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PubMed2002

Technical approaches to correction of oocyte aneuploidy.

BACKGROUND: This study describes the technical approaches used in treatment of age-related oocyte aneuploidy, the efficiency of each step of nuclear transplantation into mouse and human oocytes, and the ability of germinal vesicle (GV) transplantation to restore artificially induced ooplasmic damage. Finally, it examines the possibility of constructing viable female gametes by transferring diploid somatic cell nuclei into enucleated oocytes. METHODS: GV stage mouse oocytes were collected from unstimulated ovaries, and human GV oocytes were donated from consenting patients undergoing ICSI. Stromal (somatic) cells were isolated from uterine biopsies of consenting patients. Mouse cumulus cells were obtained after ovarian stimulation. GV ooplasts prepared by removing nuclei were transplanted either with GV nuclei or with somatic cells by micromanipulation. Grafted oocytes were electrofused and cultured to allow maturation, following which they were inseminated or analysed cytogenetically. Ooplasmic dysfunction was induced by photosensitization with a mitochondria-specific fluorescent dye. RESULTS: GV transplantation had an overall efficiency of 87 and 73% in the mouse and humans respectively. Maturation rates of 95 (mouse) and 64% (human) following reconstitution were comparable with those in control oocytes, as was the incidence of aneuploidy for five chromosome-specific probes after aneuploidy among the reconstituted oocytes. Photosensitization of oocytes significantly reduced the maturation rate to 4.2%, whereas 61.9% of oocytes matured after transfer of photosensitized GV karyoplasts into healthy ooplasts, with 52% of these mature oocytes being successfully fertilized by ICSI. Enucleated immature oocytes receiving mouse cumulus or human endometrial cell nuclei extruded a polar body in >40% of cases. Five out of seven successfully transferred aged human nuclei exhibited the expected number of signals with five chromosome-specific probes suggesting an appropriate chromosome separation in young ooplasm. CONCLUSIONS: Nuclear transplantation itself does not appear to interfere with chromosome segregation and can possibly rescue oocytes with damaged mitochondria. Finally, immature mouse ooplasm supported separation of somatic chromosomes to expected numbers, implying that haploidization may be occurring. The roles of genetic imprinting and fidelity of chromosome segregation are unknown.

Aneuploidy↗
PubMed2003

Monozygotic twinning following assisted conception: an analysis of 81 consecutive cases.

BACKGROUND: This study attempts to identify risk factors for monozygotic (MZ) twinning following assisted conception. METHODS: Eighty-one MZ twinning pregnancies that occurred over a 7-year period in our IVF/embryo transfer programme were evaluated. These were compared with 4224 non-MZ pregnancies from the same period. RESULTS: The overall incidence of MZ twinning was 1.88% (81/4305) of all clinical pregnancies. A total of 63% (51/81) of the MZ pregnancies included one or more other implantations as well. MZ twinning was unrelated to maternal age, paternal age, gonadotrophin dosage, peak estradiol and progesterone levels, number of oocytes collected, and number of embryos replaced. Patients with MZ twinning had significantly more attempts at assisted reproduction than those with non-MZ multiple pregnancy. A logistic regression analysis suggested a role, albeit not emphatic, for the zona pellucida in MZ twinning. Twenty-four of the 65 MZ cases with known placentation were monoamniotic. This incidence far exceeds that seen following spontaneous conception (P < 0.0001). CONCLUSIONS: The risk of monoamniotic twinning is increased following IVF/embryo transfer. Zona pellucida disruption appears to increase the incidence of MZ twinning. However, the overall micromanipulation data together with the unexpected placentation data suggest that zona-mediated embryo splitting is not the only mechanism of twinning under artificial conditions.

Embryo Transfer↗
PubMedDate not supplied

Intracytoplasmic sperm injection: a state of the art technique.

Of the micromanipulation techniques developed in the twentieth century, intracytoplasmic sperm injection (ICSI) has been the major breakthrough in the field of assisted fertilization. This article reviews the indications for the use of ICSI, its clinical application, the establishment of an ICSI programme including protocol and the results obtained since the introduction of ICSI and the potential risks. In addition, intracytoplasmic spermatid injection is briefly discussed.

