Species-dependent expression patterns of DNA methyltransferase genes in mammalian oocytes and preimplantation embryos

DNA methyltransferases (DNMTs) comprise a family of proteins involved in the establishment and maintenance of DNA methylation patterns in the mammalian genome. DNA methylation involves the transfer of the methyl group of the coenzyme S-adenosyl-L-methionine to the 5 position of cytosine residues within CpG dinucleotides. DNA methylation is implicated in the control of imprinted genes expression, X chromosome silencing, development of certain types of cancer, and embryonic development. DNA methylation is also believed to protect the genome from parasitic elements such as transposons, retrotransposons, and viruses. The aim of this study was to analyze the expression patterns of DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L genes in rhesus macaque (Macaca mulatta) oocytes and preimplantation stage embryos from fertilization to the hatched blastocyst stage, and to compare these results with the expression profiles in the mouse and other mammalian species. We describe species-dependent differences as well as similarities in expression patterns of DNMT genes among mammals.     

Murine transforming growth factor-beta 2 cDNA sequence and expression in adult tissues and embryos

Murine transforming growth factor-beta 2 (TGF-beta 2) cDNAs were isolated from cDNA libraries derived from a differentiated murine embryonic carcinoma cell line, PCC3. The composite cDNA sequence is 4267 nucleotides long, including a 1217 nucleotides 5'-untranslated sequence, and encodes a murine TGF-beta 2 precursor of 414 amino acids with 96% identity to its human counterpart. Several consensus polyadenylation sequences are present in the 1807 nucleotides 3'-untranslated sequence. Five TGF-beta 2 mRNA species are observed in the developing mouse fetus and they show different patterns of expression during development. TGF-beta 2 mRNA expression was also examined in adult mouse tissues, in which four of the five RNA species were observed. TGF-beta 2 mRNAs were present in all adult mouse tissues examined, except liver, and was most abundant in placenta, the male submaxillary gland and lung. The patterns of expression suggest a physiological role for TGF-beta 2 both in embryonic development and in the maintenance of adult tissues.     

牛体细胞中Gadd45a的表达与DNA甲基化修饰

目的 检测成年牛心脏、肾脏、肝脏及睾丸组织中Gadd45a基因表达情况及DNA甲基化状态,并说明二者之间的关系.结果 牛Gadd45a基因在心脏中没有表达,而在其他3种组织中有表达且略有差异.选定该基因调控区中的7个CpG位点,利用甲基化特异性PCR检测CpG位点的DNA甲基化状态,结果心脏中的甲基化程度明显高于其他3种组织.结论 DNA甲基化对牛Gadd45a基因的组织特异性表达有一定影响.     

Characterization of cytosine methylated regions and 5-cytosine DNA methyltransferase (Ehmeth) in the protozoan parasite Entamoeba histolytica

The DNA methylation status of the protozoan parasite Entamoeba histolytica was heretofore unknown. In the present study, we developed a new technique, based on the affinity of methylated DNA to 5-methylcytosine antibodies, to identify methylated DNA in this parasite. Ribosomal DNA and ribosomal DNA circles were isolated by this method and we confirmed the validity of our approach by sodium bisulfite sequencing. We also report the identification and the characterization of a gene, Ehmeth, encoding a DNA methyltransferase strongly homologous to the human DNA methyltransferase 2 (Dnmt2). Immunofluorescence microscopy using an antibody raised against a recombinant Ehmeth showed that Ehmeth is concentrated in the nuclei of trophozoites. The recombinant Ehmeth has a weak but significant methyltransferase activity when E.histolytica genomic DNA is used as substrate. 5-Azacytidine (5-AzaC), an inhibitor of DNA methyltransferase, was used to study in vivo the role of DNA methylation in E.histolytica. Genomic DNA of trophozoites grown with 5-AzaC (23 µM) was undermethylated and the ability of 5-AzaC-treated trophozoites to kill mammalian cells or to cause liver abscess in hamsters was strongly impaired.     

Size of the directing moiety at carbon 5 of cytosine and the activity of human DNA(cytosine-5) methyltransferase

M13 DNAs in which carbon 5 of each deoxycytidine residue in one strand is replaced with a bulky group are very good substrates for human DNA (cytosine-5) methyltransferase. Rate enhancements of up to 35 fold are obtained depending on the size of the moiety at C-5. The enzyme appears optimally suited to sense a methyl group in one strand at this position. Alkaline density gradient analyses of the distribution of methyl groups applied to 5-BrdCyd or 5-IdCyd substituted DNA reveal that these groups serve to direct the enzyme to methylate the unsubstituted strand.     

DNA methylation in bovine adult and fetal fibroblast cells

The aim of this project was to develop a simple screening tool to measure the DNA methylation of fibroblast cells, and to determine if differences in DNA methylation could be detected in adult and fetal fibroblast cells after serum starvation (SS). Four adult and four fetal tissue explants were collected to produce presumptive fibroblast cell cultures for this experiment. All cell lines underwent three repetitions of serum starvation for 0 (control), 2, 5, or 7 days. The DNA was extracted from the cells and analyzed for DNA methylation content using methylation sensitive restriction enzyme digestion, gel electrophoresis and image analysis. There was no difference (p = 0.11) between the DNA methylation of the adult and fetal nonclonal cell lines. A cubic trend (p = 0.09) of increased DNA methylation at 2 days of serum starvation followed by periods of decreasing DNA methylation at 5 and 7 days were observed for the adult nonclonal cell lines. A significant interaction (p = 0.03) was observed between fetal cell line and day. This simple, rapid DNA methylation assay may be beneficial when evaluating cells' DNA methylation content.     

