Biomedical subjects

Y Izaike

Publications and source records attributed to Y Izaike.

9 recordsLinked to original sources

PubMed2000

In vitro culture of bovine preantral follicles.

Bovine preantral follicles (40-100 microm diameter at collection) were collected from ovaries of slaughtered cows and cultured in vitro with one of the four treatments: follicle stimulating hormone (FSH; 100 ng/ml) alone; FSH plus epidermal growth factor (EGF; 100 ng/ml); FSH plus insulin-transferrin-selenium (ITS; +1%) or FSH plus hypoxanthine (4 mM) in tissue culture medium (TCM 199) supplemented with 10% fetal calf serum (FCS), 0.1 mg/ml sodium pyruvate, 100 IU/ml of penicillin and 100 microg/ml streptomycin. The control culture medium was TCM 199 with supplements without any treatments. Follicles of each size were cultured separately in groups of one to three in 24-well multidishes each containing 500 microl of the appropriate culture medium. Culture commenced at follicle recovery (day 1) and continued for 10 days (harvested on day 11). In each case, half the medium was removed and replaced by fresh medium every third day. Follicle diameters were recorded on days 1, 5 and 11 of the experiment. At the end of the 10-day culture period, half of the follicles were stained with trypan blue to assess their potential viability and half were stained with bisbenzimide plus propidium iodine to estimate various morphological features of the follicles. Follicles of all initial sizes, on all culture treatments, increased in diameter during in vitro cultures with the greatest increases, both in absolute and proportional size, occurring between days 1 and 5 of culture. All of the culture medium supplements caused greater increases in follicle diameters than control medium at both days 5 and 11 of culture for all initial sizes of follicles (p<0.01). The most effective culture supplements for follicles of 40-, 60- and 80-microm initial diameter were FSH alone and FSH+EGF. The size of these follicles at both days 5 and 11 of culture on both the treatments was significantly larger (p<0.01) than follicles cultured in the presence of the other two supplementary treatments. The growth of follicles of 100-microm initial diameter did not differ between culture medium supplements. None of the culture media caused follicle size to increase to the initial diameters of the next larger size category during the 10 days of culture although follicles of 100-microm diameter achieved a diameter of 120 microm, after 4 days of culture. The overall follicular viability and morphology were better with treatments than the controls in all cases; however, there was no significant difference (p>0.05) among them. From this experiment, FSH and FSH plus EGF may be recommended for in vitro culture of smaller (40, 60 and 80 microm) follicles.

Animals↗

PubMed2000

Factors affecting the survivability of bovine oocytes vitrified in droplets.

Vitrification of bovine oocytes performed using the traditional, in straw system has not given satisfactory results. Although an alternative approach based on minimizing the volume of the vitrified sample has recently resulted in a much more promising survival rate of vitrified oocytes, we attempted to examine some additional factors influencing the survival and subsequent fertilization and development rates of bovine oocytes subjected to vitrification according to the minimum drop size approach. In total, 748 bovine, in vitro matured oocytes were vitrified using VS14 vitrification solution, containing 5.5-M ethylene glycol and 1.0-M sucrose after different pre-equilibration and equilibration protocols performed at 35 degrees to 37 degrees C. Experiment 1 showed no significant toxic effect during pre-equilibration treatments of oocytes in 2%, 4% or 6% ethylene glycol solutions, except the lower cleavage rate of oocytes exposed to 6% ethylene glycol (77.2% vs. 93.9% in control, P< 0.05). In Experiment 2, 12 to 15 min of pre-equilibration treatments in 0%, 1% or 2% ethylene glycol solutions were tested, followed by 30 or 45 sec of equilibration in VS 14 solution and vitrification in droplets of medium dropped directly into liquid nitrogen. The development rate of vitrified oocytes to the blastocyst stage tended to be higher after 30-sec equilibration treatment (9.5%, 13.9% and 13.8% in groups of oocytes pre-equilibrated in 0%, 1% or 2% ethylene glycol solutions, respectively). Experiment 3 tested pre-equilibration treatments in 0%, 1%, 2%, 3%, 4%, 5% or 6% ethylene glycol solutions, followed by 30-sec equilibration and vitrification in droplets. The highest cleavage, blastocyst and hatched blastocyst rates, which were not significantly different from control, were achieved in a group of oocytes pre-equilibrated in 3% ethylene glycol solution (76%, 30% and 15% vs. 89%, 42% and 21% in control, respectively). A healthy calf was born on Feb 22 1999, after transfer of 4 morula/blastocyst stage embryos developed from oocytes vitrified in droplets after pre-equilibration in 3% ethylene glycol solution. We conclude that gentle pre-equilibration of bovine oocytes in diluted, 3% ethylene glycol solution is an important factor improving the effectiveness of vitrification in droplets of bovine oocytes.

