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PubMed1965

Autoradiographic studies of bacterial chromosome replication in amino-acid deficient Escherichia coli 15T-.

Autoradiographic experiments on amino-acid requiring strains of Escherichia coli T(-) have been performed with fully-labeled cells harvested in log-phase and after periods of amino-acid starvation. The simplest segregation of incorporated label among progeny grown on non-radioactive medium is into two packets. The result corroborates the two-unit model of E. coli DNA inferred from previous studies with partially labeled cells. Following amino-acid starvation, the distribution of label among clones derived from labeled cells indicates cells are grouped into classes having DNA contents in the ratios 1:2:4. The segregation of label among progeny isolated by micromanipulation from such starved cells supports the view that the chromosomes are brought to a state of completed synthesis with different cell classes containing different integral numbers of chromosomes. Infrequent clones interpretable as arising from cells with three chromosomes suggest that the control of replication of chromosomes lying in the same cytoplasm is on an individual basis. The block in DNA synthesis resulting from amino-acid starvation is not perfect. Nevertheless, such starvation permits characterization of the dispersive replication events for more homogeneously labeled and definable DNA units than otherwise possible. The size-frequency distribution of label among progeny following six rounds of chromosome replication is close to that expected from a model of random breakage of linear polymers. The frequency of dispersive events is estimated as 0.5 to 0.7 per chromosome per generation and is only slightly influenced by tritium decays occurring during bacterial growth.

Arginine↗
PubMed1969

Direct measurement of potential difference across the human red blood cell membrane.

The electrical potential difference across the human red cell membrane has been measured directly. A biological amplifier with neutralized input capacity was used. Human red cells in modified Ringer solution were impaled individually with 3 M KCl-filled glass microelectrodes. Movements of the microelectrodes were effected by Leitz micromanipulators. Results showed a potential difference of -8.0 +/- 0.21 (SEM) mv, the inside being negative with respect to the outside. This value is approximately that calculated by using the Nernst equation considering the intracellular and extracellular chloride concentrations.As a control, similar measurements were made on nylon microcapsules containing hemoglobin. The measured potential of -0.52 +/- 0.02 (SEM) mv, which agreed very well with the value calculated on the basis of Donnan equilibrium, was much smaller in magnitude as compared to the results for the red cell, and there was evidence of fixed charges on the microcapsule membrane. There was no evidence of this in the case of the red cell.

Electrodes↗
PubMed1993

Restoration of normal implantation rates in mouse embryos with a hatching impairment by use of a new method of assisted hatching.

OBJECTIVE: To use a newly developed mouse model for embryos with a hatching defect to develop improved methods of assisted hatching. DESIGN: We had previously demonstrated that mouse embryos with reduced cell numbers, when resident within an intact zona, exhibit a hatching deficiency, and we used these "hatching-defective" embryos to demonstrate that hatching is mediated predominantly, if not entirely, by lysis of the zona pellucida. In the present study, the hatching-defective embryos were implanted into pseudo-pregnant females to determine if implantation rates were reduced. In addition, to compensate for reduced zona lysin production, the zonae of some embryos were circumferentially thinned with acid Tyrode's solution. The implantation rates and developmental potential of hatching-defective embryos subjected to zona thinning were compared with those of the hatching-defective embryos implanted without zona thinning. SETTING: University-based basic research laboratory. RESULTS: When hatching-defective embryos, created by destruction of 1/4 of the blastomeres at the four-cell stage, were implanted into pseudopregnant female mice, no pregnancies were established, and no implantation sites could be identified after transfer of 41 embryos. However, when hatching-defective embryos were subjected to zona thinning before ET, a normal rate of implantation was observed (18 implantation events of 49 embryos transferred). Fetuses appeared morphologically normal. The difference in implantation rates between hatching-defective embryos subjected to zona thinning and those not treated by zona thinning was highly significant by chi 2 analysis. CONCLUSION: Failure of embryos with cell numbers reduced by micromanipulation to implant and restoration of normal implantation and development after circumferential zona thinning demonstrates that the developmental defect of the manipulated embryos is confined to hatching, and thus, that this procedure establishes a useful model for defective hatching. High rates of implantation after circumferential thinning of the zona shows that this procedure of assisted hatching is highly effective. These findings have important implications for use of assisted hatching in human IVF because the assisted hatching procedure is safe and effective and increases the efficiency of the normal hatching mechanism. Thus, the procedure may be useful in a wide variety of circumstances for which assisted hatching by zona opening has been found to be ineffective.

