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Biomedical subjects

H Kanagawa

Publications and source records attributed to H Kanagawa.

At least 37 records · Page 2Linked to original sources
PubMed1997

Developmental ability of mouse late 2-cell stage blastomeres fused to chemically enucleated oocytes in vitro.

The effects of different concentrations of etoposide and cycloheximide (ETO-CHXM), used for chemical enucleation of mouse oocytes, on polar body extrusion and chromatin expulsion were tested. The developmental ability of blastomeres of late 2-cell stage embryos fused to chemically enucleated oocytes of different ages or cytoplasts from different sources was also examined in vitro. Metaphase I oocytes cultured in different concentrations of ETO-CHXM (10-50 micrograms/ml/each) extruded polar bodies at rates similar to those cultured without ETO-CHXM (58.5-65.9% and 64.6%, respectively). However, low percent of the oocytes (1.7-6.2%) expressed signs of meiotic perturbation, which was manifested by blebbing of the cytoplasmic membrane and extrusion of two or more polar body-like fragments. Twenty-three percent of the chemically enucleated oocytes cultured in ETO-CHXM-free medium spontaneously fused to their polar bodies. The rates of total chromatin expulsion were similar when ETO-CHXM concentrations were 36 and 50 micrograms/ml (93.5 and 98%, respectively). The results also showed that the cleavage rates of reconstituted embryos were significantly (P < 0.001) affected by the age of the chemically enucleated oocytes. Cytoplasts of bisected oocytes that matured in vivo supported the development of 31.7% of the reconstituted embryos to the blastocyst stage. However, both cytoplasts of chemically enucleated oocytes and in vitro matured oocytes did not support the development to the blastocyst stage. A high percentage (85.5%) of the reconstituted embryos with chemically enucleated recipients displayed abnormality of the metaphase plate. These results suggest that concentrations of etoposide between 36 and 50 micrograms/ml are optimum for enucleation of mouse oocytes. Furthermore, increasing the age or reducing the cytoplasmic volume of the chemically enucleated oocytes did not improve the development of the reconstituted embryos to the blastocyst stage.

Animals↗
PubMed1997

In vitro viability of mouse 8-cell embryos vitrified in a simple solution of ethylene glycol.

A study was made to determine if ethylene glycol (EG) can be used in a simple solution to vitrify mouse 8-cell embryos and to determine the critical factors that affect its success. Mouse 8-cell embryos were vitrified after exposure to 2M and 7M EG prepared in Dulbecco's phosphate buffered saline (PBS) with 10% heat-inactivated calf serum (CS). Mouse 8-cell embryos exposed to 2M EG for 2, 5 and 10 min, and to 7M EG for 2 and 5 min had survival rates similar to the untreated controls (93.3-100%). No significant difference in their survival rates in vitro was observed. Higher room temperatures (> 24 degrees C) at exposure before cooling resulted in poor development rates to the blastocyst stage. The survival rates of embryos vitrified after 2 min in 7M EG at 18-22 degrees C room temperature did not differ significantly from the control, but embryos vitrified after 5 min had significantly lower survival rates (p < 0.0001). In conclusion, effective vitrification of mouse 8-cell embryos can be achieved by initial exposure to 2M EG for 2-10 min followed by equilibration in 7M EG for 2 min at 18-22 degrees C room temperature. This study has shown that 7M EG in PBS with 10% CS is sufficient to provide cryoprotection of vitrified mouse 8-cell embryos and that exposure of the embryos to the vitrification solution at temperatures over 24 degrees C is critical to their subsequent development in vitro.

Animals↗
PubMed1996

Post-implantation development of demi-embryos and induction of decidual cell reaction in mice.

