Single-cell analysis RNA sequencing of prenatal and neonatal gonads/ovaries at E11.5, E12.5, E14.5, E16.5, E18.5, P1 and P5

We sequenced more than 52,500 single cells from E11.5 to P5 gonads to analyze primordial follicles and wave 1 medullar follicles during mouse fetal and perinatal oogenesis. Germ cells clustered into six meiotic substages as well as dying/nurse cells. We also define genes expressed by epithelial progenitors, and clarify the similar but distinct genetic programs of bipotential-derived and epithelial-derived pre-granulosa cell progenitors. Their differentially expressed genes are candidates to control the distinctive developmental programs of wave 1 and wave 2 follicles. These observations provide a strong basis for further studies of the development, physiology, and evolutionary conservation of mammalian ovarian follicle formation. We performed single cell RNA-seq on developing prenatal gonads/ovaries atE11.5 (sex determination), E12.5 (cycle mitotically to form germline cysts), E14.5 (transition to meiosis), E16.5 (progress to pachytene),E18.5 and P1 (organelle transfer, cyst breakdown, oocyte differentiation and germ cell turnover), and P5 (primordial follicle formation). The selected time points span key events during perinatal ovarian development. After a meticulous dissection and trypsin incubation, ovaries were dissociated into single-cell suspensions. 52,542 dissociated cells were subsequently captured, loaded onto oil droplets, and used for cDNA library construction, deep sequencing, and cluster analysis. Expression information on an average of 2,700 different genes was recovered from each cell.本研究对小鼠胚胎期11.5天（E11.5）至出生后第5天（P5）的性腺开展了超过52500个单细胞的测序工作，以解析小鼠胎期及围产期卵子发生（oogenesis）过程中的原始卵泡（primordial follicles）与第一波髓质卵泡（wave 1 medullar follicles）。研究将生殖细胞（germ cells）聚类为六个减数分裂亚阶段（meiotic substages）以及凋亡/滋养细胞（dying/nurse cells）。本研究还明确了上皮祖细胞（epithelial progenitors）的表达基因，并阐明了双潜能起源（bipotential-derived）与上皮起源的前颗粒细胞祖细胞（pre-granulosa cell progenitors）之间相似但独特的遗传程序。二者的差异表达基因（differentially expressed genes）可作为调控第一波与第二波卵泡独特发育程序的候选靶点。上述研究结果为进一步探究哺乳动物卵巢卵泡形成的发育过程、生理机制及进化保守性提供了坚实基础。 本研究对发育中的产前性腺/卵巢（prenatal gonads/ovaries）开展了单细胞RNA测序（single cell RNA-seq），涵盖的时间点包括：E11.5（性别决定（sex determination）阶段）、E12.5（进行有丝分裂增殖以形成生殖细胞囊（germline cysts）阶段）、E14.5（向减数分裂（meiosis）过渡阶段）、E16.5（发育至粗线期（pachytene）阶段）、E18.5与P1（细胞器转运（organelle transfer）、生殖细胞囊破裂（cyst breakdown）、卵母细胞分化（oocyte differentiation）及生殖细胞更新（germ cell turnover）阶段），以及P5（原始卵泡形成阶段）。所选时间点覆盖了围产期卵巢发育的关键事件。经过精细解剖与胰酶孵育后，卵巢组织被解离为单细胞悬液（single-cell suspensions）。随后共捕获52542个解离细胞，将其加载至油滴中，用于构建cDNA文库（cDNA library construction）、深度测序（deep sequencing）及聚类分析（cluster analysis）。平均每个细胞可获取2700个不同基因的表达信息。