bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient co-expression of a recombinant staphopain A and its inhibitor staphostatin A in Escherichia coli
Authors: WLADYKA Benedykt , PUZIA Katarzyna , DUBIN Adam
Published: December 2004

Staphopain A is a staphylococcal cysteine protease. Genes encoding staphopain A and its specific inhibitor, staphostatin A, are localized in an operon. Staphopain A is an important staphylococcal virulence factor. It is difficult to perform studies on its interaction with other proteins due to problems in obtaining a sufficient amount of the enzyme from natural sources. Therefore efforts were made to produce a recombinant staphopain A. Sequences encoding the mature form of staphopain A and staphostatin A were PCR-amplified from Staphylococcus aureus genomic DNA and cloned into different compatible expression vectors. Production of staphopain A was observed only when the enzyme was co-expressed together with its specific inhibitor, staphostatin A. Loss of the function mutations introduced within the active site of staphopain A causes the expression of the inactive enzyme. Mutations within the reactive centre of staphostatin A result in abrogation of production of both the co-expressed proteins. These results support the thesis that the toxicity of recombinant staphopain A to the host is due to its proteolytic activity. The coexpressed proteins are located in the insoluble fraction. Ni2+-nitrilotriacetate immobilized metal-affinity chromatography allows for an efficient and easy purification of staphopain A. Our optimized refolding parameters allow restoration of the native conformation of the enzyme, with yields over 10-fold higher when compared with isolation from natural sources.
Transgenic expression of human complement regulators reduces skeletal muscle ischaemia/reperfusion injury in mice
Authors: KNIGHT Kenneth R. , SHINKEL Trixie A. , COWAN Peter J. , ROMEO-MEEUW Rosalind , d'APICE Anthony J. F. , MORRISON Wayne A.
Published: December 2004

This study aimed to explore the hypothesis that activated complement components contribute significantly to I/R (ischaemia/reperfusion) injury in skeletal muscle. After 50, 70 and 90 min of tourniquet ischaemia and 24 h of reperfusion, viability of the medial gastrocnemius muscle in CBA-C57BL/6 wild-type mice, assessed histochemically by reduction of NBT (Nitro Blue Tetrazolium) dye, was 60, 21 and 8% respectively. Skeletal muscle viability after 70 min of ischaemia and 24 h of reperfusion in transgenic mice expressing a combination of human CD46, CD55 and CD59, all inhibitors of complement activation, was 45% compared with 24% in ischaemic reperfused wild-type mice (P=0.008; n=6 per group). Muscle from sham-treated transgenic mice and wild-type littermates had no significant loss of viability relative to normal contralateral gastrocnemius muscle. A significant reduction in myeloperoxidase activity (a measure of neutrophil infiltration), xanthine oxidase activity (a source of free radicals) and water content (a measure of oedema) was observed in ischaemic reperfused muscle from transgenic mice compared with ischaemic reperfused wild-type muscle (P<0.05). Haematoxylin and eosin-stained histological sections also showed less damage and less apparent leucocyte infiltration in muscles from ischaemic reperfused transgenic mice than those from wild-type animals given the same degree of injury. Muscles from sham-treated transgenic and wild-type controls were almost identical with normal muscle. It is concluded that complement activation contributes to the pathogenesis of I/R injury in murine skeletal muscle, resulting in increased neutrophil infiltration into the injured muscle, increased free radical production and vascular permeability during reperfusion, and a net detrimental effect on muscle viability.
Computational study of hydrogen binding by metal-organic framework-5
Authors: Sagara Tatsuhiko , Klassen James , Ganz Eric
Published: December 2004

We report the results of quantum chemistry calculations on H2 binding by the metal-organic framework-5 (MOF)-5. Density functional theory calculations were used to calculate the atomic positions, lattice constant, and effective atomic charges from the electrostatic potential for the MOF-5 crystal structure. Second-order Møller-Plesset perturbation theory was used to calculate the binding energy of H2 to benzene and H2-1,4-benzenedicarboxylate-H2. To achieve the necessary accuracy, the large Dunning basis sets aug-cc-pVTZ, and aug-cc-pVQZ were used, and the results were extrapolated to the basis set limit. The binding energy results were 4.77 kJ/mol for benzene, 5.27 kJ/mol for H2-1,4-benzenedicarboxylate-H2. We also estimate binding of 5.38 kJ/mol for Li-1,4-benzenedicarboxylate-Li and 6.86 kJ/mol at the zinc oxide corners using second-order Møller-Plesset perturbation theory. In order to compare our theoretical calculations to the experimental hydrogen storage results, grand canonical Monte Carlo calculations were performed. The Monte Carlo simulations identify a high energy binding site at the corners that quickly saturated with 1.27 H2 molecules at 78 K. At 300 K, a broad range of binding sites are observed.
Bernstein mode aided anomalous absorption of laser in a plasma
Authors: Kumar Asheel , Tripathi V. K.
Published: December 2004

A laser propagating through a plasma, in the presence of an electron Bernstein wave, undergoes nonlinear mode coupling, producing a beat mode (ω+ω0, k+k0) where (ω0, k0) and (ω, k) are the frequency and wave number of the laser and the Bernstein mode. The oscillatory electron velocity associated with this beat mode couples with electron density perturbation due to the Bernstein wave to produce a nonlinear current at the laser frequency. When the beat mode is Landau damped on electrons, the nonlinear current at the laser frequency has an in-phase component with the laser field, giving rise to anomalous resistivity. The normalized anomalous resistivity is found to be maximum for q=∣k+k0∣νth∕(ω+ω0)≈0.8–0.9.
Consistent modeling of scalar mixing for presumed, multiple parameter probability density functions
Authors: Mortensen Mikael
Published: December 2004

