Mitochondrial DNA dynamics during in vitro culture and pluripotency induction of a bovine Rho0 cell line.

Large number of cellular changes and diseases are related to mutations in the mitochondrial DNA copy number. Cell culture in the presence of ethidium bromide is a known way of depleting mitochondrial DNA and is a useful model for studying such conditions. Interestingly, the morphology of these depleted cells resembles that of pluripotent cells, as they present larger and fragmented mitochondria with poorly developed cristae. Herein, we aimed to study the mechanisms responsible for the control of mitochondrial DNA replication during mitochondrial DNA depletion mediated by ethidium bromide and during the in vitro induction of cellular pluripotency with exogenous transcription factor expression in a bovine model. This article reports the generation of a bovine Rho0 mesenchymal cell line and describes the analysis of mitochondrial DNA copy number in a time-dependent manner. The expression of apoptosis and mitochondrial-related genes in the cells during mitochondrial DNA repletion were also analyzed. The dynamics of mitochondrial DNA during both the depletion process and in vitro reprogramming are discussed. It was possible to obtain bovine mesenchymal cells almost completely depleted of their mitochondrial DNA content (over 90%). However, the production of induced pluripotent stem cells from the transduction of both control and Rho0 bovine mesenchymal cells with human reprograming factors was not successful.

Impactos das biotécnicas reprodutivas no controle epigenético de genes imprinted / Impact of reproductive biotechniques on the epigenetic regulation of imprinted genes

Técnicas de reprodução assistida (TRAs) são utilizadas nas medicinas humana e veterinária com oobjetivo principal de corrigir infertilidades adquiridas ou herdadas. A transferência nuclear de célula somática(TNCS) ocupa um lugar de destaque na veterinária pela possibilidade de geração de indivíduos geneticamenteidênticos, o que permite a produção de rebanhos homogêneos de alto mérito genético, e serve como modelo deestudo para técnicas de reprogramação. Porém, a utilização de TRAs, e em especial da TNCS, é consideradaresponsável pelo aumento na geração de conceptos portadores de alterações durante e após os desenvolvimentosembrionário e fetal. A provável causa é a alteração na regulação da reprogramação epigenética devido àmanipulação de gametas e embriões no período inicial do desenvolvimento, que leva a alterações na regulaçãoepigenética de genes imprinted. Esta revisão discute como marcas epigenéticas e expressão de genes imprintedpodem influenciar no desenvolvimento de conceptos bovinos produzidos por TNCS ou inseminação artificial(IA). O entendimento dos mecanismos epigenéticos relacionados aos desenvolvimentos embrionário e fetal, emespecial daqueles relacionados à dinâmica das alterações epigenéticas envolvidas no imprinting genômico, devecontribuir para a elaboração de biotécnicas mais eficientes para a medicina regenerativa ou a produção animal.(AU)

Assisted reproductive technologies (ARTs) are usually used in both human and veterinary medicineaiming the correction of heritable or acquired infertilities. The somatic cell nuclear transfer technique (SCNT) isof particular importance in veterinary as it enables the generation of genetically identical organisms, allowingthe production of homogeneous genetically improved herds, and also serving as a model for reprogrammingstudies. However, the use of TRAs, SCNT in special, may be responsible for the increase of developmentalrelatedabnormalities in the conceptuses. Such phenotypes are probably caused by a disruption in the epigeneticreprogramming due to the manipulation of gametes and embryos during the early development period, andtherefore leading to disturbances in the epigenetic regulation of imprinted genes. The present review discusseshow the epigenetic marks and expression of imprinted genes may influence the developmental competence ofanimals generated by SCNT or artificial insemination (AI). The understanding of the epigenetic mechanismsrelated to embryonic and fetal development, and in special, of those related to the epigenetic dynamics duringgenomic imprinting may contribute to the generation of efficient ARTs to be used in both regenerative medicineand animal production.(AU)

Impactos das biotécnicas reprodutivas no controle epigenético de genes imprinted / Impact of reproductive biotechniques on the epigenetic regulation of imprinted genes

Técnicas de reprodução assistida (TRAs) são utilizadas nas medicinas humana e veterinária com oobjetivo principal de corrigir infertilidades adquiridas ou herdadas. A transferência nuclear de célula somática(TNCS) ocupa um lugar de destaque na veterinária pela possibilidade de geração de indivíduos geneticamenteidênticos, o que permite a produção de rebanhos homogêneos de alto mérito genético, e serve como modelo deestudo para técnicas de reprogramação. Porém, a utilização de TRAs, e em especial da TNCS, é consideradaresponsável pelo aumento na geração de conceptos portadores de alterações durante e após os desenvolvimentosembrionário e fetal. A provável causa é a alteração na regulação da reprogramação epigenética devido àmanipulação de gametas e embriões no período inicial do desenvolvimento, que leva a alterações na regulaçãoepigenética de genes imprinted. Esta revisão discute como marcas epigenéticas e expressão de genes imprintedpodem influenciar no desenvolvimento de conceptos bovinos produzidos por TNCS ou inseminação artificial(IA). O entendimento dos mecanismos epigenéticos relacionados aos desenvolvimentos embrionário e fetal, emespecial daqueles relacionados à dinâmica das alterações epigenéticas envolvidas no imprinting genômico, devecontribuir para a elaboração de biotécnicas mais eficientes para a medicina regenerativa ou a produção animal.

