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PubMed1980

Versatile piezoelectric driver for cell puncture.

A simple and versatile tool facilitating micropuncture of small cells is described which utilizes a commercial piezoelectric element made from a stacked column of monomorph ceramic discs. The device is able to advance complete input stage-electrode-assemblies with high speed and can be used in combination with conventional micromanipulators. Advancing characteristics as recorded optically at high magnification demonstrated less axial vibration, although faster action, than two other modern micropositioners driven by step motors. In biological experiments on selected tissues (Necturus gallbladder epithelium, Amphiuma renal distal tubule cells, rabbit and human corneal endothelium) the combined use of micromanipulator and piezo-stepper was, in all cases, superior to the use of a micromanipulator alone: the percentage of successful cell penetrations increased, cell potentials were stable for a longer time, and the durability of electrode-tips improved.

Animals↗
PubMed1995

Fertilization rates using intracytoplasmic sperm injection are greater than subzonal insemination but are dependent on prior treatment of sperm.

OBJECTIVE: To determine a suitable method of sperm preparation for use in micromanipulation. To compare the fertilization rates of sibling oocytes inseminated by intracytoplasmic sperm injection (ICSI) and subzonal insemination (SUZI). DESIGN: Two methods of gamete micromanipulation to overcome male factor infertility were compared. Within this study, three trials were conducted to determine the most suitable method of sperm preparation. This method then was used to complete the study. SETTING: Procedures were performed in a teaching hospital research environment. PATIENTS: Ninety-six patients were recruited for this study, undergoing 99 stimulation cycles. INTERVENTIONS: Sibling oocytes were inseminated by subzonal sperm or intracytoplasmic injection. MAIN OUTCOME MEASURES: Fertilization, zygote development, and pregnancy rates. RESULTS: In the absence of manipulative pretreatment of sperm (trial 1), there was no difference in normal fertilization rates between ICSI and SUZI (19% and 25%, respectively). In the second trial there again was no sperm pretreatment for SUZI but, for ICSI, a polyvinylpyrrolidone (PVP) solution was used to reduce sperm velocity and the sperm tails were incised before injection. The fertilization rates were significantly different between ICSI (44%) and SUZI (17%). In the third trial, PVP was added to the sperm used for both types of insemination and the sperm tails also were incised for the ICSI insemination. Fertilization was again significantly different (16% for SUZI and 44% for ICSI). The trial 2 method of sperm preparation then was used to complete the study (trial 4) and confirmed the results of trial 2 (21% for SUZI and 42% for ICSI). The results suggest that ICSI can give improved fertilization compared with SUZI provided the sperm are treated before injection. No significant differences were found in the development rates of zygotes suitable for transfer or cryopreservation between the two micromanipulation methods.

Cytoplasm↗
PubMed1996

Development of in vitro derived bovine embryos following pronuclear transplantation and in vitro culture.

This study was designed to evaluate the survival and development of in vitro derived bovine embryos following pronuclear transplantation and in vitro embryo culture. Bovine zygotes were produced by in vitro maturation and in vitro fertilization. Pronuclei were removed by micromanipulation and either transferred back to the same cell (Group 1) or into a previously enucleated zygote (Group 2) by electrofusion. Micromanipulated and non-micromanipulated (Group 3, control) zygotes were co-cultured with oviductal cells in a sealed modular chamber filled with 5% CO2, 5% O2 and 90% N2 at 39 degrees C for 7-8 days. Fusion rates were similar for Groups 1 and 2 (90.7 and 85.1%, respectively, P > 0.05). The percentage of embryos that cleaved was not different for Groups 1 (82.0%), 2 (90.0%) and 3 (76.9%, P > 0.05). Also, the percentage of embryos developing to the compact morula or blastocyst stage was similar (25.6, 22.5 and 22.3%, respectively, for Groups 1, 2 and 3, P > 0.05). The results of this experiment are the first to demonstrate that pronuclear transfer can be carried out successfully using bovine embryos derived from in vitro oocyte maturation and in vitro fertilization. In addition, pronuclei can be transferred from one bovine embryo to another and the reconstructed embryos develop to the compact morula and blastocyst stage in vitro. This technique, used in combination with oocyte retrieval by ultrasound-guided follicular aspiration and embryo transfer, offers the potential to study cytoplasmic inheritance in cattle directly, and to evaluate the effect of cytoplasmic inheritance on traits of economic importance.

