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PubMed2001

Application of the piezo-micromanipulator for injection of embryonic stem cells into mouse blastocysts.

Microinjection of embryonic stem (ES) cells into mouse blastocysts is one of the most important techniques for production of knockout or transgenic mice. However, skillful manipulation techniques and tremendous effort are required for this method. To overcome this difficulty, we applied a piezo-micromanipulator (PMM), which has been used for intracytoplasmic sperm injection in mice and production of cloned mice, for the injection of ES cells into blastocysts. When ES cells were injected by using a conventional method, 91% of the blastocysts were manipulated successfully. Using the PMM significantly (P < 0.01) increased the success rate of ES injection to 97%. The number of embryos manipulated in an hour increased from 9.7 embryos with the conventional method to 27.0 embryos with the PMM method. The injected ES cells did not show any detrimental effects due to a pulse from the PMM. After embryo transfer of the manipulated blastocysts, 39% of the newborns were chimeric mice with the conventional method, whereas 42% of the neonates were chimeric after the PMM method. These results indicate that microinjection of the ES cells into blastocysts is more efficient by the PMM method than the conventional method.

Animals↗
PubMedDate not supplied

[The effect of the micromanipulations used for transgenosis on mouse development].

Non-specific effects of micromanipulation techniques used for producing transgenic mice on processes of embryonic development were studied. Zygotes obtained from C57BL and BALBxDD mice were treated as follows: (1) incubated in culture medium; (2) the male pronucleus punctured with a glass microneedle; (3) microinjected with a buffer solution; and (4) DNA (mouse P-35 oncogene with human insulin gene promoter) injected into the male pronucleus. Then zygotes were transferred into oviducts of syngeneic or allogeneic pseudopregnant females. Such treatment resulted in the intrauterine death of embryos, as well as in birth of the dead or non-viable offspring with numerous defects of development. Zygote pronucleus puncturing is the most damaging manipulation, since its effect exceeds that of the zygote incubation and is comparable with the effect of buffer of DNA injections.

Animals↗
PubMed1992

Animal models in micromanipulation.

Micromanipulation is a useful technique for various studies of reproductive biology. Using this technique, we investigated the role of cAMP in the regulation of oocyte maturation. Anti cAMP serum was microinjected into oocyte cytoplasma. Immature oocytes resumed meiosis after microinjection and this result shows oocyte cAMP to be important for the intrafollicular meiotic arrest of oocyte and its decrease to cause resumption of meiosis. We also determined maturation promotion factor (MPF) activity in the mouse egg. Microinjection of cytoplasma from mature egg induced geminal vesicle break down of immature oocyte in the presence of dbcAMP. In addition, we detected MPF activity in cytoplasma of metaphase II oocyte. Some of the initial events of sperm-induced egg activation are accompanied by hydrolysis of phosphatidyl inositol which generates inositol trisphosphate (IP3). Mouse eggs microinjected with increasing concentration of IP3 revealed concentration dependent increase in conversion of the zona glycoprotein ZP2 to ZP2f. Modification of zona pellucida elicited by microinjection of IP3 are similar to those that occur following fertilisation. We also microinjected some cytoplasma of morula or 2 cell mouse embryo into 1 cell embryo of ICR mouse. After microinjection and culture, most of the embryo that reached 4 cells stage and 2 cell block of ICR mouse embryo was released significantly. These results suggest that some cytoplasmic factor(s) is a requisite for the development of embryo after fertilisation.

Animals↗
PubMed2004

Comparison of fertility between intracytoplasmic sperm injection and in vitro fertilization with a partial zona pellucida incision by using a piezo-micromanipulator in cryopreserved inbred mouse spermatozoa.