In this Brief Communication we describe a consistent method for calculating the conditional scalar dissipation (or diffusion) rate for inhomogeneous turbulent flows. The model follows from the transport equation for the conserved scalar probability density function (PDF) using a gradient diffusion closure for the conditional mean velocity and a presumed PDF depending on any number of mixture fraction moments. With the presumed β PDF, the model is an inhomogeneous modification to the homogeneous model of Girimaji [“On the modeling of scalar diffusion in isotropic turbulence,” Phys. Fluids A 4, 2529 (1992)]. An important feature of the model is that it makes the classical approach to the conditional moment closure completely conservative for inhomogeneous flows.
Nonmodal growth of three-dimensional disturbances on plane Couette–Poiseuille flows
Authors: Bergström Lars B.
Published: December 2004

The time development of three-dimensional disturbances superimposed on a variety of mean flow profiles representing plane Couette–Poiseuille flow is investigated numerically. Specifically, with y representing the wall normal coordinate, the mean flow profiles U(y) are represented by U(y)=A(1−y2)+By, where B=1 when 0⩽A⩽1∕2 and B=2A(1−A) when 1∕2⩽A⩽1. For streamwise independent disturbances, which are the most amplified ones, there is an increase of the disturbance peak amplification when the parameter A increases in the interval 1∕10⩽A⩽1∕2. In the interval 1∕2⩽A⩽9∕10, and especially for 9∕10⩽A⩽1, the disturbance peak amplification decreases rapidly when A is increased. For A close to 1, a slight reduction of A will therefore cause a strong increase of the disturbance amplification.
Demonstration of ultra-high-Q small mode volume toroid microcavities on a chip
Authors: Kippenberg T. J. , Spillane S. M. , Vahala K. J.
Published: December 2004

Optical microcavities confine light spatially and temporally and find application in a wide range of fundamental and applied studies. In many areas, the microcavity figure of merit is not only determined by photon lifetime (or the equivalent quality-factor, Q), but also by simultaneous achievement of small mode volume (V). Here we demonstrate ultra-high Q-factor small mode volume toroid microcavities on-a-chip, which exhibit a Q∕V factor of more than 106(λ∕n)−3. These values are the highest reported to date for any chip-based microcavity. A corresponding Purcell factor in excess of 200 000 and a cavity finesse of >2.8×106 is achieved, demonstrating that toroid microcavities are promising candidates for studies of the Purcell effect, cavity QED or biochemical sensing.
Long-time evolution of linearly stratified spin-up flows in axisymmetric geometries
Authors: Smirnov S. A. , Baines P. G. , Boyer D. L. , Voropayev S. I. , Srdic-Mitrovic A. N.
Published: December 2004

We present results of laboratory experiments on the evolution of continuously stratified rotating flows initiated via incremental spin-up in either cylindrical or annular geometries. It is found that the flow behavior is governed mainly by the Rossby (ε) and Burger (Bu) numbers. Cyclonic and anticyclonic eddies are formed in a large region of ε−Bu parameter space due to instability of the flow, after the Ekman layer arrest time scale. The extended Eady model of baroclinic instability, in which the sheared wall layers are taken into account, is advanced to explain this mechanism of eddy formation. This model accounts for most of the observed features of the instability, and provides a realistic estimate for the time of onset of eddy formation.
Single-mask, three-dimensional microfabrication of high-aspect-ratio structures in bulk silicon using reactive ion etching lag and sacrificial oxidation
Authors: Rao M. P. , Aimi M. F. , MacDonald N. C.
Published: December 2004

This letter describes a simple method for three-dimensional microfabrication of complex, high-aspect-ratio structures with arbitrary surface height profiles in bulk silicon. The method relies on the exploitation of reactive ion etching lag to simultaneously define all features using a single lithographic masking step. Modulation of the mask pattern openings used to define the features results in etch depth variation across the pattern, which is then translated into surface height variation through removal of the superstructure above the etched floors. Utilization of a nonisotropic superstructure removal method based on sacrificial oxidation enables definition of high-aspect-ratio structures with vertical sidewalls and fine features. The utility of the approach is demonstrated in the fabrication of a sloping electrode structure for application in a hybrid micromirror device.
Electron-irradiation enhanced photoluminescence from GaInNAs∕GaAs quantum wells subject to thermal annealing
Authors: Pavelescu E.-M. , Gheorghiu A. , Dumitrescu M. , Tukiainen A. , Jouhti T. , Hakkarainen T. , Kudrawiec R. , Andrzejewski J. , Misiewicz J. , Tkachenko N. , Dhaka V. D. S. , Lemmetyinen H. , Pessa M.
Published: December 2004

Electron irradiation of a 1.3-μm-GaInNAs∕GaAs multi-quantum-well heterostructure, grown by molecular beam epitaxy and subsequently rapid-thermal annealed, is found to induce much stronger photoluminescence than what is observed for an identical as-grown sample upon annealing. Annealing of the irradiated sample also causes a small additional spectral blueshift and reduces alloy potential energy fluctuations at the conduction band minimum. These irradiation-related phenomena are accompanied by small but discernable changes in x-ray diffraction features upon annealing, which indicate compositional and∕or structural changes in the quantum wells.
Dielectric response of the charge-ordered two-dimensional nickelate La1.5Sr0.5NiO4
Authors: Rivas J. , Rivas-Murias B. , Fondado A. , Mira J. , Señarís-Rodríguez M. A.
Published: December 2004