Animals↗
PubMed1995

Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint.

Some cells have a quality control checkpoint that can detect a single misattached chromosome and delay the onset of anaphase, thus allowing time for error correction. The mechanical error in attachment must somehow be linked to the chemical regulation of cell cycle progression. The 3F3 antibody detects phosphorylated kinetochore proteins that might serve as the required link (Gorbsky, G. J., and W. A. Ricketts. 1993. J. Cell Biol. 122:1311-1321). We show by direct micromanipulation experiments that tension alters the phosphorylation of kinetochore proteins. Tension, whether from a micromanipulation needle or from normal mitotic forces, causes dephosphorylation of the kinetochore proteins recognized by 3F3. If tension is absent, either naturally or as a result of chromosome detachment by micromanipulation, the proteins are phosphorylated. Equally direct experiments identify tension as the checkpoint signal: tension from a microneedle on a misattached chromosome leads to anaphase (Li, X., and R. B. Nicklas. 1995. Nature (Lond.). 373:630-632), and we show here that the absence of tension caused by detaching chromosomes from the spindle delays anaphase indefinitely. Thus, the absence of tension is linked to both kinetochore phosphorylation and delayed anaphase onset. We propose that the kinetochore protein dephosphorylation caused by tension is the all clear signal to the checkpoint. The evidence is circumstantial but rich. In any event, tension alters kinetochore chemistry. Very likely, tension affects chemistry directly, by altering the conformation of a tension-sensitive protein, which leads directly to dephosphorylation.

Animals↗
PubMed2000

New techniques for isolation of single prokaryotic cells.

Since the 1960s, several new attempts have been made to improve the management of single prokaryotic cells using micromanipulator techniques. In order to facilitate the isolation of pure cultures we have recently developed an improved micromanipulation method for routine work. With the aid of this method single prokaryotic cells can be picked out of a mixed community under direct visual control. The isolated aerobic or anaerobic cells can be grown in pure culture or can be subjected to single cell PCR. Other powerful and completely new approaches are the applications of laser micromanipulation systems, such as optical tweezers or laser microdissection techniques. Of the latter two methods only optical tweezers have been successfully applied to cloning prokaryotic cells.

Bacteria↗
PubMed2005

Effects of removal of necrotic blastomeres from mouse cryopreserved embryos on blastocyst formation and hatching.

To evaluate whether the developmental potential of embryos that were partially damaged after freezing and thawing can be improved by removal of necrotic blastomeres. Eight-cell mouse embryos were cryopreserved using 1,2-propanediol and sucrose as cryoprotectant with slow cooling procedure. After thawing, blastocyst formation and hatching of fully intact embryos were compared between no treatment and with laser-assisted hatching. For partially intact embryos, the effects of removal of necrotic blastomeres with micromanipulation were evaluated. Laser-assisted hatching of mouse cryopreserved fully intact embryos significantly increased blastocyst hatching (63.4% versus 48.3%, P<0.05), but had little effect on blastocyst formation (72.0% versus 70.1%, P>0.05). The removal of necrotic blastomeres from partially damaged mouse cryopreserved embryos with micromanipulation significantly increased blastocyst formation (52.9% versus 32.0%, P<0.05) and blastocyst hatching (41.2% versus 22.0%, P>0.05) compared with the control group. The developmental potential of partially damaged cryopreserved embryos can be improved by removal of necrotic blastomeres with micromanipulation.

Animals↗
PubMed2001

Implementation of self-sensing SPM cantilevers for nano-force measurement in microrobotics.

