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At least 1,279 records · Page 71Linked to original sources
PubMed2004

Use of Mammalian eggs for assessment of human sperm function: molecular and cellular analyses of fertilization by intracytoplasmic sperm injection.

PROBLEMS: Intracytoplasmic sperm injection (ICSI ) has been described as the 'cure' for male sterility because a single sperm can now be directly introduced into an egg with some chance of pregnancy. While ICSI has revolutionized the practice of assisted reproductive techniques (ART), there are few molecular and cellular studies about its safety and efficacy. Even by using ICSI, fertilization in humans succeeds only if the sperm effectively accomplishes a number of tasks including 'post-ICSI events' in fertilization. To assess the function of human sperm after ICSI, we used heterologous ICSI with human sperm into animal eggs. Egg activation, sperm decondensation and sperm centorosomal function were examined in sperm from fertile men and infertile patients. METHODS: Sperm from fertile men and infertile patients were injected into hamster, rabbit and bovine eggs by Piezo micromanipulator, and studied in decondensation of sperm nuclei, egg activation and microtubule organization. RESULTS: Decondensation human sperm head following ICSI into hamster eggs occurred initially form basal lesion, and apical portion of sperm nuclei which is surrounded by acrosome and perinuclear theca, still condensed in early pronuclear stage. Radial array of microtubules from sperm centrosome 'sperm aster' which is essential for pronuclear movement was observed in 30% rabbit eggs following ICSI with human sperm. By heterologous ICSI system with fertile human sperm and bovine eggs, 83.3% of eggs was activated and 60% eggs had sperm aster, indicating that bovine Piezo ICSI system is appropriate for assessing human sperm oocyte activation ability and human sperm centrosomal function. Oocyte activation and sperm centrosomal function were significantly low in sperm from men with globozoospermia and men with dysplasia of fibrous sheath. CONCLUSION: These assays indicate differences of the process of fertilization between in vitro fertilization and ICSI, and reflect the human sperm function especially for the 'post-ICSI events' in fertilization. More molecular and cellular analyses in fertilization by ICSI are needed for improvement of ART.

Acrosome↗
PubMed2004

Analysis of fetal DNA from maternal peripheral blood by lectin-polymerase chain reaction-single strand conformation polymorphism.

Fetal nucleated cells in maternal peripheral blood are a non-invasive source of fetal DNA for prenatal genetic diagnosis. However, the number of fetal cells present in maternal peripheral blood is very small. Therefore, fetal cell enrichment is generally considered necessary to allow detection and subsequent genetic analysis of the rare fetal cells. In the study presented here, we performed fetal cell separation from maternal blood using galactose-specific lectin to concentrate fetal nucleated red blood cells (FNRBCs), and attempted paternal diagnosis using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Fetal cell separation was performed using galactose-specific lectin on a PV-MeA coated slide. Twenty cells consisting of an NRBC and its surrounding 19 maternal cells were collected using laser microdissection for stable DNA amplification. DNA analysis was performed using three sequence tagged site markers (D13S270, D17S5, and D18S474) by PCR-SSCP. All seven cases were informative because they showed heterozygosity at least one locus in D13S270, D17S5, or D18S474, and paternal-specific bands were detected in all cases. These results suggest that our proposed lectin-laser-micromanipulation-PCR-SSCP method may contribute to the development of prenatal diagnosis.

DNA↗
PubMed1985

The variation in action of excitatory amino acids in relation to distance of iontophoretic application to spinal motoneurones.

Intracellular recordings were made from lumbosacral motoneurones of barbiturate-anaesthetized cats. DL-homocysteate (DLH) and L-glutamate were iontophoresed extracellularly over a range of distances from the impaled motoneurone. Movement of the iontophoretic electrode unit was controlled by a micromanipulator which was advanced independently of that moving the intracellular electrode. Depolarizations to DLH were first detected at a greater distance from the impaled motoneurones (mean, 383 micron) than depolarizations to L-glutamate (mean, 165 micron). As the point of application approached the soma of the motoneurone, depolarizations developed more rapidly, were larger and the latent period of the L-glutamate depolarization became shorter. Dendritic 'hot-spots' of the depolarizing action of L-glutamate were not detected.

Animals↗
PubMed1999

The planes of the utricular and saccular maculae of the guinea pig.

