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At least 1,369 records · Page 76Linked to original sources
PubMed2001

Response of the cortex to the mitotic apparatus during polar body formation in the starfish oocyte of Asterina pectinifera.

In order to understand the mechanism of unequal division, polar body formation was investigated using the oocytes of the starfish, Asterina pectinifera. Cortical actin filaments were quantitatively measured after staining the maturing oocytes with fluorescently labeled phalloidin using a computer and image-processing software. Before polar body formation, at first the actin filaments at the animal pole decreased and subsequently the animal pole bulged. On the other hand, actin filaments surrounding the animal pole increased gradually and made a cleavage furrow around the animal pole as the bulge grew. Then the furrow ingressed and finally a polar body formed. When the surface force was calculated according to the cell shape, the surface force decreased at the animal pole but the force at the contractile ring increased. When by micromanipulation the mitotic apparatus was detached and translocated to the cortex other than the animal pole, polar body formation occurred all over the cortex of the oocyte, which indicates that the response of the whole cortex to the mitotic apparatus is equal. These results indicate that the decrease in the actin filaments and surface force near the centrosome of the mitotic apparatus as well as the increase in the actin filaments and surface force at some distance of the centrosome is important for cytokinesis.

Actins↗
PubMed2004

Exposure to warmer postoperative temperatures reduces hypothermia caused by anaesthesia and significantly increases the implantation rate of transferred embryos in the mouse.

Embryo transfer (ET) is among the key factors determining the overall efficiency of transgenic technology in the mouse. A successful ET depends among other factors on the quality of the transferred embryos, foster mothers and anaesthetic reagents and on the transfer techniques. Anaesthesia-caused deaths and suboptimal ET procedures are factors which reduce the success of transgenic experiments and mouse colony maintenance. Here we compared the effects of two anaesthetic reagents-a ketamine/xylazine combination, and tribromoethanol (Avertin)-on the rates of implantation and development to term of mouse zygotes transferred into the oviducts of CD-1 foster mothers, and evaluated whether hypothermia caused by anaesthetics after the ET operation could be overcome by postoperative incubation of the foster mothers. We established two experimental groups of fosters, one of which was kept at room temperature (RT, 21 degrees C) with the other in an incubator (33 degrees C) overnight after ET. Rates of implantation, resorption and development to normal fetuses were evaluated by sacrificing the foster mothers on the 15th day of their pregnancy. Our results showed that regardless of the anaesthetic reagents used, the rates of implantation and of development to normal fetuses can be significantly improved by exposing the foster mothers to warmer temperatures (33 degrees C) immediately after the ET operation. These results may have important implications in increasing the success rate of ET with micromanipulated embryos.

Anesthetics↗
PubMed2006

Improvement of cumulus-free oocyte maturation in vitro and its application to microinsemination with primary spermatocytes in mice.

In micromanipulation experiments using immature oocytes, final ooplasmic maturation is often compromised because the oocytes are usually first freed from their nurturing cumulus cells. This study was undertaken to determine whether cumulus-free in vitro maturation (IVM) in mice could be improved by modifying IVM medium having defined components. Cumulus-free germinal vesicle (GV) stage oocytes were subjected to IVM in either alphaMEM medium, TYH medium, or a 1:1 mixture of the two (termed TaM). TYH medium produced a better maturation rate (181/196; 92.3%) than alphaMEM (184/257; 71.6%). However, alphaMEM supported better embryo development to the morula/blastocyst stage than TYH following in vitro fertilization (93.3% vs. 76.5%) or parthenogenetic activation (82.4% vs. 60.4%). Mitochondrial distribution in MII oocytes was diffuse following IVM in alphaMEM, but was aggregated with TYH. The maturation promoting factor (MPF) activity in MII oocytes was significantly higher in TYH than in alphaMEM (P<0.05). Oocytes cultured in TaM had intermediate characteristics and essentially resembled in vivo matured oocytes, with the mitochondrial distribution pattern being most typical of that condition. The highest rate of development from GV oocytes to full-term fetuses following in vitro fertilization and embryo transfer to foster mothers (23.8%) was obtained using TaM. When this IVM system was applied to MI oocytes injected with spermatocytes, offspring were first obtained without cytoplasmic replacement at MII. Thus, optimization of the culture medium can considerably improve the quality of cumulus-free oocyte IVM in mice.

