RESUMO
The epigenetic reprogramming that occurs during the earliest stages of embryonic development has been described as crucial for the initial events of cell specification and differentiation. Recently, the metabolic status of the embryo has gained attention as one of the main factors coordinating epigenetic events. In this work, we investigate the link between pyruvate metabolism and epigenetic regulation by culturing bovine embryos from day 5 in the presence of dichloroacetate (DCA), a pyruvate analog that increases the pyruvate to acetyl-CoA conversion, and iodoacetate (IA), which inhibits the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to glycolysis inhibition. After 8 h of incubation, both DCA and IA-derived embryos presented higher mitochondrial membrane potential. Nevertheless, in both cases, lower levels of acetyl-CoA, ATP-citrate lyase and mitochondrial membrane potential were found in blastocysts, suggesting an adaptative metabolic response, especially in the DCA group. The metabolic alteration found in blastocysts led to changes in the global pattern of H3K9 and H3K27 acetylation and H3K27 trimethylation. Transcriptome analysis revealed that such alterations resulted in molecular differences mainly associated to metabolic processes, establishment of epigenetic marks, control of gene expression and cell cycle. The latter was further confirmed by the alteration of total cell number and cell differentiation in both groups when compared to the control. These results corroborate previous evidence of the relationship between the energy metabolism and the epigenetic reprogramming in preimplantation bovine embryos, reinforcing that the culture system is decisive for precise epigenetic reprogramming, with consequences for the molecular control and differentiation of cells.
Assuntos
Epigênese Genética , Transcriptoma , Feminino , Gravidez , Animais , Bovinos , Acetilcoenzima A/metabolismo , Desenvolvimento Embrionário/genética , Blastocisto/metabolismo , Perfilação da Expressão Gênica , Piruvatos/metabolismoRESUMO
Several opportunities for embryo development, stem cell maintenance, cell fate, and differentiation have emerged using induced pluripotent stem cells (iPSCs). However, the difficulty in comparing bovine iPSCs (biPSCs) with embryonic stem cells (ESCs) was a challenge for many years. Here, we reprogrammed fetal fibroblasts by transient expression of the four transcription factors (Oct4, Sox2, Klf4, and c-Myc, collectively termed "OSKM" factors) and cultured in iPSC medium, supplemented with bFGF, bFGF2i, leukemia inhibitory factor (LIF), or LIF2i, and then compared these biPSC lines with bESC to evaluate the pluripotent state. biPSC lines were generated in all experimental groups. Particularly, reprogrammed cells treated with bFGF were more efficient in promoting the acquisition of pluripotency. However, LIF2i treatment did not promote continuous self-renewal. biPSCs (line 2) labeled with GFP were injected into early embryos (day 4.5) to assess the potential to contribute to chimeric blastocysts. The biPSC lines show a pluripotency state and are differentiated into three embryonic layers. Moreover, biPSCs and bESCs labeled with GFP were able to contribute to chimeric blastocysts. Additionally, biPSCs have shown promising potential for contributing to chimeric blastocysts and for future studies.
RESUMO
Besides their canonical roles as energy sources, short-chain fatty acids act as metabolic regulators of gene expression through histone posttranslational modifications. Ketone body ß-hydroxybutyrate (BHB) causes a novel epigenetic modification, histone lysine ß-hydroxybutyrylation (Kbhb), which is associated with genes upregulated in starvation-responsive metabolic pathways. Dairy cows increase BHB in early lactation, and the effects of this increase on cellular epigenomes are unknown. We searched for and identified that Kbhb is present in bovine tissues in vivo and confirmed that this epigenetic mark is responsive to BHB in bovine and human fibroblasts cultured in vitro in a dose-dependent manner. Maturation of cumulus-oocyte complexes with high concentrations of BHB did not affect the competence to complete meiotic maturation or to develop until the blastocyst stage. BHB treatment strongly induced H3K9bhb in cumulus cells, but faintly in oocytes. RNA-seq analysis in cumulus cells indicated that BHB treatment altered the expression of 345 genes. The downregulated genes were mainly involved in glycolysis and ribosome assembly pathways, while the upregulated genes were involved in mitochondrial metabolism and oocyte development. The genes and pathways altered by BHB will provide entry points to carry out functional experiments aiming to mitigate metabolic disorders and improve fertility in cattle.
