Involvement of phosphodiesterase 5 (PDE5) on lipid accumulation in bovine oocytes and embryos produced in vitro

The aim of this study was to investigate the involvement of PDE5 on lipid metabolism in bovine oocytes by assessing the effects of PDE5 inhibition during in vitro culture on lipid contents in oocytes and resulting in vitro produced (IVP) embryos, and their cryotolerance. In Experiment 1, cumulus-oocye complexes (COCs) from slaughterhouse ovaries were submitted to IVM in TCM199 supplemented with 0.4% BSA or 10% FCS associated or not with a PDE5 inhibitor (10-5M sildenafil- Sigma-Aldrich) and after 22h oocytes were denuded and stained with Nile Red (1µg/ml, 30 min) to assess cytoplasmic lipid levels measured by fluorescence intensity. In Experiment 2, 10-5M sildenafil (SDF) was included during IVM and/or IVC (SOFaa) during embryo development after IVF (TALP medium using frozen sperm from the same bull prepared by Percoll gradient). Controls were cultured without SDF and all groups were cultured with 10% FCS. After 22h IVM, 20h IVF and seven days IVC, embryos were assessed for cleavage (Day 4) and blastocyst development rates. Day 7 blastocysts (BL) were fixed and stained with Nile Red to evaluate lipids. In Experiment 3, the same groups were assessed plus two others including melatonin (10-7M) as an antioxidant during IVC in SDF treated groups. Cleavage and BL rates were determined and embryos were vitrified. After thawing, BLs were cultured for 24h to assess reexpansion and 48-72 h for hatching. Cultures were at 38.5oC under 5%CO2 in air. Statistical analyses were performed by ANOVA followed by Tukey test using SAS and significance level was 5%. In Experiment 1, SDF reduced (P 0.05). Reduction in lipids was only observed in BLs produced with SDF in both IVM and IVC (30.2; P 0.05) or reexpansion and hatching (89 and 64%, respectively, Pu003e0.05). In conclusion, PDE5 inhibition during IVM reduces lipid content in oocytes, but in embryos, inhibition is necessary during both IVM and IVC. Lipid reduction, however, did not translate into improved cryotolerance, neither did the addition of the antioxidant melatonin. PDE5 appears to be involved in lipolysis in bovine oocytes and embryos possibly related to cGMP levels and PKG activity and may be an interesting target for studies to understand lipid metabolism in oocytes and IVP embryos. To our knowledge, this is the first study to show the possible relationship between this pathway and lipid metabolism in bovine oocytes and embryos.