To establish a link between otolith anatomy and function it is necessary to know the regions of the utricular and saccular maculae, which are stimulated by any arbitrary linear acceleration stimulus. That requires accurate information about the location and orientation of the spatially extended maculae in head-fixed coordinates and referred to head-fixed landmarks (such as Reid's line). New data showing the location of the otolithic maculae in the guinea pig with respect to head-fixed stereotaxic coordinates are presented. Guinea pigs were perfused with Karnovsky's fixative and the maculae were exposed while the head was held in a guinea pig stereotaxic device. An electrolytically sharpened fine wire held in a calibrated micromanipulator was touched to points all over the surface of each macula under visual observation with the aid of a high-power operating microscope. The x, y, z coordinates of these points were plotted using a three-dimensional plotting program. Both maculae have pronounced curvature so that dorsoventral shear forces will stimulate regions of both the utricular and saccular maculae.

Acoustic Maculae↗
PubMed2001

Fertility and maternal age strategies to improve pregnancy outcome.

In humans, the live birth rate drops precipitously with increasing maternal age, and this decline is associated with increases in the incidence of oocyte and embryo aneuploidy. Preimplantation aneuploidy screening has improved pregnancy outcome by significantly lowering the miscarriage rate. Nevertheless, aneuploidy screening only identifies the affected embryos; it does not attempt to correct the underlying biologic problem. Anomalies in chromosome segregation can result from a dysfunctional first or second meiotic division in the egg or develop after fertilization during the first few mitoses of early embryonic development. In both instances, ooplasmic anomalies may account for the nuclear problem. Low cell levels of cytoplasmic proteins (e.g., cytoskeletal elements, enzymes, energy stores, cell cycle regulatory proteins) may lead to a dysfunctional division of chromosomes during egg maturation or following fertilization. Ooplasmic injection is a micromanipulation technique that has produced pregnancies in patients with a history of poor-quality, fragmented embryos. Germinal vesicle transfer is a research procedure used to investigate the ooplasmic-nuclear interplay regulating cell cycle, maturation, and fertilization. Both these techniques may prove to be effective in improving the quality of eggs from patients of advanced maternal age.

Adult↗
PubMed1997

Passive stress-strain measurements in the stage-16 and stage-18 embryonic chick heart.

The first stress-strain measurements on embryonic cardiovascular tissue are described here, obtained from cyclic uniaxial loading of the primitive ventricle. An excised ventricular segment from Hamburger/Hamilton stage-16 or stage-18 chicks (2-1/2 and 3 days of a 21-day incubation period) was mounted longitudinally between two small wires in oxygenated Krebs-Henseleit cardioplegia solution. One wire was attached to an ultrasensitive force transducer and the other to a Huxley micromanipulator controlled by remote motor drive. A real-time video tracking system calculated three myocardial surface strains based on the positions of three surface markers while the heart was deformed in a triangular wave pattern. Force transducer output was filtered, digitally sampled, and stored with strains and time. Results were plotted as strain (longitudinal, circumferential, shear, and principal) versus time, stress versus time, and stress versus longitudinal strain. The stress-strain curves were nonlinear, even at low strain levels. The hysteresis loops were large; mean hysteresis energy as a proportion of total cycle stored strain energy was 36 percent (stage 16) and 41 percent (stage 18). We created a finite element model of the ventricle and fit the model behavior to the experimental behavior to determine parameters for a stage-18 pseudoelastic strain-energy function of exponential form. The calculated exponential parameter is significantly lower than that found in corresponding uniaxial studies of mature myocardium, possibly indicating the lower fiber content of the immature tissue. The results of this study are the first step in characterizing material properties for comparisons with later developmental stages and with impaired and altered myocardium. The long-term goal is to aid in identifying the biomechanical factors regulating growth and morphogenesis.

Animals↗
PubMed1964

POLIOVIRUS: GROWTH IN NON-NUCLEATE CYTOPLASM.

Cytoplasmic fragments were produced by micromanipulation of cells from a human amnion cell line cultured on coverslips. The cultures were infected with type 1 (Mahoney) poliovirus, and incubated for 7 hours with tritiated uridine (H3U). Fluorescent antibody to the poliovirus indicated antigenic sites in a number of non-nucleate fragments. By autoradiography the incorporation of H3U was demonstrated at some of the same sites. The occurrence of poliovirus antigen at the same site as induced synthesis of RNA in non-nucleate cytoplasm of mammalian cells indicates that poliovirus infection and growth occurred independently of immediate contribution from the nucleus.

