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Biomedical subjects

Y Toyoda

Publications and source records attributed to Y Toyoda.

At least 163 records · Page 9Linked to original sources
PubMed1995

Association of p34cdc2 and cyclin B1 during meiotic maturation in porcine oocytes.

The relative levels and association of p34cdc2 and cyclin B1 have been determined in pig oocytes during meiotic progression from G2 to metaphase II (MII). Fully grown G2-arrested porcine oocytes contained large amounts of free p34cdc2 and extremely small amounts of p34cdc2-cyclin B1 complex which did not increase in amount during the GV stage. Cyclin B1 is not synthesized in measurable quantities until 23 hr after the start of maturation. During the first metaphase (MI) the amount of both cyclin B1 and the p34cdc2-cyclin B1 complex increased sharply until 35 hr. Thereafter the amount of the p34cdc2-cyclin B1 complex increased again to 45 hr, resulting in higher levels of the complex in MII than in MI. Moreover, three distinct migration forms of p34cdc2 were detected in pig oocytes by immunoblotting with anti-PSTAIRE antibody, and most of the cyclin B1-associated p34cdc2 was detected in the lower band in MI oocytes but migrated in the second band during MII. These results suggest that cyclin B1 levels in porcine oocytes differ from those in starfish, clam, and Xenopus oocytes in which considerably higher concentrations of cyclin B1 appear to be present during the GV stage and that the nature of the association between p34cdc2 and cyclin B1 changes between the first and the second metaphase.

Animals↗
PubMed1995

Acute left atrial thrombus causing cardiogenic shock following mitral valve replacement: report of a case.

We report herein the rare case of a 53-year-old woman who developed cardiogenic shock due to an acute left atrial thrombus following replacement of the mitral valve. A definitive diagnosis was not able to be made using precordial echocardiography because of the broad, flat shape of the thrombus; however, transesophageal echocardiography imaged the thrombus in detail. The patient was initially stabilized by percutaneous cardiopulmonary support after which a thrombectomy was successfully performed.

Echocardiography, Transesophageal↗
PubMed1995

Tissue and subcellular distribution of glucokinase in rat liver and their changes during fasting-refeeding.

The distribution of glucokinase in rat liver under both normal feeding and fasting-refeeding conditions was investigated immunohistochemically. Under normal feeding conditions, glucokinase immunoreactivity was observed in both nuclei and cytoplasm of parenchymal cells. The nuclei were stained intensely and evenly, whereas the cytoplasm showed weak immunoreactivity of different degrees of staining intensity depending on the location of the cells. The cytoplasm of perivenous hepatocytes was stained more intensely, though not so much more, than that of periportal hepatocytes. The cytoplasm of hepatocytes surrounding the terminal hepatic venule (THV), of hepatocytes surrounding the portal triad, and of some other hepatocytes showed a stronger immunoreactivity than that of residual hepatocytes. The nuclear immunoreactivity in hepatocytes surrounding the portal triad and in some other hepatocytes was weak or absent, and positive immunoreactivity was detected at the plasma membrane of some of these cells. After 72 h of fasting, glucokinase immunoreactivity was markedly decreased in all hepatocytes. After the start of refeeding, the cytoplasmic immunoreactivity began to increase first in the parenchymal cells surrounding the THV and extended to those in the intermediate zone followed by those in the periportal zone. In contrast, the increase in nuclear immunoreactivity started in hepatocytes situated in the intermediate zone adjacent to the perivenous zone and then extended to those in the perivenous zone followed by those in the periportal zone. Hepatocytes surrounding either THV or portal triad showed a distinctive change in immunoreactivity during the refeeding period. After 10 h of refeeding, strong immunoreactivity was observed in both the cytoplasm and the nuclei of all hepatocytes, and appreciable glucokinase immunoreactivity was detected at the plasma membrane of some hepatocytes. These findings are discussed from the standpoint of a functional role of glucokinase in hepatic glucose metabolism.

Animals↗
PubMed1995

Cryopreservation of wild mouse spermatozoa.