Mouse demi-embryos that developed from bisected morulae were transferred to recipients. The eu-blastocysts (distinct inner cell mass and well-developed trophectoderm) contained cells equal to 51% of the controls that developed from zona-free morulae. The rate of decidual cell reaction induced by the eu-blastocysts was not significantly different from that of the controls, but the size of the deciduum containing the egg cylinder was significantly smaller on Day 5.5 of pregnancy (P < 0.001). A significant increase in embryonic loss was observed from Day 7.5 to Day 9.5 in the eu-blastocysts (P < 0.05), while the controls exhibited no significant difference. Although the embryos from the eu-blastocysts showed retardation of developmental stages and decreased size, they attained normal stages and size regulation up to 90% of that of the control on Day 10.5. The pseudo-blastocysts (poorly developed inner cell mass enclosed by trophectoderm) contained cells equal to 25% of those of the controls and showed less than a 10% developmental rate to the egg cylinder stage. The trophectodermal vesicles (no enclosed cells) and nonintegrated forms (disorganized clusters of cells) contained cells less than 18% of those of the controls. They showed lower rates of decidual cell reaction than those in the controls (P < 0.05), and no egg cylinder was found in the deciduum. The results indicate that a severe decrease in the number of embryonic cells affects the regulation of embryonic development and decidual cell reaction in the uterus.

Journal Article↗
PubMed1996

Evidence for the presence of macrophage migration inhibitory factor in murine reproductive organs and early embryos.

Cytokines appear to play a critical role in the establishment of early pregnancy. In this study, we examined the mRNA expression of murine macrophage migration inhibitory factor (MIF), one of the cytokines activating macrophages, in the murine reproductive system by Northern blot analysis. It revealed that MIF mRNA was expressed in the ovary, the oviduct and the uterus during the pre-implantation period and all stages of the estrus cycle. The histological localization of MIF in the uterus was examined by an immunohistochemical method, which revealed that MIF was mostly present in the tunica muscularis. It was, moreover, demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) that MIF mRNA was expressed in the ovulated oocytes, zygotes, 2-cell embryos, 8-cell embryos and blastocysts. MIF is known to be present in T-lymphocytes and monocytes/macrophages; however, it was revealed by the present study that MIF mRNA is expressed in the reproductive organs and the early stages of embryos. Considering these results, it s suggested that MIF plays an important role in the establishment of pregnancy.

Animals↗
PubMed1996

Treatment of gastric fundal varices by balloon-occluded retrograde transvenous obliteration.

Although less common than oesophageal varices in portal hypertension, gastric fundal varices carry a higher mortality rate when they rupture. They are less amenable to sclerotherapy. We have developed a minimally invasive balloon-occluded retrograde transverse obliteration (B-RTO) procedure to treat gastric fundal varices. B-RTO involves inserting a balloon catheter into an outflow shunt (gastric-renal or gastric-vena caval inferior) via the femoral or internal jugular vein. Blood flow is then blocked by inflating the balloon, and 5% ethanolamine oleate iopamidol is injected in a retrograde manner. The embolized gastric varix subsequently disappears. B-RTO was performed in 32 patients with gastric varices. Follow-up endoscopies were performed at intervals of 2-4 months for an average observation period of 14 months. Eradication of the varices has been confirmed in 31 of 32 patients. No recurrence occurred in any patients in the follow-up period. There were no significant changes in liver function after the procedure. We conclude that B-RTO is a safe and effective procedure for the treatment of gastric fundal varices.

Adult↗
PubMed1996

Influence of oocyte aging on developmental ability of reconstituted embryos produced from oocyte cytoplast and single blastomeres of two-cell stage embryos.

The present study was conducted to investigate the influence of aging of recipient oocyte on the developmental ability of reconstituted mouse embryos produced from the cytoplast of oocytes and single blastomeres of early or late 2-cell stage embryos by electrofusion. Oocytes were obtained at 14 (newly ovulated oocytes), 18, 22 (oocyte that time passed after ovulation; aged oocyte) hr after hCG injection and oocyte cytoplast was produced by manual enucleation using a fine glass needle under the dissecting microscope. The aging of the oocytes significantly influenced on the fusion rate of the reconstituted embryos (14 hr: 42.7-47.2% vs. 22 hr: 75.3-77.6%). Similarly, the cleavage rate of reconstituted embryos increased with aging of the oocytes (14 hr: 50.8-56.3% vs. 22 hr: 82.2-90.7%). The percentage of reconstituted embryos produced from cytoplast of aged oocytes (22 hr post hCG) and single blastomeres of late 2-cell stage embryos developing to the blastocyst (20.8%) was significantly higher than that of reconstituted embryos produced by other combinations (2.0-8.2%: P < 0.01). Although cell cycle stage of donor nuclei influenced to developmental ability of reconstituted embryos, these results are probably related to the aging of the oocytes since aged oocytes can be activated more easily by electrical stimulation than newly ovulated oocytes.

