Posterior lingual mandibular bone depression.
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Adipose Tissue↗
Biomedical subjects
S Kito
Publications and source records attributed to S Kito.
Activation of primordial follicles in vitro.
The resting pool of primordial follicles in mammalian ovaries is a potential resource for the genetic manipulation of domestic animals, the preservation of endangered species, and the amelioration of some forms of infertility in humans. Exploitation of this large reservoir of follicles depends on the development of methods for activating primordial follicles to begin growth in vitro and of methods for sustaining follicular growth to the stage at which oocytes are capable of meiotic maturation, fertilization and development to live young. It has been shown that primordial follicles of rodents, cattle and primates can initiate growth in vitro, even in serum-free medium. The signals that cause primordial follicles to leave the resting pool or remain quiescent are unknown. However, of interest is the observation that in cultures of whole rodent ovaries an apparently normal number of follicles leaves the resting pool and begins to grow, whereas in cultures of isolated bovine or primate ovarian cortex almost all primordial follicles activate and develop into primary follicles. This finding suggests that non-cortical portions of the ovary may regulate the flow of follicles from the resting reservoir. In cattle, it has been difficult to sustain follicular growth beyond the primary stage and the development of methods for doing so are critical for achievement of the practical goal of use of the primordial pool for embryo production. However, the development of murine follicles in vitro from the primordial stage through oocyte maturation and fertilization, and the birth of one pup, provides encouragement for efforts to achieve similar results in large mammals.
Animals↗
Activation of bovine and baboon primordial follicles in vitro.
Mammalian ovaries contain a large pool of non-growing, primordial follicles. The ability to initiate growth of this pool of resting follicles in vitro and to maintain follicular growth to a stage when the oocyte could be matured and fertilized would increase the reproductive potential of valuable domestic animals, endangered species and infertile women. This paper summarizes our progress to date in activating primordial follicles of cattle and baboons. Pieces of ovarian cortex, rich in primordial follicles, were obtained from fetal bovine and baboon ovaries during late gestation. Pieces were maintained in organ culture in serum-free medium containing ITS+ (insulin-transferrin-selenium-linoleic acid-BSA) for up to 20 days and at various times during culture some pieces were fixed for histological morphometry. As early as 2 days of culture, the number of primordial follicles had decreased by 88% or 55%, whereas the number of primary follicles had increased 2.5- or 5-fold, compared to tissue freshly isolated from bovine or baboon ovaries, respectively (P < 0.01). In baboon cortical pieces a small number of secondary follicles developed during a 20-day culture period. The development of primary and secondary follicles was accompanied by an increase in diameter of both the granulosa cell layer and the oocyte. The addition of FSH (1, 10, or 100 ng/ml) had no effect on the development of follicles in bovine cortical pieces after 7 or 14 days of culture, relative to control cultures without FSH. These results show that a high percentage of primordial follicles from cattle and baboons can be activated to grow in serum-free medium in the absence of gonadotropins. Conditions that will support further growth in vitro of follicles from these species remain to be elucidated. The culture system we have developed could be used to develop such conditions and to explore factors that regulate the movement of primordial follicles into the pool of growing follicles.
Animals↗
Effects of volume, culture media and type of culture dish on in vitro development of hamster 1-cell embryos.
