Data_Sheet_1_Melatonin improves the vitrification of sheep morulae by modulating transcriptome.ZIP

Embryo vitrification technology is widely used in livestock production, but freezing injury has been a key factor hindering the efficiency of embryo production. There is an urgent need to further analyze the molecular mechanism of embryo damage by the vitrification process. In the study, morulae were collected from Hu sheep uterine horns after superovulation and sperm transfusion. Morulae were Cryotop vitrified and warmed. Nine morulae were in the vitrified control group (frozen), and seven morulae were vitrified and warmed with 10−5 M melatonin (melatonin). Eleven non-frozen morulae were used as controls (fresh). After warming, each embryo was sequenced separately for library construction and gene expression analysis. p < 0.05 was used to differentiate differentially expressed genes (DEG). The results showed that differentiated differentially expressed genes (DEG) in vitrified morulae were mainly enriched in protein kinase activity, adhesion processes, calcium signaling pathways and Wnt, PI3K/AKT, Ras, ErbB, and MAPK signaling pathways compared to controls. Importantly, melatonin treatment upregulated the expression of key pathways that increase the resistance of morulae against vitrification induced damage. These pathways include kinase activity pathway, ErbB, and PI3K/Akt signaling pathway. It is worth mentioning that melatonin upregulates the expression of XPA, which is a key transcription factor for DNA repair. In conclusion, vitrification affected the transcriptome of in vivo-derived Hu sheep morulae, and melatonin had a protective effect on the vitrification process. For the first time, the transcriptome profiles caused by vitrification and melatonin in sheep morulae were analyzed in single embryo level. These data obtained from the single embryo level provide an important molecular mechanism for further optimizing the cryopreservation of embryos or other cells.

胚胎玻璃化冷冻技术在畜牧生产中应用广泛，但冷冻损伤始终是制约胚胎生产效率的核心瓶颈，目前亟需进一步解析玻璃化冷冻过程造成胚胎损伤的分子机制。本研究中，研究人员在超数排卵与输精后，从湖羊子宫角采集桑葚胚（morulae）。采用Cryotop法对桑葚胚进行玻璃化冷冻并复苏：其中9枚桑葚胚归入玻璃化冷冻对照组（仅冷冻复苏组），另有7枚经10⁻⁵ M褪黑素（melatonin）处理后进行玻璃化冷冻复苏；另取11枚未冷冻的桑葚胚作为新鲜对照组。复苏后，对每枚胚胎单独开展文库构建与基因表达测序分析，以p<0.05作为筛选差异表达基因（differentially expressed genes, DEG）的阈值。结果显示，与对照组相比，玻璃化冷冻桑葚胚中筛选得到的差异表达基因主要富集于蛋白激酶活性、黏附过程、钙信号通路以及Wnt、PI3K/AKT、Ras、ErbB与MAPK信号通路。值得注意的是，褪黑素处理上调了可增强桑葚胚抵御玻璃化冷冻损伤的关键通路的表达，此类通路涵盖激酶活性通路、ErbB与PI3K/Akt信号通路。值得一提的是，褪黑素可上调XPA的表达，而XPA是DNA修复的关键转录因子。综上，玻璃化冷冻会影响体内来源湖羊桑葚胚的转录组，而褪黑素对玻璃化冷冻过程具有保护作用。本研究首次在单胚胎水平解析了绵羊桑葚胚经玻璃化冷冻及褪黑素处理后的转录组特征，本次获得的单胚胎水平转录组数据，为进一步优化胚胎或其他细胞的冷冻保存技术提供了重要的分子机制依据。