Combined single-cell RNA-sequencing and single-cell bisulfite-sequencing reveal key regulatory mechanisms associated with oocyte in vitro maturation in pig [scRNA-seq]

In vitro maturation (IVM) is a critical step in current animal in vitro embryo production protocols. However, in vitro matured oocytes often exhibit lower developmental competence than their in vivo counterparts, and the molecular mechanisms underlying successful maturation remain unclear. In this study, we compared the developmental competence up to the blastocyst stage of IVM and in vivo matured porcine oocytes (N=397) and also analysed simultaneously their gene expression and DNA methylation profiles at single cell resolution (N=40) using RNA sequencing and bisulfite sequencing. Our final aim was to find the associations between the developmental and molecular differences between the two groups of oocytes. All the oocytes were obtained by aspiration of either ovarian follicles between 3-6 mm diameter, and the subsequent IVM, or ovarian follicles from 8-10 mm diameter, with no need of maturation (in vivo matured oocytes). Significant differences were found at day 2 for cleavage rates (58.2 ± 3.0 and 45.7 ± 4.4, P = 0.010) and at day 7 for the blastocyst rates (31.4 ± 3.7 and 47.5 ± 6.6, P = 0.014) for IVM and in vivo groups, respectively, but not for the total cell number per blastocyst (48.1 ± 1.9 and 48.2 ± 2.7). Using the in vivo group as a reference, 1297 and 476 differentially expressed genes (DEGs) were down-regulated and up-regulated, respectively, in the IVM group. The up-regulated DEGs in IVM oocytes were mainly associated with organelle organization, DNA methylation, and cell cycle processes; down-regulated genes were mainly enriched in ribosomal RNA processing, protein synthesis, oxidative phosphorylation and metabolomic processes such as glycosyl and aldehyde compound pathways. The global percentage of methylation was similar between the IVM and in vivo groups, but 321, 344, and 843 differentially methylated regions (DMRs) were detected (P < 0.05) in CpG islands, promoters and coding regions, respectively. Integrative analysis of RNA-sequencing and DNA methylation data identified the main methylated regions and genes that define each group. A total of 236 loci and 296 genes defined the IVM group, while 856 loci and 688 genes were related to the in vivo group. In addition, in the IVM group, we found a higher number of negative correlations between gene expression and DNA methylation, while the in vivo group showed higher number and stronger positive correlations. Taken together, these results indicate a discrete effect of IVM on the DNA methylation landscape in mature porcine oocytes, but these changes seem to have a greater impact on gene expression regulation. Overall design: Ovaries from Landrace-Large-White gilts were collected at the slaughterhouse and transported to the laboratory (within 60 minutes) in saline solution containing 100 µg/mL kanamycin sulphate at 38.5°C, washed once in 0.04% cetrimide solution (w/v) and then twice in saline. Then, the IVM protocol was the same as in Romero-Aguirregomezcorta et al. (2024). As for the in vivo matured oocytes, the contents of follicles of 8-10 mm diameter were aspirated and washed twice in Dulbecco's PBS supplemented with 1 mg mL-1 PVA. All IVM and in vivo matured COCs were identified under a stereomicroscope and mechanically detached from the cumulus cells using an automatic pipette set to a volume of 200 microlitres. To do this, the COCs were gently aspirated and repeatedly ejected into the Petri dish until the zona pellucida was visualised free of cumulus cells at 50x magnification. Only oocytes with a visible polar body under the stereomicroscope were selected and snap frozen in LN2 and stored at -80°C until use (n = 20 IVM and 20 in vivo matured oocytes).