Telomere-to-centromere ratio of bovine clones, embryos, gametes, fetal cells, and adult cells

In 1997, Dolly, the first animal cloned from an adult cell, was born. It was announced in 1999 that Dolly might be aging faster than normal because her telomeres were shorter than age-matched control sheep. Telomeres, a repeated DNA sequence located at the ends of linear chromosomes, allow for base pair loss during DNA replication. Telomere shortening acts as a "mitotic clock," leading to replicative senescence. By using whole cell lysate and slot-blot analysis, we determined the telomere-to-centromere ratio (T/C) for bovine gametes, embryos, fetal tissues (brain, heart, lung, kidney, uterus, ovary, and skin), adult donor cells, and cloned embryos. Our data indicates a consistency in T/C among the various fetal tissues. The T/C of sperm is significantly lower than in oocytes. The T/C decreases from the oocyte to the 2-8-cell stage embryo, increases dramatically at the morula stage, and decreases at the blastocyst stage. Our data shows no significant difference in T/C between cloned embryos and in vitro fertilized (IVF) embryos, but there is a significant difference between cloned embryos and adult donor cells. In conclusion, the enucleated bovine oocyte has the ability to reestablish the telomere length of adult somatic cell donor nuclei.     

Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos

The import of exogenous DNA (eDNA) from the cytoplasm to the nucleus represents a key intracellular obstacle for efficient gene delivery in mammalian cells. In this study, cumulus cells or oolemma vesicles previously incubated with eDNA, and naked eDNA were injected into the cytoplasm of MII oocytes to evaluate their efficiency for eDNA expressing bovine embryo production. Our study evaluated the potential of short time co-incubation (5 min) of eDNA with; (1) cumulus cells, to be used as donor cells for SCNT and (2) oolemma vesicles (vesicles) to produce parthenogenic transgene expressing embryos. In addition, we included a group consisting of the injection of eDNA alone (plasmid) followed by parthenogenic activation. Two different pCX-EGFP plasmid concentrations (50 and 500 ng/μl) were employed. The results showed that embryos produced by SCNT and by vesicle injection assisted by chemical activation were able to express the eDNA in higher rates than embryos injected with plasmid alone. The lower plasmid concentration allowed the highest development rates in all groups. Using confocal microscopy, we analyzed the interaction of FITC- labeled eDNA with cumulus cells and vesicles as well as oocytes injected with labeled plasmid alone. Our images demonstrated that eDNA interacted with cumulus cells and vesicles, resulting an increase in its expression efficiency. In contrast, oocytes injected with DNA alone did not show signs of transgene accumulation, and their eDNA expression rates were lower. In a further experiment, we evaluated if transgene-expressing embryos could be produced by means of vesicle injection followed by IVF. The lower plasmid concentration (50 ng/μl) injected after IVF, produced the best results. Preliminary FISH analysis indicated detectable integration events in 1/5 of SCNT blastocysts treated. Our studies demonstrate for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA-expressing embryos can be also obtained by cytoplasmic injection vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique.     

Inhibitor profiles of alkaline phosphatases in bovine preattachment embryos and adult tissues.

The alkaline phosphatases are a small family of isozymes. Bovine preattachment embryos transcribe mRNA for two tissue-specific alkaline phosphatases (TSAP2 and TSAP3) beginning at the 4- and 8-cell stages. Whereas no mRNA has been detected in oocytes, there is maternally inherited alkaline phosphatase activity. It is not known which isozyme(s) is responsible for the maternal activity or when TSAP2 and TSAP3 form functional protein. No antibodies are available that recognize the relevant bovine alkaline phosphatases. Therefore, sensitivity to heat and chemical inhibition was used to separate the different isozymes. By screening tissues, it was determined that the bovine tissue-nonspecific alkaline phosphatase (TNAP) is inactivated by low temperatures (65C) and low concentrations of levamisole (<1 mM), whereas bovine tissue-specific isozymes require higher temperatures (90C) and levamisole concentrations (>5 mM). Inhibition by L-homoarginine and L-phenylalanine was less informative. Cumulus cells transcribe two isozymes and the pattern of inhibition suggested heterodimer formation. Inhibition of alkaline phosphatase in bovine embryos before the 8-cell stage indicated the presence of only TNAP. At the 16-cell stage the pattern was consistent with TNAP plus TSAP2 or -3 activity, and in morulae and blastocysts the pattern indicated that the maternal TNAP is fully supplanted by TSAP2 or TSAP3.     

Detection of bovine viral diarrhea virus (BVDV) in single or small groups of preimplantation bovine embryos

The objectives of this study were to develop techniques to detect BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using either virus isolation (VI) or real time quantitative polymerase chain reaction (RT-QPCR) assays. In vivo-derived and in vitro-produced bovine embryos at 7 d post-fertilization were exposed to SD-1, a high affinity strain of BVDV, for 2 h and then processed according to the International Embryo Transfer Society (IETS) guidelines prior to testing. Groups of five or two in vivo-derived embryos, and single in vivo-derived embryos, were VI positive for BVDV 100, 50, and 33% of the time, and were RT-QPCR positive 100, 75, and 42% of the time, respectively. The virus was detected by the VI technique in all of the groups of five or two in vitro-produced embryos and in all of the single in vitro-produced embryos, and it was detected in 100, 80, and 50%, using RT-QPCR. Techniques for RT-QPCR were sufficiently sensitive to detect 10 copies of viral RNA in a sample and to detect BVDV associated with single embryos. Application of this new technology, RT-QPCR, will facilitate additional studies to further assess the risk of transmission of BVDV through embryo transfer.