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PubMed1990

Micromanipulation of embryos and germ cells: an approach to gene therapy?

Recent advances in mammalian gamete and embryo micromanipulation have stimulated the scientific and medical communities, and to some degree the public at large, to become aware that treatment of genetic disease by direct alteration of the genetic code may soon be possible. Because these micromanipulation techniques result in modification of the genotype at the earliest stages of development, such "gene therapy" affects not only the conceptus itself but also its germ cells. Thus such genetic modifications are heritable and can be transmitted indefinitely to succeeding generations of progeny. In the presentation, both narrow and broad definitions of gene therapy will be considered with respect to the techniques upon which they are based, their potential for treatment of genetic disease, and their current feasibility.

Animals↗

PubMed1988

Micromanipulation studies of the asymmetric positioning of the maturation spindle in Chaetopterus sp. oocytes: I. Anchorage of the spindle to the cortex and migration of a displaced spindle.

We investigated the nature of the asymmetric positioning and attachment of Chaetopterus oocyte meiotic spindles to the animal pole cortex by micromanipulation. The manipulated spindle's behavior was analyzed in clarified oocyte fragments using video-enhanced polarized light microscopy. As the spindle was drawn towards the cell interior with a microneedle, the cell surface dimpled inwards adjacent to the outer spindle pole. As the spindle was pulled further inwards, the dimple suddenly receded indicating a rupture of a mechanical link between the cell cortex and outer spindle pole. The spindle paused briefly when released from the microneedle; then it spontaneously migrated back to the original attachment site and reassociated with the cell cortex. Positive birefringent astral fibers were seen running between the outer spindle pole and the cortex during the migration. The velocity of the spindle during its migration tended to increase as it came closer to the cortex. Velocities as high as 1.25 micron/sec. were measured. If removed too far from the attachment site cortex (greater than 35 micron), the spindle remained stationary until pushed closer to the original attachment site. Spindles, inverted by micromanipulation, migrated and reattached to the cortical site by their former inner pole; thus either spindle pole can seek out and migrate to the original attachment site. However, spindle poles pushed against other cortical regions did not attach demonstrating that there is only one unique, localized attachment site for spindle attachment.

Animals↗

PubMed1992

Atypical cells in the normal guinea pig organ of Corti as revealed by micromanipulation in SEM.

Inner ear tissue of the normal guinea pig was conductively stained (OTOTO-method) for SEM investigations. The Hensen's cells of the organ of Corti were removed using a micromanipulator inside the SEM. By this method atypical bodies of sensory and supporting cells were revealed in the apical turns of the cochlea. Atypical sensory cells showed great variations in size and shape. Several had no contact to Deiter's cells and no or only one nerve supply at their basal end. Atypical Deiter's cells showed alterations in shape and in the form of their phalangeal processes. Additionally altered parts of the organ of Corti were isolated by micromanipulation and embedded for correlative TEM-investigations.

Animals↗

PubMed1990

Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits.

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.

Animals↗

PubMed1995

Isolation of fetal cells from transcervical samples by micromanipulation: molecular confirmation of their fetal origin and diagnosis of fetal aneuploidy.

Transcervical cell (TCC) samples have been shown to contain fetal cells amenable to molecular analysis. However, the presence of 'contaminating' maternal cells limits their use for prenatal diagnoses. In this report we show that clumps of fetal cells can be isolated from transcervical samples by micromanipulation and tested by fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). Out of 129 clumps, isolated from mucus aspirates and transcervical lavages from 29 patients, 29 clumps from 11 patients were found to be exclusively of fetal origin as judged by the detection of chromosome 21-specific polymorphic DNA markers and Y-derived DNA sequences by PCR and FISH. One case of a male triploid fetus, diagnosed by the analysis of TCC samples obtained by mucus aspiration and lavage, was confirmed by testing clumps of cells isolated by micromanipulation.

Aneuploidy↗

PubMed1996

The use of piezo micromanipulation for intracytoplasmic sperm injection of human oocytes.

