Single-Cell Analysis Defines Heterogeneity And Transcriptional Dynamics Of Oocyte In Newborn Mice

The oocyte is one of the largest and most mysterious cells in mammalian organisms.After many years of inactivity in a primordial state,the oocyte progresses through growth and maturation achievinga remarkable level of specialization,while maintaining throughout development a state of totipotency.Most telling of all is the unique capability of the oocyte,in collaboration with the spermatozoon,to give rise to a fully developed organism formed from hundreds of different tissues and myriads of individual cells-a feat that has inspired intellectuals of all ages.The pool of primordial follicles determines the reproductive lifespan of the mammalian female,and its establishment is highly dependent upon proper oocyte Cyst breakdown and regulation of germ cell numbers.The mechanisms controlling these processes remain a mystery.However,it is believed that the endowment of primordial follicles in mammalian ovaries is finite.Once follicles are depleted,infertility ensues.Thus,the size of the initial endowment has consequences for fertility and reproductive longevity.Follicular endowment is comprised of various processes that culminate with the incorporation of meiosis-arrested oocytes into primordial follicles.In the past decades,it has been reported programmed cell death regulators,some growth factors and signaling molecules and activated transcription factors that affect oocyte survival or Cyst breakdown during follicle assembly.Although several key regulators in follicular development have been identified using gene tic perturbation,systematic understanding is still limited.A novel method based on single cell RNA-seq technology is being exploited to analyze the dynamics transcriptomic of individual cell.We hope single-cell RNA-seq may provide a new perspective of perinatal oocyte Cyst breakdown and the assembly and activation of primordial follicles.In our study,we used Smart-seq2 to determine the gene expression profiling of 143 single cells(120 cells passed quality control,23 cells excluded from the downstream analysis)from newborn mice ovary.Unbiased clustering(Consensus Clustering Plus)of 120 single-cell transcriptomes revealed 2 distinct populations of oocyte.We defined 2 clusters as early stage and late stage by maternal factors like zpl,zp2,zp3 and padi6.Subsequently,reconstruction of the temporal dynamics of oocyte development via pseudo-temporal ordering by Monocle.And examine the molecular identities of each populations.We find that oocytes in the newborn mice ovary exist on a continuum through the processes of Cyst breakdown and primordial follicle formation and activation.Further validated finds,we use chemical-modified siRNA knockdown some interested genes.We found that Cnpy1,a positive regulator of FGF signaling,controls Cyst breakdown and oocyte growth during ovary development.Also,cell cycle regulator Spdya suppress the Cyst breakdown and oocyte growth.In conclusions,by applying consensus cluster plus,principal component,and Monocle analyses to gene expression profiling,we can distinguish the transcriptional heterogeneity of individual cells.Single cell RNA-seq provides an unbiased and systematic view of transcriptional organization of oocytes in newborn mice,and highlights how cellular heterogeneity can be orchestrated in vivo to assure oocytes homeostasis.