256 SWITCH IN THE EXPRESSION OF GENES INVOLVED IN LIPID METABOLISM FOR IN VIVO-MATURED BOVINE OOCYTES AND BLASTOCYSTS

Abstract

The competence of oocytes to develop into healthy offspring is determined by many factors including oocyte quality, embryo culture conditions, and energy requirements. Although glucose metabolism has been studied extensively, little is known about lipid metabolism during oocyte maturation and in the blastocyst. In general, cells can obtain fatty acids (FA) by transport (1) from outside across the cell membrane or by synthesis (2) inside the cell, and loose FA by catabolic processes (3). Therefore, we analyzed mRNA abundance for (1) FA translocase (FAT/CD36) and FA transport protein (FATP1), (2) acetyl-CoA carboxylase (ACC) α and FA synthase (FAS), and (3) AMP-activated protein kinase gamma 1 (AMPK) and carnitine palmitoyltransferase I (CPT-I) in bovine oocytes during resumption of meiosis in vivo vs.in vivo blastocysts. Cyclic Holstein-Friesian cows (n = 16) were treated with oFSH and a controlled LH surge (Knijn et al. 2002 Reproduction 124, 365–375) to collect oocytes at onset of maturation, 2 h before LH, and after the start of germinal vesicle breakdown (6 h after LH). A second group of cows (n = 10) was treated with eCG/anti-eCG (Voset al. 1994 Theriogenology 41, 829–840) to flush blastocysts at Day 7 after ovulation. Following ovariectomy, oocytes from all follicles between 10 and 18 mm were denuded and stored separately at -80�C. Oocytes were selected on the basis of the relative steroid concentration in the follicular fluid, producing 3 replicate pools of 13 competent oocytes each, at both 2 h before and 6 h after LH. Four replicates of 5 expanded blastocysts grade 1 (IETS) each were stored at -80�C. Total RNA was isolated using a microspin column, and DNA was digested (Absolutely RNA Microprep Kit; Stratagene, San Diego, CA, USA) and reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). Amplification of cDNA was achieved by real-time PCR using specific primers and 2X iQ SYBR Green Supermix (Bio-Rad Laboratories) in duplicate. Relative levels of expression were analyzed using a modified delta-Ct method. Statistical analysis was done using ANOVA and the Tukey post-hoc test. Relative abundance of mRNA for FA transport and catabolism was >10 times higher during oocyte maturation than in blastocysts; CPT-I was not detected in blastocysts. In contrast, transcripts of FA synthesis were sharply increased (>5 times) during the blastocyst stage. Alterations in the expression of these mRNA indicate that they play a key role in mediating cell type-specific differences in energy requirement and that exogenous FA and FA catabolism could be a main source of energy during oocyte maturation. A high level of FA biosynthesis mRNA in blastocysts may reflect high demands of de novo long-chain FA synthesis and elongation that probably are essential for protection against stress.