We examined effects of medium volume and two different culture media (HECM-3 and HECM-4) on in vitro development of hamster embryos. Groups of 5 to 8 1-cell embryos were cultured for 72 h in either < or =100 or > or =100 microl volumes. In the first experiment, embryos were cultured in Petri dishes with 2, 5, 20, 50 or 100 microl of medium using the two media (2 x 5 factorial experiment). Optimal volumes for morula and blastocyst development were 100 microl of HECM-3 and > or =50 microl of HECM-4; in HECM-4, > or =20 microl volumes were suitable whereas in HECM-3 < or = 50 microl volumes were unsuitable. In the second experiment, embryos were cultured in 100, 200, 500 and 1000 microl of HECM-3 and HECM-4 using organ culture dishes. Controls were 100 microl drops in Petri dishes. In organ culture dishes, blastocyst development was < or =6% in HECM-3 and 33-41% in HECM-4, and suitable volumes for development to at least morulae were > or =200 microl of HECM-3, and > or =100 microl of HECM-4. In both experiments development to morula and blastocyst stages with 100 microl volume in Petri dishes was significantly higher with HECM-4 (96 and 85% in Experiment 1 and 2, respectively) than that with HECM-3 (52 and 40% in Experiment 1 and 2, respectively; P < 0.05). These results indicate that attention should be paid to both type and volume of medium and interaction with type of culture dish for optimizing development of embryos in vitro.
Journal Article↗
Gonadotropins, serum, and amino acids alter nuclear maturation, cumulus expansion, and oocyte morphology in hamster cumulus-oocyte complexes in vitro.
Glucose, lactate, and pyruvate (the substrate triad), gonadotropins, serum, and amino acids were tested on maturation of cumulus-oocyte complexes (COCs) using a simple defined medium, Tyrode's-PVA (T-PVA). In experiment 1, effects of FSH (2 microg/ml) and the substrate triad were tested using a 2 x 2 factorial design. After 12-13 h, nuclear maturation was depressed in the absence of the triad or with FSH (0-14% metaphase II [MII]) compared with the triad alone (92% MII, p < 0.05). Subsequent experiments used as the base medium Tyrode's solution with the triad (TLP-PVA): adding 10% bovine calf serum (BCS) and gonadotropins (10 microg/ml FSH, 10 microg/ml LH, or both) yielded nuclear maturation equivalent to that in medium alone (88-100% post-metaphase I [post-MI] oocytes). Responses with glutamine, or with 11 but not 20 amino acids, were equivalent to the response in BCS with gonadotropins (93-100% post-MI oocytes). Some cumulus expansion occurred in COCs matured with gonadotropins and BCS, or glutamine, or 11 amino acids, but was less extensive than for in vivo-matured COCs. Oocytes matured with gonadotropins and BCS, or glutamine, or 11 amino acids plus gonadotropins, but not medium alone, had normal-appearing first polar bodies. Another cytoplasmic marker, cortical distribution of microfilaments (detected by confocal microscopy), did not differ between in vitro- and in vivo-matured oocytes. We conclude that effects of gonadotropins on hamster nuclear maturation, cumulus expansion, and oocyte morphology are modulated by serum or amino acids; maturation conditions producing normal oocyte and cumulus morphologies are predicted to yield developmentally competent oocytes.
Actin Cytoskeleton↗
Male pronuclear formation and early embryonic development of hamster oocytes matured in vitro with gonadotrophins, amino acids and cysteamine.
Male pronuclear (MPN) formation in oocytes after in vitro maturation (IVM) was compared with that of matured follicular oocytes that had matured in vivo (controls). Cumulus-oocyte complexes were matured in vitro for 13 h in modified Tyrode's solution (TLP-PVA); cumulus-free oocytes were then incubated in 20% oviductal fluid for 3 h, and washed and capacitated spermatozoa were added. MPN formation was significantly lower (P < 0.05) in IVM oocytes 3 to 12 h after insemination (0 to 34%, respectively) than in control oocytes (range, 98-100%). Female pronuclear formation was 84-100% in controls and IVM oocytes, but spermatozoa incompletely decondensed in IVM oocytes. The addition of 10 mumol l-1 during IVM, significantly increased (P < 0.05) MPN formation (from 17% in the absence of cysteine to 47% in the presence of cysteine), but was lower than that in controls (88%). During IVM, the addition of 10% serum or gonadotrophins (FSH and LH) with or without amino acids did not support MPN formation without cysteamine, whereas the treatment with gonadotrophins and 11 amino acids plus 200 mumol cysteamine l-1 (82%) equalled controls (92%). Development of oocytes after IVM (in 0, 10, 20% serum) in TLP-PVA, gonadotrophins, 11 amino acids and 200 mumol cysteamine l-1 was compared with development in controls. Of the IVM treatments, 20% serum was inferior at fertilization, but yielded the highest percentage of fertilized oocytes developing to or beyond the four-cell stage (20% serum versus controls, respectively); fertilized oocytes, 75% versus 88%; > or = four-cell embryo, 40% versus 53%; blastocyst, 8% versus 14%. It was concluded that during IVM, gonadotrophins plus 11 amino acids interacted with cysteamine, enhancing the decondensation of spermatozoa and MPN formation; oocytes matured in this medium with 20% serum were fertilized and some developed to the blastocyst stage.
