Simple SummaryThe regulation of proteins at different time points during oocyte maturation varies. In this study, dynamic changes in oocytes at the GV, MI, and MII stages during oocyte maturation were analyzed using iTRAQ technology. Protein functions from the GV stage to the MI stage were mainly concentrated in the metabolic pathway, and protein functions from the MI stage to the MII stage were mainly concentrated in the regulation of meiosis and genetic material preparation. The results showed that there were transient dynamic changes in the proteome during yak oocyte maturation, and that the physiological functions mediated by these changes were also different. The accurate identification of the differential proteins in the three stages of GV, MI, and MII provided basic information for studying protein regulation at each time point during the maturation of yak oocytes and the molecular mechanism of in vitro maturation.AbstractThe aim of this study was to investigate protein regulation at different time points during the in vitro maturation of yak oocytes. Yak oocytes at GV, MI, and MII stages were collected during in vitro maturation, and differential proteomics sequencing was performed using iTRAQ technology. GO functional classification indicated that the differential proteins were closely associated with biological processes such as “metabolic processes”, and molecular events such as “binding” molecular-function-related categories were active. KOG analysis showed that energy-metabolism-related activities were vigorous during oocyte development from the GV phase to MI phase, and genetic material preparation activities were more active when oocytes developed from the MI stage to MII stage. KEGG pathway analysis showed that the PPAR metabolic pathway, Hippo signaling pathway, and ECM–receptor interaction and metabolic pathway were enriched from the GV to the MI stages. The PI3K-Akt, TGF-β, and phagosome pathways were enriched from the MI stage to the MII stage. These results indicate that transient dynamic changes occurred in the proteome during the maturation of yak oocytes, and the physiological functions mediated by these were also different. The accurate identification of the differential proteins in the three stages of GV, MI, and MII was helpful in further analyzing the molecular regulatory mechanism of yak oocyte maturation.