Fertilization and Early Embryogenesis
(T302) ANALYSIS OF mRNA EXPRESSION OF PUTATIVELY IMPRINTED GENES IN BOVINE BLASTOCYSTS PRODUCED IN VIVO, PARTHENOGENETICALLY AND ANDROGENETICALLY.
Ruddock, Nancy1, 2, Wilson, Katrina1, 2, Cooney, Melissa1, 2, French, Andrew1, 2, Lagutina, Irina 3, Galli, Cesare3, Holland, Michael1, 2, 1 Monash Institute of Medical Research; Monash University, Clayton, VIC, Australia2 Co-operative Research Centre for Innovative Dairy Products, Melbourne, VIC, Australia3 Laboratorio di Tecnologie della Priproduzione, Cremona, Italy
ABSTRACT- Early embryonic/fetal development is greatly influenced by the expression of imprinted genes. Our group recently showed that many putatively imprinted genes are expressed during bovine embryogenesis; however, only 1 of 3 maternally imprinted genes showed differential mRNA expression between blastocysts produced by in vitro fertilization (IVF) or parthenogenesis (PA). Here we describe mRNA expression for an additional 10 genes (Ata3, Dlk1, Gnas, Grb10, Impact, Mest isoform 1, Ndn, Nnat, Sgce, and Xist). Bovine in vivo blastocysts were flushed from superovulated cattle on day 7. PA blastocysts were produced from in vitro-matured oocytes activated with 5M calcium ionophore A23187 for 4 min followed by incubation in 2.5g/ml cytochalasin D and 10g/ml cycloheximide for 5 h. Finally, androgenetic (AN) embryos were produced by oocyte enucleation followed by IVF and pronuclear transfer as previously described previously. mRNA was isolated from 5 single blastocysts/ group with Dynabeads (Dynal, Inc.) prior to cDNA production and amplification (Clontech). Gene-specific PCR products were sequence verified. Ata3, Dlk1, and Nnat were not expressed in any blastocysts; therefore imprinting status could not be determined. Gnas and Ndn showed expression in all blastocysts and were deemed not imprinted at the blastocyst stage of development. The remaining genes showed varying expression patterns. Grb10 (paternally imprinted) showed partial imprinting with expression in all in vivo and PA blastocysts, and 3 of 5 AN blastocysts. In contrast Xist, which was only expressed in in vivo and PA blastocysts, was clearly imprinted. Impact (maternally imprinted) showed expression in all but one PA blastocyst indicating that the gene may be partially imprinted in bovine. Mest was only expressed in one PA blastocyst, possibly indicating aberrant expression in that blastocyst. Finally, Sgce was expressed in all 5 PA embryos, but not in in vivo or AN embryos. This is interesting as Sgce is maternally imprinted in adult mouse tissues. Taken collectively these data suggest that bovine imprinting is still being established during early embryo development, an idea only recently postulated for the murine Xist gene.
KEY WORDS: imprinted gene, bovine, embryo, IVF