60 EFFECTS OF AY9944 A-7 ON MEIOTIC RESUMPTION OF PORCINE OOCYTES AND CUMULUS CELL EXPANSION

Abstract

In vitro studies on mammalian oocytes have shown that follicular fluid-meiosis activating sterol (FF-MAS) can overcome the inhibitory effect of hypoxanthine (Hx) on the resumption of meiosis. FF-MAS, an intermediate in the cholesterol biosynthesis pathway, is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. AY9944 A-7, an inhibitor of Δ14-reductase and Δ7-reductase, induces accumulation of FF-MAS by inhibiting its metabolism. The aim of this study was to evaluate the effects of AY9944 A-7 on meiotic resumption of porcine oocytes, cumulus cell expansion, and gene expression related to M-phase-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and oocyte maturation in oocytes and related to cumulus expansion in cumulus cells. In experiment 1, 1136 cumulus-oocyte complexes (COCs) were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in addition to a meiotic inhibitor (Hx, 4 mM) for 44 h. Oocytes treated with 10 and 20 μM AY9944 A-7 in the presence of Hx had significantly higher GVBD and M2 rates than the control group. However, 40 μM AY9944 A-7 significantly decreased GVBD and M2 rates and increased degeneration of oocytes compared with other groups. In experiment 2, 600 COCs were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in the absence of Hx for 44 h. Cumulus expansion of 40 μM AY9944 A-7 treated group was significantly decreased compared with other groups. In experiment 3, we evaluate the effects of AY9944 A-7 on gene expression, and the experiment was replicated four times. Data on gene expression were analysed using Student’s t-test. Oocytes treated with 10 μM AY9944 A-7 increased expression of genes involved in MPF (Cyclin B and Cdc2), MAPK (C-mos), and oocyte maturation (GDF9 and BMP15). Cumulus cells treated with 10 μM AY9944 A-7 decreased cumulus expansion-related genes (Has2, Tnfaip6, Ptgs2, and Ptx-3). In conclusion, our results suggest that although 10 μM AY9944 A-7 decreased cumulus expansion-related genes, there was no difference in cumulus expansion and it induced meiotic resumption of porcine oocytes with increased MPF, MAPK, and oocyte maturation-related genes. Further studies are needed to evaluate the effect of AY9944 A-7 on porcine embryo development. This study was supported by Ministry Of Trade, Industry & Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.