Micromanipulation tasks have to be solved in the assembly of microsystems, the handling of biological cells and the handling of specimens for scanning electron microscopy. For these applications, we have developed a flexible micromanipulation station, including direct-driven robots a few cubic centimeters small. The robots are able to perform high-precise manipulation and positioning of microobjects. Force-controlled microgripping strategies are now necessary to develop robust microassembly strategies. Microgripping is different from conventional gripping in two ways. First, microparts with dimensions less than 100 microm are often fragile and can easily be damaged during gripping, thus special grasping techniques are needed. Second, the mechanics of manipulation in the microworld are much different than in the macro-world. Part interactions in the microworld are dominated by adhesive forces making it difficult to release parts during manipulation tasks. Several microgrippers that do not employ force feedback have been developed; force-controlled microgrippers are much less common. Grippers with integrated piezoresistive force sensors and with attached strain gauges have been reported. These approaches, however, are limited in their ability to resolve the gripping force. Hence, we are currently integrating self-sensing SPM cantilevers into a gripper of our microrobots. These cantilevers operate by measuring stress-induced electrical resistance changes in an implanted conductive channel in the flexure legs of the cantilever. The real-time force feedback provided by these sensors enables us to better understand the prevailing nano forces and dynamics, what is indispensable for reliable micromanipulation strategies.

Journal Article↗
PubMed1999

The effect of high concentrations of cryoprotectants on the passage of bovine viral diarrhea virus through the zona pellucida of in vitro fertilized embryos.

The effect of high concentrations of cryoprotectants on the passage of bovine viral diarrhea virus (BVDV) through the zona pellucida (ZP) of intact bovine embryos during the pre-freezing step of cryopreservation was investigated in a series of experiments. In vitro fertilized (IVF) embryos at the blastocyst stage were exposed to 10(6) TCID50 BVDV (non-cytopathic NY-1 strain) in a 30% suspension of either ethylene glycol, glycerol, DMSO, or 2 M sucrose in physiological saline for 10 min at 20 degrees C. Subsequently, the embryos were washed free of residual unbound viral particles, and the ZP of some embryos were removed by micromanipulation. Groups of ZP-intact embryos, ZP-free embryonic cells and their respective ZP were then tested separately for the presence of virus. The infectious virus was detected in association with 81% (17/21) of samples containing non-micromanipulated ZP-intact embryos which were exposed to the virus and cryoprotectants and then washed 10 times and in 83% (43/53) of the samples containing only ZP from micromanipulated embryos (P > 0.05). The virus was not found in the samples containing the corresponding embryonic cells of embryos exposed previously to the virus and cryoprotectants. It was concluded that the transfer of embryos from the isotonic PBS solution into a highly hypertonic cryoprotectant solution did not cause the passage of BVDV through ZP and its entry to embryonic cells.

Animals↗
PubMed2005

Hand-made cloning approach: potentials and limitations.

Two major drawbacks hamper the advancement of somatic cell nuclear transfer in domestic animals. The first is a biological problem that has been studied extensively by many scientists and from many viewpoints, including the cell, molecular and developmental biology, morphology, biochemistry and tissue culture. The second is a technical problem that may be responsible for 50% or more of quantitative and/or qualitative failures of routine cloning experiments and is partially the result of the demanding and complicated procedure. However, even the relatively rare documented efforts focusing on technique are usually restricted to details and accept the principles of the micromanipulator-based approach, with its inherent limitations. Over the past decade, a small alternative group of procedures, called hand-made cloning (HMC), has emerged that has the common feature of removal of the zona pellucida prior to enucleation and fusion, resulting in a limited (or no) requirement for micromanipulators. The benefits of HMC are low equipment costs, a simple and rapid procedure and an in vitro efficiency comparable with or higher than that of traditional nuclear transfer. Embryos created by the zona-free techniques can be cryopreserved and, although data are still sparse, are capable of establishing pregnancies and resulting in the birth of calves. Hand-made cloning may also open the way to partial or full automation of somatic cell nuclear transfer. Consequently, the zona- and micromanipulator-free approach may become a useful alternative to traditional cloning, either in special situations or generally for the standardisation and widespread application of somatic cell nuclear transfer.

Animals↗
PubMed1984

Disparate differentiation in mouse hemopoietic colonies derived from paired progenitors.