Animals↗
PubMed1994

Subzonal insertion of multiple sperm is a treatment for male factor infertility.

OBJECTIVE: To determine whether subzonal insemination of multiple sperm is an effective treatment for male factor infertility. DESIGN: Subzonal insemination by micromanipulation of sperm and oocytes for male factor patients. PATIENTS: Two hundred eight patients were selected on previous fertilization history and/or semen parameters. They could be assigned to one of three groups: group A had previous IVF failure, group B had a low fertilization rate with previous IVF, and group C had insufficient sperm for IVF. INTERVENTIONS: Oocytes were inseminated by subzonal insertion of multiple sperm. MAIN OUTCOME MEASURES: Fertilization and pregnancy rates. RESULTS: There have been 208 patients treated through 332 treatment cycles. Of 2,789 oocytes inseminated, 784 oocytes were fertilized (28%) and 516 of these (66%) were monospermic. This represents normal fertilization in 207 cycles (62% of total patient cycles). These 207 cycles produced an average of 2.6 +/- 1.8 embryos. The resultant 487 embryos were either transferred or cryopreserved for future transfer. A total of 42 pregnancies (21% per ET) resulted from 200 ET procedures with 397 embryos. Miscarriages or elective terminations have occurred (11/42 pregnancies, 26% to date) and 21 deliveries have resulted in 26 babies. There are nine ongoing pregnancies. Three of the pregnancies have been from cryopreserved embryos. Group B patients had the highest fertilization rate (23%) but the lowest pregnancy rate (7%). No other significant relationships between patient group and outcome were found. CONCLUSIONS: Subzonal insemination of multiple sperm is an effective treatment for some forms of male factor infertility.

Embryo Transfer↗
PubMed1995

Assisted hatching by partial zona dissection of human pre-embryos in patients with recurrent implantation failure after in vitro fertilization.

OBJECTIVE: To examine the potential of the partial zona dissection technique to promote successful implantation by assisting embryo hatching after IVF. DESIGN: The study and the control group included 72 and 82 patients, respectively, each had undergone at least three failed IVF-ET attempts. Assisted hatching was performed on four- to six-cell stage embryos by creating a slit in the zona pellucida using the partial zona dissection technique. After 90 minutes incubation (5% CO2, 37 degrees C), the embryos were transferred to the uterus. SETTING: Infertility and IVF Unit of an academic tertiary referral medical center. RESULTS: In the assisted hatching group, 230 micromanipulated embryos were replaced (3 or 4 treated embryos per patient) compared with 295 nonmanipulated embryos in the control group. Clinical pregnancy rates (PRs) were similar in the assisted hatching and control groups (n = 15; 20.8% and n = 12; 14.6%, respectively). However, the contribution of assisted hatching by partial zona dissection to successful implantation was related to the patients's age: patients older than 38 years showed a markedly higher PR after assisted hatching: 23.9% in the study group compared with only 7% of the controls. CONCLUSIONS: These results demonstrate that assisted hatching by partial zona dissection is a quick and efficient method that does not induce any visible damage to the embryos replaced. In a selected group of patients (aged over 38 years, who have failed to conceive in at least three previous IVF attempts) it significantly increases the chances for pregnancy after ET.

Adult↗
PubMed1995

Microdissection of mouse and human zona pellucida using a 1.48-microns diode laser beam: efficacy and safety of the procedure.