Animals↗

PubMed2000

Effect of lipid polarization by centrifugation at different developmental stages on post-thaw survival of bovine in vitro produced 16-cell embryos.

The developmental rate to the blastocyst stage of frozen-thawed bovine in vitro produced embryos at stages earlier than Day 6 morula is not sufficiently high for practical utilization. The present study was undertaken to determine the effect of polarization of lipid droplets in the cytoplasm of bovine in vitro produced embryos from zygotes to the 8-cell stage, by centrifugation without following micromanipulation, on the survival rate of Day 4 16-cell embryos. After centrifugation at 15,500 x g in medium containing cytochalasin D, embryos were cultured to the 16-cell stage, classified as either mostly or partially delipidated by degree of lipid droplet removal, and then frozen. Embryos centrifuged at the 2-cell stage developed to the 16-cell stage similarly to those centrifuged at the 8-cell stage. The developmental rate to blastocysts after freezing of the mostly delipidated 16-cell embryos centrifuged at the 2-cell stage was higher than that of those centrifuged at the zygote stage, those that were partially delipidated at the 2-cell stage, and those that were not centrifuged. The results demonstrate that polarization of lipid droplets at the 2-cell stage by centrifugation without micromanipulation improved the survival rate of mostly delipidated 16-cell embryos after freezing.

Animals↗

PubMed2000

Short time priming of pig cumulus-oocyte complexes with FSH and forskolin in the presence of hypoxanthine stimulates cumulus cells to secrete a meiosis-activating substance.

This study evaluated the effect of forskolin and FSH on pig oocyte maturation when cultured in a maturation inhibiting system. Ovaries from prepubertal gilts were collected at a local slaughterhouse. Oocytes were cultured in a hypoxanthine (HX 4 mM) containing M 199 for 24 or 40 h with or without forskolin and FSH treatment. After the culture, we examined germinal vesicle breakdown (GVBD) and polar body (PB) formation. Two experiments were designed. (1) Cumulus enclosed oocytes (CEO) were cultured for 24 or 40 h with or without different doses of forskolin and FSH. (2) CEO were primed by forskolin and FSH for different times and then transferred into an HX-medium for a further culture. The total culture period was 24 h. The results revealed that 4 mM HX markedly prevented pig CEO from undergoing GVBD. After 24 and 40 h culture, FSH (50-200 U/L) stimulated oocytes to resume meiosis by overcoming the inhibition of HX. Both GVBD and PB formation were increased (P < 0.002 and 0.01 respectively) after 40 h exposed to FSH. Forskolin showed a biphasic effect on CEO maturation. Within 24 h forskolin, in combination with HX, inhibited oocytes maturation. The GVBD percentage was significantly decreased compared to HX alone group (2% to 20%, P < 0.01), whereas no inhibition was observed after 40 h of culture. The second experiment showed that forskolin (3 microM) and FSH (100 U/L) priming CEO could time-dependently induce oocyte maturation by overriding the inhibition of HX. After 30 and 60 min priming by FSH or forskolin, the GVBD and PB percentage was significantly increased (P < 0.002 and 0.01 respectively). No difference of GVBD percentage was observed between FSH short time priming group and FSH long time presentation group. In conclusion, we found that forskolin and FSH in vitro can stimulate pig cumulus cells to secrete a meiosis-activating substance which induces the oocyte to overcome the inhibition of hypoxanthine and undergo GVBD.

Animals↗

PubMed1999

Cytoplasmic maturation for activation of pig follicular oocytes cultured and arrested at metaphase I.

A large population (62-90%) of pig follicular oocytes can mature to metaphase II after culture for 48 h. However, a proportion (6-22%) remain in an immature stage at metaphase I (metaphase I-arrested). The main objective of this study was to determine whether the cytoplasm of metaphase I-arrested pig oocytes is capable of being activated by sperm penetration or parthenogenetic stimulation. After culture for 48 h, oocytes without a polar body (73% were shown to be at metaphase I after staining) and those with a polar body (94% were at metaphase II) were fertilized in vitro. A total of 69% and 62% of the oocytes were activated to form a female pronucleus, respectively, and the rate of polar body extrusion induced by fertilization in the activated oocytes was 90% (the first polar body) and 95% (the second polar body), respectively. When oocytes without and with a polar body were stimulated with an electric pulse, 53% and 81% of the oocytes were activated, respectively. The rate of polar body extrusion in the activated oocytes was 73% (the first polar body) and 79% (the second polar body), respectively. In contrast, young metaphase I oocytes cultured for 24 h had low (6%) or zero activation rate after in vitro fertilization or electric pulse stimulation. However, about one-third of the young metaphase I oocytes penetrated by spermatozoa after in vitro fertilization responded to electric pulse 12 h after insemination, and almost all (93%) were activated when they were stimulated 24 h after insemination. Patterns of polypeptide synthesis and histone H1 kinase activity were similar in metaphase I-arrested and metaphase II oocytes, and were characterized by increase in a 25 kDa polypeptide and by decrease in kinase activity. Although the first step of meiotic division is impaired, these results indicate that metaphase I-arrested oocytes are mature cytoplasmically.