We analyzed the differentiation of murine hemopoietic colonies derived from paired progenitors in culture. Single progenitors were isolated by use of a micromanipulation technique from blast cell colonies cultured from the spleens of 5-fluorouracil-treated mice. Eighteen to 24 hr later, the paired progenitors were separated with a micromanipulator and cultured in methylcellulose medium containing erythropoietin and pokeweed-mitogen spleen cell conditioned medium. Six to nine days later, the two colonies derived from the paired progenitors were individually picked and differential counts were performed by using May-Grunwald-Giemsa stain. The abbreviations used here are n, neutrophil; m, macrophage; e, eosinophil; mast, mast cell; M, megakaryocyte; E, erythrocyte. Of a total of 387 pairs that could be evaluated, 68 were pairs of colonies consisting of dissimilar combinations of cell lineages such as m-nmmastEM, M-nmmastEM, nm-nmmastEM, nmmastM-nmmastEM, M-nmmastM, nmmast-nmmastM, nm-nmmastE, M-nmM, n-nmM, mM-nmM, m-nmmast, nm-nme, me-nm, mM-nm, n-ne, m-mmast, m-mM, M-nm, M-mM, E-nm, m-nm, M-m, etc. Thirty-nine were homologous pairs revealing identical lineage combinations such as nmmastEM, nmmastM, nmmast, mmastEM, nmEM, nme, nmM, mM, and nm lineages. However, in members of some of these pairs, the proportions of the individual cell lineages were significantly different. The remainder were pairs of single lineage colonies. Paired progenitors obtained from the stem cell colonies of normal mice also revealed homologous and nonhomologous expression of the cell lineages. Comparison of lineage expression in colonies derived from single progenitors with the sum of lineages expressed in pairs of colonies derived from single progenitors indicated that the diversity was not due to injury inflicted by micromanipulation. These observations provide experimental data in support of stochastic mechanisms of stem cell differentiation.

Animals↗
PubMedDate not supplied

The hemizona assay: a simplified technique.

The hemizona assay is an important diagnostic tool in assessing human sperm fertilizing potential. Previous hemizona assay results have proven that this functional test is a good predictor of fertilization in vitro and can be used in clinical practice to supply additional information in male factor subfertility cases. The objective of this study was to compare two methods for cutting human zona pellucida into equal halves (manual handcutting versus micromanipulation) in order to examine the necessity of an expensive micromanipulator in performing this assay. Comparable results for recovery rate, diameter size of the hemizonae, and sperm binding were achieved with both methods. According to these results, the use of an expensive micromanipulator is not essential in performing the hemizona assay.

Female↗
PubMed1999

[Monochorionic twins after treatment of sterility using assisted reproduction methods].

OBJECTIVE: The goal of this study has been to determine frequency of occurrence of monochorial twins within the study group consisting of 521 pregnancies conceived through the In Vitro Fertilization (IVF) programme. DESIGN: An observational study. SETTING: Sanatorium Pronatal, Na Dlouhé mezi 4/12, 147 00 Praha 4-Hodkovicky. METHODS: A condition to be included in the study group was that there was a gestation sac detected by ultrasound. The first ultrasound examination was performed transvaginally and was done between the fifth and the sixth week of each pregnancy. A multiple pregnancy has been classified as monochorial in case when gestation sac contains two yolk sacs and two fetuses. RESULTS: Within the whole study group there were 13 monochorial twins which presents 2.5% of all pregnancies. The incidence of monochorionicity among all multiple pregnancies is 6.4%, in IVF cycles without micromanipulation techniques the occurrence of monochorial twins has been 0.9%, following cryoembryotransfer 3.9%, with micromanipulation (ICSI, AH) it reaches 5.7% of all pregnancies. CONCLUSION: Monochorial twins were detected seven time more frequent in comparison within occurrence in ordinary population. The occurrence rises in connection with the number of fetuses in uterus. In comparison with simple IVF cycles we have proved higher occurrence of monochorionicity in connection with micromanipulation techniques and cryoembryotransfer.

Chorion↗
PubMed1980

A microstereotactic approach to small CNS lesions. Part I. Development of CT localization and 3-D reconstruction techniques.

