Caracterização epigenética e funcional do Transcrito Específico do X Inativo (XIST) durante o desenvolvimento embrionário inicial in vitro em bovinos

During the initial development of mammals, an event of X chromosome inactivation (XCI) occurs in female embryos, which is predominantly coordinated by epigenetic factors. In mice, it is known that the long non-coding RNA (lncRNA) X-Inactive Specific Transcript (XIST), in association with a small RNA named Rep A, are essentials to the initiation of the XCI process. However, little is known about this mechanism in domestic animals. The aim of this study was to characterize the DNA methylation and gene expression patterns of the lncRNA XIST during the early development in cattle. Three different regions of 5’ portion and first exon of XIST were evaluated for DNA methylation (named here as promoter, Rep A and DMR 1) in gametes, embryos and placenta. Regarding gene expression, it was investigated the expression pattern in individual blastomeres, as wells as strand-specific expression (sense and antisense transcription) along the gene. For DNA methylation, PCR of sodium bisulfite treated-DNA followed by DNA sequencing was used. Single cell-to-CT (Ambion) kit was used for gene expression of oocytes and individual embryonic cells. Regarding sense and antisense gene expression, genespecific primers were used for cDNA synthesis. Real-time PCR was conducted using Fast Sybr Green Master Mix (Applied Biosystems). DNA methylation patterns for DMR 1 and Rep A were determined in spermatozoa (3.33% ± 1.05 and 10.36% ± 3.73, respectively), immature oocytes (79.54% ± 6.56 and 12.29% ± 4.21, respectively) matured oocytes (89.59% ± 2.31 and 74.27% ± 8.77, respectively), 816 cell embryos (0.0% ± 0.00 and 92.18% ± 2.22, respectively), morula (0.84% ± 0.57 and 95.33% ± 0.51, respectively), inner cell mass of blastocysts (1.07% ± 0.72 and 1.97% ± 1.41, respectively), trophoectoderm of blastocysts (3.93% ± 1.24 and 0.00% ± 0.00, respectively), placenta (allantochorion) of two females [A (89.38% ± 2.70 and 26.92% ± 11.27, respectively) and B (70.92% ± 10.70 and 89.24% ± 2.50, respectively)] and placenta (allantochorion) of two males [A (94.67% ± 1.18 and