Spermatozoa of wild mice from China, Czechoslovakia, Denmark, India, Japan and Switzerland were frozen and stored at -196 degrees C. After thawing, intact oocytes were inseminated in vitro with relatively high motility frozen-thawed mouse spermatozoa from Czechoslovakia, Denmark and India, while oocytes with a partially dissected zona were inseminated with low motility frozen-thawed spermatozoa from China, Japan and Switzerland. Embryos developing to the 2-cell stage from oocytes fertilized with frozen-thawed spermatozoa were transferred to the oviducts of female recipients on the first day of pseudopregnancy (day when a vaginal plug was confirmed). Successful embryo development to the 2-cell stage was 46 to 67%. Offspring resulted from 17 to 51% of these transferred 2-cell embryos.

Journal Article↗
PubMed1995

Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor exhibit lung pathology and impaired immune response, while beta IL3 receptor-deficient mice are normal.

The receptors for IL-3, GM-CSF, and IL-5 share a common beta subunit (beta c), and mice have an additional IL-3 beta subunit (beta IL3). We have independently generated mice carrying null mutations of each molecule. beta c mutant bone marrow showed no response to GM-CSF or IL-5, whereas IL-3 stimulation of beta c or beta IL3 mutant bone marrow was normal. beta c mutant mice showed lung pathology consisting of lymphocytic infiltration and areas resembling alveolar proteinosis, and also exhibited low basal numbers of eosinophils. Infection of beta c mutant mice by Nippostrongylus brasiliensis resulted in the absence of blood and lung eosinophilia. Animals repopulated with beta c mutant bone marrow cells showed slower leukocyte recovery and reduced eosinophil numbers. These data define the role of beta c in vivo, and show a phenotype that is likely to be the cumulative effect of loss of GM-CSF and IL-5 stimulation.

Animals↗
PubMed1995

Activation of mitogen-activated protein kinase during meiotic maturation in porcine oocytes.

To investigate the involvement of mitogen-activated protein kinase (MAP kinase) in meiotic maturation of porcine oocytes, we assayed MAP kinase activity using myelin basic protein (MBP) as a substrate. MAP kinase activity was low during the germinal vesicle stage, 0-20 h of culture. An abrupt increase was observed at metaphase I (30 h of culture), and activity remained significantly higher than that at 0 h until 50 h of culture, with a transient slight decrease at the time of first polar body extrusion (40 h). Detection of the kinase activity by an in-gel phosphorylation assay confirmed that the 42 and 44 kDa MAP kinases were significantly activated in 45 h cultured oocytes but not in 0 h oocytes, and just slightly in 20 h oocytes. In immunoblotting, however, the 42 and 44 kDa bands were detected in 0, 20 and 45 h cultured oocytes. Furthermore, the signal strength of the two bands did not change during the period of culture, but shifted up to 45 h, indicating that the activation of MAP kinase depended not on the synthesis but on the phosphorylation of this enzyme. These results suggest that the activation of MAP kinase is involved in the regulation of meiotic maturation of porcine oocytes, and especially in the regulation after germinal vesicle breakdown.

Animals↗
PubMed1995

Developmental ability of zona-free mouse embryos is influenced by cell association at the 4-cell stage.

Zona-free embryos at the 4-cell stage were classified into four types according to the total points of contact between the 4 blastomeres. Types A, B, C, and D had 3, 4, 5, and 6 points of contact, respectively. Embryos of these types were cultured individually in 20 microliters of Whitten's medium. Blastocysts derived from types A and D embryos at the 4-cell stage were transferred and observed on Day 17 of gestation. The number of live fetuses was significantly lower for type A than for type D embryos (p < 0.05). In spite of the removal of the zone pellucida, both types A and D blastocysts had cell numbers similar to those in zona-intact control embryos at 96 h after insemination. However, type A embryos had fewer (p < 0.05) inner cell mass (ICM) cells than did either the zona-intact control or type D embryos. Type D embryos did not have significantly fewer ICM than did the zona-intact control. Our data show that the cell association of zona-free embryos at the 4-cell stage influences differentiation of cells within the ICM and thus affects subsequent embryonic development. We suggest that one role of the zona pellucida after fertilization, in addition to protecting the preimplantation embryo, is to maintain the cell arrangement of 4-cell stage embryos to ensure subsequent successful cell lineage.