Female↗
PubMed1988

Normal in-vivo development of marmoset monkey embryos after trophectoderm biopsy.

A procedure was developed to remove trophectoderm cells from day-8 blastocysts of the marmoset monkey. Using micromanipulative techniques, a tear was made in the zona pellucida opposite the inner cell mass which facilitated the controlled herniation of trophectoderm cells as the blastocysts developed in vitro. After 24 h (day-9 blastocysts) and 48 h (day-10 blastocysts) of culture approximately 20% and approximately 50% respectively of the blastocyst had herniated. The herniated trophectoderm was cut off by freehand and the biopsied blastocysts transferred to recipients. Normal offspring were born but pregnancies could be established from day-10 blastocysts only if the recipients were treated with human chorionic gonadotrophin during early pregnancy. One pregnancy was established after the transfer of three frozen biopsied day-10 blastocysts. Biopsies of 30-50 cells from day-10 blastocysts could be readily grown in vitro as trophoblast vesicles to in excess of 1000 cells but biopsies of less than 20 cells from day-9 blastocysts formed a monolayer of binucleated and multinucleated cells with limited cell replication. Assuming human trophectoderm cells have a similar capacity to the marmoset to grow in vitro, the application of this technique to human blastocysts would provide sufficient cells on which the preimplantation diagnosis of a genetic disorder could be made.

Animals↗
PubMed1993

IVF treatment of moderate male factor infertility: a comparison of mini-Percoll, partial zona dissection and sub-zonal sperm insertion techniques.

In this study we examined various techniques of in-vitro fertilization (IVF) for treating couples in whom the male had subnormal semen parameters. We compared two sperm preparation methods (mini-Percoll and conventional swim-up) for efficiency of recovery after preparation and for fertilization rates after IVF, and compared the suitability of partial zona dissection (PZD) and sub-zonal sperm insertion (SUZI) to patients with different types of male factor infertility. The mini-Percoll technique allowed the recovery of significantly more motile spermatozoa from the same semen sample compared to the swim-up method. More oocytes were fertilized after spermatozoa were prepared by the mini-Percoll technique. An increased number of spermatozoa recovered from an ejaculate led to an improvement in the quality of spermatozoa in the insemination droplet. Subsequently, when using the PZD technique, the fertilization rate increased when there was a higher number of spermatozoa in the patient's ejaculate. When comparing the two micromanipulation techniques, SUZI provided patients with oligoasthenzoospermia (i.e. < 10 x 10(6) spermatozoa/ml and 40% motility) with a higher chance of obtaining 2-pronculeate eggs.

Colloids↗
PubMed2002

Clonogenic culture of normal spermatogonia: in vitro regulation of postnatal germ cell proliferation.

The stem cell properties of neonatal germ cells have recently been demonstrated by in vivo transplantation. Regulation of proliferation of these cells, however, is not yet understood, and an in vitro system is needed for directly testing the action of differentiation and proliferation-related factors for germ cells. We developed an in vitro model involving micromanipulation and a single-cell clonogenic assay in which results from independent experiments on spermatogonia and gonocytes have been analyzed and compared. Neonatal germ cells can be distinguished by their large size both in vivo and in vitro in a single-cell suspension. These cells are picked up singly using a micropipette and deposited into a 96-well plate precoated with an extracellular matrix component, e.g., collagen IV. The effect of growth factors or cocultured somatic cells was assayed by counting the percentage of wells containing a colony and comparing this percentage with that of control cultures. Addition of platelet-derived growth factor significantly shifted the modal colony size for gonocytes from >16-64 to >64-128 cells/colony (P < 0.001, chi2) but had no effect on spermatogonia-derived colony size and number. For testis somatic cell underlays, there was a profound inhibition of all colony types, and immunohistochemical staining of testis cell underlays showed inhibin/activinbetaA subunit expression. This finding suggests that negative regulation of germ cell proliferation is mediated by inhibin. Addition of activin A to these cultures resulted in significant recovery (P = 0.046) of gonocyte-derived colony numbers but not spermatogonia-derived colonies, which may reflect the functional regulation by these factors observed in vivo. This proliferation assay also highlights many similarities in the regulation of gonocyte and spermatogonia proliferation in vitro, suggesting that proliferation potential is not noticeably affected by the transition of gonocytes to spermatogonia. For example, the average colony cloning efficiency was 80% for gonocytes and 76% for spermatogonia. This technology forms a basis for optimizing growth of neonatal germ cells for applications such as introduction of genetic material into the germ line to produce transgenic mice and to explore gene therapy.