Amino Acids↗
Nicotine protects against the dexamethasone potentiation of kainic acid-induced neurotoxicity in cultured hippocampal neurons.
To elucidate the neuroprotective effect of nicotine, we investigated whether nicotine may attenuate dexamethasone potentiation of kainic acid-induced neurotoxicity. Primary hippocampal culture was pre-treated with nicotine for 24 h followed by dexamethasone (10(-4) M) for 24 h. Then, cultures were exposed with kainic acid (10(-4) M) and cellular viability was determined by LDH effluxmetry. Nicotine pre-treatment (10(-9)-10(-7) M) dose-dependently attenuated dexamethasone potentiation of kainic acid-induced neurotoxicity. These results may support the epidemiological data suggesting a neuroprotective effect of cigarette smoking on Alzheimer's disease or Parkinson's disease.
Animals↗
Kinetics of sperm penetration and fertilization in vitro in hamster follicular and oviductal ova.
This study compared kinetics of in vitro sperm penetration and fertilization of in vivo matured follicular vs. oviductal hamster ova. Monospermic fertilization in cumulus-intact (C+) and cumulus-free (C-) follicular ova was highest at sperm concentrations of approximately 50 and 1.0 x 10(4) sperm/ml, respectively. The cumulus layer was a barrier to sperm penetration, but penetration of C-follicular ova was still lower than with C-oviductal ova. At 1 h of sperm:egg coincubation, 21% of C- follicular ova was penetrated vs. 77% of C- oviductal ova, while by 4 h these values were 71% and 98%, respectively. In contrast, activation of zona-free follicular and oviductal ova was not different (83-99%). However, at 2 h male pronuclear formation was lower in follicular ova (9%) than in oviductal ova (26%, P < 0.05). To test the hypothesis that lower and slower penetration with follicular ova compared with oviductal ova is due to lack of oviductal exposure, effects of hamster periovulatory oviductal fluid on penetration and fertilization kinetics of C- follicular ova were examined. Adding oviductal fluid did not improve penetration of follicular ova to levels equivalent to oviductal ova, but 3 h preincubation of follicular ova with oviductal fluid improved both zona penetration and male pronucleus formation to levels equivalent to those in oviductal ova. We conclude that kinetics of sperm penetration and fertilization in follicular ova are retarded, that these deficiencies derive from lack of ovum modulation by oviductal secretions, and that prolonged oviductal exposure of ova is essential for achieving full competence.
Animals↗
Differential expression of c-fos mRNA in rat prefrontal cortex, striatum, N. accumbens and lateral septum after typical and atypical antipsychotics: an in situ hybridization study.
