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Biomedical subjects

A Iritani

Publications and source records attributed to A Iritani.

At least 37 records · Page 2Linked to original sources
PubMed1993

Development of hamster one-cell embryos recovered under different conditions to the blastocyst stage.

One-cell stage embryos, recovered from superovulated golden hamsters (8 to 12 weeks of age) 12 hours after egg activation, were cultured in HECM-1 medium at 37 degrees C and 5% CO(2) in air. The culture conditions investigated were the time and temperature required for embro recovery, the pH shift of the washing medium, and the oxygen concentration of the gas phase during and after embryo recovery. Each condition was assessed by the developmental efficiency of the embryo as determined by morphological criteria. As the time required for embryo recovery was reduced, the developmental rates of the embryos were improved: 2.3% (3 128 ) 26.9% (35 130 ) at 5 and 3 minutes, respectively, as determined by the number of embryos developed to the blastocyst stage. No blastocysts were obtained when more than 10 minutes were required for embryo recovery. As the oxygen concentration was reduced from 40 to 20% or to 5%, rather high developmental rates were obtained even when the time required for embryo recovery was prolonged: 6.9% (9 130 ) and 21.7% (28 129 ) of the embryos developed to the blastocyst stage when they were recovered under 5% oxygen within 10 and 5 minutes, respectively. Neither the temperature during embryo recovery (37 degrees C and 25 degrees C) nor the pH shift (pH 7.22 to 7.52) of the washing medium used in embryo recovery procedures influenced the development of the embryos. These findings suggest that the developmental block in hamster embryos may involve oxidative stress, which may result from exposure to high oxygen concentration and light during the manipulation of oocytes and embryos.

Journal Article↗
PubMed1992

Cryoprotective effect of polyols on rat embryos during two-step freezing.

The cryoprotective effect of polyols on rat embryos was measured after two-step freezing, and the mechanism of action of polyols on embryo survival was examined. Rat embryos frozen in solution of polyol by two-step method at the morula stage showed higher survival than that obtained using DMSO. As the number of hydroxyl groups increased, the cryoprotective effect of the polyol increased. However, this was true only when the additive could permeate the cell membrane. Of the additives tested, four or five carbon polyols were most effective at concentrations of 0.3 or 1.0 M than two, three, six, or seven carbon polyols. The highest survival rate was obtained with adonitol, which yielded 83% embryo survival at 1.0 M and 67% even at 0.3 M. Embryos frozen in 0.3 M adonitol and transferred directly into foster mothers without any dilution of the additive after thawing developed into live young. During slow cooling below -40 degrees C, embryonic blastomeres exhibited cell fusion only in the presence of adonitol. These findings suggest that one cryoprotective action of polyols is that the hydroxyl groups act both on the cell surface and the cytoplasm to stabilize the bound water on the embryonic membrane, and that the length of the C-chain determines the permeability of the membrane to the additive.

Animals↗
PubMed1992

Effect of visual light on in vitro embryonic development in the hamster.

The effect of light exposure during collection and culture of hamster embryos on their subsequent development in vitro was examined. When embryos were collected under dark conditions (70 lux) within 10 minutes and then cultured in a HECM-1 medium in 5% CO2 in air, the developmental rates of 1-cell embryos to the 4- and 8-cell stages were 88.6% (93/105) and 66.7% (70/105), respectively. These rates were significantly higher than those under light conditions (1600 lux): 51.9% (56/108) and 34.3% (37/108). Light irradiation during the culture of 1-cell embryos suppressed subsequent development. The degree of suppression correlated inversely with duration of light irradiation, and light irradiation of 30 minutes or more completely blocked development to the 2-cell stage. When 1-cell embryos were irradiated through a yellow filter, cutting the light wavelengths to less than 500 nm, embryonic development was still suppressed. However, the degree of the suppression varied and 45.7% (53/116), 6.0% (7/116), and 0.9% (1/116) of the embryos developed to the 2-, 4-, and 8-cell stages, respectively, under 30 minute light irradiation. Inhibitory effects of light irradiation on the development of 2- and 8-cell embryos were also observed, showing an inverse correlation with duration; the developmental rates of 2-cell embryos to the 8-cell stage under 0, 10, and 30 minutes of irradiation were 85.6% (107/125), 1.6% (2/122), and 0% (0/129), respectively, and those of 8-cell embryos to the blastocyst stage were 79.8% (91/114), 74.8% (86/115), and 0% (0/110), respectively. These findings indicate that early-stage embryos are sensitive to light exposure; however, severe light exposure adversely affects the development of embryos at any stage. Thus, the protection of embryos from light exposure at all stages of embryo manipulation, from collection to culture, is essential.

