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Biomedical subjects

T Miyano

Publications and source records attributed to T Miyano.

At least 127 records · Page 7Linked to original sources
PubMed1995

Association between p34cdc2 levels and meiotic arrest in pig oocytes during early growth.

The molecules involved in determining meiotic competence were determined in porcine oocytes isolated from preantral and antral follicles of different sizes. Oocytes isolated from preantral follicles had a mean diameter of 78 microns, contained diffuse filamentous chromatin in the germinal vesicle and were incapable of progressing from the G2 to the M phase of the cycle even after 72 h in culture. Oocytes from early antral follicles had a mean diameter of 105 microns, showed a filamentous chromatin configuration and about half resumed meiosis but arrested at metaphase I (MI) when cultured. Oocytes from mid-antral (3-4 mm) and large antral follicles (5-6 mm) had mean oocyte diameters of 115 and 119 microns respectively, contained condensed chromatin around the nucleolus and progressed to metaphase II (MII) in 48% and 93% of instances respectively. Analysis of p34cdc2, the catalytic subunit of maturation promoting factor (MPF), by immunoblotting indicates that the inability of small (78 microns) oocytes to resume meiosis is due, at least in part, to inadequate levels of the catalytic subunit of MPF. On the other hand, the inability of intermediate-sized (105 microns) oocytes from antral follicles to complete the first meiotic division by progressing beyond MI appears not to be limited by levels of p34cdc2, which are maximal by this stage. We postulate that an inadequacy of molecules other than p34cdc2 limits progression of MI to MII; the acquisition of these molecules during the final stages of growth may be correlated with the formation of the perinucleolar chromatin rim in the germinal vesicle.

Amino Acid Sequence↗
PubMed1994

Effect of oviductal epithelial cells on fertilization of pig oocytes in vitro.

The incidence of polyspermy is reduced by co-culture of pig oocytes with oviductal cells. It is not known whether the effect is due to soluble factors secreted into the medium. Oviductal epithelial cell monolayers and cell-conditioned media were prepared and their effects on fertilization of pig oocytes were examined. In vitro matured pig oocytes were inseminated with ejaculated boar spermatozoa at a concentration of 1x10(5) or 1x10(6) cells/ml and co-cultured in one of 5 culture systems: an oviductal epithelial cell monolayer, a fibroblast monolayer, an oviductal epithelial cell-conditioned medium, or a fibroblast-conditioned medium, and medium alone (modified-TCM199). In all 5 systems, the majority (range 85 to 100%) of the oocytes were penetrated by sperm. When oocytes were inseminated with spermatozoa at a concentration of 1x10(5) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with oviductal epithelial cells and fibroblasts than that of the oocytes cultured without these cells. In contrast, when oocytes were inseminated with spermatozoa at a concentration of 1x10(6) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with epithelial cells than those cultured with the fibroblasts and in the control medium. The suppressive effect on polyspermy was observed in the oviductal epithelial cells-conditioned medium when oocytes were inseminated with spermatozoa at both concentrations of 1x10(5) and 1x10(6) cells/ml. The effect was absent in the fibroblasts-conditioned medium. Moreover, the effect of the epithelial cells was maintained during the culture period, whereas the proportion of monospermic oocytes co-cultured with fibroblasts showed a gradual decrease, reaching 0% after 16 h. These results suggest that a soluble factor(s) derived from the oviductal epithelial cells decreased the number of spermatozoa penetrating the oocytes without suppressing the high rate of fertilization.

Journal Article↗
PubMed1994

Identification of anti-agglutinin for spermatozoa in epididymal boar plasma.

The present report identifies epididymal boar anti-agglutinin and examines its effect on sperm motility. Boar spermatozoa from the cauda epididymidis were washed and incubated in modified Krebs-Ringer bicarbonate at 37 degrees C (5% CO2 in air). In the samples washed three or five times and then incubated for 3-5 h, higher rates (72-79%) of spermatozoa were associated with one another at the acrosomal region, mainly in groups of 2-5 cells (head-to-head agglutination), and many cells exhibited intensively flagellant and/or circular types of movement but rarely progressive motility. The addition of epididymal plasma or 25 kDa protein purified from it markedly inhibited the occurrence of head-to-head agglutination in washed spermatozoa, whereas heat treatment and subsequent removal of insoluble materials reduced the anti-agglutination activity of epididymal plasma. The percentages of progressively motile cells in the samples incubated with epididymal plasma or 25 kDa epididymal protein rose coincident with the reduction of sperm agglutination. These findings demonstrate that the 25 kDa epididymal protein is an anti-agglutinin for the cauda spermatozoa and that it effectively functions to maintain progressive motility of the cells in vitro.

