Sperm replacement in sperm-storage tubules causes last-male sperm precedence in chickens.

1. This study elucidated the last-male sperm precedence (LMSP) mechanism in chickens by examining replacement in storage tubules (SSTs) after multiple artificial inseminations (AI) and the effects of seminal plasma (SP) and male breed on sperm replacement in SSTs.2. Hens were artificially inseminated with fluorescent dye-labelled spermatozoa from White Leghorn (WL) chickens. Secondary AI was conducted 3 d later with sperm labelled with different nuclear fluorescent dye. Percentage of first and second inseminated sperm in SSTs and their logarithmic odds were calculated. The effect of SP on LMSP was examined using (1) Lake's solution-washed sperm before second insemination, and (2) SP injected continuously after first insemination. Effect of breed difference on sperm replacement was investigated using Barred Plymouth Rock (BP) sperm.3. Successive WL-sperm inseminations at three-day intervals caused > 70% stored sperm replacement in SSTs. Although SP removal from sperm from second insemination significantly decreased replacement, its intra-vaginal injection did not affect release. Secondary insemination using BP sperm significantly increased replacement.4. Sperm replacement is a major factor favouring LMSP in domestic chickens. Two fluorescent staining of sperm, and intra-vaginal multiple AI technique have enabled visualisation, differentiation, and quantification of multiple inseminated sperm stored in the SSTs.

Cell cycle-dependent dynamics of cytoskeleton involving mitochondrial redistribution in hamster embryos.

Mitochondria-cytoskeleton interactions were studied in the hamster embryos during interphase and M phase of the cell cycle. Two-cell embryos were cultured for 1 h with nocodazole, cytochalasin D or in a combination of both inhibitors and then centrifuged at 10,000 × g for 2 min. The control embryos were only centrifuged with no inhibitor treatment. Centrifuged embryos were fluorescently stained to examine the distribution of active mitochondria and nuclear configuration. In the control 2-cell embryos, most mitochondria were accumulated at the perinuclear region with some at the cell cortex. Neither each inhibitor nor centrifugation did affect the distribution of mitochondria in interphase blastomeres. However, mitochondria were spun down towards the centrifugal pole in 71% (n = 41) of the interphase blastomeres treated with centrifugation following a combination of nocodazole plus cytochalasin D, suggesting that both microtubules and microfilaments may involve in mitochondrial redistribution during interphase of the cell cycle. In contrast, when M-phase blastomeres were treated with all drug treatments applied, including cytochalasin D, mitochondria had been usually dislocated in a unipolar cluster, suggesting that microfilaments, not microtubules, may involve in the mitochondrial redistribution during M phase of the cell cycle. The data indicate that microfilaments function in mitochondrial redistribution regardless of the stages of the cell cycle and that microtubules may strongly associate with mitochondria during the interphase but dissociate from them during the M phase.

Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), the most common cause of sudden death in the young, is an autosomal dominant disease characterized by ventricular hypertrophy accompanied by myofibrillar disarrays. Linkage studies and candidate-gene approaches have demonstrated that about half of the patients have mutations in one of six disease genes: cardiac beta-myosin heavy chain (c beta MHC), cardiac troponin T (cTnT), alpha-tropomyosin (alpha TM), cardiac myosin binding protein C (cMBPC), ventricular myosin essential light chain (vMLC1) and ventricular myosin regulatory light chain (vMLC2) genes. Other disease genes remain unknown. Because all the known disease genes encode major contractile elements in cardiac muscle, we have systematically characterized the cardiac sarcomere genes, including cardiac troponin I (cTnI), cardiac actin (cACT) and cardiac troponin C (cTnC) in 184 unrelated patients with HCM and found mutations in the cTnI gene in several patients. Family studies showed that an Arg145Gly mutation was linked to HCM and a Lys206Gln mutation had occurred de novo, thus strongly suggesting that cTnI is the seventh HCM gene.

Disruption of endothelial tight junctions in a patient with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS).

An electron microscopic study revealed disruption of capillary endothelial tight junctions (TJs) in both biopsied muscle, taken at 5 years and 1 month of age, and the autopsied brain, taken at 13 years and 6 months of age, in a patient with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) and mitochondrial DNA (mtDNA) point mutation A3243G. This endothelial barrier disruption might result in vasogenic edema and systemic lactic acidosis, possibly the critical pathology of MELAS.

Intermediate filament keratin dynamics during oocyte maturation requires maturation/M-phase promoting factor and mitogen-activated protein kinase kinase activities in the hamster.