We report the dielectric response of La1.5Sr0.5NiO4, a system that experiences charge ordering above room temperature (TCO=480K) and a rearrangement of its charge-order pattern in the temperature region 160–200K. A careful analysis of the role of the electrical contacts used, sample thickness, and grain size on the experimental data allows us to determine that this material exhibits a high intrinsic dielectric constant. In addition, the temperature dependence of the dielectric constant, that shows a maximum in the region of the rearrangement of the charge-order pattern, points to a link between the two phenomena.
Operating characteristics of a traveling-wave semiconducting polymer optical amplifier
Authors: Heliotis G. , Bradley D. D. C. , Goossens M. , Richardson S. , Turnbull G. A. , Samuel I. D. W.
Published: December 2004

We report a study of light amplification in dilute solutions of the semiconducting polymer poly(9,9-dioctylfluorene-co-benzothiadiazole) (F8BT). The operating characteristics of the F8BT solution amplifier are investigated in detail, and we show that it provides a large optical gain (>40dB∕cm) across a wide (>48nm) spectral bandwidth in the green. The saturation characteristics of the amplifier are found to be in excellent agreement with theoretical predictions, allowing an estimate of the F8BT stimulated emission cross-section, σ. We find σ∼1.7×10−16cm2, comparable with the reported values for established laser dyes and other high-gain conjugated polymers.
Spin injection, diffusion, and detection in lateral spin-valves
Authors: Ji Y. , Hoffmann A. , Jiang J. S. , Bader S. D.
Published: December 2004

We demonstrate the injection of spin-polarized electrons into paramagnetic Au nanowires by driving an electric current from a ferromagnetic permalloy (Py) electrode. The nonequilibrium spin accumulation in Au results in a difference between the chemical potentials for spin-up and spin-down electrons that is detected as a field-dependent voltage signal using a second Py electrode. The magnitude of the voltage contrast (>10%) and its coincidence with the magnetic switching of the Py electrodes attest to the spin-sensitive origin of the signals. By increasing the separation of the Py injector and detector, we observe an exponential decay of the spin signals. The measurements yield a spin-diffusion length of 63±15nm and an injected spin polarization of 3% in Au at 10 K.
Meiosis and embryo technology: renaissance of the nucleolus
Authors: Maddox-Hyttel Poul , Bjerregaard Bolette , Laurincik Jozef
Published: December 2004

The nucleolus is the site of rRNA and ribosome production. This organelle presents an active fibrillogranular ultrastructure in the oocyte during the growth of the gamete but, at the end of the growth phase, the nucleolus is transformed into an inactive remnant that is dissolved when meiosis is resumed at germinal vesicle breakdown. Upon meiosis, structures resembling the nucleolar remnant, now referred to as nucleolus precursor bodies (NPBs), are established in the pronuclei. These entities harbour the development of fibrillogranular nucleoli and re-establishment of nucleolar function in conjunction with the major activation of the embryonic genome. This so-called nucleologenesis occurs at a species-specific time of development and can be classified into two different models: one where nucleolus development occurs inside the NPBs (e.g. cattle) and one where the nucleolus is formed on the surface of the NPBs (e.g. pigs). A panel of nucleolar proteins with functions during rDNA transcription (topoisomerase I, RNA polymerase I and upstream binding factor) and early (fibrillarin) or late rRNA processing (nucleolin and nucleophosmin) are localised to specific compartments of the oocyte nucleolus and those engaged in late processing are, to some degree, re-used for nucleologenesis in the embryo, whereas the others require de novo embryonic transcription in order to be allocated to the developing nucleolus. In the oocyte, inactivation of the nucleolus coincides with the acquisition of full meiotic competence, a parameter that may be of importance in relation to in vitro oocyte maturation. In embryo, nucleologenesis may be affected by technological manipulations: in vitro embryo production apparently has no impact on this process in cattle, whereas in the pig this technology results in impaired nucleologenesis. In cattle, reconstruction of embryos by nuclear transfer results in profound disturbances in nucleologenesis. In conclusion, the nucleolus is an organelle of great importance for the developmental competence of oocytes and embryos and may serve as a morphological marker for the completion of oocyte growth and normality of activation of the embryonic genome.
Mitochondria and the success of somatic cell nuclear transfer cloning: from nuclear - mitochondrial interactions to mitochondrial complementation and mitochondrial DNA recombination
Authors: Hiendleder Stefan , Zakhartchenko Valeri , Wolf Eckhard
Published: December 2004

The overall success of somatic cell nuclear transfer (SCNT) cloning is rather unsatisfactory, both in terms of efficacy and from an animal health and welfare point of view. Most research activities have concentrated on epigenetic reprogramming problems as one major cause of SCNT failure. The present review addresses the limited success of mammalian SCNT from yet another viewpoint, the mitochondrial perspective. Mitochondria have a broad range of critical functions in cellular energy supply, cell signalling and programmed cell death and, thus, affect embryonic and fetal development, suggesting that inadequate or perturbed mitochondrial functions may adversely affect SCNT success. A survey of perinatal clinical data from human subjects with deficient mitochondrial respiratory chain activity has revealed a plethora of phenotypes that have striking similarities with abnormalities commonly encountered in SCNT fetuses and offspring. We discuss the limited experimental data on nuclear–mitochondrial interaction effects in SCNT and explore the potential effects in the context of new findings about the biology of mitochondria. These include mitochondrial fusion/fission, mitochondrial complementation and mitochondrial DNA recombination, processes that are likely to be affected by and impact on SCNT cloning. Furthermore, we indicate pathways that could link epigenetic reprogramming and mitochondria effects in SCNT and address questions and perspectives for future research.
109 A BRIDGE OF SPERM TAIL BETWEEN BLASTOMERES ENHANCED PROTEIN MIGRATION IN THE RAT TWO-CELL STAGE EMBRYOS
Authors: Kashiwazaki N. , Takizawa A. , Hisamatsu S. , Akamatsu Y. , Shino M. , Sonoki S. , Inomata T.
Published: December 2004