Animals↗
PubMed1996

Development of in vitro matured/fertilized bovine embryos in a chemically defined medium: influence of oxygen concentration in the gas atmosphere.

The influence of oxygen concentrations in the gas atmosphere on the development of IVM/IVF bovine embryos was determined by culturing them in a microdrop of modified synthetic oviduct fluid medium supplemented with amino acids, insulin and PVA (mSOFai). After removing the cumulus cells at 18 hr post-insemination, presumptive zygotes were cultured in mSOFai for 104-106 hr under 5% CO2 with various O2 concentrations (2.5 to 20%). Reduced O2 (5-10%) improved the development to the morula stage, and 5% O2 gave the highest development. In the next experiment, morulae obtained after 102-104 hr of culture, were further cultured for 50 hr in mSOFai with 2mM glucose under 5 and 20% O2. An increase in the mean cell number in blastocysts, but not in the frequency of blastocysts, was observed under 5% O2. In the third experiment, zygotes were cultured for 152-154 hr in mSOFai under 5 and 20% O2, or cocultured with bovine oviduct epithelial cells in TCM199 + 10% FCS under 5% CO2 in air. Percentage of blastocysts for mSOFai in 5% O2 doubled to that for 20% O2, and was similar to that for coculture. Moreover, mean cell number in the blastocysts for mSOFai in 5% O2 was significantly higher than that for coculture. Results demonstrate that oxygen concentration critically affects embryonic development through zygotes to blastocysts, and suggest that around 5% 02 is optimal. It also indicates that bovine zygotes can be cultured up to the blastocyst stage using a chemically defined medium with rates similar to those of a conventional coculture system.

Amino Acids↗
PubMed1996

The role of macrophage migration inhibitory factor in pregnancy and development of murine embryos.

Cytokines appear to play a critical role in the establishment of pregnancy. In this study, we measured serum and amniotic fluid concentrations of macrophage migration inhibitory factor (MIF) on the maternal hosts in murine pregnancy by enzyme-linked immunosorbent assay. The MIF concentrations in the murine maternal circulation in pregnancy ranged from 10 to 200 ng/ml and the level of MIF was lowest on Day 3 of pregnancy. On Day 4, the level of MIF recovered to the non-pregnant level and remained unchanged after implantation. MIF was also detected in the amniotic fluid, but the concentration was not parallel with that of MIF in the maternal circulation. MIF in the amniotic fluid, possibly produced by embryos, may have an autocrine function for embryo development. The temporary decrease of MIF in the maternal circulation may not directly relate to the development of embryos; however, it appears as an early sign of pregnancy for the maternal host.

Amniotic Fluid↗
PubMed1995

Effects of water quality on in vitro fertilization and development of bovine oocytes in protein-free medium.

Examination was made of the effects of water quality in medium preparation on fertilization and early development of bovine in vitro matured (IVM) oocytes in a protein-free medium. The IVM oocytes were inseminated and cultured for 7 d in protein-free media prepared with 4 different types of water preparations: tap, deionized, twice-distilled, and purified water using the Milli-Q system (Milli-Q water). High frequencies (70 to 83%) of normal fertilization were obtained in media prepared with all types of water. However, the frequency of development to the blastocyst stage in media prepared with Milli-Q water (31 +/- 3%) was significantly higher than with the 3 other types of water (11 to 13%). Moreover, the effects of storage period of Milli-Q water on early development of bovine embryos was also examined. The frequency of development to the blastocyst stage in media prepared with Milli-Q water immediately after preparation (fresh Milli-Q water; 35 +/- 4%) was significantly higher than for Milli-Q water stored for 1 wk (18 +/- 4%) or 2 wk (18 +/- 3%). Effects of commercially available purified water on early development of bovine embryos were also examined. The frequency of development to the blastocyst stage in media prepared with Milli-Q water (33 +/- 5%) was significantly higher than for purified water purchased from 3 different suppliers (Brand A; 21 +/- 6%, Brand B; 21 +/- 2%, Brand C; 21 +/- 4%). Each water sample was analyzed by the measurement of electrical conductivity, organic compounds and/or inorganic ion and endotoxin concentrations to evaluate purity. Fresh Milli-Q water showed the lowest level of electrical conductivity and contained the lowest concentration of organic compounds. These results indicate that in vitro fertilization of bovine oocytes is not affected by the water quality in the preparation of medium; however, early development of bovine embryos is seriously affected by the purification method and the storage period of water used for medium preparation.

