Dynamic methylation pattern of the methyltransferase1o (Dnmt1o) 5′‐flanking region during mouse oogenesis and spermatogenesis

DNA methyltransferase1o (Dnmt1o), which is specific to oocyte and preimplantation embryo, plays a role in maintaining DNA methylation in mammalian cells. Here, we investigated the methylation status of CpGs sites in the Dnmt1o 5′‐flanking region in germ cells at different stages of oogenesis or spermatogenesis. The methylation levels of the CpG sites at the 5′‐flanking regions were hypermethylated in growing oocytes of all follicular stages, while the oocytes in meiotic metaphase II (MII) were demethylated. The methylation pattern within the CpGs sites in the 5′‐flanking region, however, was dramatically changed during spermatogenesis. We observed that there was significant non‐CpG methylation both in MII oocytes and spermatocytes. Although a low methylation level in non‐CpG sites was observed in primary and secondary oocytes, the CpA site of position 25 and CpT site of position 29 within the no‐CpG region in the 5′‐flanking region of Dnmt1o was highly methylated in MII oocytes. During spermatogenesis, the low degree of methylation at CpG sites in spermatocytes increased to a higher degree in sperm, while the high ratio of methylation in non‐CpG sites in spermatocytes decreased. Together, germ cells showed inverted methylation patterns between CpG and non‐CpG sites in the Dnmt1o 5′‐upstream region, and the methylation pattern during oogenesis did not drastically change, remaining generally hypomethylated at the MII stage. Mol. Reprod. Dev. 80: 212–222, 2013. © 2013 Wiley Periodicals, Inc.     

Varied patterns of DNA methylation change between different satellite regions in bovine preimplantation development

Global reduction of DNA methylation, a part of genome reprogramming processes, occurs in a gradual manner until before implantation and is recognized as a conserved process in mammals. Here, we reported that in bovine, satellite regions exhibited varied patterns of methylation changes when one-cell egg advanced to the blastocyst; a maintenance methylation was observed in satellite I sequences, a decrease in alpha satellites, and an increase in satellite II regions. Cloned embryos exhibited similar changes for DNA methylation in the satellite I and alpha. We also observed that the satellite I and alpha sequences were methylated more in inner cell mass region of the blastocyst whereas the satellite II showed selective demethylation in this region. Together, these findings point that individual satellite sequences carry their own methylation patterns under the pressure of global demethylation, suggesting that local methylation control system acts on the satellite regions in early bovine embryos.     

A new embryonic antigen,p67, present during mouse preimplantation development

An antibody prepared against nullipotential teratocarcinoma stem cells (A-N₁) detects cell surface antigens expressed by early mouse embryos and inhibits in vitro development of embryos in the absence of complement [Calarco and Banka, 1979]. Here we report the immunoprecipitation and electrophoretic characterization of A-N₁-detected antigens from preimplantation mouse embryos. Predominant antibody activity is directed against a 67,000-dalton glycoprotein (p67) with a mean pI of 5.3, which has not been previously described. This protein is not detected, at least as p67, after culture of embryos in tunicamycin. The p67 antigen is also expressed by pluripotential PSA1 teratocarcinoma cells but not by several different differentiated mouse cell types.     

Identification and analysis of stage-specific expression of lysosome-associated protein transmembrane 4alpha gene during development of preimplantation rabbit nuclear transfer embryo

The stage-specific expression of Lysosome-associated protein transmembrane 4alpha (LAPTM4alpha) in preimplantation rabbit nuclear transfer (NT) embryo was identified with the DDRT-PCR and reverse Northern Blot. The full length (1,364 bp) cDNA of LAPTM4alpha was screened out from cDNA library constructed with rabbit ovary and in situ hybridization (ISH) was used to trace the distribution of the LAPTM4alpha mRNA in intra-ovary, especially the follicle which proved that the LAPTM4alpha gene expression is involved in the follicles development, maturation, ovulation, luteinization, and preimplantation development in the rabbit (Oryctolagus cuniculus domestica). To our knowledge, this is the first characterization of LAPTM4alpha gene expression and mRNA distribution in the rabbit ovary and first evidence for this gene involving in follicle development and rabbit preimplantation development.     

