Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

<p>Abstract</p> <p>Background</p> <p>Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning.</p> <p>Results</p> <p>Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from <it>in vitro </it>fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping.</p> <p>Conclusion</p> <p>The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.</p

Molecular reprogramming in bovine embryos after serial somatic cell chromatin transfer

Somatic Cell Nuclear Transfer (SCNT), commonly known as cloning, is the transfer of a somatic nucleus into an enucleated oocyte to produce a clone. The chromatin structure of somatic cells permits the expression of certain genes, while silencing the rest of the genome. The cytoplasm of oocytes can reprogram a somatic nucleus by reactivating the genes necessary for embryonic development and silencing the somatic genes. However, the low efficiency of SCNT indicates that successful nuclear reprogramming is a rare event. The objectives of this study were determine the extent of transcriptional reprogramming in bovine blastocysts produced by serial rounds of chromatin transfer (from first and fourth generations), using blastocysts produced by in vitro fertilization (IVF) as controls, to identify cumulative errors in the transcriptome profile. Differentially expressed genes were studied further to determine their function in embryonic development. We identified a set of transcripts consistently misregulated in cloned blastocyst, some of which had a more marked misregulation in the embryos produced by 4 successive rounds of cloning. Among the genes significantly upregulated in both CT groups compared to IVF blastocysts were both de novo DNA methylation enzymes DNMT3A and DNMT3B. Expression patterns, structural and functional analyses were performed for DNA methyltransferases. A high structural and functional conservation was observed for DNA methyltransferases among human, mouse, and bovine species. A set of genes that participate in early embryonic development, chromatin remodeling and DNA methylation were differentially regulated in cloned embryos and had not been fully annotated at the time of the analysis. We annotated those genes and submitted them to the Bovine Genome Sequencing Consortium database. These results have important implications for the selection of models for the study of DNA methylation during early development. The present study provides a valuable data set for identifying possible cumulative errors in somatic cell chromatin transfer that could hinder nuclear reprogramming shedding light on the epigenetic role in reprogramming and cell plasticity

Comparative functional genomics of mammalian DNA methyltransferases

DNA methylation involves biochemical modification of DNA by addition of methyl groups onto CpG dinucleotides, and this epigenetic mechanism regulates gene expression in disease and development. Mammalian DNA methyltransferases, DNMT (DNMT1, DNMT3A and DNMT3B), together with the accessory protein DNMT3L establish specific DNA methylation patterns in the genome during gametogenesis, embryogenesis and somatic tissue development. The present study addresses the structural and functional conservation of the DNMT in humans, mice and cattle and the patterns of mRNA abundance of the different enzymes during embryogenesis to improve understanding of epigenetic regulation in early development. The findings showed a high degree of structural and functional conservation among the human, mouse, and bovine DNMT. The results also showed similar patterns of transcript abundance for all of the proteins at different stages of early embryo development. Remarkably, all of the DNMT with an important role in DNA methylation (DNMT1, DNMT3A, DNMT3B, and DNMT3L) show a greater degree of structural similarity between human and bovine than that between human and mouse. These results have important implications for the selection of an appropriate model for study of DNA methylation during early development in humans. © 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved

Delivering value from sperm proteomics for fertility

Fertilization of an egg by a spermatozoon sets the stage for mammalian development. Viable sperm are a prerequisite for successful fertilization and beyond. Spermatozoa have a unique cell structure where haploid genomic DNA is located in a tiny cytoplasmic space in the head, mitochondria in the midpiece and then the tail, all enclosed by several layers of membrane. Proteins in sperm play vital roles in motility, capacitation, fertilization, egg activation and embryo development. Molecular defects in these proteins are associated with low fertility or in some cases, infertility. This review will first summarize genesis, molecular anatomy and physiology of spermatozoa, fertilization, embryogenesis and then those proteins playing important roles in various aspects of sperm physiology. © Springer-Verlag 2012

Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

Background: Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the cumulative errors could reveal the epigenetic reprogramming blocks in animal cloning. Results: Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion: The present study provides adata set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying cumulative errors after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research. © 2009 Rodriguez-Osorio et al; licensee BioMed Central Ltd

