title: Allele-specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos.
abstract: During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8- to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production.
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Laboratory of Animal Reproduction, Brasília, DF, Brazil.
authors.forename: A R
authors.initials: AR
authors.lastname: Ferreira
authors.forename: G M
authors.initials: GM
authors.lastname: Machado
authors.forename: T O
authors.initials: TO
authors.lastname: Diesel
authors.forename: J O
authors.initials: JO
authors.lastname: Carvalho
authors.forename: R
authors.initials: R
authors.lastname: Rumpf
authors.forename: E O
authors.initials: EO
authors.lastname: Melo
authors.forename: M A N
authors.initials: MA
authors.lastname: Dode
authors.forename: M M
authors.initials: MM
authors.lastname: Franco
mesh_terms: D000483:Alleles; D000818:Animals; D001755:Blastocyst / Q000378:metabolism / Q000502:physiology*; D002417:Cattle; D054885:Cumulus Cells; D019175:DNA Methylation; D005260:Female; D020869:Gene Expression Profiling; D008297:Male; D008995:Monoamine Oxidase / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D012150:Polymorphism, Restriction Fragment Length; D012333:RNA, Messenger / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D049951:X Chromosome Inactivation / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D008995:Monoamine Oxidase