Cryopreservation of mouse spermatozoa has been widely applied for maintenance of transgenic and knockout lines. However, the fertility of cryopreserved spermatozoa from some inbred strains such as C57BL/6 and BALB/c is extremely poor. We have recently reported that a partial zona-pellucida incision by piezo-micromanipulator (ZIP) significantly improves the fertilization rate and subsequent embryonic development after in vitro fertilization (IVF) using cryopreserved C57BL/6 transgenic mouse spermatozoa and that inbred C57BL/6 mice could be produced by intracytoplasmic sperm injection (ICSI). These findings prompted us to compare the efficiency of fertilization and subsequent embryonic development between ICSI and IVF with ZIP (ZIP/IVF) using cryopreserved spermatozoa. In conventional IVF, BALB/cA, C57BL/6J, and B6C3F1 cryopreserved spermatozoa fertilized 19%, 0%, and 51% of oocytes, respectively. The fertilization rates of manipulated oocytes by ICSI versus ZIP/IVF using cryopreserved BALB/cA spermatozoa were 52% versus 68%, cryopreserved C57BL/6J spermatozoa were 43% versus 63%, and cryopreserved B6C3F1 spermatozoa were 58% versus 82%, respectively. In these strains, fertilization rates for ZIP/IVF were significantly higher (P < 0.05) than for other techniques. However, embryonic development to term for oocytes fertilized by cryopreserved spermatozoa was not significantly different between ZIP/IVF and ICSI in C57BL/6J and B6C3F1. The overall efficiency of mouse production in ZIP/IVF was higher than for ICSI and conventional IVF in C57BL/6J and B6C3F1. Furthermore, ZIP/IVF required approximately 3.3 times less manipulation time than did ICSI. Our results indicate that ZIP is a useful assisted reproductive technique for IVF of ova by cryopreserved spermatozoa and improves production in some mouse strains.

Animals↗
PubMedDate not supplied

[Assistance in human oocyte fertilization using micromanipulation].

In recent years different types of micromanipulation have become available to improve human gametes interaction, particularly in cases of severe sperm defect. Direct injection of one spermatozoon into the cytoplasm of the egg has not been applied in man, not only because of technical difficulties and low efficacy, but also in view of the theoretical risk of chromosomal anomalies. However, subzonal sperm injection (SUZI) and partial zona dissection (PZD) are now used routinely by a number of IVF centers. In our hands this latter method has increased significantly the fertilization rate (21.6 vs 2.9 p.cent among intact oocytes) and has allowed to obtain 3 pregnancies (of which 2 ongoing) after 28 attempts. Our results and those of the literature are discussed with respect to the risks and advantages linked to these techniques.

Female↗
PubMed1994

Micromanipulation of gametes for male factor infertility.

Techniques such as PZD, SZI, and ICSI are advantageous in cases of extreme male factor infertility. They are adjunctive techniques, applicable in the laboratory only during an IVF cycle. They can be used either simultaneously on sibling oocytes or, if indicated, individually. The present results demonstrate a lack of correlation between quantitative sperm parameters and outcome of assisted fertilization procedures. Preliminary results strongly suggest that ICSI will replace SZI and PZD for treatment of severe male factor infertility. Hundreds of viable pregnancies have been established well below the normal cut-off values for regular assisted conception procedures. Fertilization and pregnancy occurred following the use of spermatozoa without progressive motility and without normal morphology. In some patients sperm counts were correspondingly reduced and spermatozoa could be visualized only after centrifugation of the semen specimen. The current results provide evidence that spermatozoa from extremely oligoasthenoteratozoospermic men can produce normal offspring after the application of micromanipulation techniques, even when fertilization previously failed following standard IVF. Currently, investigators have not been able to identify, on the basis of conventional semen evaluations, subgroups of patients who would not benefit from microsurgical techniques. Findings published to date suggest approximately 24% clinical pregnancy rates per cycle when SZI and PZD are used together, but 35% clinical pregnancy rate per cycle for ICSI. Preliminary results suggest that the application of these techniques does not increase the risk of birth defects for subsequent offspring. However, continued observation of the potential effects of these procedures on progeny will be required.

Female↗
PubMed1996

[Assisted hatching--a useful micromanipulation technic in women after repeated failure of embryo transfer].

One reason for the low implantation rate after IVF/ET is the limiting ability of blastocysts to escape from the zona pellucida, and of nidation. This procedure can be assisted by the creation of an artificial opening in the zona pellucida before the transfer (assisted hatching). An artificial slot in the zona of 58 embryos (20 patients) was created mechanically by using Leitz Fluovert microscope and Narishige micromanipulators. From 20 embryo transfers 6 clinical pregnancies resulted (30.0% clin. preg. rate). In 4 cases women with three or more unsuccessful transfers were involved. This method should be used in classical IVF, specially in cases of repeated high quality embryo transfers which failed.

Embryo Transfer↗
PubMedDate not supplied

[Cloning leishmania at the promastigote stage using the Vonbrun micromanipulator].

