Expression pattern of oxygen and stress-responsive gene transcripts at various developmental stages of in vitro and in vivo preimplantation bovine embryos.

The present study examined the expression pattern of oxygen (O(2)) and stress-responsive gene transcripts at various preimplantation developmental stages of in vitro produced (IVP) and in vivo derived (IVD) bovine embryos. Embryos were produced in vitro from oocytes matured, fertilized and cultured in synthetic oviductal fluid (SOF) medium under low (5%) and high (20%) O(2) concentrations. In vivo embryos were derived from 18 superovulated and artificially inseminated cows. In IVP and IVD groups, embryos were collected at 2-, 4-, 8-, 16-cell morula and blastocyst stages at specific time points for gene expression analysis. The cleavage rates (69.8+/-4.8%) did not differ significantly, but blastocyst rates were significantly higher (28.5+/-3.7%) in low O(2) than those in high O(2) group (18.7+/-3.9%). Mean cell number in low O(2) (145+/-12) and high O(2) (121+/-73) IVP blastocyst were lower (P<0.05) than those of IVD blastocyst (223+/-25). The ICM ratio of IVD blastocyst (26+/-4) was lower (P<0.05) than that of IVP embryos under 5% O(2) (33+/-5) and 20% O(2) (34+/-4) concentrations, respectively. Using real time PCR, for the set of target transcripts (Glut1, Glut5, Sox, G6PD, MnSOD, PRDX5, NADH and Hsp 70.1) analyzed, there were differences in the mRNA expression pattern at 2-, 4-, 8-, 16-cell morula and Day 7 blastocyst stages between the two embryo sources. It can be concluded that, although in vitro bovine embryo culture in SOF medium under low (5%) O(2) concentration provided a more conducive environment in terms of blastocyst formation; differences in the total cell number and gene expression pattern between the IVP and IVD embryos reflected the effect of O(2) concentration.

Development and quality of porcine embryos in different culture system and embryo-producing methods.

In this study, the developmental ability and cellular composition of porcine IVF, parthenote and somatic cell nuclear transfer (SCNT) embryos were evaluated following different in vitro culture systems. Group 1, embryos were cultured in NCSU-23 with 5.55 mM D-glucose (NCSU+) until day 6 on 20% O(2) or 5% O(2) (Group 2). Group 3, embryos were cultured in D-glucose-free NCSU-23 (NCSU-) with 0.17 mM Na pyruvate/2.73 mM Na lactate for 58 h and subsequently cultured in NCSU+ until day 6 (NCSU -/+) on 20% O2 or 5% O(2) (Group 4). IVF blastocysts did not differ significantly with O(2) concentrations, but differed significantly with major energy source (glucose and pyruvate/lactate). In Group 3 and 4 IVF blastocysts, the total cell number and apoptosis rates were not significantly different with different O(2) concentrations. Blastocyst rate, total cell number and apoptosis rate in Groups 3 and 4 parthenote embryos also were not significantly different. Parthenote and SCNT, under the same culture treatment, exhibited significant differences in blastocyst and apoptosis rates (47.5 +/- 16.1 vs. 24.0 +/- 4.0 and 4.9 +/- 9.0 vs. 22.8 +/- 23.3). Apoptosis-generating rate increased in the order parthenote, IVF and then SCNT. In conclusion, in vitro development of porcine embryos was not affected by O(2) concentrations but was affected by major energy source. Even so, the concentration of each major energy source and the timing of its inclusion in culture could accomplish relatively high embryonic development, the apoptosis rate stressed that more work still needs to be done in developing a better defined culture system that could support SCNT embryos equivalent to in vivo preimplantation porcine embryos.

Cellular composition and viability of cloned bovine embryos using exogene-transfected somatic cells.

