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PubMedDate not supplied

Selection of acrosome-reacted sperm with MH61-immunobeads.

Separation of acrosome-reacted viable sperm is an important problem in the analysis of sperm function and the micromanipulation of gametes such as the microinjection of sperm into ooplasm and subzonal sperm insertion. In the present study, we collected acrosome-reacted sperm selectively using paramagnetic immunobeads, referred to as MH61-beads, which attach themselves to the head of the acrosome-reacted sperm and are recovered by a magnet. The maximum number of sperm was recovered by the MH61-beads when 3 x 10(5) sperm/100 microliters and 6 x 10(5) MH61-beads were coincubated for 1 hour. The recovery of sperm was significantly correlated with the percentage of the acrosome reaction in the capacitation medium. After incubation for 12 hours, 2.4-9.0 x 10(4) sperm could be collected, and the percentage of viable and acrosome-reacted sperm was 28-60%. To improve the recovery of the acrosome-reacted sperm, we utilized follicular fluid, which induces the acrosome reaction. Addition of follicular fluid to the capacitation medium at a final concentration of 20% induced the acrosome reaction and increased the recovery of acrosome-reacted sperm significantly. In conclusion, separation of acrosome-reacted sperm by the MH61-bead-binding method could become a useful technique in research of sperm function and gamete micromanipulation.

Acrosome↗
PubMed1996

[The first pregnancy in the Czech Republic achieved by oocyte fertilization using ICSI--intracytoplasmic sperm injection].

ICSI--an intracytoplasmic sperm injection can fertilize an oocyte in vitro with one sperm, using micromanipulation. ICSI was done on an inverted Diaphot 300 microscope with Hoffmann modulation contrast and Narishige micromanipulators. Between March and May 1995 23 ICSI cycles were performed. Of 166 oocytes (7.2 per cycle) 22% were degenerated or immature and 6% damaged during ICSI. 45% of the injected eggs were fertilized. Embryos were transferred in 65% couples. Three pregnancies resulted (20%/ET), all are viable. ICSI is suitable in cases of severe oligospermia, asthenospermia and teratospermia, immunology infertility and low cleavage rate of oocytes in the first IVF cycle.

Adult↗
PubMedDate not supplied

Assisted conception and immunological infertility.

Our work with a group involved in Assisted Conception (AC) has provided us with a perfect opportunity for making an assessment of the efficiency of the techniques used for the treatment of Male Antisperm Autoimmunization (MAA) and Female Antisperm Isoimmunization (FAI). The aim of this study is to present all the most important methods of AC used by our group for the treatment of immunological infertility, such as intrauterine insemination, intraperitoneal insemination, intrafallopian gamete transfer, in-vitro insemination and intrauterine embryo transfer, intrafallopian zygote transfer, intrafallopian embryo transfer, and gamete micromanipulation. In-vitro fecundation would appear to be particularly efficient in cases of FAI, while MAA would seem to benefit more from the use of gamete micromanipulation.

Autoimmunity↗
PubMed1997

The effect of acute experimental retinal vein occlusion on cat retinal vein pressures.

PURPOSE: Retinal ischemic damage associated with retinal vein occlusion is exacerbated by fluid extravasation and hemorrhage, which may be caused by increased permeability, elevated intravascular pressure, or both. Direct measurement of the retinal vein pressure in the cat after acute experimental retinal vein occlusion may define the role of intravascular pressures in fluid extravasation associated with this condition. METHODS: Intravenous retinal pressure measurements were obtained using a micropipette connected to a servonull device and positioned by a robot micromanipulator, while a major retinal vein near the optic disc was occluded by argon laser radiation delivered through an optical fiber positioned by a manual micromanipulator. After occlusion, retinal vein pressures were measured on both sides of the occlusion site at a controlled intraocular pressure of 20 mm Hg. RESULTS: Upstream of the occlusion site, the retinal vein pressures were not greatly elevated, although they were significantly different from controls. Downstream vein pressures were significantly lower than controls, but vascular collapse near the optic nerve was not observed. CONCLUSIONS: In retinal vein occlusion, venous pressures in a segmental retinal circulatory bed are not substantially elevated, thus implying the presence of a pressure-release mechanism and implicating vascular damage for the increased transvascular fluid flux. The lack of vascular collapse downstream of the occlusion site suggests collateral communication before a large intraocular pressure-dependent resistance segment that lies between the intraocular and extraocular vessels.

Acute Disease↗
PubMed1997

Giant cell formation in cells exposed to 740 nm and 760 nm optical traps.

