201 Transcriptional profiling of cumulus cells from FGF, LIF, and IGF1 matured oocytes identifies junctional and structural pathways as key components in oocyte maturation

The production of genetically engineered pigs for biomedical and agricultural purposes requires performing in vitro oocyte maturation and embryo culture. However, oocytes and embryos are exposed to suboptimal environment in vitro. Many advancements have been made to oocyte maturation media, including the addition of FGF2, LIF, and IGF1 (FLI), which improved the number of oocytes that reached the MII stage and doubled the number of oocytes that reached the blastocyst stage. Although it is known that adding FLI to the oocyte maturation system significantly improves oocyte development and embryonic success, the mechanisms by which FLI makes these improvements are still largely unknown. Because FLI has a significant effect on cumulus cell (CC) expansion, we analyzed the transcriptional profile of CC from oocytes matured with FLI and controls without FLI for 24 h and identified differentially expressed genes (DEGs) that may provide insight into how FLI promotes oocyte maturation and embryo development. Five CC samples from both control and FLI-matured oocytes that produced blastocysts were analyzed by RNA sequencing. The resulting data were processed by using Deseq2 to identify DEG. The data were sorted based on genes with an adjusted P-value < 0.05. Of 27 256 genes, there were 3928 DEGs with 1988 upregulated in control medium and 1940 downregulated in control medium compared with FLI. The DEGs were uploaded into DAVID, and gap, adherens, and tight junction KEGG pathways were identified as enriched in the upregulated DEGs. Preliminary results of tight junction protein 1 (TJP1) expression illustrate that there is more TJP1 expression in FLI-matured cumulus–oocyte complexes (COCs) at 42 h than controls (18 105.23 ± 521.34 RFU vs. 14 372.60 ± 293.93 RFU [one rep, 17 COCs]). Preliminary results for TJP2 showed similar expression in both control COCs and FLI COCs (7543.05 ± 238.48 RFU vs. 7462.26 ± 256.43 RFU) at 42 h. ICC is continuing to be performed in COC cultured in control or FLI medium at both 24 h and 42 h for TJP1 and TJP2, as well as, gap junction protein A5 and cadherin 8 to determine the effect FLI is having on the CC proteins that play a critical role in forming critical junctions with the oocyte. The potential increase of transcripts in FLI-cultured oocytes still needs to be confirmed with further ICC and real-time PCR analysis; however, these preliminary findings indicate that FLI treatment affects the expression of important junctional protein genes in COCs.