The present study compared the efficiency of transgenic (TG) cloned embryo production by somatic cell nuclear transfer (SCNT) with fetal-derived fibroblast cells (FFCs) which were transfected with pEGFP-N1 to in vitro-fertilized (IVF), parthenogenetic and SCNT counterparts by evaluating the rates of cleavage and blastocyst formation, apoptosis rate at different developmental stages, cell number, ploidy and gene expression in blastocysts. In SCNT and TG embryos, the rates of cleavage and blastocyst formation were significantly lower (p < 0.05) than those of IVF controls, but it did not differ between SCNT and TG embryos. In IVF control, 86.7% embryos displayed diploid chromosomal complements and the rates were significantly (p < 0.05) higher than those of SCNT and TG embryos. Most TG embryos (79%) with FFCs expressed the gene by both PCR and under fluorescence microscopy. The expression of apoptosis by TUNEL was first detected at six to eight cell stages in all embryos of IVF, SCNT and TG groups, but the expression rate at each developmental stages was significantly higher (p < 0.05) in SCNT and TG embryos than in IVF counterparts. The expression rate in inner cell mass (ICM) of TG embryos was significantly higher (p < 0.05) than in SCNT and IVF embryos. These results indicate that the high occurrence of apoptosis observed in SCNT and TG embryos compared with IVF counterparts might influence the developmental competence. Moreover, the SCNT embryos derived using non-transfected donor cells exhibited a lower apoptosis expression in ICM cells than in TG embryos derived using pEGP-N1-transfected donor cells suggesting a possible role of negative gene effect in TG embryos.

DNA methylation levels in porcine fetal fibroblasts induced by an inhibitor of methylation, 5-azacytidine.

Removal of the somatic DNA methylation pattern from donor cells and remodeling of embryonic status have been suggested as integral processes for successful nuclear transfer (NT) reprogramming. This study has investigated the effects of 5-azacytidine (5-azaC), a DNA methylation inhibitor, on global methylation changes in porcine fetal fibroblasts (PFF); this may improve NT attributable to the potential reprogramming of the methyl groups. PFF in 5th passage cultures were treated with 0, 0.5, 1.0, 2.0, and 3.0 microM 5-azaC for 96 h; 5-azaC inhibited the growth at all tested concentrations. At the higher concentrations of 5-azaC used, cells appeared to exhibit morphological changes and to become apoptotic as observed by TUNEL assay. Thus, cells were negatively affected by 5-azaC. Differences in cellular ploidy were also observed at higher concentrations. Analysis showed no considerable changes in the proportion of cells at the G1-phase of the cell cycle with 5-azaC concentrations. The fractional part of the methylated DNA of these cells was significantly reduced by 5-azaC treatment. Confocal microscopy confirmed the inhibition of methylation levels in PFF with increased concentrations of 5-azaC. Exposure to 5-azaC altered the expression of genes involved in imprinting (IGF2) or pro-apoptosis (BAX), whereas there was a reduction in the expression of the main enzyme responsible for replicating the DNA methylation pattern (DNMT1) and anti-apoptosis (BCL2L1). Therefore, 5-azaC induces a relative reduction in methylation in PFF, and cells treated with 0.5 microM 5-azaC may have enhanced potential for porcine NT.

Effects of confluent, roscovitine treatment and serum starvation on the cell-cycle synchronization of bovine foetal fibroblasts.

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.

Efficient production of cloned bovine embryos using cdc2 kinase inhibitor.

This study was carried out to compare the effects of the combination of ionomycin with a H1-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on the development of reconstituted bovine eggs. For this study, the enucleated bovine oocytes were injected with a presumptive primordial germ cell pre-treated with 1% sodium citrate, and randomly allocated into three activation groups: Group 1 (ionomycin 5 microm, 5 min), Group 2 (ionomycin + DMAP 1.9 mm, 3 h), and Group 3 (ionomycin + SPP 2 mm, 3 h). The reconstituted eggs were compared on the rates of cleavage and development with the blastocyst stage and the ploidy of embryos at 96 h post-activation. Cleavage rates and blastocyst development in Groups 1, 2 and 3 were 7 and 0%, 63 and 17%, and 53 and 14%, respectively. The chromosomal composition differed significantly (p < 0.05) among treatments. Although the embryos in Group 1 had significantly lower developments, 60% of embryos evaluated had diploid chromosomal sets. In contrast, approximately 60% of embryos in Group 2 had abnormal ploidy (21% polyploid and 38% mixoploid). In Group 3, the appearance of abnormal chromosome sets was reduced with the proportion of diploid embryos being increased to 86% (19 of 22), significantly higher (p < 0.05) than in Group 2. It can be concluded that the use of SPP with ionomycin reduces greatly the incidence of chromosomal abnormalities, and may be applicable for the activation of nuclear transplant bovine embryos.

Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection.

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 microM ionomycin for 5 min (group 3), 5 microM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 microM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

Novel method to assess gastric emptying in humans: the Pellet Gastric Emptying Test.