Journal Article↗
PubMed1992

Effects of low molecular weight oviductal factors on the development of mouse one-cell embryos in vitro.

The relationship between the oviduct and embryo development in the mouse was investigated and the period at which the influence of oviduct can be concerned in the development of mouse embryos in vitro was identified. In addition, the relative molecular weight of oviductal factors that promote embryo development was demonstrated. Mouse zygotes developed to the blastocyst stage when co-cultured with ampulla. The period of embryo co-culture significantly affected the further development of the embryos. Fewer one-cell embryos co-cultured with dissected ampullae for less than 24 h developed to blastocysts than those co-cultured for more than 28 h (P < 0.001). A high percentage of embryos co-cultured with ampullae after 24 h of culture in vitro developed to the blastocyst stage, which suggests that the influences of ampulla on the development of mouse embryos are restricted to a specific period at the two-cell stage (about 55-56 h after hCG injection) in vitro. Mouse ova that were cultured in media conditioned by ampullae could also develop to the blastocyst stage. The fractionated medium that contained low molecular weight fractions was more effective (P < 0.001) on the development of embryos to the blastocyst stage than that containing high molecular weight fractions. These results suggest that the low molecular weight oviductal factors play an important role in the development of mouse embryos at a certain critical age in vitro.

Animals↗
PubMed1991

Sexing of rat embryos with antisera specific for male rats.

Male-specific antigenicity (H-Y antigen) of rat embryos has been examined, and the feasibility of sexing rat embryos by use of H-Y antibodies has been studied. Rat H-Y antisera were produced by immunization of female Wistar rats with a homogenate of testes from male Wistar neonates. Male specificity of the antiserum (H-Y antibody) was determined by retention of cytotoxicity to male epidermal cells after absorption with female cells. After cultivation of rat embryos for 5 to 6 hr in the presence of antibody, half of the embryos were arrested at the morula stage. However, these embryos developed into blastocysts after removal of the antiserum, and then they grew into male young in recipient foster mothers. Eighty percent of the embryos that developed to blastocysts in the presence of the antiserum grew into female young.

Animals↗
PubMed1991

Fertilizing competency of multiple ovulated eggs in the domestic fowl (Gallus domesticus).

Fertilizing competency of multiple ovulated eggs in the domestic fowl was examined by fertilization in vitro and early development in culture. Normal laying hens (White Leghorn) were treated with 75 IU of PMSG for 7 days followed by injection of anterior pituitary extracts from chickens (CAPE). Ovulation began to occur 7.5 h after injection of CAPE. These hens ovulated 1-7 ova but some premature ovulation of GV stage ova were observed. In vitro fertilization of the multiple ovulated ova was examined by inseminating 10(6)-10(7) sperm onto the germinal disks in m-Ringer's solution. The gamete or zygote nuclei were detected by DNA specific fluorescence using DAPI (4',6'-diamidino-2-phenylindole) in the histological section prepared from the germinal disk. Process of fertilization was examined in the eggs incubated for 4 h after insemination in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Fertilization rate of the total multiple ovulated eggs was 55% (11/20), in which 90% (9/10) and 10% (1/10) in the eggs recovered 7.5-8.5 h and 9.0-9.5 h after CAPE injection were obtained, respectively. Normal pronuclei were formed in five eggs of those recovered 7.5-8.5 h after CAPE injection. Early development after fertilization in vitro was also examined by incubation for 12 h in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Although development in vitro was delayed compared to that in utero condition, normal development was observed in naturally and multiple ovulated eggs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Early morphological events of in vitro fertilized bovine oocytes with frozen-thawed spermatozoa.

Bovine follicular oocytes were matured and inseminated in vitro with spermatozoa capacitated in vitro. The first evidence of sperm penetration was observed at 3 h after insemination. The penetration rate increased until 5 h, and reached a maximum rate (92%) at 5 h. Decondensation of the sperm head and pronuclear formation were observed 4 h and 7 h after insemination, respectively. Female chromatins of all penetrated oocytes were activated at 3 h, and female pronuclei were formed at 7 h after insemination. Percentages of oocytes with male and female pronuclei at 9 h were 88 and 94%. Polyspermy (4, 7, 19 and 29% at 4, 5, 7 and 9 h after insemination, respectively) and abnormal development of male pronuclei (6 and 7% at 7 and 9 h after insemination, respectively) were also seen.

Journal Article↗
PubMed1991

Full-term development of bovine follicular oocytes matured in culture and fertilized in vitro.