Assisted reproductive technologies (ARTs) are usually used in both human and veterinary medicineaiming the correction of heritable or acquired infertilities. The somatic cell nuclear transfer technique (SCNT) isof particular importance in veterinary as it enables the generation of genetically identical organisms, allowingthe production of homogeneous genetically improved herds, and also serving as a model for reprogrammingstudies. However, the use of TRAs, SCNT in special, may be responsible for the increase of developmentalrelatedabnormalities in the conceptuses. Such phenotypes are probably caused by a disruption in the epigeneticreprogramming due to the manipulation of gametes and embryos during the early development period, andtherefore leading to disturbances in the epigenetic regulation of imprinted genes. The present review discusseshow the epigenetic marks and expression of imprinted genes may influence the developmental competence ofanimals generated by SCNT or artificial insemination (AI). The understanding of the epigenetic mechanismsrelated to embryonic and fetal development, and in special, of those related to the epigenetic dynamics duringgenomic imprinting may contribute to the generation of efficient ARTs to be used in both regenerative medicineand animal production.

Epigenetic anomalies associated with prenatal survival and neonatal morbidity in cloned calves

Many of the developmental anomalies observed in cloned animals are related to fetal and placental overgrowth, a phenomenon known as the “large offspring syndrome” (LOS) in ruminants. It has been hypothesized that the epigenetic control of imprinted genes, i.e. genes that are expressed in a parental-specific manner, is at the root of LOS. Our recent research has focused on understanding the epigenetic alterations to imprinted genes that are associated with assisted reproductive technologies (ART), such as early embryo in vitro culture (IVC) and somatic cell nuclear transfer (SCNT) in cattle. We have searched and identified single nucleotide polymorphisms in Bos indicus DNA useful for analysis of parental-specific alleles and their respective transcripts in tissues from hybrid embryos derived by crossing Bos indicus and Bos taurus cattle. Due to the frequency of placental anomalies in SCNT and in some IVC gestations, our initial studies focused on genes known to be necessary for trophoblast proliferation (Mammalian Achaete Scute-like Homologue 2; ASCL2) and differentiation (Heart and neural crest cell derivative 1; HAND1). ASCL2 was bi-allelically expressed prior to implantation but paternally silenced after implantation. At day 17, SCNT embryos showed more abundant ASCL2 and less abundant HAND1 transcripts. After implantation, SCNT fetal cotyledons displayed higher ASCL2 and HAND1 than AI and IVC tissues. To further investigate epigenetic anomalies, we analyzed the differentially methylated regions of other imprinted genes in cattle, i.e. SNRPN, H19 and the IGF2R. Compared with the patterns observed in vivo (AI), we observed a generalized hypomethylation of the imprinted allele and the bi-allelic expression of embryos produced by SCNT...

Epigenetic anomalies associated with prenatal survival and neonatal morbidity in cloned calves

Many of the developmental anomalies observed in cloned animals are related to fetal and placental overgrowth, a phenomenon known as the “large offspring syndrome” (LOS) in ruminants. It has been hypothesized that the epigenetic control of imprinted genes, i.e. genes that are expressed in a parental-specific manner, is at the root of LOS. Our recent research has focused on understanding the epigenetic alterations to imprinted genes that are associated with assisted reproductive technologies (ART), such as early embryo in vitro culture (IVC) and somatic cell nuclear transfer (SCNT) in cattle. We have searched and identified single nucleotide polymorphisms in Bos indicus DNA useful for analysis of parental-specific alleles and their respective transcripts in tissues from hybrid embryos derived by crossing Bos indicus and Bos taurus cattle. Due to the frequency of placental anomalies in SCNT and in some IVC gestations, our initial studies focused on genes known to be necessary for trophoblast proliferation (Mammalian Achaete Scute-like Homologue 2; ASCL2) and differentiation (Heart and neural crest cell derivative 1; HAND1). ASCL2 was bi-allelically expressed prior to implantation but paternally silenced after implantation. At day 17, SCNT embryos showed more abundant ASCL2 and less abundant HAND1 transcripts. After implantation, SCNT fetal cotyledons displayed higher ASCL2 and HAND1 than AI and IVC tissues. To further investigate epigenetic anomalies, we analyzed the differentially methylated regions of other imprinted genes in cattle, i.e. SNRPN, H19 and the IGF2R. Compared with the patterns observed in vivo (AI), we observed a generalized hypomethylation of the imprinted allele and the bi-allelic expression of embryos produced by SCNT...(AU)