To further validate the Pellet Gastric Emptying Test (PGET) as a marker of gastric emptying, a randomized, four-way crossover study was conducted with 12 healthy subjects. The study consisted of oral co-administration of enteric coated caffeine (CAFF) and acetaminophen (APAP) pellets in four treatment phases: Same Size (100 kcal), Fasted, Small Liquid Meal (100 kcal), and Standard Meal (847 kcal). The time of first appearance of measurable drug marker in plasma, t(initial), was taken as the emptying time for the markers. Co-administration of same size enteric coated pellets of CAFF and APAP (0.7 mm in diameter) revealed no statistically significant differences in t(initial) values indicating that emptying was dependent only on size and not on chemical make-up of the pellets. Co-administration of different size pellets indicated that the smaller 0.7-mm diameter (CAFF) pellets were emptied and absorbed significantly earlier than the larger 3.6-mm diameter (APAP) pellets with both the Small Liquid Meal (by 35 min) and the Standard Meal (by 33 min) (P<0.05). The differences in emptying of the pellets were not significant in the Fasted Phase. The results suggest that the pellet gastric emptying test could prove useful in monitoring changes in transit times in the fasted and fed states and their impact on drug absorption.

Follicular fluid enhances sperm attraction and its motility in human.

PURPOSE: Follicular fluid has a pivotal effect on motility and chemotaxis of spermatozoa for successful fertilization. The effect of human follicular fluid (hFF) and progesterone on attraction and motility of spermatozoa were investigated using simplified capillary assays. METHODS: Capillary tubes loaded with hFF, modified human tubal fluid (m-hTF), or m-hTF supplemented with progesterone, respectively, were used for assessments of attraction and motility of spermatozoa following culture at various time intervals. RESULTS: Number and motile ratio of spermatozoa in the tubes loaded with hFF were significantly (P < .05) higher than those with m-hTF. In the tubes loaded with m-hTF, m-hTF supplemented with progesterone, and hFF, the attracted number of spermatozoa were 34 x 10(5), 131 x 10(5), and 108 x 10(5), and motile ratio of spermatozoa was 37, 48, and 82%, respectively. CONCLUSIONS: We conclude that hFF clearly plays a crucial role in enhancing attraction and motility of spermatozoa, and progesterone has strong effect on attraction of spermatozoa.

Genomic cloning of the Hsc71 gene in the hermaphroditic teleost Rivulus marmoratus and analysis of its expression in skeletal muscle: identification of a novel muscle-preferred regulatory element.

To further our understanding of the role of stress proteins in development as well as in adaptation of fish to adverse environmental conditions, we undertook molecular analyses of stress protein encoding genes from the hermaphroditic teleost Rivulus marmoratus. We isolated a genomic clone containing the Hsc71 gene (rm-hsc71m) and its upstream sequences. rm-Hsc71m is not induced by external stress, but is enriched in a tissue-specific manner during early development. In adult, the strongest expression appeared in skeletal muscle, whereas lower expression was seen in the gill, eye and brain. To understand the regulatory basis of high muscle expression of rm-hsc71m, transfection of R.marmoratus muscle tissue was performed using 5' deletion fragments containing the rm-hsc71m promoter driving EGFP expression. An upstream region from -2.7 to -1.9 kb was identified as a muscle-specific regulatory region. Within this region, we identified at least three sites with the novel sequence TGTnACA interacting with a fish muscle factor having an M(r) of 32 000. Our data indicate that rm-hsc71m expression in skeletal muscle is controlled by a muscle-specific regulatory element containing this novel motif.

The stress-activated MAP kinase Sty1/Spc1 and a 3'-regulatory element mediate UV-induced expression of the uvi15(+) gene at the post-transcriptional level.

Exposure of Schizosaccharomyces pombe cells to UV light results in increased uvi15(+) gene expression at both the mRNA and protein levels, leading to elevated cell survival. This UV-induced expression of the uvi15(+) gene was reduced in Deltasty1 and Deltawis1 cells lacking the stress-activated protein kinase pathway, but not in DNA damage checkpoint mutants. To further understand the cellular mechanisms responsible for this UV-induced expression, the transcription rate and mRNA half-life were investigated. Transcription run-on assays revealed that the rate of uvi15(+) transcription was increased 1.8-fold regardless of Sty1 when cells were UV irradiated. The half-life of uvi15(+) mRNA was also increased 1.5-fold after UV irradiation, but it was decreased in the Deltasty1 background for both basal and UV-induced mRNAs, indicating that the stress-activated MAPK cascade can mediate UV-induced gene expression by increasing mRNA half-life. Deletion analyses identified a 54 nt element downstream of the distal poly(A) site, which was involved in the increased half-life of uvi15(+) mRNA. These results suggest that both Sty1 and the 3'-regulatory element regulate UV-induced expression of the uvi15(+) gene at the post-transcriptional level.