The aim of the present study was to examine transient expression of transgene injected into nuclei of rat 2-cell stage embryos. We also investigated the relationship between expression in both blastomeres and tail position of penetrated spermatozoa in rat 2-cell stage embryos. Rat 2-cell stage embryos were recovered from superovulated Wistar females mated with same strain mature males at 48 h after hCG injection. DNA fragments, as the transgene containing the EGFP (enhanced green fluorescent protein) gene controlled under the CMV-IE promoter, were microinjected into one nucleus of 2-cell stage embryos. After microinjection, embryos were cultured in KRB at 37.0°C in a 5% CO2 and 95% humidified air until observation. First, transient EGFP expression in 151 injected embryos was observed using a fluorescence microscope at 6 h intervals until 48 h after injection. At 6 h after microinjection fluorescent embryos were detected, and the proportion of fluorescent embryos increased over time. The rate reached maximum (84%, 52/62) at 24 h after microinjection, and several fluorescent patterns of fluorescent blastomeres in the embryos were observed. There were blastomeres with the same or different fluorescence levels and a single fluorescent blastomere. Second, to assess tail position of the penetrated sperm in the fluorescent embryos, 75 whole mount specimens were observed by inverted phase-contrast microscopy at 24 h after the injection. Also, parthenogenetic 2-cell stage embryos that never contained sperm tail were microinjected with the transgene and observed in the same manner. To obtain parthenogenetic 2-cell embryos, 80 ovulated ova were collected from non-mated females, and incubated with 2 mM 6-DMAP for 4 h. The ova were additionally cultured for 20 h in KRB at 37.0°C in a 5% CO2 and 95% humidified air. In embryos with both blastomeres fluorescent (94%, 33/35), the sperm tail existed in both blastomeres like a bridge between blastomeres. In contrast, in one embryo with a single fluorescent blastomere (4%, 1/24), the sperm tail existed in both blastomeres, and in other embryos with a single fluorescent blastomere (75%, 18/24), the sperm tail was positioned in the one blastomere. On the other hand, in 63 parthenogenic rat 2-cell embryos in which there was no sperm tail, most embryos (86%, 54/63) had a single fluorescent blastomere at 24 h after microinjection. The results indicated that the sperm tail position in the 2-cell embryos makes the protein migration variable. In conclusion, when the CMV-IE/EGFP gene was microinjected into nuclei of rat 2-cell embryos, at 24 h after the microinjection the EGFP was detected in most embryos; however, fluorescent patterns in blastomeres varied. It seems that EGFP derived from the transgene injected into one blastomere may move into another blastomere in rat 2-cell stage embryos, and that the presence of a sperm tail in both blastomeres may influence EGFP distribution.
111 CLONING AND CHARACTERIZATION OF PIG VASA HOMOLOG GENE AND ITS SPECIFIC EXPRESSION IN GERM CELL LINEAGE
Authors: Lee G.S. , Lee S.H. , Kim H.S. , Jeung E.B. , Kang S.K. , Lee B.C. , Hwang W.S.
Published: December 2004

All of the vasa homologue genes in C. elegans (Caenorhabditis elegens, a free-living soil nematode), xenopus, zebrafish, mouse, human, chicken, trout, and rat exhibited a germ line-specific expression and are used as specific molecular probes to distinguish the developmental profile of germ cells. In order to determine a useful marker for the research of germ cell commitment and development in pigs, we investigated the cloning and expression profile of porcine vasa homolog gene (Pvh). A Pvh cDNA gene of size 2172 bps (submitted to NCBI gene Bank No. AY626785) was cloned from pig ovary by reverse transcription-polymerase chain reaction (RT-PCR) amplification. The amplification was repeated three times and each RT-PCR product was sequenced. The isolated cDNA had 724 deduced amino acids with significant homology to mouse (85%) or human (91%) vasa. The Pvh sequence presents five copies of the RGG motifs and the DEAD box. By RT-PCR amplification, the expression of Pvh mRNA was restricted to the ovary and testis and was undetectable in somatic tissues including brain, whole blood, heart, lung, kidney, spleen, intestine, and liver. When analyzed by RT-PCR amplification, during pre-implantation embryo development, Pvh was transcribed in oocytes and fertilized 2-cell embryos (no difference in the expression levels between oocytes and fertilized 2-cell embryos), but not in 4-cell, 8-cell, morula and blastocyst stages. Using mouse vasa antibody (kindly donated from Dr. Noce, Japan; tested in porcine cells with porcine oocytes and mouse oocytes as positive control and with porcine brain cells as negative control), immunohistochemical analysis of fetal (Day 100) and adult gonad sections revealed that Pvh protein was specifically expressed in proliferating primordial germ cells (PGC), oocytes and spermatocytes. Interestingly, Pvh protein was not expressed in embryonic germ cells, but it was strongly expressed in freshly isolated PGC. Our results indicate that Pvh gene is specifically transcribed in pig germ cells.
This study was supported by grants from the Korean Ministry of Science and Technology (Biodiscovery) and the Biogreen 21-1000520030100000.
113 THE DISTRIBUTION OF THE LEPTIN PROTEIN WITHIN BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS MATURED AND FERTILIZED IN VITRO
Authors: Madeja Z. , Lechniak D. , Peippo J. , Switonski M.
Published: December 2004