Animals↗
PubMed1994

Docosahexaenoic acid status of patients with extrahepatic biliary atresia.

Docosahexaenoic acid (DHA) is believed to be an important long-chain polyunsaturated fatty acid (LCPUFA), which may be essential for neurofunction in infants. Patients with extrahepatic biliary atresia (EBA) may have DHA deficiency secondary to fat malabsorption. The authors investigated DHA and other LCPUFA levels in plasma and red blood cell (RBC) phospholipids of patients after the Kasai portoenterostomy and after supplementation with essential fatty acids. Ten children aged 8 to 17 months (mean, 12.6 months) comprised the study group. Five were jaundiced and five had a normal bilirubin level. The patients received 1 mL/kg of fat emulsions (10% Intralipid, containing 50% linoleic acid and 9% alpha-linolenic acid) in addition to an age-appropriate diet. Additional supplements were ursodeoxycholic acid (UDCA) (15 mg/kg/d) and taurine (100 mg/kg/d). The percentages of DHA in both plasma and RBC phospholipids of patients in the jaundiced group were significantly lower than those of normal children. Patients in the jaundice-free group had significantly lower levels of DHA and higher levels of linoleic acid in both plasma and RBC phospholipids in comparison to the normal group. This study shows that postoperative EBA patient become DHA-deficient even when supplemented with fat emulsions (largely composed of linoleic acid) that contain DHA's precursor, alpha-linolenic acid. This demonstrates a deficiency in the long-chain acid desaturase activity of these patients. It is recommended that excessive/linoleic acid intake be avoided and that all EBA patients have small amounts of DHA added to their lipid supplementation.

Biliary Atresia↗
PubMed1994

A new clinical prognostic predictor for patients with biliary atresia.

The excretion of D-glucaric acid (DGA), a metabolite of the cytochrome P-450 pathway, is considered a marker of the viability of hepatocytes. Twelve cases of biliary atresia (BA) have been investigated, correlating the level of DGA, degree of histological alteration of liver parenchyma, and clinical outcome. Urine was collected in the preoperative period. Five patients had good bile flow after the Kasai operation and demonstrated mild liver fibrosis. The urine excretion level of DGA in this group was 17.2 +/- 3.1 mumol/d (control, 15.2 + 2.2 [67od] mmol/d). Seven patients had partial bile drainage or prolonged jaundice. They also had altered liver parenchyma and a very low level of DGA in the urine preoperatively (4.2 + 2.2 mumol/d). Urine excretion of DGA reflected the degree of alteration of liver parenchyma and closely correlated with clinical outcome. Measurement of DGA before the Kasai operation provides information concerning the present hepatic function and the postoperative prognosis. Once the activity of hepatocytes deteriorates, this test is quicker than the other liver function tests. It is noninvasive and is a useful clinical prognostic predictor in BA.

Biliary Atresia↗
PubMed1994

Indwelling mesenteric venous catheterization for early detection of portal vein thrombosis: possible application to pediatric liver transplantation.

Portal vein thrombosis (PVT) is a well-recognized complication after liver transplantation, particularly in children. When it occurs early in the postoperative period, it has serious consequences, and rapid detection is essential. The purposes of this study were (1) to ascertain whether continuous monitoring of mesenteric venous pressure (MVP), via an indwelling mesenteric venous catheter, could assist in early detection of PVT and (2) to investigate the role of portography, via the catheter, in confirming this complication. An animal model of PVT was developed in pigs. At laparotomy, a heparin-coated catheter was inserted into a jejunal mesenteric vein, delivered percutaneously and connected to a pressure transducer. Conditions of PVT were simulated by progressive occlusion of the portal vein (PV) using a silastic tourniquet, and the degree of PV stenosis was assessed by Doppler ultrasound flow-velocity measurement. MVP was recorded 1 and 3 minutes after PV occlusion, and portography was performed via the indwelling catheter. There were significant increases in MVPs with all degrees of PV stenosis (P < .01, Student's t test). No significant changes in MVP were noted between 1 and 3 minutes postocclusion. Portography clearly demonstrated PV stenosis. There were no instances of PVT, despite repeated and prolonged occlusion of the PV. Progressive degrees of PV stenosis have been clearly detected by an indwelling mesenteric venous catheter in an animal model. This method may be useful for the diagnosis and treatment of PVT after pediatric liver transplantation.