Amnion↗
PubMed1986

Pancreatic zymogen granules differ markedly in protein composition.

The activities of both chymotrypsin and amylase in individual zymogen granules of rat pancreas were measured by means of micromanipulation and microfluorometric methods. The enzyme content and the ratio of amylase to chymotrypsin varied widely among granules taken from the same animal. These results are compatible with short-term nonparallel bulk secretion of the two enzymes through exocytosis. The distribution of each enzyme activity in a population of granules suggests quantal packaging of amylase and chymotrypsinogen into the granules.

Amylases↗
PubMed1999

Polymersomes: tough vesicles made from diblock copolymers.

Vesicles were made from amphiphilic diblock copolymers and characterized by micromanipulation. The average molecular weight of the specific polymer studied, polyethyleneoxide-polyethylethylene (EO40-EE37), is several times greater than that of typical phospholipids in natural membranes. Both the membrane bending and area expansion moduli of electroformed polymersomes (polymer-based liposomes) fell within the range of lipid membrane measurements, but the giant polymersomes proved to be almost an order of magnitude tougher and sustained far greater areal strain before rupture. The polymersome membrane was also at least 10 times less permeable to water than common phospholipid bilayers. The results suggest a new class of synthetic thin-shelled capsules based on block copolymer chemistry.

Chemical Phenomena↗
PubMed1986

Determination of junction avidity of cytolytic T cell and target cell.

A direct measurement of the avidity of the junction between a cytotoxic T lymphocyte and its target cell was achieved by using a biophysical approach. A micromanipulation technique was used to determine the force required to separate a cytotoxic T cell (human clone F1, with specificity for HLA-DRw6) from its specific target cell (JY: HLA-A2, -B7, -DR4, w6) prior to delivery of the lethal hit. The force required to separate the F1-JY pair is 1.5 X 10(4) dynes per square centimeter. This junction avidity for F1-JY pairs is 6 to 13 times greater than that for F1-F1 and JY-JY pairs; the F1-JY conjugate requires a stronger separating force and is more easily rejoined than the homologous cell pairs. This study provides an estimate of the avidity of cytotoxic T cells for their target cells and insights into the biophysical correlates of the molecular complexes formed in the interaction of cytotoxic T cells and their targets during the cytotoxic process.

Cell Communication↗
PubMed1983

Development of gynogenetic eggs in the mouse: implications for parthenogenetic embryos.

Mouse eggs with different genetic constitutions were prepared by micromanipulation of fertilized diploids and triploids. The diploid gynogenones, activated by the male gamete which was then removed, developed at best to about the 25-somite stage as did the genetically similar diploid parthenogenones stimulated to develop in the complete absence of the male gamete. The failure of development to term in both cases may be due to homozygosity and does not appear to be due to a lack of extragenetic contribution from spermatozoa.

Animals↗
PubMed1993

Germ-line gene modification and disease prevention: some medical and ethical perspectives.

There has been considerable debate about the ethics of human germ-line gene modification. As a result of recent advances in the micromanipulation of embryos and the laboratory development of transgenic mice, a lively discussion has begun concerning both the technical feasibility and the ethical acceptability of human germ-line modification for the prevention of serious disease. This article summarizes some of the recent research on germ-line gene modification in animal models. Certain monogenic deficiency diseases that ultimately might be candidates for correction by germ-line intervention are identified. Several of the most frequently considered ethical issues relative to human germ-line gene modification are considered in the context of professional ethics, parental responsibility, and public policy. Finally, it is suggested that there is merit in continuing the discussion about human germ-line intervention, so that this technique can be carefully compared with alternative strategies for preventing genetic disease.

Animal Experimentation↗
PubMed1996

Phenotypic and phylogenetic analyses show Microcoleus chthonoplastes to be a cosmopolitan cyanobacterium.

We used micromanipulation to isolate from their environment representative samples of seven geographically distant field populations fitting the description of Microcoleus chthonoplastes (a cyanobacterium) and obtained seven corresponding cultured strains. Samples of both field populations and cultures were phenotypically characterized by microscale techniques, and their partial 16S rRNA gene sequences were compared by denaturing gradient gel electrophoresis and in some cases by sequencing. All field populations and strains were phenotypically extremely coherent, and their 16S rRNA sequences were indistinguishable by DGGE. The sequences determined were identical or virtually identical. Thus, M. chthonoplastes represents a single, well-delimited taxon with a truly cosmopolitan distribution. Comparison with three culture collection strains originally assigned to M. chthonoplastes revealed that strain PCC 7420 belongs to the same tightly delimited group, both phenotypically and in 16S rRNA gene sequence, but that strains SAG 3192 and 10mfx do not.