A method was worked out for cloning Leishmania strains at the promastigote stage from cultures reared on two-phase nutrient medium by means of Vonbrün micromanipulator. By this method cultures of Leishmania can be obtained from one cell of these protozoans. During the cloning of Leishmania strains isolated from experimentally and naturally infected Phlebotomus papatasi and Ph. caucasicus the per cent of obtaining the clones from isolated promastigotes cultivated on a nutrient medium depends on the absolute and relative age of culture and on the growth intensity of the strain on a nutrient medium.

Animals↗
PubMed1996

Update on micromanipulation techniques for assisted conception.

This article reviews the progress in the field of micromanipulation in the past year. The major event has been the wide application of intracytoplasmic sperm injection. Other advances include haploid spermatid injection and the development of successful mouse models for intracytoplasmic sperm injection. There is now increasing awareness of the genetic basis of male infertility and its possible impact on how we offer treatment.

Animals↗
PubMed1998

Amplification of TCRbeta gene rearrangements from micromanipulated single cells: T cells rosetting around Hodgkin and Reed-Sternberg cells in Hodgkin's disease are polyclonal.

Despite accounting for only a minor fraction of all cells in Hodgkin's lymphoma tissue, the Hodgkin and Reed-Sternberg (HRS) cells represent the malignant tumor cell clone in Hodgkin's disease (HD). By far the most abundant cell type in the tumor tissue are CD4+ T cells. Some of them intimately associate with HRS cells forming rosettes around them. This study addresses the question whether the rosetting phenomenon reflects a specific interaction between T and HRS cells by asking whether the rosettes are composed of T cells expressing a restricted TCR repertoire. Single rosetting T cells were micromanipulated from frozen sections of tumor tissue in two cases of nodular sclerosing HD and one case of lymphocyte predominant HD. TCR Vbeta gene rearrangements were amplified from these single cells by PCR. Of 83 potentially functional Vbeta gene rearrangements obtained altogether from the three cases, 81 were found to be clonally unrelated. Furthermore, they did not show signs of selection of the receptor chains for recognition of common epitopes: The usage of Vbeta and Jbeta gene segments as well as the distribution of complementarity-determining region (CDR) 3 lengths was similar to what was seen in a collection of 60 Vbeta gene rearrangements from blood of healthy donors and no recurrent CDR3 amino acid motifs were found. These data suggest that the HRS cells attract CD4+ T cells nonspecifically.

Adult↗
PubMed1993

Micromanipulation of chromosomes in PTK2 cells using laser microsurgery (optical scalpel) in combination with laser-induced optical force (optical tweezers).

An optical scalpel and optical tweezer have been combined to perform intracellular microsurgery and micromanipulation in vivo. When only laser microsurgery was performed on metaphase chromosomes, the dissected sister chromatid fragments drifted off to either the side of the spindle or completely off the spindle. At anaphase the fragments separated and the two arms generally moved to their respective daughter cells. When the chromosome arm was cut during anaphase A and B, the distal chromosome fragment separated from the rest of the chromosome and moved toward the pole, following the proximal chromosome fragment. Distal chromosome fragments laser-dissected during metaphase were held together throughout anaphase using the optical trap. Optical trapping of dissected chromosome fragments during anaphase A and B inhibited movement of the chromosome fragment to its pole. As a result, the trapped chromosome fragments were (1) incorporated into the opposite daughter cell, (2) lost in the cleavage furrow during cytokinesis, or (3) eventually incorporated into the correct daughter cell. These results indicate that optical traps are effective in holding laser-dissected chromosome fragments throughout mitosis. This new tool should be useful for studies on chromosome movement and cell genetics.

Animals↗
PubMed1985

Microtubules, chromosome movement, and reorientation after chromosomes are detached from the spindle by micromanipulation.

