Allele-specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos.
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.
Ferreira
A R
AR
Embrapa Genetic Resources and Biotechnology, Laboratory of Animal Reproduction, Brasília, DF, Brazil.
Machado
G M
GM
Diesel
T O
TO
Carvalho
J O
JO
Rumpf
R
R
Melo
E O
EO
Dode
M A N
MA
Franco
M M
MM
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*
D012333:RNA, Messenger; D008995:Monoamine Oxidase