Journal Article↗
PubMed1995

Attachment, outgrowth, invasion and formation of the egg cylinder in mouse half embryos in vitro.

To investigate the effect of the decrease in the number of embryonic cells on the development and morphogenesis of mouse embryos at the peri-implantation stage, half embryos which developed from bisected morulae were morphologically classified into eu-blastocysts (eu-blasts), pseudo-blastocysts (pseudo-blasts), trophectodermal vesicles (TVs) and non-integrated forms (NIFs) and then cultured on plastic substratum, uterine-epithelial-cell monolayer and type I collagen gel. When half embryos were cultured on plastic substratum and cell monolayer, the rates of attachment and trophoblast outgrowth in the eu- and pseudo-blasts were not significantly different from those of the controls. The TVs and NIFs showed significantly lower rates of outgrowth than the controls (P < 0.01). When half embryos were cultured on type I collagen gel, no significant difference was observed in the rate of primitive endoderm formation between the eu-blasts and controls after 36 hr of culture. In the eu-blasts, however, the developmental rate to the egg cylinder at stage 8 was significantly lower than in the controls after 72 hr (P < 0.05). The pseudo-blasts revealed significantly lower rates of endoderm formation and development to the egg cylinder than the controls after 36 and 72 hr, respectively (P < 0.05). In the TVs and NIFs, the rates of outgrowth were significantly lower than those of the controls (P < 0.05) and no egg cylinder was observed. The invasion of type I collagen gel by the cytoplasmic protrusions of trophoblast cells was observed regardless of the type and developmental stage of the embryos. The results show that a decrease in the number of embryonic cells affects the formation of the primitive endoderm and the development to the egg cylinder in vitro.

Animals↗
PubMed1995

Insulin and insulin-like growth factor-I (IGF-I) stimulate the development of bovine embryos fertilized in vitro.

To study the effects of insulin and insulin-like growth factor-I (IGF-I) on early development of bovine embryos, one-cell embryos were cultured in a chemically defined, protein-free medium: modified synthetic oviduct fluid. The addition of 10 micrograms/ml insulin or 20 ng/ml IGF-I increased the percentage of morulae at Day 5 (5 days after insemination). The cell number of blastocysts which developed after 2 days culture of morulae obtained at Day 5 was increased by supplementation with insulin or IGF-I. The combination of insulin and IGF-I had neither additive nor synergistic effect on the embryonic development. These results clearly show that insulin and IGF-I improve the in vitro development of bovine embryos.

Animals↗
PubMed1995

Stimulatory effects of insulin on the development of bovine embryos fertilized in vitro.

To obtain information about the effects of insulin on the development of bovine embryos, fertilized bovine embryos in vitro were cultured in a chemically defined, protein-free medium: modified synthetic oviduct fluid (mSOF) supplemented with 1 mg/ml polyvinyl alcohol (PVA). The percentage of embryos reaching the morula stage was not affected by the addition of 5 micrograms/ml insulin alone, but insulin showed a beneficial synergistic effect with amino acids (AAs) on the embryonic development to morula. Supplementation of insulin (1-100 micrograms/ml) with mSOF+AAs did not improve the percentage of embryos developing to the blastocyst stage. However, a significant increase in cell numbers of blastocysts was observed with the addition of 1 mM glucose and 1 or 10 micrograms/ml insulin from the morula stage. These results show that insulin has a beneficial effect on the preimplantation development of bovine embryos in the presence of AAs and/or glucose, and suggest that insulin improves embryonic development by stimulating AAs transport and/or glucose uptake.