INTRODUCTION: This paper describes the use of piezo actuator technology for intracytoplasmic sperm injection of human eggs. METHODS: Piezo micromanipulation involves a simple and easily made injection pipette of very thin diameter and wall thickness, and it has recently been developed using a mouse model. No bevel or spike is required on the pipettes used. The piezo actuator attaches to conventional micromanipulators and acts to mechanically advance the pipette tip through the zona pellucida using a piezoelectric effect. The oolemma could, in some cases, also be broken using piezo. This technique was applied to 39 patients presenting for in vitro fertilization due to male-factor infertility. RESULTS: The fertilization rate of eggs injected (60.5%), damage rate (7.6%), abnormality rate (3%), and ongoing pregnancy rate beyond 8 weeks (28.2%) are comparable to those for other techniques that have been successfully reported.

Female↗

PubMed1991

New ocular micromanipulator for measurements of retinal and vitreous physiologic parameters in the mammalian eye.

A new ocular micromanipulator for introducing a microelectrode into the mammalian eye is described. Included is a technical description of the device, incorporating arched tracks and DC motors to provide for angular motion around a scleral entrance point. Applications for in vivo measurements of local PO2 in the retina and vitreous of miniature pigs and cats are shown. The new design features of this micromanipulator allow for smooth microelectrode movements over large retinal areas, facilitating the rapid collection of data in many different areas of the retina and vitreous.

Animals↗

PubMed1993

A simple micromanipulator for multiple uses in freely moving rats: electrophysiology, voltammetry, and simultaneous intracerebral infusions.

An inexpensive, easily fabricated micromanipulator is described that can be used for single-unit recording or voltammetry in freely moving rats. The basic design is configured around the standard coupling system between a plastic syringe and corresponding needle hub. The device can be used with glass or metal microelectrodes for electrophysiology or carbon-fiber or carbon-disk microelectrodes for voltammetry. With either recording technique, the micromanipulator also can accommodate a 33-ga infusion cannula, which allows drugs to be administered directly to the recording site. The entire assembly is lightweight and can be used with a head-mounted amplifier system for relatively noise-free recording.

Action Potentials↗

PubMed1995

Cystic fibrosis gene mutations do not affect sperm function during in vitro fertilization with micromanipulation for men with bilateral congenital absence of vas deferens.

OBJECTIVE: To assess the effects of cystic fibrosis transmembrane-conductance regulator (CFTR) gene mutations on sperm function and fertility in men with bilateral congenital absence of the vas deferens. DESIGN: Prospective. SETTING: Division of urologic microsurgery and associated hospital-based IVF unit. MAIN OUTCOME MEASURES: Fertilization and pregnancy rates. PATIENTS: Men referred to our fertility unit for treatment of bilateral congenital absence of the vas deferens, using sperm surgically retrieved from the epididymis with IVF and micromanipulation. RESULTS: Of 45 men with bilateral congenital absence of the vas, 54% (19/35) tested were found to be carriers of CFTR gene mutations, with one compound heterozygote. Epididymal sperm from men affected with CFTR mutations fertilized 19% (29/152) of oocytes, whereas men without mutations fertilized 22% (44/204) of oocytes. Pregnancy rates (PRs) were 36% (5/14) for cycles involving men with CFTR mutations and 33% (5/15) for other patients with congenital absence of the vas deferens but without detectable CFTR mutations. CONCLUSIONS: The presence of detectable CFTR mutations does not affect fertilization rates or PRs for men with bilateral congenital absence of the vas deferens when IVF and micromanipulation are applied.

Cystic Fibrosis↗

PubMed2003

Laser-based micromanipulation for separation and identification of individual Frankia vesicles.

In studies of symbiotic efficiency it is of great importance to identify and separate individual Frankia strains from a nodule. Therefore, a new laser-based micromanipulation technique has been developed in which individual vesicles from root nodules of two Frankia-Alnus symbioses have been successfully cut loose and separated from clusters of vesicles in sterile conditions under light microscopy using a laser scalpel and optical tweezers. Vesicles from the Alnus incana-Frankia AvCI1 symbiosis were successfully isolated and grown in culture using this technique. The DNA from both Frankia sources was amplified by polymerase chain reaction (PCR). The work shows that a combination of laser-based manipulation techniques and PCR can be used for the separation and study of individual vesicles. This novel laser-based micromanipulation technique opens up various new possibilities, for instance, to study whether several Frankia strains can grow simultaneously in the same root nodule.

Cytoplasmic Vesicles↗

PubMed1999

Rapid isolation of single microbial cells from mixed natural and laboratory populations with the aid of a micromanipulator.

