239 ALLELE-SPECIFIC EXPRESSION OF X CHROMOSOME-LINKED GENE MAO-A DURING PRE-IMPLANTATION DEVELOPMENT IN BOVINE EMBRYO

Abstract

The in vitro embryo culture might affect epigenetic mechanisms, which are involved in controlling the expression of genes related to embryonic development and inactivation of X chromosome. Female mammals have 2 X chromosomes, and males have only 1. This has led to a particular mechanism of evolution of dosage compensation, called X-chromosome inactivation, an important epigenetic event that must occur in all mammalian female embryos. During embryogenesis, at the late blastocyst development (Xue F et al. 2002 Nature Genet. 31, 216�220), 1 of the 2 X chromosomes is randomly inactivated in each cell of the inner cell mass and preferentially X paternal in trophoblast. The aim of this study was to characterize the allele-specific expression of the X chromosome-linked gene monoamine oxidase type A (MAO-A) during in vitro pre-implantation embryo development in bovine. For phenotyping of the MAO-A gene, the RT-PCR restriction fragment length polymorphism technique was used. Primers were designed flanking a single nucleotide polymorphism and the sequence of forward inner primer creating a site of restriction to the RsaI enzyme, thus allowing the detection of allele-specific expression (Bos taurus Taurus × Bos taurus indicus). Oocytes were aspirated from 9 Nelore heifers homozygous for theA allele previously genotyped. The oocytes were selected, matured in vitro, and inseminated with X-sorted sperm from a Holstein bull homozygous for the G allele. Two pools of 10 heterozygous in vitro embryos of each developmental stage, 4-cell [44 h post-insemination (p.i.)], 8- to 16-cell (72 h p.i.), morula (144 h p.i.), blastocyst (156 p.i.), and expanded blastocyst (168 h p.i.), were produced and frozen until RNA extraction. Total RNA was extracted using Invisorb^® Spin Cell RNA Mini Kit (Invitek, Berlin, Germany) according to the manufacturer’s protocol, and residual genomic DNA was removed with DNase I treatment. cDNA was done using Oligo dT primers (Invitrogen) and superscript III reverse transcriptase (Invitrogen). Nested PCR for each pool was performed and then the amplicons were digested with 10 U of RsaI enzyme (Promega, Madison, WI, USA). The products were separated by electrophoresis on a 3% agarose gel stained with ethidium bromide. The results showed that both alleles were expressionally represented in the 4-cell, 8- to 16-cell, and expanded blastocyst stages, with the X paternal allele disappearing in morula and blastocyst. We can conclude that both, maternal and paternal X chromosomes, are activated in the 2 earliest stages, inactivated in the morula and blastocyst stages, and reactivated in the expanded blastocyst stage.

This research was supported by Embrapa Genetic Resources and Biotechnology and the Brazilian National Council for Scientific and Technological Development (CNPq).