Analysis of Variance↗

PubMed1995

Histone H1 kinase activity during in vitro fertilization of pig follicular oocytes matured in vitro.

The present study was conducted to clarify the relationship between histone H1 kinase (H1K) activity and events associated with in vitro fertilization of pig follicular oocytes matured in vitro. Histone H1 kinase has been shown to be homologous with a maturation promoting factor (MPF). Cumulus-oocyte complexes obtained from prepubertal gilts were cultured for 46 h in a modified Waymouth's MB752/1 medium and were then inseminated in vitro with frozen-thawed and preincubated epididymal boar spermatozoa. At 4, 6, 8 and 10 h post insemination, the oocytes were stained with 10 microg/ml Hoechst-33342 and examined under a fluorescent microscope for the stage of fertilization, according to morphological changes of oocyte nuclear chromatin and the extent of sperm penetration. Sperm penetration was observed to occur within 4 h post insemination (20.5%), and the percentage of fertilized oocytes increased (P < 0.01) to 72.9% at 8 h post insemination. Pronuclear formation was observed from 6 h post insemination (3.3%) and the percentage increased (P < 0.01) to 46.8% at 10 h post insemination. In each examination period, H1K activities in unfertilized oocytes at metaphase-II remained unchanged (112.0 fmol/h/oocyte) and were higher (P < 0.01) than those in fertilized oocytes (30.1 fmol/h/oocyte). The H1K activity in fertilized oocytes such as oocytes emitting a second polar body, oocytes with an enlarging sperm head(s) and oocytes with multiple pronuclei did not differ significantly. These results suggest that MPF in pig oocytes is inactivated shortly after sperm penetration and is maintained at the basal level throughout pronuclear formation.

Journal Article↗

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↗

PubMed1993

Effect of follicle cells on in vitro fertilization of pig follicular oocytes.

We investigated the effect of cumulus and granulosa cells (follicle cells) on in vitro fertilization of pig follicular oocytes matured in vitro. Oocytes surrounded by cumulus and connected with a piece of parietal granulosa cells (complexes) were matured in vitro for 46hours and were then divided into 4 groups: Group I oocytes were surrounded by expanded cumulus and granulosa cells; Group II oocytes were surrounded by expanded cumulus cells; Group III were denuded oocytes; and Group IV were denuded oocytes with cumulus cells from other complexes. After incubation for 4 hours and 40 minutes with frozen, thawed and preincubated pig epididymal spermatozoa, the oocytes were cultured for 5 hours and 20 minutes. When oocytes were inseminated in the presence of cumulus cells, the penetration rates were higher (92.5% for Group II and 89.5% for Group IV) than when cumulus cells were not used for insemination (Group III, 66.8%) or when oocytes with follicle cells were inseminated (Group I, 72.3%). Denudation of follicle cells before insemination (Group III) decreased the percentage of male pronuclear formation (50.8%) compared with that of oocytes surrounded by follicle cells (66.7% for Group II and 80.2% for Group I). These results support the ability of a moderate number of follicle cells to facilitate sperm penetration of pig follicular oocytes and male pronuclear formation.

Journal Article↗

PubMed1982

A novel nuclear protein in rat ventral prostate. Androgen-dependent and age-related change.

A novel protein was found in the nuclei of rat ventral prostate. This protein has a molecular weight of about 21 kDa as measured by SDS-polyacrylamide gel electrophoresis. It showed a characteristic change between 3 and 84 weeks after birth in close association with the level of testosterone in the blood. After castration, the level of the 21-kDa protein decreased to 1/60 of normal in 7 days, but on daily injection of testosterone the level was restored to normal in 8 days and to twice the normal level in 14 days. Unlike H1 and H1(0) histone and high mobility group proteins, the 21-kDa protein was not extracted with 5% HClO4, but was partially extracted with 0.35 M NaCl. The 21-kDa protein was not found in kidney, liver, or bain, suggesting that it is specific to the ventral prostate.

Aging↗