Bunel A, Jorssen E P, Merckx E, Leroy J L, Bols P E, Sirard M A
Centre de Recherche en Biologie de la Reproduction, Faculté des Sciences de l'Agriculture et de l'Alimentation, Département des Sciences Animales, Université Laval, Québec, Quebec, Canada.
Gamete Research Center, Laboratory for Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, Wilrijk, Belgium.
Theriogenology. 2015 Jan 15;83(2):228-37. doi: 10.1016/j.theriogenology.2014.09.019. Epub 2014 Sep 18.
Studying cumulus cell (CC) transcriptome is of great interest as it could provide a noninvasive method to assess oocyte quality. In cattle, the search for quality markers has not been done with cumulus-oocyte complexes (COCs) cultured individually from maturation to blastocyst stage. Here, differences between high- and low-potential COCs were examined by transcriptomic analysis of CC biopsies obtained from COCs of 2 to 6 mm follicles (n = 249; eight replicates) before individual in vitro maturation, fertilization, and culture until Day 8 after fertilization. Each COC was individually tracked and categorized based on his fate: embryo at blastocyst stage (CC-Blast) or embryo arrested at 2- to 8-cell stage (CC-2-8-cells). Average blastocyst rates were 27.7% for individual culture and 31.2% for group control (not significantly different). For transcriptomic analysis, five cumulus biopsies per replicate were pooled for each fate. Three CC replicates underwent transcriptomic analysis using RNA microarray assay. Some clear differences in gene expression between the CC-Blast and the CC-2-8-cell groups were identified. Considering a 1.5-fold change (P < 0.05), 68 genes were differentially expressed between the CC-Blast and CC-2-8-cells. Quantitative reverse transcription-polymerase chain reaction validations were performed for 12 selected genes: six upregulated genes for each COC fate. Higher expression of 1-acylglycerol-3-phosphate O-acyltransferase 9 (AGPAT9) (lipid metabolism), Chloride intracellular channel 3 (CLIC3), Keratin 8 (KRT8), and Lumican (LUM) (molecular transport) was observed in CC-2-8-cells (P < 0.05). The CC-Blast fate analysis revealed a significantly higher expression of Glycine amidinotransferase (L-arginine:glycine amidinotransferase) (GATM) (posttranslational modification, amino acid metabolism, and free radical scavenging). This newly identified set of genes could provide new markers to distinguish COCs associated with good quality embryos from COCs with limited developmental potential.
研究卵丘细胞（CC）转录组具有重要意义，因为它可以提供一种非侵入性方法来评估卵母细胞质量。在牛中，尚未对从成熟到囊胚阶段单独培养的卵丘-卵母细胞复合体（COC）进行质量标记物的寻找。在此，通过对从2至6毫米卵泡的COC（n = 249；8个重复）中获取的CC活检组织进行转录组分析，在个体体外成熟、受精并培养至受精后第8天之前，检查了高潜力和低潜力COC之间的差异。每个COC根据其发育结果进行单独跟踪和分类：囊胚阶段的胚胎（CC-囊胚）或停滞在2至8细胞阶段的胚胎（CC-2-8细胞）。个体培养的平均囊胚率为27.7%，组对照为31.2%（无显著差异）。对于转录组分析，每个发育结果每个重复中收集5个卵丘活检组织。3个CC重复样本使用RNA微阵列分析进行转录组分析。在CC-囊胚组和CC-2-8细胞组之间鉴定出了一些基因表达的明显差异。考虑到1.5倍变化（P < 0.05），CC-囊胚组和CC-2-8细胞组之间有68个基因差异表达。对12个选定基因进行了定量逆转录-聚合酶链反应验证：每个COC发育结果各6个上调基因。在CC-2-8细胞中观察到1-酰基甘油-3-磷酸O-酰基转移酶9（AGPAT9）（脂质代谢）、氯离子细胞内通道3（CLIC3）、角蛋白8（KRT8）和亮氨酸蛋白聚糖（LUM）（分子转运）的表达较高（P < 0.05）。CC-囊胚发育结果分析显示，甘氨酸脒基转移酶（L-精氨酸：甘氨酸脒基转移酶）（GATM）（翻译后修饰、氨基酸代谢和自由基清除）的表达显著更高。这组新鉴定的基因可以提供新的标记物，以区分与高质量胚胎相关的COC和发育潜力有限的COC。