The authors describe a newly designed and utilized stereotactic methodology for the removal of central nervous system lesions as small as a few millimeters in diameter. These small lesions are detected and localized by non-invasive computerized axial tomography (GE 8800 scanner) with additional computer processing of the digital data by means of a PDP-1145 computer. Multiple computer algorithms have been developed to enhance regions of interest on CT scans by three-dimensional reconstruction and magnification techniques. This same data can then be used to calculate a stereotactic approach to a small CNS lesion. The stereotactic approach coordinates are then mated to a head fixation system modified from the Reichert-Mundinger stereotactic apparatus enabling information transfer from CT scan to the stereotactic surgical system. These small CNS lesions, as small as 5 mm, can be removed with the apparatus described herein under direct binocular 3-D vision with minimal tissue damage, through a small trephine craniotomy or burr hole. Newly designed instruments and instruments in the process of being developed are mounted on a micromanipulator attached to the Riecher-Mundinger frame for guidance at surgery. These new instruments include stereoendoscopes with xenon arc illumination, multiple tissue expanders for exposing the operative site, a radiation tracer probe, a rotary-sucker extractor, and multiple other small instruments for operating and removal of blood and tissue from small CNS lesions. The stereotactic frame accurately defines all areas of the cranium in three-dimensional coordinates, and its combination with the micromanipulator-instrument assembly enables the site of any small CNS lesion, the three-dimensional coordinates of which have been located by additional computer processing of the CT digital data, to be accurately approached by the stereotactic guide micromanipulator assembly. The instrument described herein allows removal of very small tumor burdens, and opens the possibility for successful secondary application of adjuvant immunotherapy to a CNS tumor site as described in part II of this paper.

Brain↗
PubMed2004

Comparison of bulk enucleation methods for porcine oocytes.

Cloning of mammalian oocytes requires that the recipient oocyte is enucleated to remove all genetic material associated with the chromosomes. The procedure currently used in most species requires careful micromanipulation of oocytes treated with cytochalasin B to prevent structural damage. Although functional, this procedure requires time and limits the number of oocytes available for cloning, and our ability to understand the mechanisms of nuclear reprogramming. Therefore, this study aimed at evaluating different procedures to enucleate large pools of oocytes in a time-efficient manner. Two different approaches were tested. The first approach involved centrifugation of zona-free oocytes through a percoll gradient to separate the portion containing the chromatin from the cytoplasmic portion. The second used etoposide to prevent chromatin segregation at first metaphase and resulting in the expulsion of all chromosomes in the polar body. Using the chemical approach an average enucleation rate of 39.4 +/- 7.5% was obtained, while the centrifugation approach resulted in an average enucleation rate of 66.9 +/- 6. In terms of time efficiency, the control manipulation method takes 0.11 min and the centrifugation took an average of 0.52 min per oocyte. The MPF activity at the end of procedure was estimated through the measurement of H1 activity and as expected, the etoposide-cycloheximide treated oocytes had lower H1 activity which was restored by further incubation in the maturation medium for 5 hr while the centrifugation gave a nonsignificant intermediary result. In conclusion, the results presented suggest that both the chemical and the mechanical methods are usable alternatives to micromanipulation of oocytes to generate a large number of chromosome free cytoplasm for biochemical analysis. Mol. Reprod. Dev. 67: 70-76, 2004.

Animals↗
PubMed1991

Assisted hatching of human embryos.

There are benefits as well as drawbacks of zona pellucida breaching. Narrow gaps in the zona may cause disintegration of the hatching trophectoderm, when embryos are cultured in vitro. An increase in the formation of monozygotic twins following micromanipulation may also occur, due to the forced separation of the inner cell mass during blastocyst expulsion in utero. Mouse studies indicate that one or several narrow zona openings (less than 5 microns) are detrimental. Such embryos may become trapped during hatching but may be rescued by drilling an additional larger gap elsewhere on the zona. The use of acidic Tyrode's solution for clinical assisted hatching of eight-cell embryos is currently under investigation. The findings suggest that large holes are efficient for promoting hatching (at least one-fourth of the embryos implanted thus far) and that embryos with unthinned zonae (those with normally the poorest prognosis) benefit mostly from assisted hatching. Results also indicate that embryos with the best zona morphology should be replaced without micromanipulation.