It has recently been documented that leptin regulates processes linked to reproduction including preimplantation development, embryo implantation (trophoblast invasion), and fetal growth. Transcripts for the leptin gene (LEP) and the leptin receptor gene (LEPR) have been identified in ovary, testis, placenta, endometrium, ovarian follicles, and oocytes, and also in mouse, rat, human, and bovine pre-implantation embryos. Moreover, the leptin protein was detected in mouse and human oocytes and embryos, and its localization was polarized. The distribution of regulatory proteins within oocytes and pre-implantation embryos is critical for early mammalian development, such as determination of the animal pole and the establishment of the trophoblast and the inner cell mass cells (ICM). So far there is no published evidence concerning this phenomenon in bovine oocytes and embryos. Therefore, the aim of this work was to analyze the leptin protein distribution within bovine oocytes and preimplantation embryos matured and fertilized (in vitro). The material for this work consisted of oocytes collected from slaughterhouse ovaries and sperm collected from AI bulls. In vitro oocyte maturation and fertilization were carried out according to the method described by Makarevich and Markkula (2002 Biol. Reprod. 66, 386–392). The preliminary experiment of leptin protein localization by immunofluorescent staining included immature and matured oocytes and blastocysts. Oocytes and embryos were fixed in PBS containing 4% paraformaldehyde and reacted with affinity-purified polyclonal rabbit primary antibody directed against leptin (0.1 mg/mL; Ob (Y20), Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); and then exposed to secondary goat-anti-rabbit antibody (1.0 mg/mL; Santa Cruz Biotechnology Inc.)-fluorescein isothiocyanate (FITC) conjugate. Finally, chromatin was visualized by propidium iodide staining (0.5 μg/mL). Slides were examined under a conventional fluorescence microscope (Nikon) and confocal microscope (Zeiss). The preliminary results demonstrate that the distribution of leptin differed between immature and mature oocytes: it was spherical in immature oocytes (a rim beneath the oolemma) whereas it became evenly distributed after maturation. In blastocysts, leptin signals were present inboth the trophoblast cells and in the ICM cells. This is in contrast with studies on mouse embryos which showed the presence of the LEP protein in the trophoblast only. Future experiments will include studies of embryos at the 2-cell, 4-cell, 8–16-cell, and morula stages. The present study for the first time shows the pattern of leptin protein distribution within bovine oocytes and preattachment embryos.
131 MODIFICATION OF AMINO ACID CONCENTRATIONS IN MEDIUM BY PIG EMBRYOS FROM THE ZYGOTE TO THE BLASTOCYST STAGE
Authors: Booth P. , Watson T. , Leese H.
Published: December 2004

Pre-implantation embryos can produce and consume amino acids in a manner dependent upon stage of embryonic development (Partridge and Leese 1996 Reprod. Fert. Dev. 8, 945) that may also be predictive of subsequent viability (Houghton et al. 2002 Hum. Reprod. 17, 999). To examine these relationships in the pig, the appearance or depletion of 18 amino acids from a presumptive near-physiological mixture was determined by HPLC in porcine in vitro-produced embryos from the zygote to the blastocyst stage. Cumulus oocyte complexes derived from slaughterhouse prepubertal pig ovaries were matured for 40 h in modified TCM-199 before being fertilized (Day 0) with frozen thawed semen in tris-based medium. After 6 h, presumptive zygotes were denuded and cultured in groups of 20 in NCSU medium modified to contain a physiological mixture of 18 amino acids including 0.1 mM glutamine (NCSUaa). Groups of 2–10 embryos (dependent on stage) were removed on Day 0 (1 cell), Day 1 (2- and 4-cell), Day 4 (compact morula), and Day 6 (blastocyst) and placed in 4 μL NCSUaa for 24 h. After incubation, the embryos were removed and the medium analyzed by HPLC. Each stage was replicated 3–9 times. Since amino acid profiles of 2- and 4-cell embryos were not different, data were combined. Overall, arginine (1.19 ± 0.33), glutamine (0.78 ± 0.34) and threonine (0.05 ± 0.04) were significantly (P < 0.01) depleted from the medium whereas alanine (0.21 ± 0.1), glycine (0.20 ± 0.06), asparagine (0.13 ± 0.5), lysine (0.1 ± 0.03), isoleucine (0.08 ± 0.01), valine (0.05 ± 0.01), leucine (0.04 ± 0.02), phenylalanine (0.03 ± 0.01), and histidine (0.02 ± 0.04) significantly (P < 0.05) accumulated (mean of the 4 sampling timepoints; all values pmol/embryo/h ± SEM). The difference between amino acid accumulation and depletion (balance) was approximately equivalent between Day 0 and the morula stage although turnover (sum of depletion and accumulation) steadily decreased during this period from 3.1 on Day 0 to 1.35 pmol/embryo/h at the morula stage. However, at the blastocyst stage, turnover and balance increased to 6.32 and 2.42 pmol/embryo/h, respectively, i.e. net appearance occurred. Notable changes in amino acid profile during development included decreases in accumulation of asparagine, glutamate, and glycine in the medium and the depletion of glutamine over Days 0, 1, and 4, followed by reversal of these trends by Day 6. These data suggest that pig embryos can alter the accumulation and depletion rates of amino acids in a manner that is dependent on the specific amino acid and the stage of embryonic development.
This work was supported by BBSRC.
139 BOVINE AMNIOTIC FLUID FOR THE CULTURE OF TWO-CELL MURINE EMBRYOS
Authors: Herholdt G.J.M. , Barry D.M.
Published: December 2004