Research related to intermediate filaments in mammalian oocytes remains poorly advanced. We investigated keratin reorganization in oocytes during meiotic maturation using immunofluorescence, and examined effects of inhibitors for cdc2 and mitogen-activated protein kinase kinase (MAPKK) on keratin assembly. In germinal vesicle (GV) oocytes (n = 26), large and oval-shaped aggregates of non-fibrillar keratin were found in the cortical ooplasm (designated as a 'cortical' pattern). The delicate network of keratin filaments was concentrated in the GV periphery. The large keratin aggregates began to divide into small fragments at the pro-MI/MI stage (n = 22, designated as a 'fragmented' pattern). Some keratin fragments were occasionally broken down into several granules at the peripheral region. In the MII oocytes (n = 24), the filament network was extended over the ooplasm and numerous keratin granules were scattered across the oocyte (designated as a 'granular' pattern). After 12 h of incubation with roscovitine, 66.7% of the oocytes (20/30) were at the GV stage and showed a cortical pattern of keratin. After incubation with U0126, most oocytes (83.9%, 26/31) were at the MII stage; most of them (76.9%, 20/26) showed a fragmented pattern of keratin. The increasing complexity of keratin filament network from the GV to MII stages suggests a possible role in maintaining cell integrity under physical stress after ovulation. In fact, maturation/M-phase promoting factor is necessary for such keratin reorganization, as is meiotic nuclear progression. In addition, MAPKK is involved in keratin reorganization from a fragmented pattern to a granular pattern.

Ghrelin and leptin did not improve meiotic maturation of porcine oocytes cultured in vitro.

To improve culture system for in vitro maturation (IVM) of porcine oocytes, ghrelin, leptin or growth hormone (GH), at concentration of 0, 0.5, 5, 50 and 500 ng/ml were added to the porcine follicular fluid (pFF)-supplemented medium NCSU23, and their effects on the maturation and cytoskeletal distribution of the oocytes with or without cumulus cells were compared. In the cumulus-denuded oocytes, no significant changes were noted in the maturation rate by different hormone treatments due to a marked decline in the controls. Maturation of the cumulus intact oocytes was moderately interfered by ghrelin (0.5-50 ng/ml, p < 0.01), but not significantly affected by leptin and GH. Distribution density of the cytoplasmic microtubules was decreased significantly by addition of ghrelin (by approximately 30% in 50-500 ng/ml, p < 0.01), whereas no remarkable effect was noted by leptin supplementation. High concentration (500 ng/ml) of ghrelin or leptin decreased significantly the cytoplasmic microfilaments in density (by 43% and 38%, p < 0.01, respectively). GH did not affect cytoskeletal distribution. The results suggest, in the culture system using pFF-supplemented medium that (i) ghrelin may have some inhibitory effect on the organization of microtubules and microfilaments, probably being a factor in lowered maturation rate and (ii) the addition of higher concentration of leptin may decrease microfilaments in density with no effect on meiotic maturation of the porcine oocytes.

Therapeutic silencing of an endogenous gene by siRNA cream in an arthritis model mouse.

Recent advances of biotechnology have laid the groundwork for potent and specific molecular-targeting therapies including RNA interference. The largest remaining hurdle for widespread use of this technology in skin is an effective delivery system. Here, we demonstrate an effective topical delivery system using a cream formulation containing a small-interfering RNA (siRNA) that specifically targets osteopontin (OPN). OPN is a validated target in numerous inflammatory diseases, including rheumatoid arthritis (RA). The siRNA targeting OPN was incorporated into a cream formulation GeneCream that penetrates the stratum corneum, depositing siRNA in the epidermis, dermis, and to a lesser extent, subcutaneous tissue. In addition, when the OPN siRNA cream was topically applied to the skin of a collagen antibody-induced RA mouse model, the siRNA cream prevented the occurrence of severe, irreversible damage to bone and cartilage. Thus, the siRNA cream provides effective delivery of active OPN siRNA, suggesting this formulation may represent a platform technology for delivery of siRNAs for treating various disorders including RA.

Serial measurement of uterine blood flow from mid to late gestation in growth restricted pregnancies induced by overnourishing adolescent sheep dams.