Base Sequence↗
PubMed1997

Genetic evidence for nonrandom sorting of mitochondria in the basidiomycete Agrocybe aegerita.

We studied mitochondrial transmission in the homobasidiomycete Agrocybe aegerita during plasmogamy, vegetative growth, and basidiocarp differentiation. Plasmogamy between homokaryons from progeny of three wild-type strains resulted in bidirectional nuclear migration, and the dikaryotization speed was dependent on the nuclear genotype of the recipient homokaryon. Little mitochondrial migration accompanied the nuclear migration. A total of 75% of the dikaryons from the fusion lines had both parental mitochondrial haplotypes (mixed dikaryons), and 25% had only a single haplotype (homoplasmic dikaryons); with some matings, there was a strong bias in favor of one parental haplotype. We demonstrated the heteroplasmic nature of mixed dikaryons by (i) isolating and subculturing apical cells in micromanipulation experiments and (ii) identifying recombinant mitochondrial genomes. This heteroplasmy is consistent with the previously reported suggestion that there is recombination between mitochondrial alleles in A. aegerita. Conversion of heteroplasmons into homoplasmons occurred (i) during long-term storage, (ii) in mycelia regenerated from isolated apical cells, and (iii) during basidiocarp differentiation. Homokaryons that readily accepted foreign nuclei were the most efficient homokaryons in maintaining their mitochondrial haplotype during plasmogamy, long-term storage, and basidiocarp differentiation. This suggests that the mechanism responsible for the nonrandom retention or elimination of a given haplotype may be related to the nuclear genotype or the mitochondrial haplotype or both.

Agaricales↗
PubMed1998

Microbiology of a nitrite-oxidizing bioreactor.

The microbiology of the biomass from a nitrite-oxidizing sequencing batch reactor (NOSBR) fed with an inorganic salts solution and nitrite as the sole energy source that had been operating for 6 months was investigated by microscopy, by culture-dependent methods, and by molecular biological methods, and the seed sludge that was used to inoculate the NOSBR was investigated by molecular biological methods. The NOSBR sludge comprised a complex and diverse microbial community containing gram-negative and gram-positive rods, cocci, and filaments. By culture-dependent methods (i.e., micromanipulation and sample dilution and spread plate inoculation), 16 heterotrophs (6 gram positive and 10 gram negative) were identified in the NOSBR sludge (RC), but no autotrophs were isolated. 16S ribosomal DNA clone libraries of the two microbial communities revealed that the seed sludge (GC) comprised a complex microbial community dominated by Proteobacteria (29% beta subclass; 18% gamma subclass) and high G + C gram-positive bacteria (10%). Three clones (4%) were closely related to the autotrophic nitrite-oxidizer Nitrospira moscoviensis. The NOSBR sludge was overwhelmingly dominated by bacteria closely related to N. moscoviensis (89%). Two clone sequences were similar to those of the genus Nitrobacter. Near-complete insert sequences of eight RC and one GC N. moscoviensis clone were determined and phylogenetically analyzed. This is the first report of the presence of bacteria from the Nitrospira phylum in wastewater treatment systems, and it is hypothesized that these bacteria are the unknown nitrite oxidizers in these processes.

Bacteria↗
PubMed2001

Phylogenetic and morphological diversity of cyanobacteria in soil desert crusts from the Colorado plateau.

We compared the community structures of cyanobacteria in four biological desert crusts from Utah's Colorado Plateau developing on different substrata. We analyzed natural samples, cultures, and cyanobacterial filaments or colonies retrieved by micromanipulation from field samples using microscopy, denaturing gradient gel electrophoresis, and sequencing of 16S rRNA genes. While microscopic analyses apparently underestimated the biodiversity of thin filamentous cyanobacteria, molecular analyses failed to retrieve signals for otherwise conspicuous heterocystous cyanobacteria with thick sheaths. The diversity found in desert crusts was underrepresented in currently available nucleotide sequence databases, and several novel phylogenetic clusters could be identified. Morphotypes fitting the description of Microcoleus vaginatus Gomont, dominant in most samples, corresponded to a tight phylogenetic cluster of probable cosmopolitan distribution, which was well differentiated from other cyanobacteria traditionally classified within the same genus. A new, diverse phylogenetic cluster, named "Xeronema," grouped a series of thin filamentous Phormidium-like cyanobacteria. These were also ubiquitous in our samples and probably correspond to various botanical Phormidium and Schizothrix spp., but they are phylogenetically distant from thin filamentous cyanobacteria from other environments. Significant differences in community structure were found among soil types, indicating that soil characteristics may select for specific cyanobacteria. Gypsum crusts were most deviant from the rest, while sandy, silt, and shale crusts were relatively more similar among themselves.