Animals↗
PubMed1994

Effects of ursodeoxycholic acid treatment on essential fatty acid deficiency in patients with biliary atresia.

To assess whether ursodeoxycholic acid (UDCA) treatment has any beneficial effect on essential fatty acid (EFA) deficiency in patients who have had a Kasai operation for extrahepatic atresia (EBA), responses of serum fatty acids to UDCA administration (15 mg/kg/d) were investigated in eight jaundice-free patients and in eight patients with jaundice (serum total bilirubin > or = 1.0 mg/dL). All patients were also given taurine supplementation (100 mg/kg/d). Serum fatty acid composition was determined before and 6 months after UDCA treatment. Serum total bile acid concentration and serum total bilirubin value, as a part of conventional liver function tests, were measured before and during UDCA therapy. Before UDCA treatment, the concentrations of linoleic acid and arachidonic acid were significantly lower (P > .05 for the former; P > .01 for the latter) in both the jaundice and jaundice-free groups than in the controls. After 6 months of treatment, the linoleic acid concentration significantly increased (P > .05), to the normal range, in the jaundice-free group, but not in the jaundice group. The arachidonic acid concentration did not increase significantly in either group. The serum total bile acid concentration was lower in six of the eight jaundice-free patients and in four of the eight jaundice patients. The serum total bilirubin value decreased in six of the eight jaundice-free patients and in four of the eight jaundice patients; however, the degree of improvement was not statistically significant in either group. No side effects developed, and there were no changes in blood chemistry values unrelated to liver disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic Acid↗
PubMed1994

Tubed latissimus dorsi musculocutaneous flaps for thoracic esophageal replacement: an experimental study.

Tubed musculocutaneous flap has never been used to replace a circumferential defect of the thoracic esophagus in adults or children. The aim of this study was to examine whether a tubed latissimus dorsi musculocutaneous flap (TLDMF) can be used to repair a long gap defect of the thoracic esophagus. In 14 puppies with an average weight of 4.9 kg, part of the esophagus (two vertebrae in length) was excised through a right thoracotomy. A latissimus dorsi musculocutaneous flap was introduced into the thoracic cavity. The cutaneous layer was rolled into a tube and interposed in the space of the excised esophagus. The muscle layer was wrapped around the anastomosis. Three puppies died of operative complications, such as a minor anastomotic leakage and postoperative bleeding. One died of an unknown cause. Ten puppies were studied postoperatively with a barium meal, endoscopic examination, and autopsy. The management of these puppies over 4 months is described. It proved possible to replace a long gap thoracic esophageal defect with a TLDMF, which was safe, easy to be transferred, and obviated a laparotomy. Thus, this technique might offer an alternative method to bridge a long gap in patients with esophageal atresia or severe stenosis.

Animals↗
PubMed1994

Abnormal distribution of nerve terminals in infantile hypertrophic pyloric stenosis.

Smooth muscle biopsy specimens obtained from nine infants with infantile hypertrophic pyloric stenosis (IHPS) and from three controls were studied immunohistochemically with respect to the distribution of nerve terminals and neurofilaments. To label nerve terminals and neurofilaments, monoclonal antibodies (MAb) 171B5 and 2F11 were used, respectively. In all specimens of the control group, nerve terminals were numerous in both the myenteric plexus and the muscle layer. There were abundant neurofilaments in the myenteric plexus and a moderate number in the muscle layer. In all specimens of the IHPS group, the density of nerve terminals and neurofilaments was reduced in the muscle layer. In the myenteric plexus, there was no such reduction. The results indicate poor neuronal innervation of the muscle layer in the pylorus of infants with IHPS. This poor innervation may be related to the pathogenesis of pyloric stenosis and hypertrophy.

Antibodies, Monoclonal↗
PubMed1994

Effects of hyaluronic acid on the development of 1- and 2-cell porcine embryos to the blastocyst stage in vitro.