The regional difference in the expression of c-fos mRNA induced by typical and atypical antipsychotics was determined in prefrontal cortex, striatum, N. accumbens and lateral septum in rats by in situ hybridization. Two typical antipsychotics, haloperidol (2 mg/kg) and fluphenazine (2 mg/kg), and three atypical antipsychotics, (-)sulpiride (100 mg/kg), clozapine (20 mg/kg) and OPC-14597 (40 mg/kg), were used. Brains were fixed with 4% paraformaldehyde 45 min after drug administration (i.p.). Brain sections of 30 microns-thickness were made in a cryostat and hybridized with 35S-labelled for c-fos oligonucleotide probe. These sections were apposed to X-ray films and the autoradiograms were semi-quantitatively analysed by computer-assisted densitometry. All antipsychotics used increased c-fos mRNA expression in N. accumbens shell, a region of the forebrain associated with limbic systems. On the other hand, two typical antipsychotics (haloperidol and fluphenazine) that cause a high incidence of acute motor side effects increased the expression of c-fos mRNA in the dorsolateral striatum, an extrapyramidal region primarily involved in motor control. Only clozapine induced c-fos mRNA in the medial prefrontal cortex and lateral septum. These results strongly suggest that the shell region of N. accumbens may be a common site of therapeutic action of antipsychotics.
Animals↗
Maturation of hamster oocytes under chemically defined conditions and sperm penetration through the zona pellucida.
This study aimed to achieve high frequencies of nuclear maturation and penetrability through the zona pellucida of hamster oocytes cultured under protein-free conditions. Completion of nuclear maturation by cumulus-intact, immature oocytes (79% metaphase II stage) was depressed (37%; p < 0.05) by adding four amino acids (glutamine, isoleucine, methionine and phenylalanine) reported necessary for nuclear maturation of cumulus-free oocytes. Following in vitro maturation, cumulus cells were removed and oocytes were inseminated with capacitated sperm, but after 6 h sperm:egg co-incubation, only 24% of in vitro matured oocytes were penetrated compared with 60% of in vivo matured oocytes (p < 0.05). Time required for zona lysis by alpha-chymotrypsin was not significantly different among in vitro and in vivo matured oocytes and 1-cell embryos. Addition to the maturation medium of soybean trypsin inhibitor or fetuin, both known to inhibit the zona reaction in vitro, did not improve penetrability of in vitro matured oocytes, implying that in hamsters, unlike other rodent species, a premature zona reaction is unlikely to be responsible for inhibiting sperm penetration. When oocytes were incubated with 20% periovulatory oviductal fluid (OF) for another 3 h after maturation, penetration was significantly improved (60% vs 37% with and without OF, respectively; p < 0.05), but was not equivalent to penetration of in vivo matured follicular oocytes similarly treated with OF (84%, p < 0.05). However, zona penetration was further improved by increasing sperm concentration from 1.0 x 10(4) (66%) to 5.0 or 10.0 x 10(4) sperm/ml (89%, p < 0.05). This study shows that nuclear maturation of hamster oocytes can occur in chemically defined medium, and indicates that a deficiency in the zona of in vitro matured oocytes can be overcome by preincubation with OF and insemination at high sperm concentration.
Amino Acids↗
[An oral repeated dose toxicity study of a new antineoplastic agent S-1 in dogs. I. A 13-week repeated dose toxicity study. II. An ophthalmologic toxicity recovery study].
