The impact of transcription inhibition during in vitro maturation on the proteome of bovine oocytes†

Abstract

Proper oocyte maturation is a prerequisite for successful reproduction and requires the resumption of meiosis to the metaphase II stage (MII). In bovine oocytes, nuclear maturation has been shown to occur in in vitro maturing cumulus-enclosed oocytes (COCs) in the absence of transcription, but their developmental capacity is reduced compared to transcriptionally competent COCs. To assess the impact of transcription during in vitro maturation of bovine COCs on the quantitative oocyte proteome, a holistic nano-LC–MS/MS analysis of GV oocytes and MII oocytes matured with or without addition of the transcription inhibitor actinomycin D (ActD) was carried out. Analyzing eight biological replicates for each of the three groups, a total of 2018 proteins were identified. These could be clearly classified into proteins depending or not depending on transcription during oocyte maturation. Proteins whose abundance increased after maturation irrespective of transcription inhibition—and hence independent of transcription—were related to the cell cycle, reflecting the progression of meiosis, and to cellular component organization, which is crucial for cytoplasmic maturation. In contrast, transcription-dependent proteins were associated with cell–cell adhesion and translation. Since a high rate of protein synthesis in oocytes has been shown to correlate with their developmental competence, oocyte maturation in transcriptionally impaired COCs is apparently disturbed. Our experiments reveal that impaired transcription during in vitro maturation of COCs has a substantial effect on specific components of the oocyte proteome, and that transcription is required for specific classes of oocyte proteins predominantly involved in translation.

Protein Cargo of Extracellular Vesicles From Bovine Follicular Fluid and Analysis of Their Origin From Different Ovarian Cells

“…Moreover, more than 91% of GC-unique proteins also overlapped with human EV-proteomes. Taking in account published oocyte and CC proteomic analysis (41,42) and our data, it may be suggested that a significant part of ffEVs likely originated from GC and also from CC and oocyte. Hence, ffEVs may have diverse origins because around 40% of the proteins identified in ffEVs were neither detected in GC nor in CC (41).…”

“…More recent study by Gegenfurtner et al (42) reported the identification of 1960 proteins in bovine oocyte (42). By comparing ffEV and oocyte lists of proteins, we found that 217 out of 322 ffEV proteins were also present in bovine oocytes.…”

“…Interestingly, marked differences in gene expression values were observed between the oocyte and follicular cells for all the ribosomal proteins detected in ffEVs, and all ribosomal genes showed the lowest mRNA abundance in oocyte (Figure 7A, Supplementary Table 5). At protein level, 57 small and large ribosome subunit proteins were identified in ffEVs, and 54 of them were common to list of 75 ribosomal proteins identified in bovine oocytes (42) as shown by Venn diagram (Figure 7B).…”

“…Interestingly, most of these proteins were unique to ffEVs and were not here detected in GC, which means significant enrichment of ribosomal proteins in ffEVs. However, 54 of ffEV ribosomal proteins were identified in bovine oocytes (42). Previous studies demonstrated that EVs released from different cells contain a substantial amount of coding and non-coding RNAs.…”

“…The list of official symbols of ffEV proteins was compared with lists produced from either bovine or human EV-proteomes. In addition, the list of proteins identified here in bovine ffEVs was compared with lists of proteins recently reported in bovine oocyte and cumulus cells (41,42).…”