Transcriptome profiles of porcine oocytes and their corresponding cumulus cells reveal functional gene regulatory networks
Type of DegreeMaster's Thesis
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The oocyte acquires developmental competence as it progresses through folliculogenesis. It does so, by communicating with the surrounding cumulus cells in a bidirectional fashion. Analyses of single oocytes are essential for further clarification of these molecular mechanisms. Standard protocols to obtain RNA for single-cell RNA-Seq involve RNA extraction kits that select for mRNAs or cell lysis procedures, which present limitations when the oocyte is the cell of interest. We adapted the phenol-chloroform procedure for the purification of total RNA from single oocytes with modifications that included the use of Phasemaker™ tubes, a second chloroform wash, and the precipitation of the RNA with glycogen in microcentrifuge tubes. We profiled the total RNA of single oocytes (bovine and porcine), and observed distinct peaks for small RNAs, 18S, and 28S. We amplified the total mRNA and observed DNA fragments longer than 5000 bp in length, suitable for single-cell RNA sequencing. We applied this approach to sequence the transcriptome of 17 prepubertal porcine oocytes and their corresponding cumulus cells. We hypothesized that the transcript profiles of the cumulus cells and oocyte display distinct gene regulatory networks within the oocyte and cumulus cells. We quantified 7277 genes expressed in the cumulus-oocyte complex. Independent clustering of co-expressed genes revealed critical biological functions for the oocyte, such as regulation of transcription, stem cell population maintenance, and insulin receptor signaling pathway. In cumulus cells translation, cellular response to insulin stimulus, and regulation of transcription emerged as critical functions among co-expressing genes. In summary, we developed an approach to extract total RNA from single oocytes. The sequencing of single oocytes and their surrounding somatic cells revealed coordinated expression of hundreds of genes that formed functional regulatory networks.