Activation method does not alter abnormal placental gene expression and development in cloned pigs.

Nuclear transfer efficiency is low and is thought to be caused by inadequate placental development. The objective of this study was to identify differentially expressed transcripts in pig placentas derived from in vivo fertilization, in vitro fertilization or nuclear transfer at Day 30 of gestation. Three activation methods were compared: electrical fusion/activation, electrical fusion/activation followed by treatment with reversible proteasomal inhibitor, MG132 or electrical fusion followed by activation with Thimerosal/DTT. Extraembryonic membranes were collected 30 days after artificial insemination (IVV) or embryo transfer (IVF and NT). Extraembryonic membrane cDNAs labeled with Cy5 and a reference cDNA labeled with Cy3 were hybridized to a pig reproductive tissue-specific 19,968 spot cDNA microarray. Images acquired and assessed by using Genepix Pro 4.0 were analyzed by Genespring 7.3.1. ANOVA (P < 0.05) identified 227 differentially expressed transcripts between the five treatments and 0 between the three activation methods. The nuclear transfer groups were pooled and compared to in vivo samples, identifying 34 up- and 19 down-regulated transcripts (>2-fold change, P < 0.05). Ten transcripts were validated by real-time PCR. UPTI, PAG2, and GLUD1 protein was quantified by Western blot and densitometry verified that UPTI and PAG2 proteins had an expression pattern that mirrored mRNA abundance (P < 0.05). Localization patterns were also determined for UPTI, PAG2, GLUD2 and 14-3-3 gamma in Day 35 extraembryonic membranes. Observed differences in gene and protein expression in nuclear transfer extraembryonic membranes indicate that an impaired fetal-maternal interface, and not the activation method, may be causing defects observed in cloned pigs.