Activins↗
PubMed1996

Single-cell analysis of T-cell receptor-gamma rearrangements in large-cell anaplastic lymphoma.

Large-cell anaplastic lymphomas (LCAL) are characterized by their distinctive morphology together with expression of the CD30 antigen. In addition, a chromosomal translocation, t(2;5) (p23; q35), can be detected in most cases. A significant proportion of LCALs carry rearrangements of the T-cell receptor-gamma (TCR-gamma) locus and display a T-cell phenotype. In about a third of the cases, another type of non-Hodgkin-lymphoma precedes LCAL. Early transformations of non-Hodgkin's lymphoma into LCAL might escape clinical detection in a significant number of cases. The existence of clonally related lymphoid cells within the lymph node infiltrates must be claimed in these cases. Recently, a small-cell-predominant variant of LCAL was described in which only few large tumor cells expressing the CD30 antigen are found together with numerous small lymphocytes, which are frequently CD30-. This observation in particular prompted us to investigate the clonal relationship of the tumor cell compartment and admixed small lymphocytes in one case of common LCAL with T-cell genotype. For this purpose, we chose to amplify rearranged TCR-gamma sequences from single cells isolated from immunostained frozen sections by using a micromanipulator. A total of 119 cells were investigated. Amplification products were obtained in 17 of 79 CD3+ cells, 12 of 30 CD30+ cells, and three of 10 CD20+ cells. The nucleotide sequences were determined in 28 cells by nonradioactive sequencing. In 11 CD30+ cells, the predominant rearrangement of TCR-gamma was identified. No clonal diversity was observed. The small CD3+ lymphocytes were unrelated to the anaplastic CD30+ tumor cells. This report describes a method to analyze rearrangements of the TCR-gamma in single cells isolated from immunostained frozen sections. Application of this technique revealed an absence of clonal diversity in a case of LCAL and documented the polyclonal nature of admixed small CD3+ lymphocytes.

Cell Separation↗
PubMed2004

Near-field-magnetic-tweezer manipulation of single DNA molecules.

We have developed an instrument for micromanipulation of single DNA molecules end labeled with 3-microm-diameter paramagnetic particles. A small, permanent magnet that can be moved as close as 10 microm to the particle being manipulated can generate forces in excess of 200 pN, significantly larger than obtained in other recent "magnetic-tweezer" studies. Our instrument generates these forces in the focal plane of a microscope objective, allowing straightforward real-time observation of molecule extension with a position resolution of approximately 30 nm. We show how our magnetic manipulation system can be combined with manipulation and force measurement using glass micropipettes to allow rapid switching between measurements in fixed-force and fixed-extension ensembles. We demonstrate the use of our system to study formation of DNA loops by an enzyme which strongly binds two copies of a specific 6-base-pair sequence.

DNA↗
PubMed2004

Nucleosome interactions in chromatin: fiber stiffening and hairpin formation.