S-1, an antineoplastic formulation of a fluorinated pyrimidine derivative containing tegafur (FT), CDHP, and potassium oxonate (Oxo) in a molar ratio of 1:0.4:1, was recently developed by Taiho Pharmaceutical Co., Ltd., with the aim of prolonging the effective plasma concentration of 5-fluorouracil (5-FU) over that producted by FT alone and reducing its dose-limiting gastrointestinal toxicity. As a part of the S-1 toxicity study, a 13-week repeated dose toxicity study and a recovery study of opthalmologic effects were conducted in dogs. The following results were obtained. All S-1 doses are expressed in terms of their FT content. 1. Concerning the general condition, dark brown pigment was deposited on the sclera of the eye in all S-1 treated groups starting at the second week of treatment, and clouding of the cornea was noted in the 3 mg/kg/day group starting after 3-4 weeks of treatment. In the 3 and 6 mg/kg/day groups, general signs such as salivation, reduction in spontaneous movements, and sedation appeared, and 1 male and 2 females of the 3 mg/kg/day group died or were moribund 4-5 weeks after treatment began. All animals of the 6 mg/kg/day died or were sacrificed within 2 weeks of the start of the study. 2. Food consumption and body weight were reduced in the groups administered 1 mg/kg/day or more S-1. 3. No apparent drug-induced changes were observed on electrocardiography, urinalysis, fecal occult blood test, hematological examination, liver and kidney function tests, or ocular mucosa infection tests. 4. Blood biochemical examinations showed decreases in creatinine and chloride levels in males, an increase in LDH activity, and decreases in the albumin level and A/G ratio in females of the 3 mg/kg/day group. 5. Organ weighing showed that the relative weight of the kidney was increased in males and females of the 3 mg/kg/day group. 6. Histopathological examination revealed melanin deposition in the conjunctiva or cornea and atrophy inflammation, neutrophil infiltration, and neovascularization in the corneal epithelium. Atrophy of lymphatic tissues, such as the thymus, spleen and various lymph nodes, and changes in the reproductive system such as aspermatogenesis and uterine atrophy, which are commonly observed side effects of anticancer drugs, were also noted. 7. In the group administered FT, vacuolation of the cerebral fornix and commissura anterior was observed in 1 animal, but no changes were observed in other examinations. 8. The toxic effects of S-1 appeared primarily in the eyes, lymphatic tissues, and reproductive organs, and deaths were ascribed to weakening due to exacerbation of the general effects accompanied by disorders of immunological function. The NOAEL of S-1 in this study was estimated to be the dose that delivered less than 0.5 mg/kg/day in both males and females. 9. Changes in the eye observed after S-1 administration are such as pigmentation of the sclera and white turbidity of the cornea. Though it may be produced vision decreased, these changes are considered to be unaccompanied by functional disorders and to be reversible.
Administration, Oral↗
NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, increases extracellular GABA in the striatum of the freely moving rat.
We investigated the effect of an NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), on the levels of endogenous GABA in the rat striatum using in vivo microdialysis. Rats were perfused with the artificial CSF containing L-NMMA (0.1, 0.3 and 1.0 mM) or its inactive isomer D-NMMA (1.0 mM) for 1 h. Infusion of L-NMMA, but not its D-isomer, dose-dependently increased GABA concentration. Co-infusion with tetrodotoxin (1 microM) did not antagonize the increase of GABA induced by L-NMMA. These results show that decreased NO activity enhances GABA release even in the absence of depolarization of GABA neurones. We conclude that NO may be directly acting on GABA nerve terminals and tonically inhibiting GABA release or synthesis under basal conditions.
Animals↗
Characterisation of extracellular amino acids in striatum of freely moving rats by in vivo microdialysis.
To investigate the characteristics of extracellular amino acids released from the striatum, we performed in vivo microdialysis in non-anaesthetised, freely moving rats. Amino acids were determined after precolumn derivatisation with o-phthalaldehyde by high-performance liquid chromatography and fluorescence detection. The omission of Ca2+ in the perfusion medium partially decreased the basal concentration of aspartate and glutamate. This shows that a small fraction of basal concentration of aspartate and glutamate is of neuronal origin. The effect of high K+ and veratrine stimulation was evaluated in the presence or absence of Ca2+ or tetrodotoxin (1 microM). High K+ and veratrine caused a remarkable increase in the aspartate and glutamate efflux. The omission of Ca2+ only partially decreased K(+)-stimulated aspartate and glutamate efflux. Tetrodotoxin completely antagonised veratrine-stimulated aspartate and glutamate efflux. Although glycine and taurine releases were stimulated by high K+ and veratrine, their release was not always antagonised with Ca2+ omission or tetrodotoxin inclusion. Thus, the neuronal origin of stimulated release of glycine and taurine is unclear. Although tetrodotoxin sensitivity and Ca2(+)-dependency are regarded as a basic criterion for classical neurotransmitters in microdialysis experiments, they should not be adapted to the physiological characteristics of the release of amino acids.