Functional genomics of HMGN3a and SMARCAL1 in early mammalian embryogenesis

Background: Embryonic genome activation (EGA) is a critical event for the preimplantation embryo, which is manifested by changes in chromatin structure, transcriptional machinery, expression of embryonic genes, and degradation of maternal transcripts. The objectives of this study were to determine transcript abundance of HMGN3a and SMARCAL1 in mature bovine oocytes and early bovine embryos, to perform comparative functional genomics analysis of these genes across mammals. Results: New annotations of both HMGN3a and SMARCAL1 were submitted to the Bovine Genome Annotation Submission Database at BovineGenome.org. Careful analysis of the bovine SMARCAL1 consensus gene set for this protein (GLEAN_20241) showed that the NCBI protein contains sequencing errors, and that the actual bovine protein has a high degree of homology to the human protein. Our results showed that there was a high degree of structural conservation of HMGN3a and SMARCAL1 in the mammalian species studied. HMGN3a transcripts were present at similar levels in bovine matured oocytes and 2-4-cell embryos but at higher levels in 8-16-cell embryos, morulae and blastocysts. On the other hand, transcript levels of SMARCAL1 decreased throughout preimplantation development. Conclusion: The high levels of structural conservation of these proteins highlight the importance of chromatin remodeling in the regulation of gene expression, particularly during early mammalian embryonic development. The greater similarities of human and bovine HMGN3a and SMARCAL1 proteins may suggest the cow as a valuable model to study chromatin remodeling at the onset of mammalian development. Understanding the roles of chromatin remodeling proteins during embryonic development emphasizes the importance of epigenetics and could shed light on the underlying mechanisms of early mammalian development. © 2009 Uzun et al; licensee BioMed Central Ltd

Functional genomics of HMGN3a and SMARCAL1 in early mammalian embryogenesis

Background: Embryonic genome activation (EGA) is a critical event for the preimplantation embryo, which is manifested by changes in chromatin structure, transcriptional machinery, expression of embryonic genes, and degradation of maternal transcripts. The objectives of this study were to determine transcript abundance of HMGN3a and SMARCAL1 in mature bovine oocytes and early bovine embryos, to perform comparative functional genomics analysis of these genes across mammals. Results: New annotations of both HMGN3a and SMARCAL1 were submitted to the Bovine Genome Annotation Submission Database at BovineGenome.org. Careful analysis of the bovine SMARCAL1 consensus gene set for this protein (GLEAN_20241) showed that the NCBI protein contains sequencing errors, and that the actual bovine protein has a high degree of homology to the human protein. Our results showed that there was a high degree of structural conservation of HMGN3a and SMARCAL1 in the mammalian species studied. HMGN3a transcripts were present at similar levels in bovine matured oocytes and 2-4-cell embryos but at higher levels in 8-16-cell embryos, morulae and blastocysts. On the other hand, transcript levels of SMARCAL1 decreased throughout preimplantation development. Conclusion: The high levels of structural conservation of these proteins highlight the importance of chromatin remodeling in the regulation of gene expression, particularly during early mammalian embryonic development. The greater similarities of human and bovine HMGN3a and SMARCAL1 proteins may suggest the cow as a valuable model to study chromatin remodeling at the onset of mammalian development. Understanding the roles of chromatin remodeling proteins during embryonic development emphasizes the importance of epigenetics and could shed light on the underlying mechanisms of early mammalian development. © 2009 Uzun et al; licensee BioMed Central Ltd

Effects of culture media and inhibitors on biology of porcine early embryonic development in vitro

In vitro-production of porcine embryos is crucial for biomedical and agricultural research, however, current culture systems for porcine embryos are sub-optimal, and the developmental potential of in vitro-produced embryos is not well studied. The objectives of this study were to evaluate whether PZM-3 or NCSU-23 better support porcine embryo development and the effects of α-amanitin and cycloheximide on porcine embryonic development. Briefly, porcine presumptive zygotes were produced after in vitro maturation/fertilization (IVM/IVF) and cultured either in NCSU-23 or PZM-3 culture media. Transcript levels of BAX and BCL2L1 genes from blastocysts were detected by using Real-time PCR. The effects of α-amanitin and cycloheximide were evaluated for the role of inhibiting transcription and translation during early porcine embryogenesis. Results showed that both cleavage and blastocyst rates decreased significantly in NCSU-23 group compared as PZM-3 group. However, BAX and BCL2L1 transcript levels were similar in blastocysts cultured in both PZM-3 and NCSU-23 media. When porcine embryos cultured in PZM-3, cleavage rates were significantly decreased in the present of cycloheximide and both α-amanitin and cycloheximide treatments completely inhibited the blastocyst formation. Results showed that PZM-3 medium better supported porcine early embryonic development than NCSU-23 medium, and the inhibition of embryonic genome activation does not completely stop embryo cleavage but prevents development to the blastocyst stage. © 2008 Elsevier B.V. All rights reserved