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，以解析小鼠胚胎期及围产期卵子发生过程中的原始卵泡与第一波髓质卵泡发育情况。生殖细胞可聚类为六个减数分裂亚阶段，同时包含凋亡细胞与滋养细胞。本研究同时鉴定了上皮祖细胞的表达基因，并阐明了双潜能起源与上皮起源的前颗粒细胞祖细胞所共享但又存在差异的遗传程序。二者的差异表达基因可作为调控第一波及第二波卵泡独特发育程序的候选靶点。上述研究结果为哺乳动物卵巢卵泡形成过程的发育生物学、生理学及进化保守性研究提供了坚实的理论基础。本研究针对发育中的胚胎期性腺/卵巢开展了单细胞RNA测序（single cell RNA-seq），采样时间点涵盖E11.5（性别决定阶段）、E12.5（有丝分裂增殖以形成生殖细胞囊）、E14.5（向减数分裂过渡阶段）、E16.5（进展至粗线期）、E18.5及P1（细胞器转移、生殖细胞囊破裂、卵母细胞分化与生殖细胞更新阶段），以及P5（原始卵泡形成阶段）。所选取的时间点覆盖了围产期卵巢发育的关键事件。研究团队通过精细解剖与胰酶消化，将卵巢组织解离为单细胞悬液。最终共捕获52542个解离后的细胞，将其加载至油滴微环境中，随后开展cDNA文库构建、深度测序与细胞聚类分析。每个细胞平均可检测到2700个不同基因的表达信息。