RESUMO
Embryonic and placental development is highly orchestrated by epigenetic processes. Disruptions in normal placental development, commonly observed in pregnancies produced by nuclear transfer, are associated with abnormal gene expression and altered epigenetic regulation of imprinted and vital placental genes. The objective of this study was to evaluate expression and epigenetic regulation of the imprinted gene TSSC4 in cotyledonary and intercotyledonary tissues from day 60 pregnancies produced by embryo transfer (ET), in vitro fertilization (IVF) and nuclear transfer (NT) in cattle. TSSC4 expression was reduced by 30% in cotyledons at 60days of gestation in the NT group. The proximal promoter region of TSSC4 showed an increase in the permissive histone mark (H3K4me2) and a reduction in the inhibitory histone mark (H3K9me2) in the cotyledons produced by NT, in relation to cotyledons produced by embryo transfer. Interestingly, H3K9me2 was also significantly reduced in cotyledons produced by IVF, compared to the ET controls. DNA methylation, in CpG-rich regions located at the proximal promoter region and the coding region of TSSC4 did not differ. These results suggest that the reduction in TSSC4 expression, observed following NT, can not be explained by the histone changes investigated in the proximal promoter region of the gene, or by changes in methylation in three regions evaluated. Also, a decrease in the levels of H3K9 dimethylation in IVF samples, indicate that in vitro culturing could corroborate with the alterations seen in the NT group.
Assuntos
Bovinos/genética , Transferência Embrionária/métodos , Fertilização in vitro/métodos , Técnicas de Transferência Nuclear , Placenta/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Especificidade de Órgãos , Gravidez , Proteínas Supressoras de Tumor/genéticaRESUMO
Abnormal placental development is frequent in nuclear transfer (NT) pregnancies and is likely to be associated with altered epigenetic reprogramming. In the present study, fetal and placental measurements were taken on Day 60 of gestation in cows with pregnancies produced by AI, IVF and NT. Placentas were collected and subjected to histological evaluation, the expression of genes important in trophoblast differentiation and expression of the placental imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2), as well as chromatin immunoprecipitation (ChIP) for histone marks within the promoter of PHLDA2. Fewer binucleated cells were observed in NT cotyledons, followed by IVF and AI cotyledons (P<0.05). Expression of heart and neural crest derivatives expressed 1 (HAND1), placental lactogen (PL), pregnancy-associated glycoprotein 9 (PAG-9) and PHLDA2 was elevated in NT cotyledons compared with AI cotyledons. Expression of PHLDA2 was higher in IVF than AI samples (P<0.05). ChIP revealed an increase in the permissive mark dimethylation of lysine 4 on histone H3 (H3K4me2), surprisingly associated with the silent allele of PHLDA2, and a decrease in the inhibitory mark H3K9me2 in NT samples. Thus, genes critical for placental development were altered in NT placentas, including an imprinted gene. Allele-specific changes in the permissive histone mark in the PHLDA2 promoter indicate misregulation of imprinting in clones. Abnormal trophoblast differentiation could have resulted in lower numbers of binucleated cells following NT. These results suggest that the altered expression of imprinted genes associated with NT are also caused by changes in histone modifications.
Assuntos
Expressão Gênica , Código das Histonas , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Técnicas de Transferência Nuclear/veterinária , Placenta/metabolismo , Alelos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bovinos , Feminino , Histonas/genética , Proteínas Nucleares/genética , Lactogênio Placentário/genética , Lactogênio Placentário/metabolismo , Placentação/fisiologia , Gravidez , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Trofoblastos/metabolismoRESUMO
Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585±34,775 vs. 595,579±31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179±45,617 vs. 498,771±33,231) and blastocysts (816,627±40,235 vs. 765,332±51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.