We use Monte Carlo simulations to study attractive and excluded volume interactions between nucleosome core particles in 30-nm chromatin fibers. The nucleosomes are treated as disklike objects having an excluded volume and short-range attraction modeled by a variant of the Gay-Berne potential. The nucleosomes are connected via bendable and twistable linker DNA in the crossed linker fashion. We investigate the influence of the nucleosomal excluded volume on the stiffness of the fiber. For parameter values that correspond to chicken erythrocyte chromatin, we find that the persistence length is governed to a large extent by that excluded volume whereas the soft linker backbone elasticity plays only a minor role. We further find that internucleosomal attraction can induce the formation of hairpin configurations. Tension-induced opening of such configurations into straight fibers manifests itself in a quasiplateau in the force-extension curve that resembles results from recent micromanipulation experiments. Such hairpins may play a role in the formation of higher-order structures in chromosomes like chromonema fibers.

Chromatin↗
PubMed2005

Formation of loops in DNA under tension.

We study the formation of loops along a DNA molecule under applied tension, as might occur in single-DNA micromanipulation experiments with proteins which are able to simultaneously bind two DNA sites. We consider the case of "bare" DNA in the loop, which forms a "teardrop" shape, and the case where a single DNA-bending protein produces a "kink" in the middle of the loop; the presence of a right-angle kink in the loop reduces its bending energy by a factor of 3. Using the bending energy plus an estimate of the free energies associated with fluctuations and the elasticity of the extended nonlooped DNA, we obtain a probability distribution for loops as a function of loop size and force. Force strongly suppresses formation of all loops, but suppresses large loops more severely than small ones. This quenching effect of force is reduced in the presence of a kink in the loop. We also calculate the speed at which length is absorbed into loops between arbitrary positions along the DNA (i.e., for non-sequence-specific loop forming proteins). The speed of retraction of the molecule decays as a stretched exponential function of the force with characteristic force scales depending on the geometry of the loops.

Computer Simulation↗
PubMed1986

Microscopic observation and video recording of cellular response to sudden changes in environment.

A method to record responses of single cells to rapid changes in their environment is described. A rectangular glass microcapillary is filled with the cell suspension under investigation and placed under a light microscope. With the aid of a micromanipulator a smaller microcapillary with a narrowed tip, which was still over 10 times wider than the cell diameter, is driven into the larger, cell containing, microcapillary. The environment of the cells is changed by injecting a new medium via the smaller capillary and the cell responses are recorded using a video recording system. The usefulness of the system has been examined by studying the time course of the osmotic swelling of erythrocytes after water injection and the shape transformations caused by lysophosphatidylcholine.

Erythrocytes↗
PubMed1994

Adhesion strength of human ligament fibroblasts.

The adhesive interactions of cells with other cells and the extracellular matrix (ECM) play a fundamental role in the organization of cells in differentiated organs, cell motility, and the healing process. The adhesion characteristics of ligament fibroblasts depend on the expression of cell surface molecules and their interaction with the ECM. Although many receptors mediating the effects of ECM components on ligament cell function remain poorly defined, it is known that fibronectin (FN) allows ligament cells to adhere through the VLA-5 receptor (alpha 5 beta 1). A direct measurement of the adhesion between anterior cruciate ligament (ACL) or medial collateral ligament (MCL) fibroblasts and fibronectin matrix proteins was achieved by using a micromanipulation technique to determine the force required to detach an ACL or MCL cell from fibronectin-coated glass. We have found that the adhesion strength is not random, but has well-defined functional relationships with the FN concentration and the seeding time (time allowed for the cell to establish attachment). The adhesion strength (i.e., force required to detach) of ACL cells shows a stronger dependence on FN concentration (1, 2, and 5 micrograms/ml) for short seeding times (15-30 min) than for long seeding times (38-75 min). For MCL cells, the effect of the seeding time on adhesion strength was apparent for all concentrations. For all the seeding times studied and FN concentrations used, MCL cells had higher adhesion strength than ACL cells. The adhesion strengths of ACL and MCL fibroblasts to FN are correlated to cell adhesion area.(ABSTRACT TRUNCATED AT 250 WORDS)

Anterior Cruciate Ligament↗
PubMed2001

Species-specific, nested PCR-restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts.

Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.

Animals↗
PubMed1966

Mutants of Escherichia coli variably resistant to bacteriophage T1.

