RNA-seq of knocked-out oocytes and directly induced oocyte-like cells (DIOLs)

During female germline development oocytes become a highly specialized cell type, and form a maternal cytoplasmic store of crucial factors during oocyte growth. Oocyte growth is triggered at the primordial-primary follicle transition accompanied with dynamic changes in gene expression, yet the gene regulatory network underpinning oocyte growth remains elusive. Here we identified a set of transcription factors sufficient to trigger oocyte growth. By dissection of the change in gene expression and functional screening using an in vitro oocyte development system, we identified 8 transcription factors, each of which was essential for the primordial-primary follicle transition. Surprisingly, enforced expression of these transcription factors swiftly converted pluripotent stem cells to oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription factor-induced oocyte-like cells were formed without PGC specification, epigenetic reprogramming or meiosis, establishing that oocyte growth and lineage-specific de novo DNA methylation is separable from the preceding epigenetic reprogramming in PGCs. This study identifies a core set of transcription factors for orchestrating oocyte growth, and also provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine. polyA+RNA was harvested from mouse oocytes and directly induced oocyte-like cells (DIOLs). Extracted polyA+RNA were used for the directional (strand-specific) RNA-seq library construction.

在雌性生殖系发育进程中，卵母细胞会分化为高度特化的细胞类型，并在卵母细胞生长阶段构建关键因子的母源性细胞质储存库。卵母细胞生长起始于原始-初级卵泡转换阶段，伴随基因表达的动态变化，然而支撑卵母细胞生长的基因调控网络仍未得到阐明。本研究鉴定出一组足以触发卵母细胞生长的转录因子(transcription factors)。通过解析基因表达变化并利用体外卵母细胞发育系统开展功能筛选，我们鉴定出8个转录因子，每一个均对原始-初级卵泡转换至关重要。令人意外的是，对这些转录因子进行强制表达后，可快速将多能干细胞转化为具备受精能力且可后续发生卵裂的类卵母细胞。此类由转录因子诱导产生的类卵母细胞无需经过原始生殖细胞(PGC)特化、表观遗传重编程或减数分裂即可形成，这表明卵母细胞生长与谱系特异性从头DNA甲基化可与原始生殖细胞中先行发生的表观遗传重编程相互分离。本研究鉴定出一组调控卵母细胞生长的核心转录因子，同时也提供了一种替代性卵质来源，该来源可作为生殖生物学与生殖医学领域的独特研究材料。研究人员从小鼠卵母细胞与直接诱导类卵母细胞(directly induced oocyte-like cells, DIOLs)中提取聚腺苷酸化RNA(polyA+ RNA)，将所提取的聚腺苷酸化RNA用于构建定向（链特异性）RNA测序文库。