Cyanobacteria↗
PubMed2003

Characterization and in situ carbon metabolism of phototrophic consortia.

A dense population of the phototrophic consortium "Pelochromatium roseum" was investigated in the chemocline of a temperate holomictic lake (Lake Dagow, Brandenburg, Germany). Fluorescence in situ hybridization revealed that the brown epibionts of "P. roseum" constituted up to 37% of the total bacterial cell number and up to 88% of all green sulfur bacteria present in the chemocline. Specific amplification of 16S rRNA gene fragments of green sulfur bacteria and denaturing gradient gel electrophoresis fingerprinting yielded a maximum of four different DNA bands depending on the year of study, indicating that the diversity of green sulfur bacteria was low. The 465-bp 16S rRNA gene sequence of the epibiont of "P. roseum" was obtained after sorting of individual consortia by micromanipulation, followed by a highly sensitive PCR. The sequence obtained represents a new phylotype within the radiation of green sulfur bacteria. Maximum light-dependent H(14)CO(3)(-) fixation in the chemocline in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that there was anaerobic autotrophic growth of the green sulfur bacteria. The metabolism of the epibionts was further studied by determining stable carbon isotope ratios (delta(13)C) of their specific biomarkers. Analysis of photosynthetic pigments by high-performance liquid chromatography revealed the presence of high concentrations of bacteriochlorophyll (BChl) e and smaller amounts of BChl a and d and chlorophyll a in the chemocline. Unexpectedly, isorenieratene and beta-isorenieratene, carotenoids typical of other brown members of the green sulfur bacteria, were absent. Instead, four different esterifying alcohols of BChl e were isolated as biomarkers of green sulfur bacterial epibionts, and their delta(13)C values were determined. Farnesol, tetradecanol, hexadecanol, and hexadecenol all were significantly enriched in (13)C compared to bulk dissolved and particulate organic carbon and compared to the biomarkers of purple sulfur bacteria. The difference between the delta(13)C values of farnesol, the major esterifying alcohol of BChl e, and CO(2) was -7.1%, which provides clear evidence that the mode of growth of the green sulfur bacterial epibionts of "P. roseum" in situ is photoautotrophic.

Bacteria↗
PubMed2004

Biogeography, evolution, and diversity of epibionts in phototrophic consortia.

Motile phototrophic consortia are highly regular associations in which numerous cells of green sulfur bacteria surround a flagellated colorless beta-proteobacterium in the center. To date, seven different morphological types of such consortia have been described. In addition, two immotile associations involving green sulfur bacteria are known. By employing a culture-independent approach, different types of phototrophic consortia were mechanically isolated by micromanipulation from 14 freshwater environments, and partial 16S rRNA gene sequences of the green sulfur bacterial epibionts were determined. In the majority of the lakes investigated, different types of phototrophic consortia were found to co-occur. In all cases, phototrophic consortia with the same morphology from the same habitat contained only a single epibiont phylotype. However, morphologically indistinguishable phototrophic consortia collected from different lakes contained different epibionts. Overall, 19 different types of epibionts were detected in the present study. Whereas the epibionts within one geographic region were very similar (Dice coefficient, 0.582), only two types of epibionts were found to occur on both the European and North American continents (Dice coefficient, 0.190). None of the epibiont 16S rRNA gene sequences have been detected so far in free-living green sulfur bacteria, suggesting that the interaction between epibionts and chemotrophic bacteria in the phototrophic consortia is an obligate interaction. Based on our phylogenetic analysis, the epibiont sequences are not monophyletic. Thus, the ability to form symbiotic associations either arose independently from different ancestors or was present in a common ancestor prior to the radiation of green sulfur bacteria and the transition to the free-living state in independent lineages. The present study thus demonstrates that there is great diversity and nonrandom geographical distribution of phototrophic consortia in the natural environment.

Betaproteobacteria↗