OBJECTIVE: To investigate the efficacy and safety of a small and affordable 1.48-microns continuous wave diode laser for zona pellucida (ZP) microdissection. DESIGN: Mouse and human oocytes and zygotes were submitted to ZP drilling. The hole characteristics and possible laser-induced structural alterations of the neighboring cytoplasm were investigated with scanning and transmission electron microscopy. The safety of the procedure was checked on control and drilled zygotes by determining their ability to develop in vitro and in vivo. SETTING: Collaborative study between three Swiss academic centers. INTERVENTIONS: The collimated diode laser beam was delivered through a 45x objective of an inverted microscope and focused through the culture dish and culture medium in 1- to 3-microns spots. MAIN OUTCOME MEASURE: Safety assessment of the laser drilling procedure. RESULTS: The 1.48-microns radiation achieves a rapid, precise, and easily controlled lysis of the ZP without any micromanipulative handling of the eggs. Different shapes of holes can be produced by varying the laser beam intersection site on the ZP, laser power, and irradiation time. The energy needed to drill holes of a given diameter is greater for zygotes than for oocytes. Safety of the drilling procedure is confirmed by the lack of damage at the ultrastructural and biologic levels. CONCLUSIONS: The low-cost 1.48-microns diode laser allows an easy, objective-driven, nontouch microdissection of the ZP. The procedure is safe, as drilled embryos give rise to normal and fertile offspring.

Animals↗
PubMed1996

Birth of healthy children after preimplantation diagnosis of common aneuploidies by polar body fluorescent in situ hybridization analysis. Preimplantation Genetics Group.

OBJECTIVE: To perform preimplantation diagnosis of common aneuploidies by polar body analysis and fluorescent in situ hybridization technique using probes specific for chromosomes X, 18, and 13/21. DESIGN: The first and/or second polar bodies were removed and studied by fluorescent in situ hybridization to detect and avoid fertilization and transfer of oocytes with common aneuploidies. SETTING: The Reproductive Genetics Institute's IVF program at Illinois Masonic Medical Center. PATIENTS: One hundred ninety-three couples of advanced maternal age (34 to 46 years) under-going IVF treatment volunteered to be part of a clinical trial on preimplantation polar body diagnosis of common aneuploidies. INTERVENTIONS: Using micromanipulation procedures, the first and second polar bodies were removed after their extrusion from the oocytes. MAIN OUTCOME MEASURE: Fluorescent in situ hybridization signals specific for chromosomes X, 18, and 13/21. RESULTS: In 235 IVF cycles performed in 193 couples, 1,293 oocytes were biopsied and subjected to fluorescent in situ hybridization analysis, with fluorescent in situ hybridization results available in 993 oocytes (76.8%). Of 993 oocytes with fluorescent in situ hybridization results, 665 (67%) were predicted to be normal based on the chromosomes studied; 460 embryos resulting from these oocytes were transferred in 187 treatment cycles, resulting in 12 births of healthy children and 18 ongoing pregnancies after confirmation of the polar body diagnosis by chorionic villus sampling or amniocentesis. CONCLUSION: Polar body fluorescent in situ hybridization analysis may be used for preimplantation diagnosis of common aneuploidies in IVF patients of advanced maternal age.

Adult↗
PubMed2001

Splitting and biopsy for bovine embryo sexing under field conditions.

Improvements on embryo micromanipulation techniques led to the use of embryo bisection technology in commercial embryo transfer programs, and made possible the direct genetic analysis of preimplantation bovine embryos by biopsy. For example, aspiration and microsection, allow bovine embryos sexing by detection of male-specific Y-chromosome in a sample of embryonic cells. We report on the application of the methodologies of splitting and biopsy of bovine embryos in field conditions, and on the results of embryo sex determination by the polymerase chain reaction (PCR). Pregnancy rates achieved with fresh bisected or biopsied embryos (50 to 60%) were similar to the fresh intact embryos (55 to 61%). The PCR protocol used for embryo sexing showed 92% to 94% of efficiency and 90 to 100% of accuracy. These results demonstrate these procedures are suitable for use in field conditions.

Animals↗
PubMed2002

Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment.

