Siberian Sturgeon Oocyte Extract Induces Epigenetic Modifications of Porcine Somatic Cells and Improves Developmental Competence of SCNT Embryos.

Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from the germinal vesicle (GV) stage Siberian sturgeon oocytes prior to their use as nuclear donor for SCNT would improve in vitro development. A reversible permeability protocol with 4 µg/mL of digitonin for 2 min at 4°C in order to deliver Siberian sturgeon oocyte extract (SOE) to porcine fetal fibroblasts (PFFs) was carried out. As results, the intensity of H3K9ac staining in PFFs following treatment of SOE for 7 h at 18°C was significantly increased but the intensity of H3K9me3 staining in PFFs was significantly decreased as compared with the control (p<0.05). Additionally, the level of histone acetylation in SCNT embryos at the zygote stage was significantly increased when reconstructed using SOE-treated cells (p<0.05), similar to that of IVF embryos at the zygote stage. The number of apoptotic cells was significantly decreased and pluripotency markers (Nanog, Oct4 and Sox2) were highly expressed in the blastocyst stage of SCNT embryos reconstructed using SOE-treated cells as nuclear donor (p<0.05). And there was observed a better development to the blastocyst stage in the SOE-treated group (p<0.05). Our results suggested that pre-treatment of cells with SOE could improve epigenetic reprogramming and the quality of porcine SCNT embryos.

Activation-Induced Cytidine Deaminase Induces DNA Demethylation of Pluripotency Genes in Bovine Differentiated Cells.

Activation-induced cytidine deaminase (AID) is the only enzyme that has been suggested as a putative DNA demethylase in mammals. However, very little is known about AID function as DNA demethylase of bovine differentiated cells toward pluripotent state. To investigate the effect of AID on DNA demethylation, bovine AID complementary DNAs were transfected into bovine differentiated cells, which were mostly methylated in the promoter regions of pluripotency genes. As a result, AID-transfected bovine cells started to transform into colonies at day 19 of transfection. The colonies derived from the transfected cells showed positive alkaline phosphatase (AP) staining and expression of pluripotency genes (OCT-3/4, NANOG, SOX2) and pluripotency-related antigens (SSEA-4, TRA1-60, TRA1-81), which have been widely used to characterize human embryonic stem cells. In particular, the levels of OCT-3/4 and NANOG expression were significantly increased in the AID-transfected cells when compared with the control and empty vector-transfected cells (p < 0.05). Finally, DNA demethylation in the promoter regions of pluripotency genes (OCT-3/4, NANOG) was significantly increased compared with the control (p < 0.05). These results demonstrate that the induction of the AID gene into bovine differentiated cells improves DNA demethylation and expression of pluripotency genes.