207 GANGLIOSIDE IMPROVES THE MEIOTIC MATURATION AND ANTI-APOPTOTIC EFFECTS DURING IN VITRO MATURATION OF PORCINE OOCYTES

Abstract

Gangliosides are sialic-acid-glycosphingolipids expressed in almost all vertebrate cells. These play a role in regulating cell differentiation and signal molecules of cellular signaling, including interactions with molecules of signal transduction pathways, such as receptors tyrosine kinases (RTK). Gangliosides perform critical functions through interactions with proteins as receptors in cell‐cell recognition, such as the epidermal growth factor (EGF) receptor (EGFR). Recent studies investigated functional a link between gangliosides-derived RTK signaling and embryo development until blastocyst in mice. However, there is no report on the directly relationship of GM3 or GD1a and oocyte maturation in pigs. Our studies have verified an interaction mechanism between EGF receptor signaling and gangliosides (GM3 and/or GD1a) in porcine oocyte during meiotic maturation and further development. The GM3 expression increased in mouse oocytes that were in vitro-matured (IVM) under apoptotic conditions. To induce the apoptotic conditions of oocytes, we used H₂O₂. We first confirmed that the expression of GM3 in oocytes of H₂O₂-treated groups (1 mM) was gradually increased compared with those of the control group (P < 0.05). We investigated the anti-apoptotic effect of GM3 as the inhibition apoptosis in porcine oocyte IVM by treatment of GM3 (5 or 10 μM) after pretreatment with H₂O₂. Next EGFR protein levels and meiotic maturation were investigated by Western blot analysis and acetic-orcerin staining, respectively, of IVM oocytes. The proportion of germinal vesicle arrested oocytes at 22 h was significantly increased (P < 0.05; 41.6 ± 1.5% v. 25.0 ± 0.0% in the untreated group) in EGF (10 ng mL^–1) + GD1a (10 μM) treated group. To confirm the completion of meiotic maturation, we investigated the proportion of metaphase II (MII) oocytes at 44 h. The MII oocytes were increased (P < 0.05; 89.9 ± 3.6% v. 57.4 ± 5.3% in the untreated group) in oocytes cultured with EGF+GD1a. Interestingly, p-EGFR protein levels of matured oocytes were significantly increased (P < 0.05) by the EGF+GD1a treatment in the maturation process. In addition, EGF+GD1a treatment increased the proportion of normal pronucleus formation (2PN of 43.1 ± 5.2%) and increased (P < 0.05; 50.4 ± 6.1% v. 27.2 ± 2.7% in the untreated group) pre-implantation developmental potential until blastocyst. These results suggest that EGF+GD1a treatment improved the developmental competence of embryos via enhanced meiotic maturation of porcine oocytes by inducing EGFR activation. We confirmed that GM3 has anti-apoptotic effects during porcine oocyte maturation periods. Furthermore, these data will be helpful for better understanding the receptor mechanisms during oocyte maturation in pigs.