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Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genomes after fertilization to establish a totipotent state for normal development. In the present study, the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, during in vitro fertilization (IVF) of bovine oocytes on subsequent embryonic development were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse bovine ovaries were matured in vitro and subjected to IVF in a defined medium supplemented with 0 (control), 5, 50, and 500 nM TSA for 18 h. After IVF, presumptive zygotes were cultured in modified synthetic oviductal fluid (mSOF) medium until 168 h postinsemination (hpi). Some oocytes were immunostained using antibody specific for histone H4-acetylated lysine 5 at 10 hpi. Cleavage, blastocyst development and cell number of inner cell mass (ICM) and trophectoderm (TE) of blastocysts were assessed. TSA treatment enhanced histone acetylation that was prominent in decondensed sperm nuclei. TSA did not affect the postfertilization cleavage, blastocyst rates, and TE cell number. However, it significantly enhanced ICM cell number (p < 0.05). These results indicate that TSA treatment during IVF of bovine oocytes does not affect blastocyst development but alters the cell number of ICM, suggesting that overriding epigenetic modification of the genome during fertilization has a carryover effect on cell proliferation and differentiation in preimplantation embryos. Thus, further environmental quality controls in assisted reproductive technologies are needed in terms of factors which affect chromatin remodelling.

Abstract. Mouse embryonic stem (ES) cells consist of heterogeneous populations with differing abilities to proliferate and differentiate. We previously demonstrated that the expression level of platelet endothelial cell adhesion molecule 1 (PECAM1)/CD31 was positively correlated with the undifferentiated state of mouse ES cells. In order to screen for a novel gene(s) involved in ES cell pluripotency, we performed an oligo microarray analysis and identified that B-box and SPRY domain containing protein (BSPRY) was expressed at high levels in PECAM1-positive cells. Two splice isoforms of BSPRY, BSPRY-1 and BSPRY-2, were expressed in undifferentiated ES cells and in blastocysts. Knockdown of BSPRY-1/2 in ES cells significantly reduced the number of undifferentiated colonies and caused increased expression of primitive ectoderm marker gene Fgf5. The overexpression of BSPRY-2 reciprocally increased the number of undifferentiated ES cells in the presence of LIF. Similarly, injection of BSPRY-1/2 siRNAs into 2-cell embryos caused developmental retardation and degeneration of embryos, and a significant decrease in the number of cells, especially in the inner cell mass (ICM), was observed at the blastocyst stage. Furthermore, microinjection of a BSPRY-1 expression vector into pronuclear stage embryos resulted in an increase in the hatching blastocysts rate after 120 h of culture. These results suggest that BSPRY-1 and BSPRY-2 are associated with both ES cell pluripotency and early embryonic development. E mbryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocysts and maintain the pluripotency to differentiate into three germ layers. Recent studies have demonstrated that mouse ES cells consist of heterogeneous populations with differing abilities to proliferate and differentiate [1][2][3]. We previously reported that there was wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM1) [1]. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that marker genes for undifferentiated ES cells were highly expressed in PECAM1-positive cells. PECAM1-positive ES cells injected into 8-cell host embryos could contribute to the epiblast in a chimeric embryo, but PECAM1-negative ES cells failed to differentiate into epiblast cells. These results suggested that the PECAM1 expression level was positively correlated with the undifferentiated state of ES cells.In order to search for a novel gene(s) involved in ES cell pluripotency, we analyzed gene expression profiles in PECAM1-positive and PECAM1-negative populations using an oligo microarray and isolated several genes that were expressed at high levels in PECAM1-positive cells. We focused upon one of these genes, which encodes mouse B-box and SPRY domain containing protein (BSPRY) protein, because in situ hybridization analysis revealed that Bspry was also expressed at the ICM of blastocysts [4]. These results suggest that BSPRY may be involved in not only ES cell pluripotency but also early emb...

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Abstract. In the present study, we examined the timing of onset, intensity, and mosaicism of embryonic gene expression in bovine nuclear transfer (NT) embryos. The relationship between gene expression and early embryonic development was also examined. To monitor the gene expression of NT embryos, we produced NT embryos with bovine transfected fibroblasts carrying a firefly luciferase gene under the control of a chicken β-actin promoter, an expression system that has previously been shown to be representative of embryonic gene expression in mice. Photon count imaging showed that luciferase luminescence began in NT embryos with fibroblasts 48 hours post fusion (hpf) and reached a plateau at the 4-to 8-cell stage at 60 hpf. Only 4-to 8-cell NT embryos luminescent by 60 hpf developed to the blastocyst stage. At 60 hpf, strongly luminescent embryos developed to the blastocyst stage at a higher rate (P<0.05) than embryos with weak or absent luminescence. However, embryos with mosaic luminescence developed at a much lower rate (P<0.05) than those with wholeembryo luminescence, even if the embryos exhibited strong luminescence. Our results indicate that precise and uniform embryonic gene expression at the 4-to 8-cell stage at 60 hpf may be closely related to development of bovine NT embryos to the blastocyst stage.