BACKGROUND AND OBJECTIVE: Optical trapping is becoming a useful and widespread technique for the micromanipulation of cells and organelles. Giant cell formation following optical trapping was studied to detect the potential adverse effects. STUDY DESIGN/MATERIALS AND METHODS: The nuclei of preselected single CHO cells were exposed to 740 nm and 760 nm laser microbeam generated by a titanium-sapphire tunable laser at 88 and 176 mW and different time exposures. The irradiated single cells were recorded and observed morphologically following exposure. Giant cells were tabulated and photographed. RESULTS: The irradiated cells either failed to divide, or they underwent nuclear proliferation to form giant cells through endoreduplication. CONCLUSION: Giant cells were induced by both 740 nm and 760 nm. The frequency of giant cell formation was higher for the longer time exposures and at the higher power densities. The use of an optical etalon to remove intracavity mode beating and high peak powers of the titanium-sapphire laser caused a significant reduction in the formation of giant cells.

Animals↗
PubMed2001

DNA separations in microfabricated devices with automated capillary sample introduction.

A novel method is presented for automated injection of DNA samples into microfabricated separation devices via capillary electrophoresis. A single capillary is used to electrokinetically inject discrete plugs of DNA into an array of separation lanes on a glass chip. A computer-controlled micromanipulator is used to automate this injection process and to repeat injections into five parallel lanes several times over the course of the experiment. After separation, labeled DNA samples are detected by laser-induced fluorescence. Five serial separations of 6-carboxyfluorescein (FAM)-labeled oligonucleotides in five parallel lanes are shown, resulting in the analysis of 25 samples in 25 min. It is estimated that approximately 550 separations of these same oligonucleotides could be performed in one hour by increasing the number of lanes to 37 and optimizing the rate of the manipulator movement. Capillary sample introduction into chips allows parallel separations to be continuously performed in serial, yielding high throughput and minimal need for operator intervention.

Automation↗
PubMed1991

Handed asymmetry, handedness reversal and mechanisms of cell fate determination in nematode embryos.

Embryos of the nematode Caenorhabditis elegans exhibit left-right asymmetry with an invariant handedness. The embryonic cell lineage is asymmetrical: although the animal is generally bilaterally symmetrical with only a few left-right asymmetries, many of its contralaterally analogous cells arise via different lineages on the two sides of the embryo. Larvae and adults also exhibit left-right asymmetries with a handedness that is normally invariant. The frequency of animals with opposite handedness was increased among the progeny of adults exposed to the mutagen ethyl methanesulphonate and among animals that developed from embryos treated in early cleavage with chitinase to destroy the egg shell. Reversal of embryonic handedness was accomplished directly by micromanipulation at the 6-cell stage, resulting in mirror-image but otherwise normal development into healthy, fertile animals with all the usual left-right asymmetries reversed. This demonstrates that (1) the handedness of cell positions in the 6-cell embryo dictates handedness throughout development; (2) at this stage the pair of anterior blastomeres on the right is equivalent to the pair on the left; and (3) the extensive differences in fates of lineally homologous cells on the two sides of the animal must be dictated by cellular interactions, most of which are likely to occur early in embryogenesis and appear to have been conserved in widely diverged nematode species.

Animals↗
PubMed1993

Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections.

Germinal centres are areas of intense B lymphocyte proliferation inside primary B cell follicles in spleen and lymph nodes. Rearranged V genes from single human B cells, isolated from histological sections of two such structures by micromanipulation, were amplified and sequenced. Cells from the follicular mantle were clonally diverse and largely expressed germline V genes. Germinal centres were dominated by a few large B cell clones dispersed throughout these structures and exhibiting intraclonal diversity by ongoing somatic hypermutation. Pronounced counterselection of replacement mutations seen in one of the germinal centres may indicate a late phase of the germinal centre reaction. A polyclonal population of activated B cells expressing unmutated antibodies in the dark zone of the other germinal centre may represent the initial founder cells.

Adult↗
PubMed2004

Microscopy of biological sample through advanced diffractive optics from visible to X-ray wavelength regime.