Animals↗
PubMed1995

Influence of nocodazole on the development of donor blastomeres from 16-cell stage bovine embryos in nuclear transfer.

The aim of the present study was to establish a reliable procedure with nocodazole treatment for the synchronous cleavage of blastomeres of bovine embryos used as nuclear donors for nuclear transfer. Sixteen-cell stage embryos derived from in vitro-maturation, fertilization and culture were used. In three initial experiments, embryos were incubated in mTCM-199+FCS with various concentrations (0-20 microM) of nocodazole under 5% CO2 in air. The concentrations required to arrest the blastomeres in the mitotic phase were examined. The effects of 10 microM nocodazole were also examined by observation of the division rate of blastomeres after the removal of nocodazole. Ninety percent (90%) of the blastomeres were arrested in the mitotic phase when embryos were exposed to 10 and 20 microM nocodazole. Exposure to 10 microM nocodazole had the highest blastomere-cleavage rate (47%). When the exposure period to 10 microM nocodazole was prolonged to 36 hr, the division rate of the blastomeres decreased. Furthermore, the effects of 2 culture conditions (mTCM-199 under 5% CO2 in air vs modified synthetic oviduct fluid medium under 5% CO2, 5% CO2 and 90% N2) were compared on the division rate of blastomeres of embryos exposed to 10 microM nocodazole for 12 hr. When the embryos were exposed to nocodazole in mSOF, the division rate of blastomeres was improved to about 60%. The blastomeres produced by this treatment condition were used as nuclear donors and the developmental potential of the reconstituted embryos was investigated. The developmental rate to the blastocyst stage was 30.1% (58/193). Five embryos were transferred to 5 recipient cows and 2 of the 5 recipients (40%) became pregnant. Subsequently, one normal calf was born.

Animals↗
PubMed1995

Evidence for the production of platelet-activating factor by murine embryos and its putative role in the maternal physiology.

Thrombocytopenia has been shown to be an initial maternal response to conception. We investigated a potent bioactive lipid, platelet-activating factor (PAF), as one of the candidates derived from embryos to induce the decrease of platelet counts in maternal blood. First, we examined the potential of murine embryos to liberate PAF. In brief, twenty to thirty murine two-cell embryos of C57BL/6 mice were cultured in Whitten's medium for 24 h. The supernatant of the medium was collected and the total was extracted. After developing the lipid on thin-layer chromatography, the area corresponding to authentic PAF was scraped off and the lipid was extracted. The embryo-derived PAF was quantified by platelet aggregation using rabbit whole blood, and the presence of PAF was further confirmed by the addition of a PAF-receptor antagonist, SRI63-441. The results showed that a significant amount of PAF (ca. 10 ng/ml) was recovered from the lipid extract, whereas little platelet-aggregating activity was observed in the presence of SRI63-441. Second, the physiological function of murine platelets was examined using authentic PAF. When PAF (up to 83 micrograms/ml) was added to the murine platelets suspended in the whole blood, it neither induced platelet aggregation nor reduced the number of platelets. Considering these results, it is considered that PAF released from the embryos does not directly act on maternal platelets, but transduces its signal to the maternal host via some other bioactive substance.

Animals↗
PubMed1995

Electrofusion of zona-free mouse embryonic cells in electrolytes and their development in vitro.

