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Candidate gene expression patterns in rabbit preimplantation embryos developed in vivo and in vitro.

ABSTRACT: PURPOSE: The levels and timing of expression of genes like BCLXL, HDAC1 and pluripotency marker genes namely, OCT4, SOX2, NANOG and KLF4 are known to influence preimplantation embryo development. Despite this information, precise understanding of their influence during preimplantation embryo development is lacking. The present study attempts to compare the expression of these genes in the in vivo and in vitro developed preimplantation embryos. METHODS: The in vivo and in vitro developed rabbit embryos collected at distinct developmental stages namely, pronuclear, 2 cell, 4 cell, 8 cell, 16 cell, Morula and blastocyst were compared at the transcriptional and translational levels using Real Time PCR and immunocytochemical studies respectively. RESULTS: The study establishes the altered levels of candidate genes at the transcriptional level and translational level with reference to the zygotic genome activation (ZGA) phase of embryo development in the in vivo and in vitro developed embryos. The expression of OCT4, KLF4, NANOG and SOX2 genes were higher in the in vitro developed embryos whereas and HDAC1 was lower. BCLXL expression had its peak at ZGA in in vivo developed embryos. Protein expression of all the candidate genes was observed in the embryos. BCLXL, KLF4 and NANOG exhibited diffused localisation whereas HDAC1, OCT4, and SOX2 exhibited nuclear localisation. CONCLUSIONS: This study leads to conclude that BCLXL peak expression at the ZGA phase may be a requirement for embryo development. Further expression of all the candidate genes was influenced by ZGA phase of development at the transcript level, but not at the protein level.

SUBMITTER: Henderson GR

PROVIDER: S-EPMC4096891 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Candidate gene expression patterns in rabbit preimplantation embryos developed in vivo and in vitro.

Henderson Gibence Rose Winnie GR Brahmasani Sambasiva Rao SR Yelisetti Uma Mahesh UM Konijeti Suman S Katari Venu Charan VC Sisinthy Shivaji S

Journal of assisted reproduction and genetics 20140424 7

<h4>Purpose</h4>The levels and timing of expression of genes like BCLXL, HDAC1 and pluripotency marker genes namely, OCT4, SOX2, NANOG and KLF4 are known to influence preimplantation embryo development. Despite this information, precise understanding of their influence during preimplantation embryo development is lacking. The present study attempts to compare the expression of these genes in the in vivo and in vitro developed preimplantation embryos.<h4>Methods</h4>The in vivo and in vitro devel ...[more]

PMID: 24760721

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Project description:BACKGROUND: The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos. RESULTS: The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4) were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A) exhibited the highest degree of homology (96.4%). Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A) was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1. The results of quantitative real-time PCR experiments showed that POU5F1 mRNA was abundantly present in oocytes and zygotes, but it was gradually reduced until the activation of the embryonic genome, thereafter a continuous increase in POU5F1 mRNA level was observed until blastocyst stage. By using the XYClone laser system the inner cell mass (ICM) and trophoblast portions of embryos were microdissected and examined separately and POU5F1 mRNA was detected in both cell types. CONCLUSION: In this study we provide a comparative sequence analysis of the regulatory region of rabbit POU5F1 gene. Our data suggest that the POU5F1 gene is strictly regulated during early mammalian development. We proposed that the well conserved CR4 region containing the DE-2A enhancer is responsible for the highly conserved ESC specific gene expression. Notably, we are the first to report that the rabbit POU5F1 is not restricted to ICM cells only, but it is expressed in trophoblast cells as well. This information may be well applicable to investigate further the possible phylogenetic role and the regulation of POU5F1 gene.

Project description:Vitrification is an economically effective method for embryo cryopreservation in human and livestock animals; however, it carries the risk of damage by the exposure to severe oxidative stress. The present study was conducted to evaluate the effect of leptin at different levels on the in vitro development of fresh and vitrified preimplantation embryos in a rabbit model. Normal embryos at morulae stage were randomly cultured for 2 h with 0, 10, 20 or 100 ng/mL of leptin, then were cultured for further 48 h as freshly or after vitrification. Thereafter, developed blastocysts form the best leptin level in fresh and vitrified embryos along with their controls were allocated to analyze the pro-oxidant (malondialdehyde, MDA; nitric oxide, NO), antioxidant (total antioxidant capacity, TAC; superoxide dismutase, SOD; glutathione peroxidase, GPx), apoptotic (Bcl-2 associated X protein, BAX; heat shock 60kD protein member 1, HSP60; tumor necrosis factor alpha, TNFα) and developmental (sex determining region Y box protein 2, SOX2; Nanog homeobox protein, NANOG; Octamer-binding protein 4, OCT4) biomarkers. Results indicate that expanding and hatching rates of embryos were significantly higher at 20 ng/mL leptin than the other levels, while vitrification had an independent suppression effect on the in vitro development rates. The MDA and NO were significantly higher, while TAC, SOD and GPx were significantly lower in the vitrified than fresh embryos. In contrast, leptin treatment significantly decreased the pro-oxidant biomarkers and increased the antioxidant biomarkers in both fresh and vitrified embryos. Vitrification significantly increased the antiapoptotic biomarkers, and decreased the developmental biomarkers in embryos. In contrast, leptin decreased the BAX and TNFα, increased the HSP60, and moreover, ameliorated the reduction of developmental biomarkers in the vitrified embryos. These results conclude that leptin could be used as antiapoptotic and antioxidant promotor to support the in vitro embryonic development, particularly under oxidative stress emerged from cryopreservation programs.

Project description:The surge in the number of gene expression studies and tendencies to increase the quality of analysis have necessitated the identification of stable reference genes. Although rabbits are classical experimental model animals, stable reference genes have not been identified for normalization. The aims of this study were to compare the expression profiles of the widely used reference genes in rabbit oocytes and preimplantation stage embryos, and to select and validate stable ones to use as reference.Quantitative real time PCR method was used to evaluate 13 commonly used references (Actb, Gapdh, Hprt1, H2afz, Ubc, Ppia, Eef1e1, Polr2a, Tbp, G6pdx, B2m, Pgk1, and Ywhaz) and POU5F1 (Oct4) genes. Expressions of these genes were examined in multiple individual embryos of seven different preimplantation developmental stages and embryo types (in vivo and in vitro). Initial analysis identified three genes (Ubc, Tbp, and B2m) close to the detection limit with irregular expression between the different stages. As variability impedes the selection of stable genes, these were excluded from further analysis. The expression levels of the remaining ten genes, varied according to developmental stage and embryo types. These genes were ranked using the geNorm software and finally the three most stable references (H2afz, Hprt1, and Ywhaz) were selected. Normalization factor was calculated (from the geometric averages of the three selected genes) and used to normalize the expressions of POU5F1 gene. The results showed the expected expression patterns of the POU5F1 during development.Compared to the earlier studies with similar objectives, the comparison of large number of genes, the use of multiple individual embryos as compared to pools, and simultaneous analyses of in vitro and in vivo derived embryo samples were unique approaches in our study. Based on quantification, pattern and geNorm analyses, we found the three genes (H2afz, Hprt1, and Ywhaz) to be the most stable across developmental stages and embryo types, and the geometric averages of these genes can be used for appropriate normalization.

Project description:Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.

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Candidate gene expression patterns in rabbit preimplantation embryos developed in vivo and in vitro.

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Candidate gene expression patterns in rabbit preimplantation embryos developed in vivo and in vitro.

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