1 ABERRANT DNA METHYLATION IN PORCINE IN VITRO-, PARTHENOGENETIC-, AND NUCLEAR TRANSFER-PRODUCED BLASTOCYSTS

Abstract

Aberrant DNA methylation of in vitro-, parthenogenetic-, and nuclear transfer-derived embryos has been implicated in the low developmental competence of early embryos. Demethylation of the genome occurs immediately after fertilization and continues through the blastocyst stage. Remethylation or reprogramming of the genome occurs around the time of implantation and is maintained in somatic tissues. The aim of this study was to analyze DNA methylation in porcine gametes and blastocysts. Differential DNA methylation hybridization was conducted to analyze the methylation status of the Bstu I site (CGCG) in the gamete and blastocyst epigenomes. Germinal vesicle oocytes were aspirated from ovaries collected at an abattoir, sperm was isolated from a fresh ejaculate, and blastocysts were derived and collected from in vivo, in vitro, nuclear transfer, and parthenogenetic sources. Genomic clones were selected from a porcine CpG Island library based on the presence of a Bstu I site. The inserts from these clones were PCR amplified and spotted on glass slides. DNA was digested with Mse I, ligated to linkers, and digested with Bstu I. Fragments with methylated Bstu I sites remained intact whereas fragments with unmethylated Bstu I sites were cut. Intact fragments were amplified by PCR and labeled with amino allyl-dUTP. Liver DNA served as the reference and was labeled with Cy5; the other samples were labeled with Cy3. An Axon Genepix 4000B scanner (Axon Instruments, Inc., Union City, CA, USA) was used to scan the slides. Initial analysis of the microarray image was performed with GenePix Pro 4.0 software. Additional analysis, performed by using Genespring 7.0 ANOVA (P < 0.05), identified 221 clones as being significantly different in at least one of the biological conditions of the gametes or the blastocysts. Forty-six clones were sequenced and BLAST analysis identified 18 clones that were unique, 16 clones that had no similarity, and 12 clones that had similarity to multiple genes. Ribosomal (RPS20, RPL18) and protoporphyrinogen oxidase (PPOX) genes were identified in several clones. Components of the immune system (CCRs, TLRs), a transcription factor (ATF2), and an embryo-specific gene (WNT8B) were also identified. A condition tree was created according to the standard correlation similarity measure for the spots identified as significantly different. The condition tree shows that the methylation profiles are most similar in the germinal vesicle oocyte, parthenogenetic blastocyst, nuclear transfer blastocyst, in vitro-produced blastocyst, and sperm. In vivo-produced blastocysts grouped separately from the other samples. These results are consistent with previous studies that have shown that gametes undergo demethylation after fertilization on through the blastocyst stage when the genome is remethylated. Additionally, these results suggest that the reprogramming events that occur during the development of the in vivo-produced blastocysts are less likely to occur in in vitro-, nuclear transfer-, and parthenogenetic-produced blastocysts.

This work was funded by a grant from the NIH (RR13438) and Food for the 21st Century.