小鼠植入前胚胎致密化与囊胚形成的分子调控

哺乳动物胚胎植入前的发育中致密化和囊胚形成分别标志着第一次、第二次细胞分化（即细胞命运决定）的起始,是胚胎正常发育的必要条件。因此对影响致密化和囊胚形成的蛋白及调节因子的研究尤为重要。本文探讨了与致密化相关的细胞黏附蛋白、连接蛋白、细胞骨架等分子和囊胚形成相关的紧密连接蛋白、钠钾三磷酸腺苷激酶等分子的一系列调控,以及致密化和囊胚形成在细胞命运决定中的重要作用。     

Paradoxical methylation of the tyrosine hydroxylase gene in mouse preimplantation embryos

The mouse tyrosine hydroxylase (Th) gene is located in an evolutionarily conserved imprinted gene cluster on distal chromosome 7. It is associated with a CpG island that spans the promoter of the gene. Using a bisulfite sequencing method we show that the Th promoter is fully methylated in both male and female mouse germ cells and in human spermatozoa, suggesting that it belongs to the newly identified category of CpG islands, the similarly methylated regions (SMRs). Contrary to other tissue-specific gene sequences, the mouse Th promoter escapes the initial wave of genome demethylation during the first few cell cycles, but becomes demethylated between the morula and the blastocyst stages. This unusual methylation ontogeny may be a characteristic of the SMRs and/or related to the localization of the Th gene in an imprinted gene cluster.     

DNA methyltransferase is actively retained in the cytoplasm during early development.

The overall DNA methylation level sharply decreases from the zygote to the blastocyst stage despite the presence of high levels of DNA methyltransferase (Dnmt1). Surprisingly, the enzyme is localized in the cytoplasm of early embryos despite the presence of several functional nuclear localization signals. We mapped a region in the NH(2)-terminal, regulatory domain of Dnmt1 that is necessary and sufficient for cytoplasmic retention during early development. Altogether, our results suggest that Dnmt1 is actively retained in the cytoplasm, which prevents binding to its DNA substrate in the nucleus and thereby contributes to the erasure of gamete-specific epigenetic information during early mammalian development.     

Changes in cAMP phosphodiesterase activity and cAMP concentration during mouse preimplantation development.

Cyclic nucleotide phosphodiesterase (PDE) activity and cAMP amounts were measured in mouse preimplantation embryos at the 1-cell, 2-cell, 8-cell/morula, and mid-blastocyst stages. PDE activity remained constant between the 1-cell and 2-cell stages. It decreased by the 8-cell stage and continued to decrease by the mid blastocyst stage to about 14% of the 1- and 2-cell values. By contrast, cAMP amounts remained essentially constant at 0.05 fmole/embryo (0.3 microM) from the 1-cell to the blastocyst stage and increased to 0.175 fmole in the fully expanded blastocyst that was close to hatching. Measurements of embryo volume indicated that intracellular volume remained essentially constant up to the blastocyst stage. The morphological changes in cell shape that accompany differentiation of the trophectoderm and that are coupled with blastocoel expansion decreased the intracellular volume. This decrease resulted in an increase in the cAMP concentration to about 0.4 microM by the mid-blastocyst stage. Previous studies indicate that either cAMP or TGF-alpha/EGF can stimulate the rate of blastocoel expansion. Although TGF-alpha/EGF can elevate cAMP levels in other cell types, TGF-alpha, at a concentration that maximally stimulates the rate of blastocoel expansion, did not elevate cAMP in blastocysts. Thus, it was unlikely that elevation of cAMP is the mechanism by which TGF-alpha stimulates the rate of blastocoel expansion.     

Effect of flanking matrix attachment regions on the expression of microinjected transgenes during preimplantation development of mouse embryos

The efficiency of transgenic animal production would increase if microinjected embryos with a successfully integrated transgene could be identified prior to transfer. It is possible to detect microinjected DNA in embryos. However, no reliable system is able to distinguish between transgenes merely present as extrachromosomal DNA and those that have been integrated into chromatin. The experiments reported here were designed to determine if the inclusion of matrix attachment regions (MARs) would enhance the efficiency of transgenic embryos identification using a selection scheme based on the expression of green fluorescent protein (GFP). Pronuclei of mouse embryos were microinjected with GFP reporter gene under the control of three different promoters and flanked or not by three different MAR elements. Transgene expression profiles were followed by direct visualization of GFP in cultured microinjected embryos. Embryos at different developmental stages were classified according to their GFP expression and groups with the same expression pattern were transferred into oviducts of pseudopregnant female mice. Fetuses were collected between days 12–15, and their genomic DNA was purified and analyzed to detect transgene integration. We did not find any statistically significant difference between the percentage of transgenic fetuses produced from GFP-positive or GFP-negative embryos transferred at 4-cell, morula, or blastocyst stage. However, when MAR elements were included in the construct, we found that GFP-positive embryos transferred at the 2-cell stage produced a significantly higher percentage of transgenic fetuses than GFP-negative embryos, but MAR sequences did not completely eliminate false positives.     

