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
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)
Assuntos
Animais , Feminino , Bovinos , Epigênese Genética , Bovinos/embriologia , Bovinos/genética , Técnicas de Reprodução Assistida/veterinária , Metilação de DNA/genética , Técnicas de Cultura Embrionária/veterináriaRESUMO
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.
Assuntos
Feminino , Animais , Bovinos , Bovinos/embriologia , Bovinos/genética , Epigênese Genética , Metilação de DNA/genética , Técnicas de Reprodução Assistida/veterinária , Técnicas de Cultura Embrionária/veterináriaRESUMO
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.
RESUMO
The aim of the present study was to evaluate the effects of sperm motility enhancers and different IVF times on cleavage, polyspermy, blastocyst formation, embryo quality and hatching ability. In Experiment 1, sex-sorted X chromosome-bearing Bos taurus spermatozoa were incubated for 30min before 18h fertilisation with hyperactivating factors, namely 10mM caffeine (CA), 5mM theophylline (TH), 10mM caffeine and 5mM theophylline (CA+TH); and untreated spermatozoa (control). In Experiment 2, matured B. taurus oocytes were fertilised using a short (8h) or standard (18h) fertilisation length, comparing two different fertilisation media, namely synthetic oviducal fluid (SOF) fertilisation medium (SOF-FERT) and M199 fertilisation medium (M199-FERT). Cleavage and blastocyst formation rates were significantly higher in the CA+TH group (77% and 27%, respectively) compared with the control group (71% and 21%, respectively). Cleavage rates and blastocyst formation were significantly lower for the shortest fertilisation time (8h) in M199-FERT medium (42% and 12%, respectively). The SOF-FERT medium with an 8h fertilisation time resulted in the highest cleavage rates and blastocyst formation (74% and 29%, respectively). The SOF-FERT medium produced the highest embryo quality (50% Grade 1) and hatching rate (66%). Motility enhancers did not affect polyspermy rates, whereas polyspermy was affected when fertilisation length was extended from 8h (3%) to 18h (9%) and in M199-FERT (14%) compared with SOF-FERT (6%). We conclude that adding the motility enhancers CA and TH to sex sorted spermatozoa and Tyrode's albumin lactate pyruvate (TALP)-Sperm can improve cleavage and embryo development rates without increasing polyspermy. In addition, shortening the oocyte-sperm coincubation time (8h) resulted in similar overall embryo performance rates compared with the prolonged (18h) interval.
Assuntos
Cafeína/farmacologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Bovinos , Separação Celular , Feminino , Fertilização in vitro/efeitos dos fármacos , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Citometria de Fluxo , MasculinoRESUMO
Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.
Assuntos
Blastocisto/fisiologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Técnicas de Transferência Nuclear/veterinária , Animais , Bovinos , Técnicas de Cultura Embrionária/métodos , FemininoRESUMO
Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.