Follicular oocytes were cultured for 28h in vitro and 91% of the oocytes reached the the second metaphase in culture. The penetration rate after insemination in vitro using frozen-thawed spermatozoa was 81%. After cultivation for 48h in vitro, 18% of the in vitro fertilized oocytes developed to the three- to four-cell stages and 21% of these developed to the six- to eight-cell stages. Following in vivo culture in the rabbit oviduct, 18% of six- to eight-cell and 5% of three- to four-cell embryos developed to the blastocyst stage. To confirm the full developmental competence, 11 blastocysts were transferred to recipient cows, and six (55%) cows became pregnant or delivered calves.

Journal Article↗
PubMed1991

Separation of mounting-inducing pheromones of vaginal mucus from estrual heifers.

There is evidence that mucus of the female bovine genital tract contains pheromones that induce physiological and behavioral responses in other animals. To study these pheromones, vaginal mucus was collected from heifers either at estrus or during diestrus. The mucus was then applied to the hindquarters of the same animal during diestrus or to the hindquarters of herdmates during diestrus. The behaviors of the treated animal and its herdmates were then observed. To attempt to isolate the mounting-inducing substance, mucus was dialyzed or separated on ion-exchange resins. Diestrous heifers to which their own estrual mucus has been applied were nearly always mounted by herdmates (P less than .01). But, heifers to which another's estrous mucus had been applied were not mounted. This suggests that vaginal mucus contains not only estrus-related pheromones, but also individual distinctive odors. The dialyzable fraction of vaginal mucus and the neutral fraction, prepared by ion-exchange chromatography of the dialyzable solution of vaginal mucus, had a mounting-inducing activity on the herdmates, as did the application of an animal's own vaginal mucus. These findings suggest that mounting-inducing pheromones are relatively low molecular weight, neutral substances.

Animals↗
PubMed1991

Structural analysis of repetitive DNA sequences in the goat growth hormone gene region.

Two clones that contain goat growth hormone (gGH) genes were isolated from goat genomic library using goat growth hormone cDNA as a probe. One clone CgGH contained gGH1 gene, and another clone, EgGH, contained gGH2 and gGH3 genes. The clone EgGH contained 491 bp inserted sequence, just upstream of the gGH3 gene, which was not present in the clone CgGH. From the sequencing data of the flanking regions of these gGH genes, short interspersed repetitive sequences (SINES) were found. Four SINES's existed in the CgGH clone and eight SINES's existed in the EgGH clone. The number of the repetitive DNA sequences in the goat genome was estimated about 10(2) copies from the analysis of the reassociation rate. As the number of gGH genes (gGH1, gGH2, and gGH3) per genome was different among individual goats, DNA fragments containing gGH1 gene, and that containing gGH2 and gGH3 genes, were estimated to be allelic on the goat chromosome.

Animals↗
PubMed1990

Early nuclear events of in vitro fertilization in the domestic fowl (Gallus domesticus).

In vitro fertilization in the domestic fowl (Gallus domesticus) was investigated by observation of the early nuclear events. Ova retrieved from the fimbria following ovulation were inseminated in vitro with 10(6)-10(7) spermatozoa in Dulbecco's modified Eagle's medium (DMEM) for 10 min and then further incubated in DMEM + albumen for 1, 2, 3, or 4 hr. These eggs were histologically examined by epifluorescent microscopy after staining with 4',6'-diamidino-2-phenylindole (DAPI). Nuclei of spermatozoa at various stages of transformation were observed in the ova incubated for 1-3 hr. Close pairing of two pronuclei, presumed to be male and female juxtaposition, was detected in ova incubated for 4 hr. These data provide direct evidence for the in vitro fertilization of fowl eggs and suggested that the early process of in vitro fertilization is comparable to that of in vivo fertilization.

Animals↗
PubMed1990

Ooplasmic transfusion: prophase germinal vesicle oocytes made developmentally competent by microinjection of metaphase II egg cytoplasm.

Approximately one fourth of all human oocytes collected for in vitro fertilization are of immature origin. Even when these oocytes undergo nuclear maturation, fertilization, and cleavage in vitro, transfer of such embryos rarely results in pregnancy reaching delivery. We hypothesized that human embryos derived from prophase I oocytes were developmentally incompetent because they lacked a factor(s) found in in vivo matured oocytes. Using micromanipulation techniques in monkeys, we removed ooplasm from metaphase II oocytes and injected it into prophase I oocytes. After nuclear maturation, oocytes were transferred to the fallopian tube for fertilization. After ooplasmic transfusion, prophase I oocytes resulted in a delivery rate of 13%. When metaphase II ooplasm was heated or exposed to ribonuclease A before microinjection into prophase I oocytes, it lost effectiveness in conferring developmental competence.

Animals↗
PubMed1989

Identification of puberty-accelerating pheromones in male mouse urine.

