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. Overall design: 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.