Techniques for sex determination of bovine embryos have evolved from karyotyping of older preimplantation embryos some 25 years ago to the current variety of widely used polymerase chain reaction (PCR) protocols. Although highly accurate, most PCR protocols for sex determination have included an electrophoresis step. The present work is a retrospective study utilizing a unique PCR protocol to sex bovine embryos without use of electrophoresis in a commercial embryo transfer program. Both in vivo and in vitro-derived embryos were produced by conventional techniques and biopsied between 7 and 8 days of age with a steel blade attached to a mechanical micromanipulator. Males constituted 49.0% of 3964 in vivo and 53.0% of 1181 in vitro-derived embryos subjected to PCR. Based on ultrasound fetal sexing and on calvings, the accuracy of sex determination was 98.7% for male embryos and 94.4% for females, with no samples producing an undetermined outcome. Pregnancy rates following transfer of biopsied Grade 1 embryos were lower than control, intact embryos as follows: 8, 6 and 16% points for in vivo, in vitro and in vivo frozen embryos, respectively. Pregnancy rates were similar for all stages of in vivo-derived embryos, whereas the pregnancy rate was significantly lower for in vitro-derived morulae compared to all stages of blastocysts. The sex ratio was significantly skewed in favor of females among in vitro-derived morulae, and in favor of males among in vitro expanded blastocysts. The sex ratio of in vivo expanded blastocysts was significantly skewed in favor of female embryos. No seasonal variation in either pregnancy rate or sex ratio was detected. There was no evidence that DNA contamination influenced the PCR assay during the duration of the study. The assay was sensitive to single blastomeres from male embryos, whereas it was not sensitive to Percoll-centrifuged or accessory sperm cells.

Animals↗
PubMed2002

Effective extra-cellular recording from vertebrate neurons in culture using a new type of micro-electrode array.

We describe the fabrication and use of a new type of extracellular micro-electrode array mounted on a flexible transparent polyimide substrate that can be rapidly moved from one part of a culture of vertebrate neurons (rat nodose) to another, which permits co-culture of glia under the neurons and is easily and rapidly replaceable in the event of damage. The array can be mounted on a micromanipulator and moved into place whenever and wherever recordings with or without stimulation are needed. The basic electrode system consists of 20-30 microm diameter gold electrodes, with or without platinisation, exposed to the cells through openings in the polyimide and joined to the recording or stimulating circuitry through gold tracks embedded in the polyimide. If rigid control over neuron placement has been achieved the patterns of electrodes can be matched to the neuron positions.

Animals↗
PubMed2002

Mitotic brain cells are just as prone to mitochondrial deletions as neurons: a large-scale single-cell PCR study of the human caudate nucleus.

Mitochondria are considered a key element in the process of organismic aging, because of their fundamental role in cellular energy generation. In the course of oxidative phosphorylation, harmful free radicals are continuously produced damaging the mitochondrial (mt) genome. One of the consequences is the occurrence of large-scale deletions in mtDNA molecules. The 4977 bp common deletion accumulates exponentially with age, in a mosaic pattern, especially in postmitotic tissues. In order to investigate whether certain cell characteristics underlie this pattern of distribution, and to look for possible age-related changes, two cell types in the caudate nucleus of the human brain from five young and five senescent subjects were analysed by single-cell PCR.MAP2-positive neurons and GFAP-positive astrocytes were isolated by micromanipulation. For each of the 10 cases, at least 30 cells of each type were collected and subjected to PCR individually. Screening for the presence of the common deletion yielded no significant differences in relative distribution, neither between astrocytes and neurons, nor between healthy young and old humans. Our results imply that the age-dependent increase of the common deletion cannot come about by an increase of independent deletion events in a greater proportion of cells, and that mitotic rate is not a major cellular risk factor for deletion accumulation in the caudate nucleus.

Adolescent↗
PubMed1994

Human preimplantation diagnosis: needs, efficiency and efficacy of genetic and chromosomal analysis.