In order to facilitate the isolation of pure cultures from natural habitats we have developed a method for the isolation of single microbial cell clones from a mixed population, e.g. the flora of the termite gut, with the aid of a modern micromanipulator. The separated single prokaryotic or eukaryotic cells were grown after transfer in culture media or they were used for single cell PCR. The micromanipulator was also applied for the removal of nuclei from protozoa, of which the SSU rDNA was directly amplified.

Animals↗

PubMed1997

Cut out or poke in--the key to the world of single genes: laser micromanipulation as a valuable tool on the look-out for the origin of disease.

The optical micromanipulation systems UV(ultraviolet)-Laser Microbeam and Optical Tweezers Trap, already proven to be powerful tools for 'non-contact' micro-manipulation of gametes, cells and organelles, have now made their way into the nanocosmos of genes and molecules. Force measurements of DNA transcription have been performed and selective DNA molecule micromanipulation gives insight into single molecule behaviour. Retrievement of selected single cells without contamination is an import prerequisite for further processing with modern methods of molecular biology. Laser micro-dissection allows to precisely eliminate any unwanted material or to isolate pieces of chromosomes or single cells of interest with high accuracy and efficiency. This enables the cell or chromosome specific molecular analysis of genes and genetic defects underlying disease, such as cancer or infection. This review article gives an overview of current topics of laser microbeam application in biological or medical research and advanced molecular diagnosis.

Animals↗

PubMed1979

Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers.

We have used micromanipulation to study the attachment of chromosomes to the spindle and the mechanical properties of the chromosomal spindle fibers. Individual chromosomes can be displaced about the periphery of the spindle, in the plane of the metaphase plate, without altering the structure of the spindle or the positions of the nonmanipulated chromosomes. From mid-prometaphase through the onset of anaphase, chromosomes resist displacement toward either spindle pole, or beyond the spindle periphery. In anaphase a chromosome can be displaced either toward its spindle pole or laterally, beyond the periphery of the spindle; however, the chromosome resists displacement away from the spindle pole. When an anaphase half-bivalent is displaced toward its spindle pole, it stops migrating until the nonmanipulated half-bivalents reach a similar distance from the pole. The manipulated half-bivalent then resumes its poleward migration at the normal anaphase rate. No evidence was found for mechanical attachments between separating half-bivalents in anaphase. Our observations demonstrate that chromosomes are individually anchored to the spindle by fibers which connect the kinetochores of the chromosomes to the spindle poles. These fibers are flexible, much less extensible than the chromosomes, and are to pivot about their attachment points. While the fibers are able to support a tensile force sufficient to stretch a chromosome, they buckle when subjected to a compressive force. Preliminary evidence suggests that the mechanical attachment fibers detected with micromanipulation correspond to the birefringent chromosomal spindle fibers observed with polarization microscopy.

Animals↗

PubMed1992

Treatment of male infertility by gamete micromanipulation.

Over the past decade, in vitro fertilization (IVF) has become a routine and acceptable tool in the treatment of infertility. However, major limitations in solving certain infertility problems still remain. Male infertility is one area in which only a small fraction of patients have benefited from IVF. Union of male and female gametes, either in vivo or in vitro, requires sperm penetration through the cumulus oophorus and the zona pellucida. Failure of fertilization despite the increased number of spermatozoa introduced into the oocyte's vicinity by IVF, has been shown to be directly related to abnormalities in sperm cell morphology and motility. The improved technology for micromanipulation of gametes has made it possible to circumvent the oocyte barriers to sperm penetration, thereby greatly reducing the number of normal sperm cells needed to achieve fertilization. This paper reviews the three major micromanipulative strategies which have been developed over the past five years, patient selection for the different procedures and the methods of sperm preparation to improve the yield of the applied technique.

Humans↗

PubMed1998

Treatment of severe male infertility by micromanipulation-assisted fertilization: news and views.