Reporter constructs showing proper globin gene switching in cultured erythroid cell lines.

Recent advances in the study of globin gene switching in the context of complete gene locus have contributed greatly to our understanding of developmental regulation mechanism of globin gene expression. However, it is not clear yet whether the cluster is sufficient in proper gene switching when the globin genes are replaced with conventional reporter genes. Furthermore, even though erythroid-specific and ubiquitous transcription factors involved in erythroid-specific globin gene expression have been characterized and some plausible globin gene switching models have been suggested, any specific factor directly involved in globin gene switching is not yet identified. In this study, as an effort to further understand globin switching mechanism and to identify globin switching factors, we constructed reporter vectors by juxtaposing several putative regulatory elements in human beta-globin locus to conventional reporter genes and analyzed their stage-specific expression in erythroid cell lines. At the end, we demonstrated that gammabeta-type constructs, in which both gamma-type and beta-type globin reporter genes were linked in cis below beta-globin locus control region (LCR), show proper stage-specific reporter gene expression in erythroid cell lines and also recapitulate globin switching in cell hybrids.

Molecular detection of TEL-AML1 transcripts as a diagnostic tool and for monitoring of minimal residual disease in B-lineage childhood acute lymphoblastic leukemia.

The chromosomal translocation t(12;21) (p12;q22) which results in the TEL-AML1 fusion gene is the most frequent genetic rearrangement in childhood B-lineage acute lymphoblastic leukemia (ALL). The rearrangement in this locus, however, is only rarely observed by routine karyotypic analysis. We established a nested-reverse transcriptase-polymerase chain reaction (nested-RT-PCR) technique for the detection of the TEL-AML1 transcript, and used this to investigate the incidence of the rearrangement, and to characterize the disease present in TEL-AML1-positive B-lineage ALL patients. The TEL-AML1 fusion transcript was detected in nine of fourteen patients. These patients were relatively homogeneous in that they were young and had low presenting leukocyte counts, both features of which are associated with a favorable prognosis. Furthermore, we could detect the TEL-AML1 transcript in the peripheral blood of t(12;21)-positive patients and we used this to assess minimal residual disease (MRD) in patients during chemotherapy. The data demonstrate that nested-RT-PCR is a suitable tool for diagnosing t(12;21)-positive ALL, that these patients constitute a clinically distinct subgroup of ALL patients, and that the method could also be used to monitor MRD in these patients.

Functional characterization of TEL/AML1 fusion protein in the regulation of human CR1 gene promoter.

The TEL/AML1 fusion gene occurs in childhood B-cell acute lymphoblastic leukemia (ALL) as a result of the translocation of human chromosome 12;21. Using reporter gene assays, we have functionally characterized TEL, AML1 and TEL/AML1 fusion proteins in the regulation of the human CR1 gene. Analysis of transcription activities showed that AML1 increased the CR1 promoter activity and that TEL repressed the basal activity of the promoter. Increased activities of the CR1 promoter by AML1 protein were reduced by the TEL protein in a concentration-dependent manner. When TEL/AML1 and AML1 proteins are present in cells at the same time, the TEL/AML1 protein inhibits the transactivation activities of AML1 protein on the human CR1 promoter even though TEL/AML1 retains the transactivation domain of AML1. A mutation analysis of the human CR1 promoter revealed that the binding sites for TEL and AML1 are necessary for the action of TEL and TEL/AML1, respectively. Thus, production of the TEL/AML1 protein by translocation of human chromosome 12;21 may contribute to leukemogenesis by the specific inhibition of AML1-dependent activation of myeloid promoters.

AML1, the target of chromosomal rearrangements in human leukemia, regulates the expression of human complement receptor type 1 (CR1) gene.