One of the limitations of existing assisted reproduction practices is that couples at genetic risk to their offspring have to face the abortion of an affected fetus following prenatal diagnosis. This is not acceptable as a measure to avoid a congenital disease in many communities or ethnic groups, where there is a great need for a method to diagnose and avoid the affected embryo before implantation and establishment of the pregnancy. In fact, preimplantation diagnosis is needed also for those who accept prenatal diagnosis as an option to avoid the birth of an affected child, because in most of the cases the couples are at high (25-50%) risk of having a child with a recessive or dominant disease, leading to their unfortunate experience of undergoing two or more abortions of wanted pregnancies. Two methods for preimplantation genetic diagnosis (PGD) have been recently developed and implemented in the framework of IVF. PGD can be performed by micromanipulation and biopsy of the first polar body before fertilization, or by blastomere biopsy before implantation of the pre-embryo. Another potentially realistic approach is blastocyst biopsy, which is still under development and has not yet been tested in clinical practice. Available data suggest that preimplantation diagnosis is safe, as no detrimental effects have been observed in studies on the viability of biopsied pre-embryos. Genetic analysis of biopsied gametes and blastomeres is now possible by DNA analysis, while enzyme analysis and preimplantation diagnosis of chromosomal disorders are still at the research stage. The accuracy of DNA analysis in preimplantation diagnosis is clear from available data on the outcome of preimplantation diagnosis: eight children free of genetic disease have been born following preimplantation diagnosis of cystic fibrosis, haemophilia A and other X-linked conditions. However, two misdiagnoses have been also described, showing the need for further development and improvement in the accuracy, efficiency and efficacy of DNA analysis in single cells. A particularly important implication for assisted reproduction practices can be expected from the further development and improvement of methods for preimplantation cytogenetic analysis. Although the efficiency and efficacy of these methods are not yet acceptable for application in clinical practice, considerable progress has been made, providing clear evidence for their feasibility in the near future. In spite of the high cost of the preimplantation diagnostic technique at present, its development is highly justified for high risk families as it provides a wider range of options for avoiding the risk of having an affected child.(ABSTRACT TRUNCATED AT 400 WORDS)

Blastomeres↗
PubMed2003

Stretching DNA and RNA to probe their interactions with proteins.

When interacting with a single stretched DNA, many proteins modify its end-to-end distance. This distance can be monitored in real time using various micromanipulation techniques that were initially used to determine the elastic properties of bare nucleic acids and their mechanically induced structural transitions. These methods are currently being applied to the study of DNA enzymes such as DNA and RNA polymerases, topoisomerases and structural proteins such as RecA. They permit the measurement of the probability distributions of the rate, processivity, on-time, affinity and efficiency for a large variety of DNA-based molecular motors.

DNA↗
PubMed2005

Construction and fertilization of reconstituted human oocytes.

Construction of artificial gametes may be made possible by transferring somatic cells into enucleated oocytes and inducing chromosomal halving of their nuclei. This study examines the possibility of constructing viable human gametes, and their potential for participation in normal fertilization. Spare germinal vesicle-stage oocytes were donated by consenting patients undergoing intracytoplasmic sperm injection (ICSI). Approximately 62% of in-vitro matured oocytes survived enucleation and subsequent cumulus cell injection. Following micromanipulation and subsequent activation, about 40% of the reconstituted oocytes yielded two pronuclear-like entities. This was not accompanied by extrusion of a polar body, but resulted in the formation of two 'putative haploid' pronuclei. Therefore selective removal of a female pronucleus marker was required to restore a balanced ploidy. Male pronuclei were identified by association with sperm mitochondria. Additional pronuclei were then removed, allowing further cleavage. Zygotes derived were 'putatively haploid' in approximately 38% of cases with a limited number of chromosomes assessed. However, on karyotypic analysis, blastomeres isolated from cleaving embryos showed a chaotic distribution of chromosomes. Oocytes could induce 'putative haploidization' of transplanted somatic cell nuclei independently of donor cell gender. Fertilization of artificial oocytes was followed by embryonic cleavage despite blastocyst development and chromosomal content possibly being compromised.

Cell Culture Techniques↗
PubMed2001

In vitro fertilisation of mouse oocytes reconstructed by transfer of metaphase II chromosomes results in live births.