The aim of this report is to demonstrate a unified version of microscopy through the use of advanced diffractive optics. The unified scheme derives from the technical possibility of realizing front wave engineering in a wide range of electromagnetic spectrum. The unified treatment is realized through the design and nanofabrication of phase diffractive elements (PDE) through which wave front beam shaping is obtained. In particular, we will show applications, by using biological samples, ranging from micromanipulation using optical tweezers to X-ray differential interference contrast (DIC) microscopy combined with X-ray fluorescence. We report some details on the design and physical implementation of diffractive elements that besides focusing also perform other optical functions: beam splitting, beam intensity, and phase redistribution or mode conversion. Laser beam splitting is used for multiple trapping and independent manipulation of micro-beads surrounding a cell as an array of tweezers and for arraying and sorting microscopic size biological samples. Another application is the Gauss to Laguerre-Gauss mode conversion, which allows for trapping and transfering orbital angular momentum of light to micro-particles immersed in a fluid. These experiments are performed in an inverted optical microscope coupled with an infrared laser beam and a spatial light modulator for diffractive optics implementation. High-resolution optics, fabricated by means of e-beam lithography, are demonstrated to control the intensity and the phase of the sheared beams in x-ray DIC microscopy. DIC experiments with phase objects reveal a dramatic increase in image contrast compared to bright-field x-ray microscopy. Besides the topographic information, fluorescence allows detection of certain chemical elements (Cl, P, Sc, K) in the same setup, by changing the photon energy of the x-ray beam.

Escherichia coli↗
PubMed1986

Effect of cutting the zona pellucida on the pronuclear transplantation in the mouse.

A new and reliable pronuclear transplantation procedure for the mouse egg has been developed by McGrath and Solter ('83). To overcome the technical difficulties of such a procedure, especially in uniformly preparing enucleation pipettes and in reducing damages during micromanipulation, we have examined the effect of cutting the zona pellucida of the eggs. By making a slit in the zona of an egg, the time for pipetting and exchange of pronuclei between eggs was shortened because the sharp tip of the pipette was not necessary. Although the proportion of pregnant recipients and young obtained after transfer of pronuclear transplanted eggs cultured for 1 day or 3 days was quite low, it was significantly increased (70% for pregnancy rate and 32% for the young) following transfer of eggs cultured for 4 days. These values were comparable with those after transfer of unoperated eggs cultured to morulae and blastocysts.

Animals↗
PubMed1988

Fertilization of zona-drilled mouse oocytes treated with a monoclonal antibody to the zona glycoprotein, ZP3.

Opening a small aperture in the zona pellucida of mouse oocytes by using micromanipulation and a stream of acidified Tyrode's solution (zona drilling) improved the efficiency of in vitro fertilization at low sperm concentrations without adversely affecting development to the blastocyst stage. Zona drilling also permitted in vitro fertilization and development when sperm penetration through the zona was blocked by a monoclonal antibody to the protein core of the zona glycoprotein, ZP3. These results provide a direct demonstration that sperm entry occurs through the aperture and also suggest that zona drilling of human oocytes may offer a therapeutic approach when autoantibodies to the zona pellucida are suspected as a cause of infertility.

Animals↗
PubMed1995

Adhesiveness of human ligament fibroblasts to laminin.

The adhesiveness of fibroblasts from the human anterior cruciate and medial collateral ligaments to the laminin molecule was studied, with particular emphasis on the intrinsic differences between fibroblasts from the two ligaments. Cellular adhesion strength, adhesion area, laminin concentration, and seeding time were examined. Cell adhesion to laminin anchored with poly-D-lysine to a cleaned cover glass was measured with a micropipette micromanipulation system after seeding. The adhesion strength of fibroblasts from the anterior cruciate ligament to laminin was greater than and significantly different from that of fibroblasts from the medial collateral ligament, depending on the laminin concentration. Fibroblasts from the anterior cruciate ligament also exhibited an increase in adhesion strength, dependent on laminin concentration of as much as 30 micrograms/ml, at which the laminin receptors were thought to be saturated. Fibroblasts from the medial collateral ligament did not show such an increase except at laminin concentrations of 5-10 micrograms/ml. There was no significant difference in adhesion area between fibroblasts from the two ligaments except after 45 minutes at a laminin concentration of 40 micrograms/ml. For both, the adhesion to laminin showed little correlation to seeding time during periods of as long as 60 minutes. Measurements of adhesion area also failed to show a significant correlation to seeding time for fibroblasts from either ligament at laminin concentrations of 20 and 40 micrograms/ml. Adhesion strength normalized by adhesion area had no correlation to seeding time.(ABSTRACT TRUNCATED AT 250 WORDS)

Anterior Cruciate Ligament↗
PubMed2001

Effect of blastomere sex and fluorescent labelling on the development of bovine chimeric embryos reconstituted at the four-cell stage.