Assuntos
Ácido 3-Hidroxibutírico , Células do Cúmulo , Epigênese Genética , Histonas , Lisina , Oócitos , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/farmacologia , Animais , Bovinos , Células do Cúmulo/metabolismo , Feminino , Histonas/metabolismo , Humanos , Lisina/metabolismo , Oócitos/metabolismoRESUMO
Metabolic and molecular profiles were reported as different for bovine embryos with distinct kinetics during the first cleavages. In this study, we used this same developmental model (fast vs slow) to determine if the relationship between metabolism and developmental kinetics affects the levels of acetylation or tri-methylation at histone H3 lysine 9 (H3K9ac and H3K9me3, respectively). Fast and slow developing embryos presented different levels of H3K9ac and H3K9me3 from the earliest stages of development (40 and 96 hpi) and up to the blastocyst stage. For H3K9me3, both groups of embryos presented a wave of demethylation and de novo methylation, although it was more pronounced in fast than slow embryos, resulting in blastocysts with higher levels of this mark. The H3K9ac reprogramming profile was distinct between kinetics groups. While slow embryos presented a wave of deacetylation, followed by an increase in this mark at the blastocyst stage, fast embryos reduced this mark throughout all the developmental stages studied. H3K9me3 differences corresponded to writer and eraser transcript levels, while H3K9ac patterns were explained by metabolism-related gene expression. To verify if metabolic differences could alter levels of H3K9ac, embryos were cultured with sodium-iodoacetate (IA) or dichloroacetate (DCA) to disrupt the glycolytic pathway or increase acetyl-CoA production, respectively. IA reduced H3K9ac while DCA increased H3K9ac in blastocysts. Concluding, H3K9me3 and H3K9ac patterns differ between embryos with different kinetics, the second one explained by metabolic pathways involved in acetyl-CoA production. So far, this is the first study demonstrating a relationship between metabolic differences and histone post-translational modifications in bovine embryos.
Assuntos
Blastocisto , Processamento de Proteína Pós-Traducional , Acetilação , Animais , Blastocisto/metabolismo , Bovinos , Histonas/metabolismo , MetilaçãoRESUMO
The effect of inbreeding depression on sperm motility is well documented, but its influence on sperm morphometry has been scarcely examined to date. Here, we combined the use of computer-assisted sperm morphometry analysis (CASMA) with a SNP-based genomic approach to determine and characterize the effect of inbreeding on the sperm shape of a highly inbred cattle population. We determined seven morphometric parameters on frozen-thawed sperm samples of 57 Retinta bulls: length (L, µm), width (W, µm), area (A, µm2 ), perimeter (P, µm), ellipticity (ELI; L/W), elongation (L-W)/(L + W) and perimeter-to-area shape factor (p2a; P2 /4 × π × A). The comparison of highly inbred (HI) and lowly inbreed (LI) individuals based on runs of homozygosity (ROH) inbreeding values (F ROH ) showed no differences between groups. An additional two-step unsupervised sperm subpopulation analysis based on morphometric parameters showed significant differences in the abundance of different sperm subpopulations between groups (p < 0.05). This analysis revealed that HI bulls harbored a higher percentage of narrow-head sperm as opposed to the higher percentage of large- and round-headed sperm detected in LI. A further genomic characterization revealed 23 regions differentially affected by inbreeding in both groups, detecting six genes (SPAG6, ARMC3, PARK7, VAMP3, DYNLRB2, and PHF7) previously related to different spermatogenesis-associated processes.
Assuntos
Bovinos/genética , Depressão por Endogamia/genética , Endogamia , Espermatozoides/ultraestrutura , Animais , Animais Endogâmicos , Variação Biológica Individual , Forma Celular , DNA/genética , Estudos de Associação Genética , Genótipo , Haplótipos/genética , Masculino , Cabeça do Espermatozoide/ultraestruturaRESUMO
Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knock-out. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts.
RESUMO
In many cell types, epigenetic changes are partially regulated by the availability of metabolites involved in the activity of chromatin-modifying enzymes. Even so, the association between metabolism and the typical epigenetic reprogramming that occurs during preimplantation embryo development remains poorly understood. In this work, we explore the link between energy metabolism, more specifically the tricarboxylic acid cycle (TCA), and epigenetic regulation in bovine preimplantation embryos. Using a morphokinetics model of embryonic development (fast- and slow-developing embryos), we show that DNA methylation (5mC) and hydroxymethylation (5hmC) are dynamically regulated and altered by the speed of the first cleavages. More specifically, slow-developing embryos fail to perform the typical reprogramming that is necessary to ensure the generation of blastocysts with higher ability to establish specific cell lineages. Transcriptome analysis revealed that such differences were mainly associated with enzymes involved in the TCA cycle rather than specific writers/erasers of DNA methylation marks. This relationship was later confirmed by disturbing the embryonic metabolism through changes in α-ketoglutarate or succinate availability in culture media. This was sufficient to interfere with the DNA methylation dynamics despite the fact that blastocyst rates and total cell number were not quite affected. These results provide the first evidence of a relationship between epigenetic reprogramming and energy metabolism in bovine embryos. Likewise, levels of metabolites in culture media may be crucial for precise epigenetic reprogramming, with possible further consequences in the molecular control and differentiation of cells.