The relationship between chromosome movement and microtubules was explored by combining micromanipulation of living grasshopper spermatocytes with electron microscopy. We detached chromosomes from the spindle and placed them far out in the cytoplasm. Soon, the chromosomes began to move back toward the spindle and the cells were fixed at a chosen moment. The microtubules seen in three-dimensional reconstructions were correlated with the chromosome movement just prior to fixation. Before movement began, detached chromosomes had no kinetochore microtubules or a single one at most. Renewed movement was always accompanied by the reappearance of kinetochore microtubules; a single kinetochore microtubule appeared to suffice. Chromosome movements and kinetochore microtubule arrangements were unusual after reattachment, but their relationship was not: poleward forces, parallel to the kinetochore microtubule axis (as in normal anaphase), would explain the movement, however odd. The initial arrangement of kinetochore microtubules would have led to aberrant chromosome distribution if it persisted, but instead, reorientation to the appropriate arrangement always followed. Observations on living cells permitted us to place in sequence the kinetochore microtubule arrangements seen in fixed cells, revealing the microtubule transformations during reorientation. From the sequence of events we conclude that chromosome movement can cause reorientation to begin and that in the changes which follow, an unstable attachment of kinetochore microtubules to the spindle plays a major role.

Animals↗
PubMed1992

Orientation and segregation of a micromanipulated multivalent: familiar principles, divergent outcomes.

We studied the orientation and segregation of a particular quadrivalent in living grasshopper spermatocytes. Quadrivalents were detached from the spindle by micromanipulation, then placed and bent as desired. The detached quadrivalents reattach and orient on the spindle. Their orientation is determined by the same principles that apply to ordinary chromosomes in mitosis and meiosis, but the outcome is different. Certain characteristics of the quadrivalent lead to a variety of orientations rather than the single one typical of ordinary chromosomes. Two kinetochores in the quadrivalent are linked to the others by unusually long, flexible chromosome arms. These kinetochores may face either the same pole or opposite poles and tend to orient initially to the pole toward which they face. Consequently, the initial orientation of the flexibly linked kinetochores is variable, and, moreover, they frequently reorient. In contrast, the other two kinetochores are as rigidly connected as those in a small bivalent and so display the typical back-to-back arrangement. Usually, this arrangement leads quickly to a stable orientation of the two kinetochores to opposite poles. Sometimes, however, the back-to-back arrangement changes to a side-by-side arrangement so that the orientation of both kinetochores to the same pole is favored. The combined effect of this diverse behavior is that the quadrivalent has four stable orientations, each leading to a different distribution of chromosomes in anaphase. The result is genetic chaos. Ironically, this chaos is produced by the same mechanisms that, in ordinary bivalents and mitotic chromosomes, produce a single stable orientation and genetically appropriate chromosome distribution.

Aneuploidy↗
PubMed1988

Effects of the zona pellucida, agar embedding, and culture on the survival of micromanipulated bovine embryos after freezing and thawing.

Three experiments were undertaken to compare how the integrity of the zona pellucida, embedding in agar, and incubation in the sheep oviduct, respectively, affected the survival of bovine embryos after micromanipulation, freezing, and thawing. In Experiment 1, the proportions of whole day 7 embryos that continued their development in vitro after being frozen without a zona pellucida (N = 18), or with the zona pellucida opened over one-half (N = 14), or one-fourth (N = 14) of its circumference, or left intact (N = 17) did not differ significantly (44, 43, 36, and 59%, respectively). Experiment 2 compared the survival of bisected day 7 embryos frozen with or without embedding in agar cylinders. In vitro, one of eight half-embryos (17%) survived in each group. In vivo, the survival rate was 0% (N = 18) after freezing half-embryos in an agar cylinder and (11%) (N = 18) for halves frozen without agar. In Experiment 3, embryos were bisected on day 5 or 6, embedded in agar, then cultured for 2 days or 1 day, respectively, in the sheep oviduct before freezing them on day 7. Of the 54 half-embryos transferred to the sheep oviducts, 27 (50%) were recovered, frozen, thawed, and transferred to recipients, resulting in the birth of 17 calves (63%), of which six pairs were identical twins. This survival rate was significantly higher than for control embryos collected at day 7, bisected, and frozen without agar embedding (16%; N = 12).(ABSTRACT TRUNCATED AT 250 WORDS)

Agar↗
PubMed1995

Sperm stimulants can improve fertilization rates in male-factor cases undergoing IVF to the same extent as micromanipulation by partial zona dissection (PZD) or subzonal sperm insemination (SUZI): a randomized controlled study.