The objective of the experiment was to evaluate bovine amniotic fluid as an alternative medium for culture of two-cell murine embryos. Variability in embryo development rate and high purchase prices of medium supplements such as fetal bovine serum have prompted the search for a serum alternative. Amniotic fluid was collected postmortem from first-trimester bovine fetuses, pooled, heat inactivated at 56°C for 30 min and stored at −20°C until used. Two-cell mouse embryos were collected from (Balb/C × C57BL6) F1 females superovulated with 10 IU PMSG and 10 IU hCG. The experiment was performed to evaluate the effect of commercial fetal bovine serum (F4135, Sigma, South Africa) supplementation to amniotic fluid. Treatments consisted of (1) bovine amniotic fluid, (2) bovine amniotic fluid supplemented with 10% fetal bovine serum, (3) M16 (M7292, Sigma) supplemented with 10% fetal bovine serum, and (4) Medium 199 (M4530, Sigma) supplemented with 10% fetal bovine serum. The latter two media were controls. Twenty-four hours before use the culture media were supplemented with 1% antibiotic antimycotic solution (A5955, Sigma). Media were equilibrated for 24 hours at 37°C and 5% CO2 before use. Embryos were cultured in 50-μL droplets with oil overlay at 37°C in 5% CO2. A minimum of 5 and maximum of 10 embryos per droplet were allowed. Six replications per treatment were done giving a total of 292 embryos in treatment (1), 318 in treatment (2), 304 in treatment (3), and 303 in treatment (4). The embryos were monitored under an inverted microscope (Olympus model IX70) at 24-h intervals for 72 h for blastocyst formation. The differences between embryo growth in the different culture media were assessed by one-way analysis of variance. All culture media supported the development of mouse embryos to the hatched blastocyst stage. A higher (P < 0.05) number of embryos hatched in M16 (64.6%) and Medium 199 (55.0%) supplemented with 10% fetal bovine serum than in frozen bovine amniotic fluid (12.2%) and frozen bovine amniotic fluid supplemented with 10% fetal bovine serum (17.8%). M16 was superior to all other treatments in supporting embryo development up to the morula stage. More than twice the number of embryos (94.9%) reached the morula stage in M16 than in frozen bovine amniotic fluid with (37.4%) or without (29.1%) serum supplementation. Bovine amniotic fluid obtained from postmortem first trimester fetuses supported the development of two-cell mouse embryos; however, embryo development in frozen fetal fluid was lower (P < 0.05) than that obtained in the control media. Fetal bovine serum, when added to amniotic fluid, did not increase the development rate. It seems likely that freezing the amniotic fluid had an adverse effect on in vitro embryo development.
142 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN KSOM, CR1AA, OR KSOM/CR1aa
Authors: Mello M.R.B. , Ferguson C.E. , Lima A.S. , Wheeler M.B.
Published: December 2004

In vitro embryo culture is an important step of in vitro production of bovine embryos. It has been shown that IVF-derived bovine embryos cultured in KSOM or CR1aa have high development rates. In our laboratory, we have observed that 8-cell embryos are morphologically superior when embryos are cultured in KSOM whereas blastocysts are morphologically superior when embryos are cultured in CR1aa. Based on these observations, we hypothesized that development of IVF-derived bovine embryos can be improved by sequential use of these media (KSOM and CR1aa). The aim of this experiment was to compare the in vitro development of bovine embryos cultured in KSOM, CR1aa or KSOM/CR1aa supplemented with BSA at Day 0 and BSA and FBS at Day 3. In order to accomplish the sequential culture, fertilized oocytes where cultured in KSOM to the 8-cell stage and then transferred to CR1aa for further development. Oocytes were purchased from Bomed (Madison, WI, USA), and after 22 hours of maturation were fertilized with frozen-thawed semen for 5 hours at 39°C in 5% CO2. After fertilization, the presumptive zygotes were denuded from cumulus cells by votexing and were randomly allotted to one of 3 treatments: (1) cultured only in KSOM (n = 110), (2) cultured only in CR1aa (n = 102), and (3) cultured in KSOM in the first 3 days and then in CR1aa from Day 3 to Day 9 (n = 110). The embryo culture was carried out in 50-μL droplets of medium that were placed in an airtight modular incubator filled with 5% CO2, 5% O2 and 90% N2. The embryos were evaluated on Days 6 to 9 post insemination. All embryo developmental rates were calculated from presumptive zygotes. The Day 6 morula rates were 52%, 40%, and 47% for KSOM, CR1aa, and KSOM/CR1aa, respectively. The Day 7 blastocyst rates for KSOM (40%), CR1aa (25%), and KSOM/CR1aa (30%) were not significantly different; however, Day 9 hatched blastocyst rates were significantly higher (P < 0.05) for KSOM (22%) compared to CR1aa (9%) but not different from KSOM/CR1aa (14%). Regarding embryo quality, Day 7 transferable embryos rates (Grade 1 and Grade 2) were 35%, 25%, and 30%, respectively for KSOM, CR1aa, and KSOM/CR1aa; however, no significant difference was observed. These results indicate that IVF-derived bovine embryos can develop in KSOM, CR1aa, or KSOM/CR1aa with no significant difference among morula, blastocyst and hatched blastocyst rates. However, the combination of KSOM and CR1aa during in vitro culture did not decrease the morula and blastocyst rates.
150 ENVIRONMENT OF THE EARLY EMBRYO AND ITS EFFECT ON DEVELOPMENT AND POSTNATAL LIFE
Authors: Watkins A. , Wilkins A. , Papenbrock T. , Osmond C. , Hanson M. , Fleming T.
Published: December 2004

