Conselho Nacional de Desenvolvimento Científico e Tecnológico / Somatic cell nuclear transfer (SCNT) has been used in recent years for various purposes; to produce copies of genetically superior animals or genetically modified animals. Mainly as a research tool, trying to understand aspects related to cell reprogramming. Despite past several years since birth first animal cloned from a somatic cell, the efficiency remains low. The causes are multifactorial and may involve species particularities, the cell used as nuclear donor or enucleated cytoplasts. Currently it is known that the main problem found in cloned embryos is related to an incomplete cellular reprogramming, which in turn compromises the expression of genes required for normal embryonic development. In the present work we studied different cell cycle associations between cytoplasts (oocytes) and donor cells and its relation with a histone deacetylase inhibitor on the reprogramming of pig embryos produced by SCNT. Oocytes were enucleated at MII or TII (pre-activated) and the associated with previously synchronized cells in G1/G0 and G2/M phase of the cell cycle using or not a histone deacetylase inhibitor Scriptaid in the first hours of embryo culture. The results showed cell cycle incompatibilities between TII cytoplasts and G1/G0 cells even as MII cytoplasts G2/M cells regarding cell reprogramming. Scriptaid treatment showed a cell cycle dependent effect, since the improvement in embryo development was observed when using MII cytoplasts, regardless of the cell used as the donor nucleus. Although widely used in order to improve cell reprogramming in cloned embryos, it was demonstrated the relationship between inhibition of deacetylation with cell cycle interactions between and cytoplasts donor cells on the development of pig embryos produced by SCNT. / A clonagem por transferência nuclear de células somáticas (SCNT) têm sido utilizada nos últimos anos para diversos fins, tanto para a produção de cópias de animais geneticamente superiores quanto para a produção de animais geneticamente modificados. Principalmente como ferramenta de pesquisa básica, buscando compreender aspectos relacionados a reprogramação celular. Apesar de passados vários anos desde o nascimento do primeiro animal clonado a partir de uma célula somática, a eficiência da técnica ainda permanece baixa. As causas são multifatoriais, podem envolver características da especie com que se está trabalhando bem como o tipo de célula utilizada como doadora de núcleo e citoplasto utilizado com receptor. Atualmente sabe-se que um dos principais problemas encontrados em embriões provenientes de clonagem estão relacionados a uma incompleta reprogramação da célula utilizada como doadora de núcleo, o que acaba por comprometer a expressão de genes necessários ao desenvolvimento embrionário normal. No presente trabalho foram estudadas diferentes associaçoes de ciclo celular entre citoplastos (oócitos) receptores e células doadoras de núcleo e sua relação com inibidores de deacetilase sobre a reprogramação de embriões suínos produzidos por SCNT. Oócitos foram enucleados no estágio de MII ou TII (pré-ativados) e associados a células previamente sincronizadas nas fases G1/G0 ou G2/M do ciclo celular, para cada grupo ainda foi utilizado ou não o tratamento com o inibidor de deacetilase Scriptaid nas primeiras horas de cultivo embrionário. O resultados obtidos permitem afirmar que existem incompatibilidades de ciclo celular entre citoplastos em TII e células em G1/G0 assim como entre citoplastos em MII e células em G2/M quanto à reprogramação celular. O tratamento com Scriptaid mostrou ter um efeito ciclo celular dependente, uma vez que só foi observada melhora no desenvolvimento embrionário quando utilizado com citoplastos em MII, independente da célula utilizada como doadora de núcleo. Apesar de amplamente utilizados com a finalidade de melhorar a reprogramação celular em embriões clonados, foi demostrado pela primeira vez a relação da inibição da desacetilação com diferentes interações de ciclo celular entre citoplastos e células doadoras de núcleo sobre o desenvolvimento de embriões produzidos por SCNT.

Bone has several crucial functions including mechanical support of movement, hematopoiesis, maintenance of mineral homeostasis, and energy regulation. Bone also undergoes continuous remodeling to maintain its structural integrity, which suggests it has strong respiration and energy consumption capability. It has been shown that during development, bones, in particular, osteoblasts, rely on glucose uptake for proper skeletal development. However, the effect of energy utilization on osteocytes’ function is currently unknown. Osteocytes are terminally differentiated osteoblasts and are deeply embedded into the mineralized matrix of bone. Previous studies have shown that PTH promotes bone anabolism, in part, by stimulating osteoblasts anaerobic glycolysis while suppressing glucose oxidation through the TCA cycle. In osteocytes, PTH suppresses Sost expression (the gene encoding a potent inhibitor of bone formation) by inducing HDAC4/5 nuclear translocation and MEF2C inhibition. Recently, Scriptaid, an HDAC complex inhibitor, has been shown to induce Mef2 expression and exercise-like adaptation in mouse muscles. In myocytes, Scriptaid disrupts the HDACs co-repressor complex and induces nuclear export of HDAC4/5 with MEF2 activation. This will subsequently increase the expressions of several genes related to energy utilization such as Glut4 and Pdk4. Thus we hypothesized that Scriptaid might regulate Sost and Glut4 expression in osteocytes. To investigate the effect of Scriptaid on osteocytes, we treated a mouse osteocytic cell line, Ocy454-12H, with Scriptaid. Unexpectedly, Scriptaid potently suppressed Sost, whereas it increased Glut4 expression. Scriptaid stimulated osteocyte respiration and glucose consumption rate. Mechanistically, Scriptaid treatment of Ocy454-12H induced nuclear translocation of Hdac5 whereas it did not affect Hdac4. Silencing of Hdac5 expression with shRNA increased Sost basal expression and blocked Sost suppression induced by Scriptaid. However, Glut4 up-regulation by Scriptaid was independent of the HDAC4/5-MEF2C pathway. Glut4 luciferase reporter assays demonstrated that two additional transcription factors binding sites, O/E&NF1 and C/EBPα, may mediate Scriptaid-induced Glut4 up-regulation. Taken together, these data demonstrate that in osteocytes Scriptaid suppresses Sost expression through regulating HDAC5-MEF2C signaling. However, Scriptaid increases Glut4 expression through Hdac5-independent mechanisms, and dependent on O/E&NF1 and C/EBPα.