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

Abstract

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), 8- 16 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 88.78% ± 5.40, respectively) and B (94.44% ± 1,58 and 92.22% ± 2.07, respectively)]. The methylation profile in spermatozoa and matured oocytes for XIST promoter region were 3.12% ± 1.68 e 46.67% ± 10.58, respectively. The methylation patterns found here suggest that these regions are transient differently methylated regions (tDMRs), which are reprogrammed in different time, and are not regions that could give an imprinted character to the XIST. Moreover, Rep A is a candidate for epigenetic marker in in vitro fertilization (IVF) due to it is reprogramming during oocyte maturation. XIST RNA was detected in matured oocytes and individual cells of embryos of 2-cell until morula stages, suggesting the presence of transcripts of both maternal and embryonic origins. Moreover, sense and antisense transcripts were detected at the first and last exons of the XIST locus in embryos and testicular tissue, which may be one or more different transcripts. The molecular characterization of XIST gene in cattle is an initial and important step to understand the events related to XCI during embryogenesis in order to improve the assisted reproductive techniques.