We have investigated the impact of mouse early embryo in vitro culture environment on (a) short-term blastocyst development and (b) long-term postnatal growth and physiology after embryo transfer. In vitro-developed blastocysts, cultured from the 2-cell stage, had reduced inner cell mass (ICM) and trophectoderm (TE) cell numbers when compared to in vivo-derived blastocysts at 96 h post-hCG (n = 13–39, P < 0.05). Despite the retardation in blastocyst development, the ICM:TE ratio was equivalent in both treatment groups. Using embryo transfer techniques, we compared the postnatal development of embryos cultured in vitro from the 2-cell to the blastocyst stage (termed “in vitro” mice) with offspring generated from blastocysts developed in vivo, but which also underwent embryo transfer (termed “in vivo” mice). These two treatment groups were in turn compared with mice derived from naturally mated mothers, which had their mean litter size at birth adjusted to a size comparable with that of the in vitro and in vivo mice (a mean of 6 animals) and which had not been transferred. All data were analyzed using a multilevel random effects regression model which took into account between-mother and within-mother variation in litter size for parameters measured from individual animals. No significant differences in birth weight were observed between in vitro and in vivo offspring. However, in vitro offspring were significantly lighter than in vivo offspring in a gender-dependent manner at 2 weeks of age (males, P = 0.009) and at 6 and 11 weeks of age (females, P = 0.037 and 0.035, respectively). In addition, at 4 weeks of age, the in vivo males became significantly lighter when compared to the naturally mated males (P = 0.034). At 8 weeks of age, the in vivo females had a significantly elevated systolic blood pressure when compared to the in vitro females (P = 0.003); however, at 21 weeks of age, both in vitro males and females had a significantly elevated blood pressure when compared to in vivo offspring (P < 0.003). At 8, 15, and 21 weeks of age, offspring derived from transferred embryos developed with significantly elevated systolic blood pressure when compared to non-embryo transfer offspring (P < 0.05). No significant differences in serum angiotensin-converting enzyme activity (a potent regulator of systolic blood pressure) was observed between the treatment groups. Significantly altered liver:body weight ratios were observed between the in vitro and in vivo males, and between the in vitro and the naturally mated (6) females (P < 0.038). All of the above data are independent of litter size. These data support the hypothesis that early embryo environment can influence postnatal growth and cardiovascular physiology.
This research was funded by an MRC research grant to TPF, and by a DOHaD studentship.
156 ENHANCED BOVINE EMBRYONIC DEVELOPMENT AFTER MICROFLUIDIC CUMULUS CELL REMOVAL POST-FERTILIZATION
Authors: Reeder A. , Monson R. , Beebe D. , Lindsey B. , Rutledge J.
Published: December 2004

Microfluidic technologies are increasingly being used in cell biology and embryology research. In order to manipulate an embryonic environment microfluidics take advantage of miniscule media amounts. With the use of pressure heads and laminar flow profiles, a presumptive zygote can be gently manipulated in a microfluidic device for removal of the supporting cumulus cells post-fertilization. Presumptive embryos were assigned at random to three cumulus removal treatments at 48 h post-fertilization: vortexing (3 min), handstripping (with 135-μm-ID stripping pipette), and microfluidics. Blastocyst rates were determined through Day 8 post-fertilization. Rates were analyzed by the GENMOD procedure in SAS (SAS Institute, Inc., Cary, NC, USA), accounting for replicates and treatment. Kinetics of development were also impacted as larger proportions of embryos in the microfluidic group reached the blastocyst stage before embryos of the vortex or hand-stripping treatments. These data suggest that cumulus cell removal in a gentle fashion is associated with enhanced embryonic development in the bovine.

Table 1.
Comparison of cumulus cell removal techniques on bovine blastocyst rates
162 EMBRYO QUALITY AND COLOR IN HOLSTEIN FRIESIAN HEIFERS AND COWS IN RELATION TO SERUM PARAMETERS
Authors: Leroy J.L.M.R. , Goossens L. , Geldhof A. , Vanholder T. , De Vliegher S. , Opsomer G. , Van Soom A.
Published: December 2004

Bovine embryo freezability is closely linked with quality and differs between breeds (Visintin JA et al. 2002 Theriogenology 57, 345–359). Dark embryos have a poor cryotolerance resulting in low pregnancy rates (Hill BR and Kuehner LF 1998 Theriogenology 49, 168), and are characterized by excessive accumulation of lipid droplets which in turn may be influenced by the biochemical composition of the embryonic environment. Earlier work revealed that Holstein Friesian (HF) cows yield significantly darker embryos compared to Belgian Blue beef cows (Leroy JLMR et al. 2004 Reprod. Fert. Dev. 16, 211). In this field examine we aimed to study the differences in embryo quality and color between HF cows (producing milk) and heifers (non-producing) in relation to four serum parameters that have previously been linked with embryo quality and fertility: urea, total protein (TP), total cholesterol (TC), and triglycerides (TG). Blood samples were collected from HF cows (n = 54) and HF heifers (n = 33) prior to embryo flushing on Day 7 after superovulation and subsequent insemination. Serum was stored frozen until assay with commercial photometric assays. Embryos were scored individually by the same operator for quality (excellent to poor: score 1 to 4) and color: light (L), medium (M), or dark (D) using a binocular stereomicroscope (40×). Independent Student's t-test and chi-square test were used when appropriate. Heifers and cows yielded on average (± SEM) 5.1 ± 0.82 and 6.1 ± 0.71 embryos per flushing, respectively. Significantly more cow embryos were classified as dark compared to heifer embryos (L, M, D: 20.4%, 55.5% and 24.1% vs. 70.8%, 27.4%, and 1.8%, for cow and heifer embryos, respectively) (P < 0.05). Significantly more heifer embryos showed an excellent morphological quality (62.5% of heifer embryos compared to 13.1% of cow embryos) (P < 0.05). Serum concentrations (± SEM) of urea (4.5 ± 0.2 vs. 2.8 ± 0.2 mM), TP (7.59 ± 0.07 vs. 6.57 ± 0.09 g/dL) and TC (183 ± 5.3 vs. 105 ± 3.8 mg/dL) were significantly higher and serum concentrations of TG (17.2 ± 0.6 vs. 23.8 ± 0.9 mg/dL) significantly lower in cows compared to heifers (P < 0.05). In this study, embryo color (i.e. lipid content) and quality as well as urea, TP, TC, and TG serum concentrations were clearly influenced by the production parameter (cow = producing vs. heifer = non-producing). These findings imply that producing milk (parity = 0 or >0) may be an important confounder. Therefore, this variable should be taken into account when studying multiple variables influencing embryo quality or color simultaneously. Further analyses are ongoing to reveal which factors at the cow level are affecting the embryo quality and color.
This research was funded by IWT-Flanders (Grant no. 13236).
178 ESTABLISHMENT OF MOUSE PLURIPOTENT STEM CELLS GENERATED FROM PRIMORDIAL GERM CELLS
Authors: Shim S.W. , Song S.J. , Shim H.S. , Uhm S.J. , Lee B.Y. , Kim S.B. , Song H. , Cho S-.G. , Chung H.M. , Chung K.S. , Lee H.T.
Published: December 2004