Assuntos
Blastocisto/enzimologia , Blastocisto/metabolismo , Ciclo do Ácido Cítrico , Metilação de DNA , Animais , Blastocisto/citologia , Bovinos , Meios de Cultura/metabolismo , Desenvolvimento Embrionário/genética , Metabolismo Energético , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Ácidos Cetoglutáricos/metabolismo , Gravidez , Ácido Succínico/metabolismoRESUMO
Orchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming. Recently, several strategies have been applied in order to improve reprogramming efficiency, mainly focused on removing repressive epigenetic marks such as histone methylation from the somatic nucleus. Herein we used the specific and non-toxic chemical probe UNC0638 to inhibit the catalytic activity of the histone methyltransferases EHMT1 and EHMT2. Either the donor cell (before reconstruction) or the early embryo was exposed to the probe to assess its effect on developmental rates and epigenetic marks. First, we showed that the treatment of bovine fibroblasts with UNC0638 did mitigate the levels of H3K9me2. Moreover, H3K9me2 levels were decreased in cloned embryos regardless of treating either donor cells or early embryos with UNC0638. Additional epigenetic marks such as H3K9me3, 5mC, and 5hmC were also affected by the UNC0638 treatment. Therefore, the use of UNC0638 did diminish the levels of H3K9me2 and H3K9me3 in SCNT-derived blastocysts, but this was unable to improve their preimplantation development. These results indicate that the specific reduction of H3K9me2 by inhibiting EHMT1/2 during nuclear reprogramming impacts the levels of H3K9me3, 5mC, and 5hmC in preimplantation bovine embryos.
Assuntos
Reprogramação Celular/genética , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Histona Metiltransferases/genética , Animais , Blastocisto , Bovinos , Diferenciação Celular , Clonagem de Organismos/métodos , Transferência Embrionária/métodos , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Técnicas de Transferência Nuclear , Oócitos/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional/genética , Quinazolinas/farmacologiaRESUMO
Cryopreservation of in vitro-derived bovine embryos is a crucial step for the widespread reproduction and conservation of valuable high-merit animals. Given the current popularity of bovine in vitro embryo production (IVP), there is a demand for a highly efficient ultra-low temperature storage method in order to maximize donor ovum pickup (OPU) turn-over, recipient availability/utilization and domestic/overseas commercial trading opportunities. However, IVP bovine embryos are still very sensitive to chilling and cryopreservation, and despite recent progress, a convenient (simple and robust) protocol has not yet been developed. At the moment, there are two methods for bovine IVP embryo cryopreservation: slow programmable freezing and vitrification. Both of the aforementioned techniques have pros and cons. While controlled-rate slow cooling can easily be adapted for direct transfer (DT), ice crystal formation remains an issue. On the other hand, vitrification solved this problem but the possibility of successful DT commercial incorporation remains to be determined. Moreover, simplification of the vitrification protocol (including warming) through the use of an in-straw dilution without the use of a microscope is a prerequisite for its use under farm conditions. This review summarizes the bovine IVP embryo cryopreservation achievements, strengths and limitations of both freezing systems and prospective improvements to enhance cryosurvival, as well as perspectives on future directions of this assisted reproductive technology.
Assuntos
Criopreservação/veterinária , Técnicas de Cultura Embrionária/veterinária , Animais , Bovinos , Criopreservação/métodos , Técnicas de Cultura Embrionária/métodos , Transferência Embrionária/métodos , Transferência Embrionária/veterinária , Embrião de Mamíferos/fisiologia , Feminino , Fertilização in vitro/veterinária , Congelamento , Gravidez , VitrificaçãoRESUMO
Epigenetic mechanisms allow the establishment and maintenance of multiple cellular phenotypes from a single genomic code. At the initiation of development, the oocyte and spermatozoa provide their fully differentiated chromatin that soon after fertilization undergo extensive remodeling, resulting in a totipotent state that can then drive cellular differentiation towards all cell types. These remodeling involves different epigenetic modifications, including DNA methylation, post-translational modifications of histones, non-coding RNAs, and large-scale chromatin conformation changes. Moreover, epigenetic remodeling is responsible for reprogramming somatic cells to totipotency upon somatic cell nuclear transfer/cloning, which is often incomplete and inefficient. Given that environmental factors, such as assisted reproductive techniques (ARTs), can affect epigenetic remodeling, there is interest in understanding the mechanisms driving these changes. We describe and discuss our current understanding of mechanisms responsible for the epigenetic remodeling that ensues during preimplantation development of mammals, presenting findings from studies of mouse embryos and when available comparing them to what is known for human and cattle embryos.(AU)