The interaction between nucleus and cytoplasm can be explored through nuclear transfer. We describe here another tool to investigate this interaction: MII meiotic apparatus transfer (MAT) between mouse oocytes. In this study, the MII oocyte meiotic apparatus or spindle from C57BL/6 mice, a black strain, was transferred into an enucleated metaphase oocyte from Kunming mouse, a white strain. The results showed that the enucleation rate by treating oocytes with 3% sucrose was 100%, but the electrofusion efficiency was very low, with only 17.6% of reconstructed karyoplast-recipient cytoplasm pairs fused. When the fused oocytes were exposed to spermatozoa from C57BL/6 mice, 9 of 11 (82%) were fertilised. Eight reconstructed embryos at 1- to 4-cell stages were transferred into the oviducts of two synchronously pregnant Kunming strain fosters and one delivered two normal C57BL/6 offspring. This study indicates that MII meiotic apparatus or spindle sustains normal structure and function after micromanipulation and electrofusion. MAT provides a model for further research on the application of this technique to assisted human reproduction.

Animals↗
PubMed2005

Transplantation of male pronucleus derived from in vitro fertilization of enucleated oocyte into parthenogenetically activated oocyte results in live offspring in mouse.

In this study, inter-strain reconstructed embryos were produced by combining the female pronucleus of Kunming mouse (white) with male pronucleus of C57BL/6 strain (black). Metaphase II (MII) oocytes of Kunming mouse were enucleated and the zona pellucida was removed. Then, the enucleated oocytes were inseminated by capacitated sperm of C57BL/6 mouse in vitro. At the same time, MII oocytes of Kunming mouse were artificially activated using strontium chloride solution, which did not contain cytochalasin B. Finally, we removed the male pronucleus derived from C57BL/6 sperm and injected it into a parthenogenetically activated one-pronucleus oocyte by micromanipulation. The reconstructed 2-cell embryos were transplanted into the oviducts of 22 foster mother mice, each receiving about 20 embryos. In the end, seven healthy and live pups were born from one recipient.

Animals↗
PubMed2004

Micromechanical analysis of the binding of DNA-bending proteins HMGB1, NHP6A, and HU reveals their ability to form highly stable DNA-protein complexes.

The mechanical response generated by binding of the nonspecific DNA-bending proteins HMGB1, NHP6A, and HU to single tethered 48.5 kb lambda-DNA molecules is investigated using DNA micromanipulation. As protein concentration is increased, the force needed to extend the DNA molecule increases, due to its compaction by protein-generated bending. Most significantly, we find that for each of HMGB1, NHP6A, and HU there is a well-defined protein concentration, not far above the binding threshold, above which the proteins do not spontaneously dissociate. In this regime, the amount of protein bound to the DNA, as assayed by the degree to which the DNA is compacted, is unperturbed either by replacing the surrounding protein solution with protein-free buffer or by straightening of the molecule by applied force. Thus, the stability of the protein-DNA complexes formed is dependent on the protein concentration during the binding. HU is distinguished by a switch to a DNA-stiffening function at the protein concentration where the formation of highly stable complexes occurs. Finally, introduction of competitor DNA fragments into the surrounding solution disassembles the stable DNA complexes with HMGB1, NHP6A, and HU within seconds. Since spontaneous dissociation of protein does not occur on a time scale of hours, we conclude that this rapid protein exchange in the presence of competitor DNA must occur only via "direct" DNA-DNA contact. We therefore observe that protein transport along DNA by direct transfers occurs even for proteins such as NHP6A and HU that have only one DNA-binding domain.

Animals↗
PubMed2000

A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes.

Factors affecting the efficiency of animal cloning remain to be elucidated. Enucleation of recipient oocytes is a critical step in cloning procedures and typically is performed by aspirating a portion of the cytoplasm underlying the first polar body. Enucleation is evaluated using epifluorescence after Hoechst staining for DNA, which may disrupt functions of the cytoplast, especially mitochondria. Mitochondrial DNA in Dolly and other cloned sheep has been shown to derive exclusively from recipient oocytes. Not only might evaluation of the aspirated karyoplast portion inadequately reflect the state of the cytoplast, it is also time consuming. Here we report a reliable, noninvasive technique for spindle imaging and enucleation of oocytes using a new microscope, the Pol-Scope. The efficiency of enucleation was 100%, and only 5.5% of the oocytes' mitochondria entered the karyoplast upon Pol-Scope-directed removal of the spindle. Moreover, Pol-Scope imaging of spindles and micromanipulation did not compromise the developmental competence of reconstituted oocytes and cytoplasts.

Animals↗