PURPOSE: Our purpose was to evaluate the efficacy of direct insemination (IVF), micromanipulation by partial zona dissection (PZD), and subzonal sperm insemination (SUZI) using sperm-treated with pentoxifylline (PF) +/- 2-deoxyadenosine (2DA). RESULTS: The overall fertilization rate achieved was similar for all three fertilization techniques (33.1, 30.2, and 26.9% for IVF, SUZI, and PZD, respectively). Patients who had reduced fertilization in previous IVF attempts showed improved fertilization with sperm stimulants, either PF alone or PF in combination with 2DA in standard IVF. In certain cases, SUZI or PZD gave significantly improved fertilization rates in comparison to IVF. CONCLUSION: Selective use of sperm stimulants in IVF can achieve fertilization for the majority of male-factor cases. However, PZD and SUZI techniques are useful, especially when sperm stimulants fail to achieve fertilization or achieve poor fertilization in direct insemination.

Deoxyadenosines↗
PubMed1999

Changes in the mechanical properties of fibroblasts during spreading: a micromanipulation study.

Cell morphology is controlled in part by physical forces. If the main mechanical properties of cells have been identified and quantitated, the question remains of how the cell structure specifically contributes to these properties. In this context, we addressed the issue of whether cell rheology was altered during cell spreading, taken as a fundamental morphological change. On the experimental side, we used a novel dual micromanipulation system. Individual chick fibroblasts were allowed to spread for varying amounts of time on glass microplates, then their free extremity was aspirated into a micropipet at given pressure levels. Control experiments were also done on suspended cells. On the theoretical side, the cell was modeled as a fluid drop of viscosity mu, bounded by a contractile cortex whose tension above a resting value was taken to be linearly dependent on surface area expansion. The pipet negative pressure was first adjusted to an equilibrium value, corresponding to formation of a static hemispherical cap into the pipet. This allowed computation, through Laplace's law, of the resting tension (tau 0), on the order of 3 x 10(-4) N/m. No difference in tau 0 was found between the different groups of cells studied (suspended, adherent for 5 min, spread for 0.5 h, and spread for 3 h). However, tau 0 was significantly decreased upon treatment of fibroblasts with inhibitors of actin polymerization or myosin function. Then, the pressure was set at 30 mmH2O above the equilibrium pressure. All cells showed a biphasic behavior: (1) a rapid initial entrance corresponding to an increase in surface area, which was used to extract an area expansion elastic modulus (K), in the range of 10(-2) N/m; this coefficient was found to increase up to 40% with cell spreading; (2) a more progressive penetration into the pipet, linear with time; this phase, attributed to viscous behavior of the cytoplasm, was used to compute the apparent viscosity (mu, in the range of 2-5 x 10(4) Pa s) which was found to increase by as much as twofold with cell spreading. In some experiments the basal force at the cell-microplate interface was quantitated with flexible microplates and found to be around 1 nN, in agreement with values calculated from the model. Taken together, our results indicate a stiffening of fibroblasts upon spreading, possibly correlated with structural organization of the cytoskeleton during this process. This study may help understand better the morphology of fibroblasts and their mechanical role in connective tissue integrity.

Actins↗
PubMed1993

Micromanipulation of gametes and embryos.

The procedures described in this chapter are applicable to problems of oogenesis, fertilization, and early development. Many important procedures have not been described (e.g., pronuclear exchange). However, nearly every experimental manipulation thus far published for the mouse either directly involves the techniques included here or is closely related to them. Therefore, if all of the skills outlined in this chapter are successfully mastered, it should be possible to adapt that knowledge to most or all known experimental protocols. Successful establishment of micromanipulation is certain to have a significant and lasting positive impact on nearly any research program in mammalian development.

Animals↗
PubMed1984

Micromanipulation of sheep morulae to produce monozygotic twins.

Donor ewes were treated with an intravaginal sponge containing 30 mg fluorogestone acetate (FGA) which was removed 12 days later. On the mornings of days 10, 11, and 12, each animal received 30 mg horse anterior pituitary (HAP) extract. Donors were mated twice daily during oestrus. Egg recovery was attemped seven days after progestagen with-drawal, when late morula/early blastocysts could be expected. The zona pellucida was opened with the aid of a micromanipulator, and the embryo was removed by gentle positive pressure from an evacuating pipette. After removal from the zona pellucida, the embryo was bisected using a fine glass needle; each demi-embryo was immediately placed either into the original zona pellucida or into one from an evacuated oocyte. The original opening was closed and both halves were transferred bilaterally into previously synchronized recipients. From 17 embryos split and transferred to 17 ewes, nine became pregnant and produced seven sets of monozygotic twins and two singletons.

Journal Article↗