Animals↗
PubMed1995

Synchronous sperm penetration of zona-free mouse eggs in vitro.

To synchronize sperm penetration of zona-free eggs immediately after insemination, zona-free eggs preloaded with Hoechst-33342 were inseminated under various conditions. Insemination was mostly conducted at 10 sperm/microliter. In preliminary experiments, fatty acid-free BSA (FAF) was more satisfactory for sperm penetration than fraction V BSA, and FAF was used in the following experiments. Only 26% of zona-free eggs were fertilized at 10 min after insemination when the eggs were inseminated immediately after zona removal and preloading. However, egg preincubation significantly improved the penetration rate (1 h preincubation: 63%, 2 h preincubation: 82% penetrated 10 min after insemination). Some eggs preincubated for 2 h were already penetrated at 3 min (7%), and the rate gradually increased in a time-dependent manner (3 min: 7%, 5 min: 30%, 10 min: 80%). The rate further improved as the sperm concentration was increased; the maximal level was obtained at 160 sperm/microliters. At 160 sperm/microliters, 54% of the eggs were penetrated at 3 min and 78% were at 5 min. These results indicate that it is possible to synchronize the sperm entry and that not only sperm but zona-free eggs should be preincubated before insemination. These data are considered valuable for investigating early events in fertilization.

Animals↗
PubMed1995

Increased calcium sequestration by sarcoplasmic reticulum in small muscular arteries in young spontaneously hypertensive rats.

1. Sarcoplasmic reticulum (SR) in small resistance arteries plays a role in the regulation of the cytosolic free calcium concentration by sequestration of calcium from cytoplasm. 2. To examine the contribution of calcium (Ca2+) sequestration by the SHR to both contraction and relaxation in young spontaneously hypertensive rats (SHR), we measured evoked tension before and after depletion of SR Ca2+ stores in the rings of the first branch of superior mesenteric artery in 5 week old SHR and age-matched Wistar-Kyoto (WKY) rats. Contractile responses were induced by 40 mmol/L potassium and 10 mmol/L caffeine before and after the treatment with ryanodine or thapsigargin, which depletes SR calcium stores. 3. The magnitude of potassium-induced contraction was almost the same in both strains. 4. Ryanodine and thapsigargin did not affect the resting tension and almost decreased caffeine-induced contraction in both strains. 5. After the treatment with ryanodine or thapsigargin, the magnitude and the rate of potassium-induced contraction were augmented greatly in SHR, but not in WKY. 6. The relative relaxation rate after exposure of potassium was significantly slowed in SHR by ryanodine or thapsigargin, but only slightly in WKY. 7. These results suggest that Ca2+ sequestration by the SR in SHR was greater than in WKY. Therefore, it is concluded that SR plays an important role in preventing the development of hypertension in SHR via a buffering effect on the elevation of cytosolic free Ca2+.

Animals↗
PubMed1995

Sex-linked differences in developmental potential of single blastomeres from in vitro-fertilized 2-cell stage mouse embryos.

Single blastomeres were isolated from 2-cell mouse embryos and analyzed for their sex using the partially deleted Y chromosome as a marker. Sex identification of 83% of the embryos was achieved with conventionally Giemsa-stained chromosome preparations. The other half-embryos were cultured individually and were transferred to pseudopregnant recipient females. The implantation rate was significantly higher in the male half-embryos. Although 6% of the half-embryos were male. The male half-embryos contained more blastomeres in the blastocyst stage than female ones. These facts suggests that the potential for the development of male half-embryos into fetuses is different from that of female half-embryos.

Animals↗
PubMed1995

Serum factor(s) stimulating cumulus expansion in porcine oocyte-cumulus complexes matured and fertilized in vitro.