Amino Acids↗
Behavioural and neurochemical effects of OPC-14597, a novel antipsychotic drug, on dopaminergic mechanisms in rat brain.
OPC-14597 is a new antipsychotic drug with a unique pharmacological profile. In a behavioural study in rats OPC-14597 did not show cataleptogenic activity even at the highest dose (40 mg/kg, i.p.), whereas it antagonized apomorphine-induced stereotypy dose-dependently (0.5-40 mg/kg). In vivo microdialysis showed that extracellular dopamine (DA) in the striatum was decreased significantly after OPC-14597 administration at higher doses of 10 and 40 mg/kg. Similar results were obtained in extracellular dopamine concentration in the frontal cortex, although the changes in DOPAC and HVA concentrations were smaller than those in the striatum. OPC-14597 also antagonized DA increase induced by the DA autoreceptor antagonist (+)-AJ76. These results that OPC-14597 acts either as an antagonist at postsynaptic dopamine receptors or as an agonist at presynaptic dopamine autoreceptors.
Animals↗
A placebo-controlled double-blind study of epalrestat (ONO-2235) in patients with diabetic neuropathy.
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Adult↗
Effect of caerulein on expression of the immediate-early genes c-fos and zif/268 in the rat brain.
The effect of caerulein, an analog of cholecystokinin-8, on expression of the immediate-early genes c-fos and zif/268 was studied in the rat brain using Northern blot analysis and an in situ hybridization technique. Intraperitoneal injection of caerulein did not change the basal c-fos and zif/268 expression. Administration of the convulsant, pentylenetetrazole (PTZ), caused a dramatic increase of c-fos and zif/268 mRNAs in the hippocampus and dentate gyrus. Pretreatment with caerulein suppressed the PTZ-induced c-fos and zif/268 expression. It is considered that systemically administrated caerulein modifies neuronal activities by exerting a suppressed effect on induction of the immediate-early genes.
Animals↗
[Secondary autonomic diseases--diabetes mellitus].
Diabetic autonomic neuropathy is an established disease entity. Nevertheless, it was not until 1945 that diabetic autonomic neuropathy became a target for extensive systemic research works. This is partly because of insidious progress of this disorder and lack of precise techniques of examinations. In this review article, signs and symptoms of diabetic autonomic disorders characteristic of each visceral organ are described. Among them, such symptoms as painless myocardial infarction and hypoglycemia without warning symptoms deserve special attention. It is essential for clinical doctors to realize that autonomic neuropathy is the most important part for understanding diabetic pathology.
Autonomic Nervous System Diseases↗
Identification and characterization of specific binding sites of [3H]spermidine in synaptic membranes of rat brain.
Synaptic membranes of rat brain contained specific binding sites of [3H]spermidine (SPD) that exhibited an inverse temperature dependency, structure selectivity, reversibility, saturability, low affinity and high density with an uneven distribution profile. The affinities were not significantly different from each other in the rodent brain, while the highest density was found in the medulla-pons among the central structures examined with progressively lower densities in the midbrain, striatum, cerebellum, hypothalamus, hippocampus and cerebral cortex. The binding was insensitive to digestion by various proteases and glycosidases but sensitive to potentiation by phospholipases. A clear correlation was seen between the abilities of several natural and synthetic polyamines to displace [3H]SPD binding and to potentiate [3H] (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine binding to open cation channels associated with an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors. Treatment of brain membranes with deoxycholic acid resulted in a significant solubilization of [3H]SPD binding sites. Furthermore, [3H]SPD markedly associated with the acidic phospholipid phosphatidylserine irrespective of the presence of synaptic membranes in a manner sensitive to inhibition by a variety of calmodulin antagonists. These results suggest that endogenous polyamines may play a stimulatory role in neuronal responses mediated by the NMDA receptor ionophore complex through an interaction between their positive charges and negative charges of membranous phosphatidylserine in rat brain.
Animals↗