Recent progress in cell micromanipulation has made it possible to achieve fertilization even with highly deficient male gametes, by injecting them directly to oocyte cytoplasm. This technique, known as intracytoplasmic sperm injection (ICSI), has proved efficient in most types of sperm pathology. However, ICSI fails when injected spermatozoa are not capable of supporting the process of oocyte activation or when they carry genetic abnormalities incompatible with normal embryonic development. Abnormalities of oocyte activation and the transmission of genetic anomalies to the progeny are also two major concerns relating to the clinical use of ICSI. The use of micromanipulation-assisted fertilization in cases of male germ cell maturation arrest, namely fertilization by round spermatid injection (ROSI), has brought preliminary clinical results, and the improvement of ROSI clinical efficacy remains a major challenge for future research.

Acrosome Reaction↗

PubMed1977

Combined micromanipulation, culture and immunofluorescent techniques for isolation of the coccal organisms comprising the "corn cob" configuration of human dental plaque.

The present study describes methods to 1) selectively isolate Corn Cob Configurations (CCC) from dental plaque by micromanipulation; 2) obtain pure cultures of the coccal constituent; 3) determine by immunofluorescent procedure which organisms originated from the CCC. Using a de Fonbrune micromanipulator, CCC specimens were isolated from supragingival plaque samples. The viability of one specimen thus obtained was established by observing growth on a slide culture. One set of CCC specimens was transferred to broth and incubated aerobically immediately upon collection. Another set was transferred to prereduced transport medium and later plated on blood agar for aerobic and anaerobic culturing. A total of 10 coccal strains were thus isolated. Antisera produced in rabbits against the 10 strains were used to localize these coccal organisms on plaque smears by using the indirect fluorescent antibody technique. Of the 10 antisera tested, 2 produced against streptococcal strains consistently gave a positive immunofluorescent reaction with the coccal component of CCC in the plaque smears; the corresponding streptococci were therefore considered to be CCC forming strains in vivo.

Bacteria↗

PubMed1975

The potentiality of antibody-producing cells. II. Evidence for two antibody molecules of different specificities secreted by micromanipulated bispecific mouse spleen cells.

Bispecific PFC appearing on the 4th day after immunization of mice with sheep erythrocytes conjugated with trinitrophenyl hapten (TNP-SRBC) were identified by their capacity to lyse native SRBC and TNP-conjugated horse erythrocytes (TNP--HoRBC) simultaneously in an open carboxymethylcellulose medium. Individual PFC thus detected, were micromanipulated into two successive media containing the indicators SRBC and TNP--HoRBC. Out of 103 transferred double cells ninety-two (89 per cent) remained double in the second medium and of the ninety-two transferred into the third medium, fifty-four (58 per cent) provoked a double lysis, eighteen (19 per cent) provoked a single lysis and twenty (23 per cent) ceased to lyse. On the other hand, when the second medium contained the indicators and the soluble specific inhibitor (TNP--BSA), out of 156 transferred double PFC, 125 (80 per cent) became single and only nineteen (12 per cent) remained double. Of these cells, 136 were transferred into the third medium (not containing inhibitor) and here again sixty-five (48 per cent) became double, forty-nine (36 per cent) remained single and twenty-two (17 per cent) ceased to provoke any lysis. Other double PFC were micromanipulated from the original revealing medium into two successive media containing only one indicator and the homologous (second medium) or the heterologous third medium) soluble inhibitor (TNP--BSA or soluble SRBC antigen), in order to see whether a soluble inhibitor suppresses only the corresponding specific lysis. Out of 186 double PFC transferred into the media containing the indicator and the corresponding inhibitor 147 (79 per cent) were specifically inhibited, whereas out of 176 double PFC transferred into the third medium (in which about 20 per cent of the cells cease to function) containing one indicator and the unrelated inhibitor, 117 (66-6 per cent) lysed the indicator in spite of the presence of the unrelated inhibitor. Since a specific inhibitor suppresses the lysis of the corresponding indicator, whereas its presence does not interfere with the lysis of the unrelated indicator in the majority of double PFC, it can be concluded that these cells secrete two types of antibody molecules possessing different specificities.

Animals↗

PubMed1993

[Micromanipulation of human gametes within the scope of assisted reproduction].

Poor function of spermatozoa accounts for half of all human infertility. Micromanipulation methods within the framework of in vitro fertilization treatment may enhance fertilization of the egg and may allow the establishment of an ongoing pregnancy in cases of severe male infertility. This review presents different methods of micromanipulation which are routinely applied in male factor infertility and for preimplantation diagnosis.

Embryo Implantation↗