The development rate of bovine chimeric embryos reconstituted at the 4-cell stage is relatively low. If chimerism is to be used as an approach in producing transgenic livestock, it is important to investigate whether this rate is affected by the sex of the blastomeres being combined and if all blastomeres survive equally well. In Experiment 1, blastomeres from 4-cell stage embryos were inserted into surrogate zonae pellucidae either in pairs to reconstitute 4-cell chimeras, or as the original sets of four to make handled controls. The development of chimeras with one pair of blastomeres labelled with PKH26-GL was also investigated. The rate of development into blastocysts was similar in chimeras with unlabelled blastomeres (23%) and in those in which one pair of blastomeres was labelled (26%) and was lower (P < 0.001) than in the handled and IVF control groups (43 and 58%, respectively). Labelled cells were distributed approximately evenly between ICM and trophoblast. In Experiment 2, the effect of sex differences between pairs of blastomeres in chimeras was investigated; chimeras were reconstituted from pairs of blastomeres taken from 4-cell embryos in which the remaining pair was sexed by PCR. No significant differences according to the sex of constituent blastomeres were detectable (mixed sex, 27%; males, 24%; females, 21%; P > 0.05). These results suggest that, in addition to the negative effects of micromanipulation, factors other than the sex of the blastomeres are involved in the reduced rate of development of chimeric bovine embryos. They also confirm the usefulness of PKH26-GL labelling for tracking the progeny of cleaving bovine blastomeres at least to the blastocyst stage.

Animals↗
PubMed1990

Subzonal transfer of multiple sperm (MIST) into early human embryos.

Microinsemination sperm transfer (MIST) is a technique whereby sperm are transferred into the perivitelline space (PVS) with the aid of a micromanipulator. MIST is now used to investigate whether blastomere membranes of early human embryos are capable of fusing with the sperm as in the metaphase II oocyte. Between 10 and 30 sperm were transferred into 11 donated human embryos between pronuclear and 16 cell stage. After culture for 6-24 hr in vitro, the embryos were fixed for transmission electron microscopy (TEM). Both acrosome-intact and acrosome-reacted sperm were located in the PVS and between blastomeres. Sperm in the PVS were sometimes penetrating the inner regions of the zona. Sperm-blastomere membrane fusion was not observed, but sperm tail incorporation by phagocytosis was occasionally evident. Sperm heads incorporated into blastomeres were often located in membrane-bound vesicles. Both acrosome-intact and acrosome-reacted sperm heads were found in vacuoles. Acrosome-reacted sperm heads were lying passively in vacuoles or were undergoing degenerative changes at their surfaces. Sperm chromatin decondensation was not observed in any of the sperm heads that were detected in the blastomeres. The evidence presented clearly shows that sperm heads are incapable of expanding their chromatin to form typical male pronuclei following MIST into early human embryos.

Blastomeres↗
PubMed1993

Nuclear transfer and electrofusion in bovine in vitro-matured/in vitro-fertilized embryos: effect of media and electrical fusion parameters.

In this study, micromanipulation and electrofusion conditions for the cloning of in vitro-produced bovine embryos (here after termed IVM/IVF embryos) derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes were established. The effect of DC field strength on the fusion rate was tested in a model system using pronuclear stage embryos in which a cytoplasmic vesicle was removed and reinserted. Efficient fusion (80%) was obtained by applying a pulse of 1.75 kV/cm for 40 microseconds. In vitro development of manipulated pronuclear stage embryos was as efficient as that of unmanipulated control embryos. Different fusion media were compared in the cloning procedure, using IVM oocytes as recipients and blastomeres from day 6 IVM/IVF donor embryos. Zimmermann cell fusion medium reduced the lysis of nuclear transfer embryos compared to F300 (5% vs. 25%). The effects of drugs disrupting the microfilaments and microtubuli were determined. Neither the addition of cytochalasin B (CCB) for 1 hr in the postfusion medium nor incubation of donor blastomeres with nocodazole had a significant effect on the fusion or cleavage rate of the nuclear transfer embryos. Additional experiments demonstrated that there was no difference in developmental potential between nuclear transfer embryos allowed to develop in vitro or in vivo and that the embryos gave a 15% pregnancy rate in recipient cattle.

Animals↗
PubMed2000

Microisolated mouse osteoclasts express VIP-1 and PACAP receptors.

