZI Xiangdong(College of Life Science and Technology,Southwest Minzu University,Chengdu 610041,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2020年第2期288-298,共11页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家重点研发计划项目(2018YFD0502303)
中央高校基本科研业务费专项资金项目(2015NZYTD02)。