Gas chromatography-mass spectrometry was employed to identify the two volatile amines in male mouse urine. These amines were much less concentrated in urine of castrated males. The identified amines, isobutylamine and isoamylamine, were assayed for the potential of puberty acceleration in postweaning female mice. A total of 105 young female mice were exposed to one of the following five odors: distilled water (control), 0.1 M isobutylamine, 0.1 M isoamylamine, a mixture of 0.05 M isobutylamine and 0.05 M isoamylamine, or fresh male mouse urine. The mixture of these amines accelerated the vaginal opening of young females. Except for the control, all experimental odors accelerated the first vaginal estrus in ICR strain mice.

Amines↗
PubMed1989

Production of identical twins by separating two-cell rat embryos.

Rat identical twins were produced from two-cell embryos. In the presence of cytochalasin B, rat two-cell embryos could be separated efficiently into two blastomeres by micromanipulation. Isolated blastomeres, embedded in agar cylinders and cultivated in ligated rat oviducts for 3 days, developed to the morula or blastocyst stage. After removing the agar, pairs of developed one-half embryos were transferred into Day 1 oviducts or Day 4 uteri of pseudopregnant rats. The percentage of embryos, separated either in the presence or absence of cytochalasin B, that developed into live fetuses was higher in cases of uterine transfer than in cases of oviduct transfer (38% vs. 18%, 31% vs. 15%, respectively). Throughout the present experiment, nine pairs of identical twins were successfully produced. This is the first report of the production of identical rat twins by separating two-cell embryos.

Animals↗
PubMed1989

Motility and fertilizing ability of rat epididymal spermatozoa washed by a continuous gradient of Percoll.

Removal of epididymal fluids from epididymal sperm suspension is an important step for the study of sperm motility, capacitation, and the acrosome reaction. The technique of washing should minimize damage to viable spermatozoa but at the same time efficiently remove debris, non-sperm cells, and biological fluids. We examined sperm motility and fertilizability in vitro of rat epididymal spermatozoa after washing with Percoll continuous gradient. Nine milliliters (ml) of 50% N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid (HEPES) buffered Percoll solution was centrifuged at 20,000 g for 45 minutes to form a continuous gradient. One hundred to 300 microliters of sperm suspension was loaded onto the surface of the gradient and centrifuged at 150 g and 1,500 g for 10 minutes. Two main layers of spermatozoa were formed, one of high (lower layer) and one of low (upper layer) motility. At centrifugation 1,500 g, the sperm density and motility in the lower layer were greater than at 150 g. Spermatozoa from both layers at 150 g and at 1,500 g were diluted with modified Krebs-Ringer's bicarbonate solution (mKRB) and preincubated for 5 hours. Superovulated eggs collected from 21-25-day-old Wistar strain immature rats were introduced into the preincubated sperm suspension for insemination and fixed 5-5.5 hours later for observation of fertilization. Spermatozoa from both layers, 150 g and 1,500 g, showed the same fertilizability in vitro as control spermatozoa. From these results we conclude that Percoll gradients can be used for washing rat epididymal sperm for the study of sperm physiology including fertilization.

Animals↗
PubMed1989

Developmental potential of rabbit oocyte matured in vitro: the possible contribution of prolactin.

The present study was undertaken to define hormonal conditions for in vitro maturation that support subsequent fertilization and embryonic development. Follicular oocytes were recovered from nonstimulated rabbit ovaries and cultured for 12 h in Brackett's medium supplemented with or without hormones. Matured oocytes were inseminated in vitro and transferred 12 h later to Ham's F-10 medium supplemented with 20% fetal calf serum. The initial cleavage frequency of matured oocytes in Brackett's medium was comparable to the frequency of development for in vitro-matured oocytes under various hormonal conditions. However, the addition of estradiol (E2, 1 microgram/ml) to incubation medium containing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) increased significantly (p less than 0.001) the percentage of embryos achieving morula or blastocyst formation (16/98, 16.3%), as compared to the mature oocytes in medium containing LH, LH plus FSH, or no hormone. The addition of prolactin (PRL) to the maturation medium increased the percentage of development to organized embryos in a dose-dependent manner. In vitro-matured oocytes in medium containing LH, FSH, and PRL exhibited a significantly (p less than 0.001) lower incidence of developmental competence (5/95, 5.3%) than oocytes matured in the presence of E2 in conjunction with pituitary hormones (43/89, 48.3%). These results demonstrate that hormonal composition in the environment of the oocyte is critical for acquisition of developmental capacity. PRL as well as E2 appears to be an important constituent in the process of oocyte maturation, promoting preimplantation embryonic development.

Animals↗