The human CR1 gene is expressed specifically in hematopoietic cells. It is suggested that some cell-type specific factors which involve in gene-specific activation or repression exist in cells according to the result that the gene expression varies differently depend on differentiation stage. Here, we demonstrate that the integrity of a polyomavirus enhancer core sequence, 5'-TGTGGT-3', is critical to the human CR1 promoter activity. AML1 is a site-specific DNA-binding protein that recognizes the enhancer core motif TGTGGT. We show that the AML1 binds specifically to this site and activates the human CR1 promoter. Furthermore, we demonstrate that the Ets binding site (GGAA) located 2 bp upstream of the AML1 site is also involved in the regulation of the human CR1 promoter activity. Point mutations of either the AML1 or the Ets binding site that abolish the binding of the respective factors result in significant decreases of the human CR1 promoter activity. These results suggest that AML1 and Ets proteins direct the expression of the human CR1 promoter.

Characterization of the human CR1 gene promoter.

The human CR1 is a single chain membrane glycoprotein that is a member of the group of regulators of the complement activation system. In order to clarify the regulatory mechanisms of human CR1 gene expression, the 5'-flanking region of the human CR1 gene was isolated and its promoter was characterized. The CR1 expression was found to be transcriptionally up-regulated in HL60 cells by stimulation with DMSO. The cloned CR1 gene promoter was sequenced and computer analyzed. The potential promoter region lacks a distinct TATA box sequence. The transcription initiation site was determined by primer extension and several possible regulatory elements for transcription were found in the promoter region.

Design of compounds that increase the absorption of polar molecules.

Hydrophilic drugs are often poorly absorbed when administered orally. There has been considerable interest in the possibility of using absorption enhancers to promote absorption of polar molecules across membrane surfaces. The bile acids are one of the most widely investigated classes of absorption enhancers, but there is disagreement about what features of bile acid enhancers are responsible for their efficacy. We have designed a class of glycosylated bile acid derivatives to evaluate how increasing the hydrophilicity of the steroid nucleus affects the ability to transport polar molecules across membranes. Some of the glycosylated molecules are significantly more effective than taurocholate in promoting the intestinal absorption of a range of drugs, showing that hydrophobicity is not a critical parameter in transport efficacy, as previously suggested. Furthermore, the most effective glycosylated compound is also far less damaging to membranes than the best bile acid absorption promoters, presumably because it is more hydrophilic. The results reported here show that it is possible to decouple absorption-promoting activity from membrane damage, a finding that should spark interest in the design of new compounds to facilitate the delivery of polar drugs.

Expression of yeast O6-methylguanine-DNA methyltransferase (MGMT) gene.

We cloned the full genomic DNA of yeast (Saccharomyces cerevisiae) O6-methylguanine-DNA methyltransferase (MGMT) gene and examined its expression. The expression of yeast MGMT gene is decreased when cells reach stationary phase and cannot be induced by the pretreatment with alkylating agents, methylmethanesulfonate (MMS) or N-methyl-N'-nitroso-N-nitrosoguanidine (MNNG). The transcription initiation site was determined by primer extension analysis. This analysis showed that the authentic start codon is the ATG at position +32 from transcription initiation site.

In vitro definition of the yeast RNA polymerase I enhancer.

In vitro conditions are reported under which an EcoRI-HpaI fragment of the Saccharomyces cerevisiae ribosomal gene spacer will enhance transcription from an adjacent RNA polymerase I promoter. Enhancement is largely independent of orientation and distance and is proportional to copy number. Mapping experiments reveal that two separate regions of the EcoRI-HpaI fragment are independently capable of promoter stimulation. These regions appear to correspond to elements which have been shown by previous workers to cause enhancement in vivo. Using the detergent Sarkosyl to limit the number of rounds of transcription from each promoter, we found that the degree of enhancement is similar whether one or many rounds of transcription occur. This finding supports a model in which the enhancer increases the number of stable promoter complexes but does not alter the loading of polymerase on an active promoter. Once the stable promoter complex is formed, the enhancer can be physically severed from the promoter with no loss of enhancement. Likewise, the upstream activation region of the promoter can be severed from the core promoter domain once the stable complex has been formed. These results are interpreted to mean that the enhancer functions only to assist stable complex formation and, once that is accomplished, the enhancer is dispensable.

In vitro definition of the yeast RNA polymerase I promoter.

The structure of the ribosomal gene promoter from Saccharomyces cerevisiae has been analyzed in a whole cell in vitro extract. The promoter contains at least two essential domains, an upstream domain located at the 5' boundary near position -150 and a core promoter domain around the site of transcription initiation at +1. The upstream domain augments transcription in vitro but is not absolutely required. Maintenance of correct spacing between the two domains is critical. The in vitro analysis agrees well with prior in vivo analysis and it appears that the yeast promoter has a structure very similar to that of vertebrate ribosomal gene promoters.