Mitotic chromosome assembly despite nucleosome depletion in Xenopus egg extracts.

The nucleosome is the fundamental structural unit of eukaryotic chromatin. During mitosis, duplicated nucleosome fibers are organized into a pair of rod-shaped structures (chromatids) within a mitotic chromosome. However, it remains unclear whether nucleosome assembly is indeed an essential prerequisite for mitotic chromosome assembly. We combined mouse sperm nuclei and Xenopus cell-free egg extracts depleted of the histone chaperone Asf1 and found that chromatid-like structures could be assembled even in the near absence of nucleosomes. The resultant "nucleosome-depleted" chromatids contained discrete central axes positive for condensins, although they were more fragile than normal nucleosome-containing chromatids. Combinatorial depletion experiments underscored the central importance of condensins in mitotic chromosome assembly, which sheds light on their functional cross-talk with nucleosomes in this process.

Inferring the choreography of parental genomes during fertilization from ultralarge-scale whole-transcriptome analysis.

Fertilization precisely choreographs parental genomes by using gamete-derived cellular factors and activating genome regulatory programs. However, the mechanism remains elusive owing to the technical difficulties of preparing large numbers of high-quality preimplantation cells. Here, we collected >14 × 10(4) high-quality mouse metaphase II oocytes and used these to establish detailed transcriptional profiles for four early embryo stages and parthenogenetic development. By combining these profiles with other public resources, we found evidence that gene silencing appeared to be mediated in part by noncoding RNAs and that this was a prerequisite for post-fertilization development. Notably, we identified 817 genes that were differentially expressed in embryos after fertilization compared with parthenotes. The regulation of these genes was distinctly different from those expressed in parthenotes, suggesting functional specialization of particular transcription factors prior to first cell cleavage. We identified five transcription factors that were potentially necessary for developmental progression: Foxd1, Nkx2-5, Sox18, Myod1, and Runx1. Our very large-scale whole-transcriptome profile of early mouse embryos yielded a novel and valuable resource for studies in developmental biology and stem cell research. The database is available at http://dbtmee.hgc.jp.

B-box and SPRY domain containing protein (BSPRY) is associated with the maintenance of mouse embryonic stem cell pluripotency and early embryonic development.

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.

Gene expression profiling of mouse embryonic stem cell subpopulations.

We previously demonstrated that mouse embryonic stem (ES) cells show a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM1) and that the level of expression is positively correlated with the pluripotency of ES cells. We also found that PECAM1-positive ES cells could be divided into two subpopulations according to the expression of stage-specific embryonic antigen (SSEA)-1. ES cells that showed both PECAM1 and SSEA-1 predominantly differentiated into epiblast after the blastocyst stage. In the present study, we performed pairwise oligo microarray analysis to characterize gene expression profiles in PECAM1-positive and -negative subpopulations of ES cells. The microarray analysis identified 2034 genes with a more than 2-fold difference in expression levels between the PECAM1-positive and -negative cells. Of these genes, 803 were more highly expressed in PECAM1-positive cells and 1231 were more highly expressed in PECAM1-negative cells. As expected, genes known to function in ES cells, such as Pou5f1(Oct3/4)and Nanog, were found to be upregulated in PECAM1-positive cells. We also isolated 23 previously uncharacterized genes. A comparison of gene expression profiles in PECAM1-positive cells that were either positive or negative for SSEA-1 expression identified only 53 genes that showed a more than 2-fold greater difference in expression levels between these subpopulations. However, many genes that are under epigenetic regulation, such as globins, Igf2, Igf2r, andH19, showed differential expression. Our results suggest that in addition to differences in gene expression profiles, epigenetic status was altered in the three cell subpopulations.

Differences in gene expression patterns between somatic cell nuclear transfer embryos constructed with either rabbit granulosa cells or their derivatives.

Successful production of offspring by somatic cell nuclear transfer (SCNT) is affected by the nature of the donor cells used. The purpose of this study was to determine whether characteristic changes induced in donor cells by culture conditions influenced gene expression patterns in the resultant SCNT embryos. Rabbit granulosa cells (rGC) were cultured under different conditions, either with or without hCG, and the two derivative cell types obtained (named respectively cGC+ and cGC-) were used as donor cells for SCNT. There were characteristic differences between fresh rGC and the two derivative cell types: p450scc expression and progesterone secretion were both higher in cGC+ than in cGC-; expression of bmp4 and fgfr2 was decreased in cGC+ and cGC- compared with rGC; and cGC+ and cGC- cell types gained collagenIV expression. Use of fresh rGC, or cGC+ and cGC- derivative cells, did not alter either the developmental potencies of SCNT oocytes or cell numbers at the blastocyst stage. The expression patterns of four genes (bmp4, fgfr2, gata4, oct3/4) in SCNT embryos and in fertilized embryos were analyzed by quantitative RT-PCR. We found that oct3/4 was expressed in all embryos. The expression patterns of the other three genes showed considerable variation between the different types of embryo: bmp4 was found in most fertilized embryos but only some of rGC and none of cGC+ and cGC- derived SCNT embryos; fgfr2 was present in fertilized embryos but was present in some rGC and cGC- NT embryos and in all cGC+ NT embryos; gata4 was not expressed in fertilized embryos but was present in a few rGC and cGC+ NT embryos and in most cGC- NT embryos. Our results suggest that the gene expression patterns in SCNT embryos derived from granulosa donor cells are affected by characteristic changes to the cells during in vitro culture.