We have investigated the factor(s) present in serum that stimulate cumulus expansion (accumulation of hyaluronic acid in the intercellular space of cumulus cells) and male pronucleus (mPN) formation in porcine oocytes matured and fertilized in vitro. After 24 hr of culture, oocyte-cumulus complexes that matured in the serum of pig, cow, rabbit and rat showed an expansion of the surrounding cumulus cell matrices. The cumulus expansion stimulating effect is not species-specific. Porcine serum was fractionated by ultracentrifugation at 220,000 x g at 10 degrees C for 48 hr, resulting in four fractions. The oocyte-cumulus complexes that matured in the 2nd fraction exhibited a marked expansion of the cumulus cell layers, including the corona radiata. Complexes cultured in the remaining fractions, however, showed very little or no expansion. There was no expansion of the oocytectomized complexes matured in the 2nd fraction. Serum was not effective in the formation of the male pronucleus. The active factor(s) was heat stable at 100 degrees C for 30 min and showed a relatively strong activity at a molecular weight of > 1.0 and < 6.5 kDa. These results indicate that serum contains a low molecular weight, heat stable factor(s) that acts on the oocyte to stimulate cumulus expansion.

Animals↗
PubMed1995

Role of protein kinase C in relationship between Ca2+ and contractile elements in rat alpha-toxin-permeabilized mesenteric artery.

Phorbol ester, which activates protein kinase C (PKC), modulates vasoconstrictor-induced tension in vascular smooth muscle. Recently, Staphylococcal aureus alpha-toxin, which produces too small pores in the plasma membrane to allow passage of proteins, such as PKC, is used to investigate the signal transduction system in vascular smooth muscle cells. In order to elucidate the role of PKC on vascular smooth muscle contraction, we examined whether PKC activation influences the relationship between intracellular Ca2+ ([Ca2+]i) and tension in Wistar rat superior mesenteric artery (SMA) using vascular smooth muscle permeabilized with Staphylococcal alpha-toxin. [Ca2+]i was clamped at specified values (10(-8.5)-10(-4) mol/L) using EGTA-Ca2+ buffer. In alpha-toxin non-treated rings of SMA, isometric tension was evoked by 10 mmol/L caffeine and 10-30 mmol/L external potassium (high K+) in the absence or presence of phorbol 12, 13-dibutyrate (PDBu), a PKC activator, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and staurosporine (PKC inhibitors). PDBu significantly augmented caffeine- and high K(+)-evoked contractions. H-7 and staurosporine significantly attenuated caffeine- and high K(+)-evoked contractions augmented by PDBu. Moreover, H-7 significantly suppressed high K(+)-induced contraction in the absence of PDBu. In alpha-toxin permeabilized artery, PDBu shifted the [Ca2+]i-force relationship curve to the left. These results suggest that PKC activates vascular smooth muscle contraction by increasing the sensitivity of the contractile apparatus to Ca2+.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

17 alpha-Hydroxyprogesterone suppresses neovascularization induced by HPLC-purified ovarian hyaluronic acid-like glycosaminoglycan in mice.

HPLC-purified glycosaminoglycans (hpGAG) prepared from extracts of non-luteal mouse (JcL:ICR strain) ovaries were assayed for neovascularization by implanting Elvax films, containing test samples, on the lateral wall of the sheath of m. rectus abdominis in adult female mice of the same strain. Neovascularization occurred in a dose-dependent manner, and was characterized by capillary outgrowth extending into the tissue surrounding the implant. The single major peak of purified GAG on a column of TSK gel DEAE got out of order after treatment with streptococcal hyaluronidase or nitrous acid. The activity of this fraction was also greatly reduced when treated with streptococcal hyaluronidase or nitrous acid. When hpGAG was embedded in the implant with 17 alpha-hydroxyprogesterone at a dose of 20 micrograms/film, neovascularization induced by means of hpGAGs was suppressed. Progesterone at a dose of 50 micrograms/film did not suppress the neovascularization induced by ovarian hpGAG. These findings suggest that 17 alpha-hydroxyprogesterone suppresses the angiogenic activity of hyaluronic acid-like hpGAG in the ovary.