Carta, Guy R. (Rutgers, The State University, New Brunswick, N.J.), and Vernon Bryson. Mutants of Escherichia coli variably resistant to bacteriophage T1. J. Bacteriol. 92:1055-1061. 1966.-Mutants resistant to bacteriophage T1 were isolated from ultraviolet (UV)-irradiated cultures of Escherichia coli B/r, a UV-resistant variant. Bacterial populations derived from some of these mutants were partially but not completely resistant to the bacteriophage. Such mutants, designated variably resistant (B/r/1v), could not be obtained from E. coli B. Phage-free mutant populations taken from different stages in growth consisted of significantly different proportions of T1-resistant and T1-sensitive cells. The growth stage-dependent range of variation exceeded 1,000-fold. In broth cultures, the highest proportion of resistant cells consistently appeared at mid-log phase, and the highest proportion of sensitive cells at lag and stationary phases. Comparable evidence for environmentally dependent changes in host-cell phenotype was obtained by efficiency of plating and cloning efficiency analysis tests. Micromanipulation showed that, in clones growing in the presence of phage T1, sensitive bacteria appeared with high frequency and underwent lysis.

Bacteriolysis↗
PubMed2005

Variable gp43 secretion by Paracoccidioides brasiliensis clones obtained by two different culture methods.

The main objectives of this study were to obtain clones of Paracoccidioides brasiliensis by two methods (micromanipulation and plating assay) and to determine if the secretion of the 43-kDa glycoprotein (gp43) is dependent on the clonal culture. The results show that the secretion of gp43 is not dependent on clonal cultures. Clones that originally were secretors of this molecule, after subculturing, lost this characteristic; on the other hand, clones that originally did not secrete gp43 began to secrete gp43 after subculturing.

Antigens, Fungal↗
PubMed2005

Activation induced cytidine deaminase expression in lymphocyte predominant Hodgkin lymphoma.

BACKGROUND: The lymphocytic and histiocytic (L&H) cells of lymphocyte predominant Hodgkin lymphoma (HL) originate from germinal centre B cells and carry mutated V gene rearrangements, usually with intraclonal diversity. It is unclear whether intraclonal V gene diversification by somatic hypermutation, which is strictly dependent on the enzyme activation induced cytidine deaminase (AID), is restricted to the early phase of lymphoma clone expansion and later silenced, or whether it remains active throughout malignant proliferation. AIMS: To analyse whether AID is expressed in L&H cells as an indicator of active somatic hypermutation in the tumour cells. METHODS: L&H cells from lymphocyte predominant HL cases and centroblasts from lymphadenites were micromanipulated and analysed for AID expression by quantitative real time polymerase chain reaction. RESULTS: The AID transcription level was higher than background in three of the six lymphocyte predominant HL cases, although it was lower than that seen in centroblasts. CONCLUSIONS: Somatic hypermutation may remain active in L&H cells in a considerable proportion of cases, increasing the risk of acquiring further transforming mutations.

Cytidine Deaminase↗
PubMed2003

Probing complex RNA structures by mechanical force.

RNA secondary structures of increasing complexity are probed combining single molecule stretching experiments and stochastic unfolding/refolding simulations. We find that force-induced unfolding pathways cannot usually be interpreted by solely invoking successive openings of native helices. Indeed, typical force-extension responses of complex RNA molecules are largely shaped by stretching-induced, long-lived intermediates including non-native helices. This is first shown for a set of generic structural motifs found in larger RNA structures, and then for Escherichia coli's 1540-base long 16S ribosomal RNA, which exhibits a surprisingly well-structured and reproducible unfolding pathway under mechanical stretching. Using out-of-equilibrium stochastic simulations, we demonstrate that these experimental results reflect the slow relaxation of RNA structural rearrangements. Hence, micromanipulations of single RNA molecules probe both their native structures and long-lived intermediates, so-called "kinetic traps", thereby capturing -at the single molecular level- the hallmark of RNA folding/unfolding dynamics.

Computer Simulation↗