Pluripotent stem cells have been generated from two embryonic sources: ES cells generated from ICM of blastocyst stage embryos, and embryonic germ (EG) cells generated from primordial germ cells (PGCs). Both ES and EG cells are pluripotent and exhibit important characteristics such as high alkaline phosphatase (AP) activity, multicellular colony formation, normal and stable karyotype, continuous passaging ability, and capacity to differentiate into three embryonic germ layers. This study was performed to establish the culture system for mouse EG cells derived from mouse PGCs. PGCs collected from the genital ridge of Day 11.5, 12.5, and 13.5 mouse embryos (C57BL/6 × DBA/2) were cultured and subsequently passaged on mitotically inactivated STO feeder cell layer. Cells were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 100 IU/mL of penicillin, 100 μg/mL of streptomycin, 1,000 units/mL of leukemia inhibiting factor (LIF), 6 ng/mL of SCF, and 10 ng/mL of bFGF at culture conditions of 5% CO2 in air, 95% relative humidity, 37°C temperature. Cells were routinely passaged every 3–4 days. Over a period of 7–10 days in primary culture, PGCs proliferated to form small, densely packed, multicellular colonies consisting of AP-positive cells that morphologically resembled undifferentiated ES cells. RT-PCR analysis confirmed mRNA expression of transcription factors Oct-4 and Nanog in these cells. Cultured cells could be maintained on the feeder cell layer for at least 10 passages and still retain normal karyotype. These results suggest that cell lines derived from mouse primordial germ cells are presumably EG cell lines and could be useful for transgenic animal production and ES cell study.
180 EFFICIENT GENE SILENCING USING siRNA IN MOUSE AND MONKEY ES CELLS AND DIFFERENTIATION
Authors: Takada T. , Nemoto K. , Yamashita A. , Katoh M. , kondo Y. , Torii R.
Published: December 2004

Gene silencing by RNA interference (RNAi) using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool for silencing specific genes in various organisms. As compared with transgene-based RNAi using shRNA expression vectors, chemically synthesized siRNAs have an advantage in that they do not modify genome organization. This nature is ideal as a differentiation method for embryonic stem (ES) cells. The objective of this work was to develop an efficient method to repress a specific gene expression in mouse and monkey ES cells using chemically synthesized siRNA, and to investigate whether this transient gene silencing can be used as a new differentiation method of ES cells. First, we tried to knock down the expression of enhanced green fluorescent protein (EGFP) gene in GFP-expressing mouse ES cells. We found that lipofection was effective to deliver siRNA into mouse ES cells. Suppression of EGFP expression was observed by fluorescence microscopy after 24 h of transfection and the silencing continued at least for 5 days. FACS analysis revealed that 86% of cells showed suppression of EGFP at 48 h after transfection. Then we tried to suppress endogenous gene Oct4 that plays an important role in the maintenance of pluripotency and the lineage commitment in mouse ES cells. We performed RT-PCR analysis and western blotting to assay for Oct4 mRNA and protein at 24, 48, 72, and 96 h after transfection. Hand1 and Cdx2, transcription factors implicated in trophoblast differentiation, were also analyzed. RT-PCR analysis showed a reduced level of Oct4 mRNA at 24–96 h. Reduction of Oct4 protein was confirmed by western blotting and the reduced level was still maintained at 96 h after transfection. RT-PCR analysis also showed up-regulation of Hand1 and Cdx2 concomitant with the suppression of Oct4. Furthermore, ES cells changed their morphology into a large and flattened shape that is characteristic of trophoblast cells. These results suggested that the transient suppression of Oct4 induced differentiation of mouse ES cells to trophectoderm cells as expected. Therefore our data imply that the chemically synthesized siRNA can be used to differentiate ES cells. Next, we tried to suppress EGFP and Oct4 in monkey ES cells. In monkey ES cells, we found that the Sendai virus (hemagglutinating virus of Japan, HVJ) envelope was suitable to deliver siRNA into cells. With this method, we detected efficient silencing of EGFP and Oct4 by fluorescence microscopy, RT-PCR analysis, and western blotting. In the case of monkey ES cells, however, no morphological change was observed by Oct4 suppression at 96 h after transfection. These results suggest possible diversity between murine and primate ES cells in the differentiation process.
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture (14580798, 13358014 and 14380382).