Porcine Ovary Oocyte Maturation and Analysis

Porcine ovaries were collected from a local abattoir and transported to our laboratory within 2 h in PBS at 4 °C. The immature COCs were aspirated from the follicles with 3–6 mm in diameter, and then 100–120 COCs were cultured in 500 μL TCM-199 medium with EGF (10 ng/mL) for 44 h to acquire the mature COCs. The denuded oocytes (DOs) were obtained by removing the cumulus cells from the mature COCs with 1 mg/mL hyaluronidase. One ampulla at follicular phase was cultured in 200 μL TCM-199 medium, supplemented with 150 immature or mature COCs, 150 DOs, 10 ng/mL TGFB1, and/or 1 μM SD208 (TGFBR1 inhibitor) for 3 h. The ampullae were collected at the end of culture, and the epithelial cells were scraped by the edge of a microscope slide. In some experiments, the epithelial cells were isolated from the ampullae at follicular and corpus rubrum phases. The samples were stored at -80 °C for mRNA and protein analysis.
OOX cumulus cells were produced by microsurgically removing the oocytes, but not the zona pellucida, from the immature COCs. Groups of 100–120 COCs or OOX cumulus cells were cultured in the medium, supplemented with EGF, growth differentiation factor 9 (GDF9, 500 ng/mL, human), bone morphogenetic protein 15 (BMP15, 500 ng/mL, human), and/or fibroblast growth factor 8B (FGF8, 100 ng/mL, human) in 500 μL medium. After 44 h culture, cumulus cells were collected for gene and protein analysis. All the cultures were incubated at 39 °C under 5% CO₂.