Defective uncoupled differentiations after nuclear reprogramming. Bos taurus

Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extraembryonic patterns, as evidenced by morphological and molecular ‘‘uncoupling’’. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters redifferentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way. Overall design: Amplified material from each extra-embryonic tissue was indirectly labelled using "random" hexamers. One independent target per tissue was generated and hybridised onto one array. 10 measurements per reproduction mode were generated.

体细胞核移植（Somatic cell nuclear transfer, SCNT）是目前已有的最高效的细胞重编程技术，在牛科动物研究中尤为如此。尽管在第7天进行胚胎移植后的数周内，体细胞核移植囊胚的表现与对照组相当甚至更优，但在着床前即可观察到胚胎伸长异常与原肠胚形成缺陷。为探究胚胎与胚胎外组织互作的发育意义，本研究对伸长期与原肠胚形成期组织的形态学与分子特征展开了分析。在第18天时，本研究对30个体细胞核移植孕体与20个对照组孕体（人工授精Artificial Insemination, AI与体外胚胎生产In Vitro Production, IVP各10个）进行了比较；其中半数体细胞核移植孕体外观正常，另一半则呈现出非典型胚胎伸长与原肠胚形成异常的特征。体细胞核移植还与胚胎及胚胎外模式的高度不一致发生率相关，这种不一致可通过形态学与分子层面的"解偶联"现象得以证实。胚胎伸长似乎是继发受到影响；30个孕体中仅3个出现了形状异常伸长的情况，且与对照组相比，其基因表达差异极少。不过，部分此类差异可能与微绒毛形成缺陷或细胞外基质组成异常相关，进而可能影响胚胎外组织的功能。与胚胎伸长过程不同，原肠胚形成阶段存在胚胎缺陷，这些缺陷很可能影响了下胚层、上胚层或其分化的早期阶段。在考虑体细胞核移植孕体的体细胞起源时，即每株牛耳成纤维细胞的重编程效率（低效组：0029、中效组：7711、高效组：5538），研究发现胚胎异常或严重的胚胎-胚胎外组织解偶联，相比伸长缺陷，与着床阶段的胚胎丢失相关性更强。另一方面，第18天时体细胞核移植与对照组孕体之间的胚胎外组织差异，与分子可塑性（高效/高可塑性）以及后续的妊娠丢失相关。最后，由于体细胞核移植重编程可改变体内的再分化过程，其以独特的方式揭示了细胞与组织间受时空限制的互作关系。实验整体设计：采用"随机"六聚体对每一份胚胎外组织的扩增产物进行间接标记。每份组织制备一个独立的靶标，并将其与一张基因芯片进行杂交。每种生殖模式均生成10组检测数据。