267 EFFECT OF FOLLICULAR FLUID CONCENTRATION ON IN VITRO DEVELOPMENT OF PORCINE OOCYTES AND THE EXPRESSION OF GENES RELATED TO CUMULUS EXPANSION AND EMBRYO QUALITY

Abstract

It is well known that the presence of porcine follicular fluid (PFF) in in vitro maturation media enhances the developmental competence of porcine oocytes. However, it is also suggested that the action of PFF can be modulated positively or negatively by its components. In this study, we investigated the effects of PFF concentration (10 v. 1%) and protein-free media (PFF 0%) on the maturation of porcine oocytes in vitro, and analysed the difference in gene expression in the resulting cumulus cells and blastocysts after parthenogenetic activation. Three groups were tested: 1) 10% PFF: TCM-199 + 10% PFF (n = 638); 2) 1% PFF: TCM-199 + 3.05 mM d-glucose + 1% PFF (n = 418); and 3) 0.1% polyvinyl alcohol: TCM-199 + 3.05 mM d-glucose + 0.1% polyvinyl alcohol (n = 693). Cumulus–oocyte complexes were cultured for 20 to 22 h in the respective media that contained gonadotrophin (1 µg mL–1), epidermal growth factor (10 ng mL–1), cysteine (0.57 mM), sodium pyruvate (0.91 mM), insulin (5 µg mL–1), and 9-cis retinoic acid (5 nM). They were then cultured for an additional 20 to 22 h without hormonal supplements. Data was analysed by one-way ANOVA using the SAS program (SAS Institute Inc., Cary, NC, USA). No significant difference in oocyte maturation rate was observed. However, significantly higher (P < 0.05) proportions of embryos developed in the blastocyst stage when the oocytes were matured in 10% PFF group (45%) than in the 1% PFF group (31.1%). The total cell numbers were not significantly different among groups (52 ± 1.3 v. 54.6 ± 3.1 v. 54.4 ± 2.5, respectively). In addition, the expression of matrix molecule (HAS2, GREM1), steroidogenesis (HSD3B), epidermal growth factor signalling (AREG, BTC), and cell cycle regulator (CCND2) genes were upregulated in the cumulus that was obtained from oocytes that matured in 10% PFF. The expression of the anti-apoptotic gene (BclxL) was upregulated, and the expression of the pro-apoptotic gene (Bax) and metabolism-related genes (GLUT1, LDHA) were downregulated in blastocysts that developed from the 10% PFF group. Therefore, it can be concluded that supplementation of 10% PFF during in vitro maturation improves embryo development by increasing matrix molecules and maturation-enabling factors in the cumulus and by reducing apoptosis. This study was supported by IPET (No. 311011-05-1-SB010), MKE (No. 10033839-2012-21), the Research Institute for Veterinary Science, the BK21 Program, and the TS Corporation.