Animals↗
PubMed2003

Factors affecting premature chromosome condensation of cumulus cell nuclei injected into rat oocytes.

To date, production of cloned rats by somatic cell nuclear transfer (NT) has not yet been successful. Inducing premature chromosome condensation (PCC) of injected cell nuclei in recipient cytoplasm is considered essential for successful mouse cloning by the Honolulu method. In the present study, some factors affecting PCC of rat cumulus cell nuclei injected into rat oocytes were examined. Wistar female rats (young: 4 to 5-week-old, mature: > or =10-week-old) were superovulated by injections of eCG and hCG, and oocytes recovered 14 or 17 h after hCG injection were received with cumulus cell nuclei using piezo-driven micromanipulator. When the oocytes were recovered 14 h post-hCG injection from young rats and the nuclear injection into oocytes was completed within 45 min, PCC was observed in 44-49% of NT oocytes. In the case of oocytes from mature rats, PCC occurred in 11-19% of the NT oocytes. Oocytes recovered 17 h post-hCG injection did not support PCC of the injected nuclei (0-7%) regardless of the donor age. Treatment of oocytes with a neutral cysteine protease inhibitor, N-acetylleucylleucylnorleucinal, slightly increased the incidence of PCC (48 vs 37%). Comparison of rat strains for oocyte donors indicated that proportions of NT oocytes undergoing PCC in Wistar and LEW oocytes (41-46%) were higher than those in Donryu and F344 oocytes (17-25%). Thus, ability of rat oocytes to promote PCC of the injected nuclei is dependent on the characteristics of oocytes, such as age or strain of donor rats, and timing of oocyte recovery.

Animals↗
PubMed2003

Effect of activation methods for bovine oocytes after intracytoplasmic injection.

A principal nuclear transfer procedure is to inject a donor cell into the perivitelline space in an enucleated oocyte and then electric fusion is performed (cell fusion method). The effects of activation methods in reconstructed oocytes for the serum-starved somatic cell cloning procedure were investigated in this study by means of intracytoplasmic injection (i.c.i.). Bovine oocytes were enucleated at 18-22 h for in vitro maturation, and subsequently the nucleus of cumulus cell collected from Japanese Black Bulls (JBCC) after 5-7 days of starved culture was injected into the recipient cytoplast with a piezo-micromanipulator. At 1 h after i.c.i., reconstructed oocytes were stimulated with ethanol (ET) or calcium ionophore (CaI) as the first activation treatment, followed by cycloheximide (CHX) or 6-dimethylaminopurin (DMAP) treatment as the second activation. In the experiment on the first activation method, the proportion of reconstructed oocytes developing to the blastocyst stage was significantly (p<0.01) higher in the ET activation method than that with CaI (10.5% and 4.7%, respectively). And the experiment on the second activation method after ET treatment showed similar proportions of blastocyst development in both CHX and DMAP treatments (5.9% and 2.8%, respectively). The present results indicated that combined activation treatment with ET and CHX was efficient for reconstructed bovine oocytes by i.c.i.

Animals↗
PubMed2004

Improved survival of vitrified in vivo-derived porcine embryos.

An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 microg/ml cytocharasin B at 12000 x g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 +/- 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 +/- 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.

Animals↗
PubMed2004

Catalase in manipulation buffer enhances the developmental competence of DNA-injected embryos.

To improve the efficiency of transgenesis, we investigated the effects of a radical scavenger during microinjection on the development to blastocysts or pups of mouse pronuclear embryos, microinjected with the enhanced green fluorescent protein (EGFP) transgene. When embryos were microinjected in medium containing 0-1,000 units/ml catalase, the developmental rate to blastocysts was significantly higher (P<0.01) in 100-units/ml catalase (81%) than those in 0 and 1,000 units/ml (56 and 65%). To investigate the ontogenetic ability of DNA-injected embryos, EGFP-injected embryos manipulated under 0 or 100 units/ml catalase were transferred separately to recipient mice. The proportion of fetuses derived from EGFP-injected embryos manipulated under 100 units/ml catalase (29%) was significantly higher (P<0.05) than that manipulated under 0 units/ml catalase (19%). Furthermore, the numbers of transgenic pups were 17 in 100 units/ml catalase and 14 in 0 units/ml catalase. The results of the present study indicate that scavenging reactive oxygen species during in vitro micromanipulation is beneficial for the development of DNA-injected embryos.