Animals↗
PubMed1993

Chemical removal of the outside of the zona pellucida of day 3 human embryos has no impact on implantation rate.

Two hundred eighteen consenting patients entered a randomized study of the application of chemical zona pellucida thinning on their day 3 embryos, prior to uterine transfer. Of those control patients (n = 108), whose embryos remained unmanipulated, 40 (37.0%) have ongoing/delivered pregnancies, while in the experimental group (n = 110), whose embryos had their zonae pellucidae chemically thinned, there are 49 patients (44.6%) who have ongoing/delivered pregnancies. Although this difference is not significant, clearly the application of this micromanipulative intervention has not been detrimental, and this bodes well for routine application of embryonic micromanipulation procedures in general. Certain patient subgroups were studied including older women, those with elevated basal follicle stimulating hormone levels, patients with embryos of differing zona thickness, and patients with embryos of differing uniformity of zona thickness. No significant influence of chemical removal of the outside of the zona on the implantation rate of embryos in any of these subgroups was observed other than a marginally significant (P = 0.095) improvement of implantation of embryos with less than 4.0 microns variation in zona thickness when chemical zona thinning was applied. Failure of chemical zona thinning to enhance human embryo implantation significantly, compared to assisted hatching by complete zona drilling, strongly suggests that the bilayered human zona pellucida needs to be fully breached, unlike that of the mouse.

Acids↗
PubMed2004

Stage-specific formation of the equine blastocyst capsule is instrumental to hatching and to embryonic survival in vivo.

Early embryonic development in the horse is characterized by the formation of an unusual acellular glycoprotein "capsule" between the trophectoderm and the overlying zona pellucida. This structure is first detected between days 6 and 7 after ovulation and completely envelops the spherical conceptus until as late as day 23 of gestation. In the present study, a micromanipulator was used to remove the capsule from 15 embryos on day 6-7 after ovulation. None of these denuded embryos developed into ultrasonographically detectable pregnancies after surgical transfer into recipient mares whereas four of six control embryos handled and transferred similarly but without capsule removal developed normally, thereby demonstrating clearly a role for the capsule in embryonic survival. In addition, observation of the embryonic investments after embryo collection and during micromanipulation led to the hypothesis that hatching of the horse embryo from its zona pellucida is assisted by the force of the expanding capsule, which causes the attenuating zona to literally burst open.

Animals↗
PubMed1994

Embryo survival after pronuclear microinjection and trophectoderm biopsy.

OBJECTIVE: Our purpose was to compare murine embryo development after pronuclear microinjection of a gene construct, followed by trophectoderm biopsy at the blastocyst state, with development after a single micromanipulation, and with cultured controls. STUDY DESIGN: alpha-Myosin heavy-chain gene sequence was microinjected into the murine embryo pronucleus and cultured to blastocyst. After trophectoderm biopsy the embryos were allowed to re-expand. Re-expanded embryos were transferred to pseudopregnant females; implantation and live birth rates were recorded. In this study group the rates were compared with three control groups of embryos simultaneously cultured after (1) pronuclear microinjection only, (2) trophectoderm biopsy only, and (3) non-micromanipulated, culture only. RESULTS: A total of 1222 embryos were divided among the four groups. In the study group 472 embryos underwent pronuclear microinjection and trophectoderm biopsy. Of these, 203 (43%) reached the blastocyst stage and underwent biopsy; 183 (38.8%) re-expanded after biopsy. Of 275 pronuclear microinjected only (control 1) embryos, 113 (41.1%) reached the blastocyst stage. Of 336 embryos 148 (44.0%) reached the blastocyst stage and underwent trophectoderm biopsy only (control 2); 129 (39.2%) survived biopsy. The cultured only group (control 3) consisted of 139 pronuclear embryos; 67 (48.2%) developed to the blastocyst stage. CONCLUSIONS: Murine embryos can survive two micro-manipulations, pronuclear microinjection followed by trophectoderm micro-biopsy. Although there is minimal effect of these procedures on embryonic development in vitro, the live birth rate is tenuous.

Animals↗