17-alpha-Hydroxyprogesterone↗
PubMed1995

In vitro fertilization and embryo development of Japanese field voles (Microtus montebelli).

Optimal conditions for in vitro fertilization of Japanese field voles (Microtus montebelli) were analysed. The medium used was a modified Krebs-Ringer bicarbonate devised for in vitro fertilization in rats. Ovulated eggs and epididymal spermatozoa were co-incubated in vitro at 37 degrees C under 5% CO2 in air for 6 h, and the eggs were fixed with 2.5% (w/v) glutaraldehyde, stained with 0.25% (v/v) acetolacmoid and examined for evidence of fertilization at the pronuclear stage. Although the fertilization rate with spermatozoa preincubated at 1-2 x 10(8) cells ml-1 for 2 h was very low (1-13%), it was significantly increased (43-51%, P < 0.05) when spermatozoa were preincubated at a lower concentration (1-2 x 10(7) cells ml-1). Furthermore, the fertilization rate was significantly higher with 1 mmol hypotaurine l-1 (74.0%) than without hypotaurine (44.4%, P < 0.05). Fertilization rates of spermatozoa preincubated at 1-2 x 10(7) cells ml-1 for 0.5 or 2 h were similar (69.0% and 73.6%), but a longer preincubation (10 h) resulted in a significantly lower fertilization rate (56.8%, P < 0.01). Vole spermatozoa preincubated for 2 h penetrated the zona pellucida 2 h after insemination, and the sperm heads became decondensed 3 h after insemination. At 6 h after insemination, male and female pronuclei were found in most penetrated eggs. When the eggs were left in the fertilization medium without washing and cultured for 96 h after insemination, they developed to two-cell (82.6%), four-cell (60.9%), eight-cell (23.2%) and morula/blastocyst (8.7%) stages in modified Krebs-Ringer bicarbonate supplemented with 1 mmol hypotaurine l-1.

Animals↗
PubMed1995

Decrease of histone H1 kinase activity in relation to parthenogenetic activation of pig follicular oocytes matured and aged in vitro.

Changes of histone H1 kinase activity before and after electrical stimulation were connected with the ability of cytoplasm of pig oocytes to be activated parthenogenetically when matured and aged in vitro. Cumulus-oocyte complexes were collected from prepubertal gilts and cultured in a modified Waymouth's MB752/1 medium. The first mature oocytes were observed after 30 h of culture. After 36 h of culture, about 65% of oocytes had matured (reached metaphase II stage with the first polar body). When oocytes matured after 36 h of culture were stimulated with an electric pulse and subsequently cultured for 10 h, only 7% became parthenogenetically activated (formation of a female pronucleus). When oocytes matured for 60 h and 72 h underwent the same treatment, significantly more became activated parthenogenetically (46% and 57%, respectively). Oocytes matured for 72 h but not stimulated electrically also exhibited high spontaneous parthenogenetic activation (24%). Activation of oocytes resulted either in the formation of a female pronucleus(ei) or in fragmentation of oocytes. Fragmentation in stimulated and nonstimulated oocytes increased significantly after 72 h of culture (37% and 18%, respectively). Histone H1 kinase activity in immature oocytes at the germinal vesicle stage was low (17.2 fmol h-1 per oocyte). However, when oocytes were cultured for 36 and 48 h, histone H1 kinase activity increased significantly (168.2 and 138.5 fmol h-1 per oocyte, respectively). Prolonged culture (60 h and 72 h) resulted in a significant decrease in histone H1 kinase activity (94.3 and 49.3 fmol h-1 per oocyte, respectively). When oocytes cultured for up to 72 h were electrically stimulated, histone H1 kinase activity in activated oocytes (oocytes that formed a female pronucleus and fragmented oocytes) was significantly lower (24.7 mol h-1 per oocyte) than that in nonactivated oocytes (99.9 mol h-1 per oocyte). The present data clearly indicate that the gradual decrease of histone H1 kinase activity is correlated with ageing of oocytes matured in vitro and with their ability to be parthenogenetically activated.

Animals↗