Animals↗
PubMed2005

Effects of partial removal of cytoplasmic lipid on survival of vitrified germinal vesicle stage pig oocytes.

This study was designed to investigate whether the partial removal of cytoplasmic lipid from immature pig oocytes prior to vitrification had any positive effects on subsequent maturation, fertilization and early development. Oocytes at the germinal vesicle stage were partially freed from cumulus cells and centrifuged, and then polarized cytoplasmic lipid was removed by micromanipulation. When cultured for 44-48 h, significantly fewer of the centrifuged oocytes reached metaphase II (M-II) than did the non-centrifuged oocytes (approximately 53% vs approximately 68%, respectively); however, no further reduction in the M-II rate was observed when centrifuged oocytes were then delipated prior to culture (approximately 47%). To evaluate their sensitivity to the equilibration and vitrification solutions containing ethylene glycol, non-centrifuged, centrifuged, and delipated oocytes were cultured continuously for several minutes in those solutions, then washed and cultured further; no significant differences in the M-II rates (approximately 20-27%) were observed among the three treatment groups. When oocytes were vitrified and then warmed, significantly more delipated oocytes reached M-II in culture (approximately 15%) than did the non-delipated oocytes, whether centrifuged or not (approximately 4% in each group). When delipated, vitrified and matured oocytes were microsurgically injected with frozen-thawed spermatozoa, approximately 39% were activated and male pronucleus formation was observed in approximately 40% of activated oocytes; none developed beyond the 4-cell stage. These results show that maturation in vitro of vitrified pig oocytes can be promoted by partial removal of cytoplasmic lipid prior to vitrification and that the vitrified oocytes can be fertilized, although the embryonic development obtained in this study was limited.

Animals↗
PubMed2004

Differences in pO2 peaks of a murine fibrosarcoma between carbon-ion and X-ray irradiation.

We measured and compared the oxygen partial pressure (pO(2)) profiles in experimental tumors after irradiation with carbon ions and with X-rays. The NFSa fibrosarcomas grown in the hind legs of C3H male mice received isoeffect single doses of carbon ions or X-rays. Coaxial oxygen microelectrodes of high spatial resolution were inserted into the tumor with 20 microm steps by a computerized micromanipulator. The number of pO(2) peaks that reached 15 mmHg were at least 0.45 per 3,000 microm in unirradiated tumors and significantly increased to 1.55 per 3,000 microm as early as day 1 of carbon-ion irradiation (p < 0.001). The tumors that received X-ray irradiation also significantly increased pO(2) peaks, but as late as day 3. The time course of pO(2) peak appearance in the present study coincides with a previous report where reoxygenation was measured by paired growth delay assay. The pO(2) peaks appeared selectively in peripheral regions of X-ray irradiated tumors, but they appeared rather homogeneously in the tumor after carbon-ion irradiation. It is concluded that carbon-ion irradiation reoxygenated the NFSa fibrosarcomas earlier in time and deeper in space than the X-ray irradiation did.

Animals↗
PubMed1989

Small fenestra stapedotomies with and without KTP laser: a comparison.

The results of 33 small fenestra stapedotomies performed using conventional techniques were compared with the results of 33 stapedotomies performed using the argon or KTP laser. The ossicular chain was reconstructed using a Teflon wire piston of 0.6 mm diameter, and follow-up was at least 1 year. Over-closure of the air-bone gap or closure to within 10 dB was accomplished in 91% of the laser-treated group versus 72% of the conventionally treated group (p less than 0.10). The hearing results were statistically better in the laser group (p less than 0.05). Transient delayed vestibular symptoms, lasting from 1 to 3 weeks, were present in 39% of the laser-treated group and in 12% of the patients treated by conventional techniques (p less than 0.05). The KTP laser stapedotomy, using a micromanipulator mounted on the microscope, is a safe, efficient technique that reduces some of the technical difficulties associated with conventional stapes surgery. The main advantage of the laser is that it enables the surgeon to make an atraumatic, bloodless opening in a fixed or mobile stapes footplate without mechanical manipulation of the stapes. Using a lower wattage to vaporize the footplate and waiting several seconds between laser bursts may decrease the incidence of postoperative vestibular symptoms. The use of the KTP laser in stapes surgery represents a major advance in surgery for otosclerosis.