Cellular and subcellular localization of stathmin during oocyte and preimplantation embryo development

Stathmin is a 19 kDa cytosolic phosphoprotein, proposed to act as a relay integrating diverse intracellular signaling pathways involved in regulation of cell proliferation, differentiation, and function. To gain further information about its significance during early development, we analyzed stathmin expression and subcellular localization in mouse oocytes and preimplantation embryos. RT‐PCR analysis revealed a low expression of stathmin mRNA in unfertilized oocytes and a higher expression at the blastocyst stage. A fine cytoplasmic punctuate fluorescent immunoreactive stathmin pattern was detected in the oocyte, while it evolved toward an increasingly speckled pattern in the two‐cell and later four‐ to eight‐cell embryo, with even larger speckles at the morula stage. In blastocysts, stathmin immunoreactivity was fine and intense in inner cell mass cells, whereas it was low and variable in trophectodermal cells. Electron microscopic analysis allowed visualization with more detail of two types of stathmin immunolocalization: small clusters in the cytoplasm of oocytes and blastocyst cells, together with loosely arranged clusters around the outer membrane of cytoplasmic vesicles, corresponding to the immunofluorescent speckles in embryos until the morula stage. In conclusion, it appears from our results that maternal stathmin is accumulated in the oocyte and is relocalized within the oocyte and early preimplantation embryonic cell cytoplasm to interact with specific cytoplasmic membrane formations. Probably newly synthesized, embryonic stathmin is expressed in the blastocyst, where it is localized more uniformly in the cytoplasm mostly of inner cell mass (ICM) cells. These expression and localization patterns are probably related to the particular roles of stathmin at the successive steps of oocyte maturation and early embryonic development. They further support the proposed physiologic importance of stathmin in essential biologic regulation. Mol. Reprod. Dev. 53:306–317, 1999. © 1999 Wiley‐Liss, Inc.     

Expression and regulation of genes associated with cell death during murine preimplantation embryo development

The newly fertilized preimplantation embryo depends entirely on maternal mRNAs and proteins deposited and stored in the oocyte prior to its ovulation. If the oocyte is not sufficiently equipped with maternally stored products, or if zygotic gene expression does not commence at the correct time, the embryo will die. One of the major abnormalities observed during early development is cellular fragmentation. We showed previously that cellular fragmentation in human embryos can be attributed to programmed cell death (PCD). Here, we demonstrate that the PCD that occurs during the 1-cell stage of mouse embryogenesis is likely to be regulated by many cell death genes either maternally inherited or transcribed from the embryonic genome. We have demonstrated for the first time the temporal expression patterns of nine cell death regulatory genes, and our preliminary experiments show that the expression of these genes is altered in embryos undergoing fragmentation. The expression of genes involved in cell death (MA-3, p53, Bad, and Bcl-xS) seems to be elevated, whereas the expression of genes involved in cell survival (Bcl-2) is reduced. We propose that PCD may occur by default in embryos that fail to execute essential developmental events during the first cell cycle. Mol. Reprod. Dev. 51:243–253, 1998. © 1998 Wiley-Liss, Inc.     

Preimplantation embryo development in the mouse: Role of histidine decarboxylase

The present study determines the effect of a specific and an irreversible inhibitor of histidine decarboxylase (HDC), α-fluoromethylhistidine (α-FMH) on the mouse preimplantation embryo development in vitro. The embryo culture technique was used to assess the effect of α-FMH. Embryos recovered at 0800–0900 hr (AM) on day 3 of pregnancy were 4–8 cells, whereas those recovered at 1600–1630 hr were mostly 8-cell compacted embryos. Of the day 3-AM embryos, 81.3 ± 4.3% developed to blastocysts within 48 hr when cultured in the medium alone, but addition of α-FMH (0.19 or 0.38 mM) drastically reduced the blastocyst formation to 26.6 ± 7 or 16.8 ± 4.3%. Most of them were arrested before the compaction stage. Addition of L-histidine, the substrate for HDC, did not alter the inhibition of blastocyst formation in the presence of α-FMH (37.2 ± 10.9%). Of the day 3-PM embryos, 99.3 ± 0.7% developed to blastocyst stage when cultured in the medium alone and addition of α-FMH (0.19 or 0.38 mM) did not affect the embryo development (92.1 ± 4.3 or 81.9 ± 9.9% developed to blastocysts). The birth of healthy young following transfer of these blastocysts into pseudopregnant mice indicates normal development of the embryos under this condition. The results suggest that histamine synthesis may be required for the process of compaction and thus the formation of blastocyst.