First round of DNA replication is essential for transcriptional reprogramming of somatic nuclei in mouse oocytes. Mus musculus

Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24-36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle. Overall design: 13 samples were analyzed. Aph-IVF: Aphidicolin not treated, in vitro fertilization, 1 biological rep Aph-IVF96: Aphidicolin not treated, in vitro fertilization (96h), 1 biological rep Aph-IVFBla: Aphidicolin not treated, in vitro fertilization blastocyst, 1 biological rep Aph+IVF: Aphidicolin treated, in vitro fertilization, 1 biological rep Aph+IVF96: Aphidicolin treated, in vitro fertilization (96h), 1 biological rep Aph+IVFBla: Aphidicolin treated, in vitro fertilization blastocyst, 1 biological rep Aph-SCNT: Aphidicolin not treated, somatic cell nuclear transfer, 1 biological rep Aph-SCNT96: Aphidicolin not treated, somatic cell nuclear transfer (96h), 1 biological rep Aph-SCNTBla: Aphidicolin not treated, somatic cell nuclear transfer blastocyst, 1 biological rep Aph+SCNTBla: Aphidicolin treated, somatic cell nuclear transfer blastocyst, 1 biological rep B6C3F1-Ooc: Aphidicolin not treated, B6C3F1 oocytes, 1 biological rep B6C3F1-Cum: Aphidicolin not treated, B6C3F1 cumulus cells, 2 biological rep

卵母细胞介导的细胞核重编程，其诸多结构与机制层面的必要条件仍未明确。此前有研究指出，小鼠合子中体细胞核的转录重编程可在24至36小时内完成，这一速度远快于其他重编程体系，由此引发了一个疑问：仅通过暴露于活化的小鼠卵质中，是否足以实现细胞核的重编程？为此，我们阻断了核移植后随之发生的DNA复制与胞质分裂，以评估二者对于克隆小鼠胚胎中关键多能性基因Oct4（Pou5f1）与Nanog的转录重编程的必要性。通过转录组与等位基因特异性分析，我们发现，在未发生DNA复制的核移植卵母细胞中，数百种mRNA的表达水平升高，但Oct4与Nanog的表达并无此变化。首轮DNA复制的进行对于Oct4与Nanog的转录重编程是必需的，但并不充分；而胞质分裂及其伴随的细胞间相互作用则并非转录重编程所必需。在体外受精产生的胚胎中，也观察到了与克隆胚胎相似的反应。我们的研究结果将重编程的发生与卵母细胞介导的核重编程此前未被认知的必要条件——DNA复制——联系了起来。除非同时存在DNA复制，否则仅靠核移植无法立即实现体细胞向多能性基因表达模式的转变。因此，本研究有助于认识到：不断追求更快的重编程方法，可能会遭遇由细胞周期所决定的极限。
整体实验设计：共分析13个样本。
Aph-IVF：未添加阿非迪霉素（Aphidicolin）的体外受精组，1次生物学重复
Aph-IVF96：未添加阿非迪霉素的体外受精组（培养96小时），1次生物学重复
Aph-IVFBla：未添加阿非迪霉素的体外受精囊胚组，1次生物学重复
Aph+IVF：添加阿非迪霉素的体外受精组，1次生物学重复
Aph+IVF96：添加阿非迪霉素的体外受精组（培养96小时），1次生物学重复
Aph+IVFBla：添加阿非迪霉素的体外受精囊胚组，1次生物学重复
Aph-SCNT：未添加阿非迪霉素的体细胞核移植（somatic cell nuclear transfer, SCNT）组，1次生物学重复
Aph-SCNT96：未添加阿非迪霉素的体细胞核移植组（培养96小时），1次生物学重复
Aph-SCNTBla：未添加阿非迪霉素的体细胞核移植囊胚组，1次生物学重复
Aph+SCNTBla：添加阿非迪霉素的体细胞核移植囊胚组，1次生物学重复
B6C3F1-Ooc：未添加阿非迪霉素的B6C3F1品系卵母细胞组，1次生物学重复
B6C3F1-Cum：未添加阿非迪霉素的B6C3F1品系卵丘细胞组，2次生物学重复