Fenestration, Labyrinth↗
PubMed1990

Benign lesions of the larynx: should the laser be used?

The use of the carbon dioxide (CO2) laser in the treatment of patients with benign laryngeal lesions, excluding respiratory papillomatosis, has been questioned because of potential adverse thermal effects on surrounding tissue. We question whether wound healing and subsequent quality of voice would be better if the surgeon used the "cold technique" with microlaryngeal instruments. Since the advent, in 1987, of a small-spot (0.3 mm) CO2 laser micromanipulator and more precise microlaryngeal instruments, we have redefined our use of the CO2 laser for benign laryngeal lesions. Over the past 4 years, in a series of 68 consecutive patients with vocal cord nodules, polyps, polypoid changes, or granulomas, the CO2 laser was useful for mucosal micro-flap dissection techniques and for vascular lesions. Smaller pedunculated lesions, such as vocal cord nodules, were more efficiently removed with the new microlaryngeal instrumentation. The combined selective use of a microspot CO2 laser at low-power settings (1 to 3 W), with 0.1-second pulses, and with precise microlaryngeal instruments will give the best results.

Adolescent↗
PubMed1991

Soft-tissue complications of laser surgery for recurrent respiratory papillomatosis.

Twenty-two patients with recurrent respiratory papillomatosis underwent 105 carbon dioxide (CO2) laser microlaryngoscopic and tracheobronchoscopic operations from July 1986 through February 1990. All soft-tissue complications, whether intraoperative or delayed secondary to laser surgery, were retrospectively analyzed. The intraoperative laser-related soft-tissue complication rate was zero. Two of the 22 patients acquired slight unilateral true vocal cord scar tissue and 1 patient developed a small posterior laryngeal web. The delayed soft-tissue complication rate was 13.6%, which compares favorably with published reports of 28.7% and 45%. This low complication rate has resulted from the selection of appropriate CO2 laser emission parameters and the use of the microspot micromanipulator, which help minimize lateral and/or deep thermal damage at the site of laser impact.

Adult↗
PubMed1992

Stapedectomy techniques in residency training.

The KTP-532 laser has decreased the technical difficulty involved in teaching and performing a stapedectomy in our residency program. Use of this laser has resulted in improved hearing and a decreased number of major and minor complications compared to an equal number of large fenestra stapedectomies performed with hand-held instruments. The major disadvantages of the KTP-532 laser are its cost and limited availability, and the inconvenience of a micromanipulator. The laser should not be relied upon entirely in performing a stapedotomy on a thick footplate. The University of Texas Medical Branch experience in training residents in both large and small fenestra stapedectomy is reported.

Adolescent↗
PubMed1989

Hemopoietic stem cells: stochastic differentiation and humoral control of proliferation.

The central feature of hemopoiesis is life-long, stable cell renewal. This process is supported by hemopoietic stem cells which, in the steady state, appear to be dormant in cell cycling. The entry into cell cycle of the dormant stem cells may be promoted by such factors as interleukin-1, interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). Once the stem cells leave G0 and begin proliferation, the subsequent process is characterized by continued proliferation and differentiation. While several models of stem cell differentiation have been proposed, micromanipulation studies of individual progenitors suggest that the commitment of multipotential progenitors to single lineages is a random (stochastic) process. The proliferation of early hemopoietic progenitors requires the presence of interleukin-3 (IL-3), and the intermediate process appears to be supported by granulocyte/macrophage colony-stimulating factor (GM-CSF). Once the progenitors are committed to individual lineages, the subsequent maturation process appears to be supported by late-acting, lineage-specific factors such as erythropoietin and G-CSF. Synthesis of a hemopoietic factor may take place in different cell types and is regulated by multiple factors. The physiological regulator of erythropoiesis is erythropoietin, which, by a feedback mechanism, provides fine control of erythrocyte production. Feedback mechanisms for leukocyte production have not been identified. It is possible that there is no feedback regulator of leukopoiesis. In this model, leukocyte production in the steady state is maintained at a genetically determined level. When an infection occurs, the bacterial lipopolysaccharides may augment the production of interleukin 1 alpha and beta, tumor necrosis factor, macrophage colony-stimulating factor, etc.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

The tumor cells in nodular lymphocyte-predominant Hodgkin disease are clonally related to the large cell lymphoma occurring in the same individual. Direct demonstration by single cell analysis.

