Sphingosine-1-phosphate (S1P) has a many function involved proliferation, differentiation and survival of many cells. In this study, to investigate whether S1P improve the developmental competence of porcine embryos, 50 nM of S1P were supplemented during in vitro maturation (with EGF or without EGF) medium and/or in vitro culture (IVC) medium. Addition of S1P was significantly increased the rate of oocytes reaching metaphase II (MII) compared to the control (83.5 vs. 64.1%) in without EGF medium, but not with EGF medium (89.5 vs. 84.6%). When treated with 1 μM of N1N-dimethylsphingosine (DMS), a sphingosine kinase inhibitor which is blocked endogenous generation of S1P, the meiotic progression rates to MII stage (without EGF: 45.2 and with EGF: 66.7%) were significantly decreased and degeneration rates (without EGF: 51.2 and with EGF: 30.1%) were increased in both medium compared to control group during IVM periods. Also, the rates of blastocyst formation was significantly increased in the S1P treated group compared to control group (29.0 vs. 19.2%) of EGF supplemented medium, whereas there were no effect in the EGF free medium (9.0 vs. 10.5%). After 12 h IVM, the phosphorylation of ERK1 and ERK2, which is major signaling pathway of MAP kinase, were increased in the S1P group than that of control or DMS group. When supplemented of S1P during IVC, the rates of blastocyst formation and total cell number (30.2% and 40.6) were significantly increased in S1P-treated group compared with control (20.1% and 32.5), DMS (12.3% and 25.1), and S1P plus DMS group (24.7% and 33.6). The percentage of apoptosis nuclei in the S1P group was significantly decreased than other groups. Also, the rates of blastocyst formation (26.7 vs. 14%) and total cell number (42.8 vs. 32.5) were significantly increased in the S1P group than those of control group when S1P added during the entire IVM and IVC periods. Taken together, our results indicate that S1P supplementation in IVM and/or IVC medium affects beneficial effect of meiotic maturation and subsequent developmental competence of porcine embryos.

ABSTRACT   INTRODUCTION   MATERIALS AND METHODS    Treatment of SlP and DMS    In Vitro Maturation (IVM)    In Vitro Fertilization (IVF) and Culture (IVC)    Aceto-Orcein Staining    Western Blot Analysis    TUNEL and Cell Counting    Statistical Analysis   RESULTS    Effect of S1P Treatment during IVM of Porcine Oocytes on Meiotic Maturation and Embryo Development    S1P Induces MAP Kinase Phosphorylation in In Vitro Matured Porcine Oocytes    Effect of S1P during IVC on Embryo Development and Apoptosis    Effect of S1P during IVM and IVC Periods on Embryo Development   DISCUSSION   REFERENCES

• Hyo-Sang Lee(lDevelopment and Differentiation Research Center, Korea Research Institute of Bioscience and Biotechnology, Oregon National Primate Research Center, Oregon Health & Science University, 505 N. W. 185(th) Avenue, Beaverton, OR 97006, USA)
• Deog-Bon Koo(Department of Biotechnology, College of Engineering, Daegu University, Gyeongsan 712-714, Korea) Corresponding Author