Skeletal tissue contains a network of nerve fibers expressing several neuropeptides, including vasoactive intestinal peptide (VIP) and the related peptide pituitary adenylate cyclase activating peptide (PACAP). These peptides have been demonstrated to regulate osteoclast formation and osteoclast activity. Using atomic force microscopy and by analysing changes of the intracellular calcium concentrations, we have recently demonstrated that multinucleated rat osteoclasts have cell membrane binding sites recognising VIP and PACAP. In the present study, we have further studied the expression of VIP receptor subtypes in mouse bone marrow cultures and isolated osteoclasts. A micromanipulation technique was used to isolate pure populations of osteoclasts formed in PTH-stimulated mouse bone marrow cultures. By reverse transcriptase polymerase chain reaction (RT-PCR), we studied the expression of mRNA for VIP-1, VIP-2, and PACAP receptors. The purity of the microisolated osteoclasts was determined by studying the expression of specific mRNA associated with the phenotypic trait of osteoclasts or osteoblasts/stromal cells. In this study, we show that mouse osteoclasts express VIP-1 and PACAP, but not VIP-2, receptor mRNA.

Acid Phosphatase↗
PubMed1998

The dynamics and regulation of mesenchymal cell fusion in the sea urchin embryo.

Cell-cell fusion occurs in a wide variety of developmental contexts, yet the mechanisms involved are just beginning to be elucidated. In the sea urchin embryo, primary mesenchyme cells (PMCs) fuse to form syncytial filopodial cables within which skeletal spicules are deposited. Taking advantage of the optical transparency and ease of micromanipulation of sea urchin embryos, we have developed methods for directly observing the dynamics of PMC fusion in vivo. A fraction of the PMCs was labeled with fluorescent dextran and transfer of the dye to unlabeled PMCs was followed by time-lapse, fluorescence microscopy. Fusion was first detected about 2 h after PMCs began to migrate within the blastocoel. Fusion proceeded in parallel with the assembly of the PMC ring pattern and was complete by the early gastrula stage. The formation of a single, extensive PMC syncytium was confirmed by DiI labeling of fixed embryos. When single micromeres were isolated and cultured in unsupplemented seawater, they divided and their progeny underwent fusion. This shows that the capacity to fuse is autonomously programmed in the micromere-PMC lineage by the 16-cell stage. PMC transplantations at late embryonic stages revealed that these cells remain fusion-competent long after their fusion is complete. At late stages, other mesenchyme cells (blastocoelar cells) are also present within the blastocoel and are migrating and fusing with one another. Fusion-competent blastocoelar cells and PMCs come into contact but do not fuse with one another, indicating that these two cell types fuse by distinct mechanisms. When secondary mesenchyme cells convert to a skeletogenic fate they alter their fusogenic properties and join the PMC syncytium, as shown by transfer of fluorescent dextran. Our analysis has provided a detailed picture of the cellular basis and regulation of mesodermal cell fusion and has important implications regarding molecular mechanisms that underlie fusion.

Animals↗
PubMed2006

Nuclear transfer in nonhuman primates.

The nonhuman primate is a highly relevant model for the study of human diseases, and currently there is a significant need for populations of animals with specific genotypes that can not be satisfied by the capture of animals from the wild or by conventional breeding. There is an even greater need for genetically identical animals in vaccine development or tissue transplantation research, where immune system function is under study. Efficient somatic cell nuclear transfer (SCNT) procedures could provide a source for genetically identical nonhuman primates for biomedical research. SCNT offers the possibility of cloning animals using cultured cells and potentially provides an alternative approach for the genetic modification of primates. The opportunity to introduce precise genetic modifications into cultured cells by gene targeting procedures, and then use these cells as nuclear donors in SCNT, has potential application in the production of loss-of-function monkey models of human diseases. We were initially successful in producing monkeys by NT using embryonic blastomeres as the source of donor nuclei and have repeated that success. However, when somatic cells are used as nuclear donor cells, the developmental potential of monkey SCNT embryos is limited, and somatic cell cloning has not yet been accomplished in primates. High rates of in vitro development to blastocysts, comparable with in vitro fertilization controls, and successful production of rhesus monkeys by NT from embryonic blastomeres suggests that basic cloning procedures, including enucleation, fusion, and activation, are consistent with the production of viable embryos. Although modifications or additional steps in SCNT are clearly warranted, the basic procedures will likely be similar to those extant for embryonic cell NT. In this chapter, we describe detailed protocols for rhesus macaque embryonic cell NT, including oocyte and embryo production, micromanipulation, and embryo transfer in nonhuman primates.

Animals↗