Large cell lymphoma (LCL) sometimes occurs concurrently or subsequently in patients with nodular lymphocyte-predominant Hodgkin disease (NLPHD). Although there is evidence of a clonal relationship between LCL and NLPHD, there has been no direct demonstration that the lymphocytic and histiocytic (L&H) cells in NLPHD are related to the tumor cells in LCL. We identified 2 cases of NLPHD with an associated LCL. Single L&H cells, the Reed-Sternberg cell variants in NLPHD, were isolated from immunostained tissue sections by micromanipulation, and the immunoglobulin heavy chain gene (IgH) complementarity determining region (CDR) III of the cells was amplified by the polymerase chain reaction (PCR). The products were compared with those obtained from microdissected LCL cells using polyacrylamide gel electrophoresis and nucleotide sequencing. The IgH CDRIII sequences from the L&H cells were related to each other, but also showed nucleotide substitutions, consistent with a germinal center origin. The sequences from the L&H cells also were related to those from the corresponding LCL cells. We have provided direct evidence through sequence analysis of the IgH CDRIII that the L&H cells are clonally related to the corresponding LCL arising in 2 cases of NLPHD.

Adult↗
PubMed2005

Two regions of mode selection in resonators with biprismlike elements.

A resonator structure in which one reflector is replaced by a biprismlike reflecting surface is investigated theoretically. It is shown that such a modification leads to two regions of parameters, each with different regimes of mode selection. The first region has an improved laser power output because of the nearly flat-top mode shape. In the second region the biprism is inverted, with the result that the main oscillating mode can be the first odd mode. The line singularity contained in such a mode is one example of singular beams that are employed in various fields, such as micromanipulators and advanced high-resolution metrology.

Journal Article↗
PubMed2004

Transformation systems in insects.

Genetic transformation is an important technology that provides unique opportunities to find, isolate, and analyze genes, as well as to create organisms with unique functional characteristics. Insect biologists have been developing genetic transformation technologies that rely extensively on transposable elements. A number of class II transposable elements isolated originally from insects have been converted into broad host range insect gene vectors. Class II transposable elements are particularly amenable to gene vector development, although they suffer from some limitations such as low rates of recombination. Use of these gene vectors requires the physical introduction of the vectors into developing insect embryos by microinjection. Microinjection methods vary to accommodate the unique physical and developmental characteristics of the target insects. All methods rely on the use of fine glass needles in conjunction with micromanipulators and a microscope. A serious constraint on the use of existing systems can be the inefficiency of successfully delivering the gene vectors to the germ cells of the developing embryo. The general method for vector delivery to insect germ cells is described, as well as variations that are useful under some conditions.

Animals↗
PubMed2004

Molecular single-cell PCR analysis of rearranged immunoglobulin genes as a tool to determine the clonal composition of normal and malignant human B cells.

Owing to the nearly limitless diversity of immunoglobulin (Ig) variable-region gene rearrangements, such rearrangements represent ideal clonal markers for B-lineage cells. This chapter describes an approach to isolate single cells from frozen tissue sections by microdissection using a hydraulic micromanipulator and the subsequent amplification of rearranged IgH and Igkappa genes from the cells in a seminested polymerase chain reaction (PCR) approach. The amplification of a priori unknown V-gene rearrangements is made possible by the usage of a collection of V-gene family-specific primers recognizing nearly all V-gene segments together with primer mixes for the J-gene segments. By sequence comparison of V-gene amplificates from distinct cells, the clonal relationship of the B-lineage cells can unequivocally be determined. As a large part of the V-gene rearrangements is amplified, the approach is also useful to address additional issues, such as V-, D-, and J-gene usage and the presence and pattern of somatic mutations.

Animals↗