The influence of increasing the physical electrofusion parameters, direct current (DC) pulse strength, pulse duration, pulse number, alternating current (AC) voltage and alignment time, in electrolytes on the rates of fusion, degeneration and development of zona-free mouse 2-cell embryos were examined. Furthermore, the effects of physiological saline and mannitol as fusion media and various mouse strains were also evaluated. Dulbecco's phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum was used as the main fusion solution. A significant increase in the rate of fusion (P < 0.05) was obtained by increasing pulse strength from 30 to 300 V/mm. The embryos fused at the pulse strengths of 30 to 70 V/mm had significantly higher development rates to blastocysts compared with those fused at 100 to 300 V/mm (P < 0.05). There were no significant differences in the rates of fusion, degeneration and development to blastocysts when the pulse duration was increased from 30 to 90 microseconds. Although fusion rates were increased (P < 0.05) by increasing the pulse number up to 4, a significant decrease (P < 0.05) in development to blastocysts was observed when the pulse number was 5. Application of AC voltage prior to the DC pulse tended to increase the fusion rate (89.2-93.8%), compared with fusion with the DC pulse only (75.0%). Prolongation of alignment time from 5 to 15 sec had no effect on the fusion rate. Under the optimum conditions (2 pulses of DC of 70 V/mm, 70 microseconds pulse duration and AC of 5 V/mm for 5 sec), no significant difference was obtained in the fusion and development rates in different mouse strains, nor were fusion and development rates significantly different among PBS, physiological saline and mannitol solutions (P > 0.05).

Animals↗
PubMed1995

Influence of time after the removal of nocodazole from nuclear donors on the development of reconstituted embryos in bovine nuclear transplantation.

The present study examined the influence of post-cleavage time of nuclear donors on the development of reconstituted embryos in bovine nuclear transfer. Blastomeres of 16-cell stage embryos derived from in vitro-maturation, fertilization and culture were used as nuclear donor source. They were treated with 10 microM nocodazole for 12 hr. Blastomeres that cleaved within 3 hr after the removal of nocodazole were used for the study. Metaphase II (M-II) oocytes were used as recipient cytoplasm. In experiment 1, donor blastomeres at 6, 11 and 15 hr after the removal of nocodazole and donor blastomeres not treated with nocodazole were transferred into ethanol-exposed and enucleated oocytes. The reconstituted embryos produced by donor blastomeres at 6 hr after the removal of nocodazole had a significantly higher developmental rate to the blastocyst stage than those at 15 hr and the untreated groups (P < 0.01). In experiment 2, blastomeres at 6 hr after the removal of nocodazole used as nuclear donors were transferred into ethanol-exposed and enucleated M-II oocytes. The reconstituted embryos with ethanol-exposed and enucleated oocytes as recipient cytoplasm had a significantly higher rate of initial-cleavage (P < 0.05) and development to the blastocyst stage (P < 0.01) than non ethanol-exposed and enucleated M-II oocytes. These results demonstrate that the development of reconstituted embryos was improved when cleaved donor blastomeres after the removal of nocodazole were immediately transferred (at 3-6 hr post-cleavage) into activated enucleated oocytes by exposure to ethanol.

Animals↗
PubMed1995

Effects of supplementation of the maturation media with insulin on in vitro maturation and in vitro fertilization of bovine oocytes.

This study was carried out to determine the effects of supplementation of the maturation media with insulin on in vitro maturation and fertilization of bovine oocytes. In Experiment 1, cumulus-intact bovine oocytes were cultured in a maturation medium (TCM-199 containing 10% fetal calf serum, 0.02 U/ml follicular stimulating hormone and 1 microgram/ml estradiol-17 beta) with or without insulin supplementation (10 micrograms/ml). The maturation and fertilization rates of oocytes and subsequent embryonic development to the blastocyst stage were not affected by the treatment with insulin in the presence of serum and the hormones during the maturation period. In Experiment 2, to avoid the effects of serum and the hormones, a serum- and hormone-free maturation medium (TCM-199 containing 1 mg/ml polyvinyl alcohol) was used. In the absence of serum and hormones during the maturation period, the maturation rate was not affected by treatment with insulin, but the fertilization rate was improved. In Experiment 3, when denuded oocytes were inseminated together with cumulus cells cultured in serum- and hormone-free maturation medium supplemented with insulin, the fertilization rate was increased. These results demonstrate that the addition of insulin to the serum- and hormone-free maturation medium improves the fertilization rate of bovine oocytes in vitro, and suggest that insulin may stimulate the secretion of sperm capacitating agent (s) from cumulus cells.

Animals↗