The purpose of this study was to evaluate the ability of hyaluronic acid to improve the development of 1- and 2-cell porcine embryos to the blastocyst stage in a simple medium. In Experiment 1, we confirmed the ability of Whitten's medium supplemented with 15 mg/ml BSA to support the development of porcine embryos to the blastocyst stage under our experimental conditions. Embryos collected from oviducts were cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in humidified air up to 6 d. After 2 d of culture, 82 and 78% of embryos reached the 4-cell stage or beyond in TCM199 supplemented with 10% fetal calf serum (FCS) and in Whitten's medium with BSA, respectively. However, no embryo developed to the morula stage in TCM199 after 6 d of culture. On the other hand, 26 and 15% of embryos developed to the morula and the blastocyst stage in Whitten's medium, respectively. In Experiment 2, we determined whether supplementation of hyaluronic acid in Whitten's medium would improve the development of porcine embryos to the blastocyst stage. After 6 d of culture, development of the embryos to the blastocyst stage was best supported in Whitten's medium with 4 mg/ml BSA and 0.5 mg/ml hyaluronic acid (70%). The proportion of degenerated embryos was lower in the presence than in the absence of hyaluronic acid. These results indicate that the supplementation of Whitten's medium with hyaluronic acid improves the development of 1- and 2-cell porcine embryos to the blastocyst stage.

Journal Article↗
PubMed1994

In vitro growth and maturation of pig oocytes.

Preantral follicles containing oocytes of 70-89.5 microns in diameter were isolated from pig ovaries and cultured in collagen gel for up to 16 days, in the presence of serum, FSH and oestradiol. Formation of follicular antra occurred as the culture proceeded. The oocytes had been enclosed by granulosa cells and contacts between the oocytes and processes of the enclosing cumulus cells were maintained over the culture period. After 16 days of culture, 30-40% of the oocytes were of normal appearance, and the diameters of about half of these oocytes were larger than 100 microns. When the oocytes grown in vitro were liberated from the follicles and cultured for a further 48 h in modified Krebs-Ringer bicarbonate solution, 6, 30 and 60% of the oocytes larger than 90, 100 and 110 microns underwent germinal vesicle breakdown, respectively. Progression to metaphase II was observed in 40% of oocytes that were over 110 microns in diameter, whereas no oocyte less than 90 microns in diameter resumed meiosis. The relationship between the size and meiotic competence of oocytes was similar for oocytes grown in vitro or in vivo. Oocytes grown and matured in vitro were penetrated by spermatozoa and formed a female pronucleus, but decondensation of the sperm head was incomplete. The results demonstrate for the first time that pig oocytes from preantral follicles can grow up to their final size, acquire meiotic competence, and be penetrated by spermatozoa in vitro.

Animals↗
PubMed1993

Acquisition of maturational competence in in vitro grown mouse oocytes.

The maturational competence of the in vitro grown mouse oocytes of various sizes was examined, and was compared to that of oocytes of equivalent chronological age which grew in vivo. Oocyte-granulosa cell complexes isolated from 10-day-old female mice were cultured for 10 days in vitro in the presence of 2 mM hypoxanthine to maintain the meiotic arrest. When the in vitro grown oocytes were denuded and cultured for further 24 h in the hypoxanthine-free medium, 9, 37, and 100% of oocytes of 60-64.5, 65-69.5, and over 70 microns in diameter underwent germinal vesicle breakdown (GVBD), respectively. Of those oocytes which underwent GVBD, 19, 37 and 68% of oocytes larger than 60, 65, and 70 microns progressed to metaphase II, respectively. No oocyte less than 60 microns resumed meiosis. The parallelism found in the oocyte size and maturational competence was similar to that of their in vivo counterparts collected from 20-day-old mice. A significant proportion of the in vitro grown oocytes, however, formed a pronucleus-like structure(s) after maturation culture, indicating that they could not maintain a normal arrest at metaphase II.

Animals↗
PubMed1993

Ultrastructural changes in the microvilli as a marker of small bowel rejection.

Small bowel rejection occurs in patches; therefore, to detect bowel rejection in situations where a stomal biopsy appears normal under the light microscope, we studied the ultrastructural changes in bowel microvilli following heterotopic small bowel transplantation in rats. Thin sections of bowel biopsy specimens, which appeared normal under a light microscope, were selected and examined under an electron microscope for microvillous changes. Microvillous changes were seen in both the syngeneic and allogeneic groups on the 2nd and 4th postoperative days, but these changes were absent in the syngeneic group by the 8th postoperative day, although they were still detected in the allogeneic group. Thus, following small bowel transplantation, electron microscopic analysis is recommended to confirm rejection with a patchy distribution after the 8th postoperative day.

Animals↗