253 DIFFERENTIAL GENE EXPRESSION IN IN VIVO-DERIVED v. IN VITRO-PRODUCED EQUINE BLASTOCYSTS AS DETERMINED BY RT-qPCR

Abstract

Although in vitro production of equine embryos has greatly evolved in recent years, there are still substantial differences between in vitro-produced and in vivo-derived equine embryos. Fundamental insight into these differences could lead to optimization of equine assisted reproductive techniques. Reverse transcription quantitative real-time PCR (RT-qPCR) is a highly specific and sensitive tool to compare mRNA expression levels of specific genes and was used in this study to determine differences in gene expression between equine in vivo and in vitro embryos. In vivo embryos (n = 8) were derived by uterine flushing of artificially inseminated mares at 7 days after ovulation. For the production of the in vitro embryos (n = 8), oocytes from slaughtered mares were matured in DMEM-F12-based medium (Galli et al. 2007 Anim. Reprod. Sci. 98, 39-55) in 5% CO₂ in air (maturation rate: 57%), fertilized by intracytoplasmic sperm injection, and cultured in DMEM-F12 with 10% fetal calf serum in 5% CO₂, 5% O₂, and 90% N² for 9.5 days (cleavage rate: 74%; blastocyst rate: 7%). RNA was extracted from single early to expanded blastocysts and amplified and converted into cDNA with the WT-Ovation RNA Amplification System (NuGEN, San Carlos, CA, USA). Based on the presumed gene functions and differential gene expression as determined in a previously performed suppression subtractive hybridization (SSH; Smits et al. 2009 Reprod. Dom. Anim. 44, 75), 5 genes [brain expressed X-linked 2 (BEX2), Mps one binder kinase activator-like 3 (MOBKL3), fatty acid binding protein 3 (FABP3), minichromosome maintenance complex component 7 (MCM7), and ornithine decarboxylase (ODC)] were selected for quantification by RT-qPCR with the KAPA SYBR^® FAST qPCR Kit (Kapa Biosystems, Belgium) on the iCycler iQ Real-Time PCR Detection System (Bio-Rad, Nazareth, Belgium). All data were normalized with previously determined stable reference genes (beta actin, ubiquitin C, ribosomal protein L32, and glyceraldehyde-3-phosphate dehydrogenase) and statistically analyzed by means of a Mann-Whitney test. The fact that all genes were expressed at greater levels in the in vivo-derived blastocysts than in the in vitro-produced blastocysts confirmed the results of the SSH. This difference was highly significant for MOBKL3, BEX2, and ODC (P < 0.005), significant for FABP3 (P < 0.05), and not significant for MCM7. These genes have already been shown to be important for embryonic cell survival (ODC), oocyte maturation and pre- implantation development (MOBKL3) in mice, regulation during embryonic development (BEX2) and fetal development (FABP3) in human, and genome replication in eukaryotes (MCM7) (Pendeville et al. 2001 Mol. Cell Biol. 21, 6549-6558; Han et al. 2005 Nucleic Acids Res. 33, 6555-6565). In conclusion, 4 genes (MOBKL3, BEX2, ODC, and FABP3) with greater expression levels in in vivo-derived equine blastocysts have been identified. Whether the up-regulation of these genes is important for normal embryonic differentiation in the horse embryo is currently under investigation.