Uterine blood flow (UtBF) is a major regulator of transplacental fetal nutrient supply. The aim was to serially measure uterine blood flow from mid to late pregnancy in a paradigm of relatively late onset placental and fetal growth restriction. Singleton bearing adolescent dams was fed high (H) or control (C) nutrient intakes to induce putatively compromised or normal pregnancies, respectively. A perivascular flow probe was attached to the uterine artery of the gravid horn on Day 83 of gestation and UtBF was then recorded continuously for 2h, three times weekly until approximately Day 135, when pregnancies were either terminated or ewes allowed to deliver at term (approximately Day 145). Pregnancy outcome was determined at term in contemporaneous ewes without UtBF assessment. Placental and fetal weights were lower (P<0.001) in H compared with C intake groups and were independent of flow probe surgery and monitoring. Uterine blood flow was lower in H compared with C groups at the first assessment (Day 88, P<0.001) and was positively correlated with adjusted fetal weight at term, irrespective of treatment group (P<0.01). UtBF increased throughout the second half of gestation in both groups. Linear regression analysis of UtBF against day of gestation revealed that the slope was equivalent (5.5 vs. 5.3ml/min per day) and the mean intercept lower (212 vs. 370ml/min, P<0.001) in H compared with C groups, respectively. This study demonstrates the feasibility of serially measuring UtBF within the same individual sheep for a protracted period during the second half of gestation. UtBF was already lower at mid gestation in putatively growth restricted compared with control pregnancies, ahead of any reduction in placental and fetal weight, but increased similarly during the second half of gestation in both groups. These data are commensurate with the reported decrease in placental angiogenic growth factor expression at mid gestation, and, indicate that attenuated UtBF is an early defect in this adolescent paradigm.

High genetic divergence in miniature breeds of Japanese native chickens compared to Red Junglefowl, as revealed by microsatellite analysis.

A wide diversity of domesticated chicken breeds exist due to artificial selection on the basis of human interests. Miniature variants (bantams) are eminently illustrative of the large changes from ancestral junglefowls. In this report, the genetic characterization of seven Japanese miniature chicken breeds and varieties, together with institute-kept Red Junglefowl, was conducted by means of typing 40 microsatellites located on 21 autosomes. We drew focus to genetic differentiation between the miniature chicken breeds and Red Junglefowl in particular. A total of 305 alleles were identified: 27 of these alleles (8.9%) were unique to the Red Junglefowl with high frequencies (>20%). Significantly high genetic differences (F(ST)) were obtained between Red Junglefowl and all other breeds with a range of 0.3901-0.5128. Individual clustering (constructed from combinations of the proportion of shared alleles and the neighbour-joining method) indicated high genetic divergence among breeds including Red Junglefowl. There were also individual assignments on the basis of the Bayesian and distance-based approaches. The microsatellite differences in the miniature chicken breeds compared to the presumed wild ancestor reflected the phenotypic diversity among them, indicating that each of these miniature chicken breeds is a unique gene pool.

Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons.

Dendritic spines serve as preferential sites of excitatory synaptic connections and are pleomorphic. To address the structure-function relationship of the dendritic spines, we used two-photon uncaging of glutamate to allow mapping of functional glutamate receptors at the level of the single synapse. Our analyses of the spines of CA1 pyramidal neurons reveal that AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors are abundant (up to 150/spine) in mushroom spines but sparsely distributed in thin spines and filopodia. The latter may be serving as the structural substrates of the silent synapses that have been proposed to play roles in development and plasticity of synaptic transmission. Our data indicate that distribution of functional AMPA receptors is tightly correlated with spine geometry and that receptor activity is independently regulated at the level of single spines.

A 2-stage procedure combining maxillary advancement by distraction technique with mandibular setback surgery in patients with cleft lip and palate.

A 2-stage procedure combining maxillary advancement by distraction technique with mandibular setback surgery was used to correct jaw deformities in 5 patients with severe maxillary retrusion secondary to cleft lip and palate. First, a Le Fort I maxillary osteotomy was performed. Immediately after maxillary distraction, the distraction device was removed. The advanced maxilla was fixed with miniplates after adjusting the length and direction of advancement, and mandibular setback surgery was performed simultaneously to obtain a normal occlusal relationship. This 2-stage procedure resulted in stable occlusion and a markedly improved facial profile.

Establishment and characterization of a new human functional cell line from a choriocarcinoma.

A new human functional tumor cell line, designated as T3M-3, has been established from a xenotransplanted choriocarcinoma grown in nude mice. One of the biggest problems of the in vitro culture of these tumor cells using the xenotransplanted tumors had been the dense contamination of fibroblasts of host nude mouse origin. In the present study, these fibroblasts were completely removed by incubating the cells with antiserum raised against nude mouse spleen cells. The cell line established from the remaining tumor cells has been successfully propagated in vitro for as long as 4 years. These cells show the morphology of epithelioid cells containing a prominent nucleus with one or two large nucleoli. The cells grow in a monolayered sheet with the population-doubling time of 19 hr. The cells show perfect tumor takes when they are reinoculated into nude mice. Chromosomal analysis revealed that the cell is a human aneuploid one with a hypotriploid mode. These cultured cells maintained well the function of secreting large amounts of human chorionic gonadotropin, progesterone, and estrogen. The secretion of human chorionic gonadotropin and progesterone by these cells is enhanced by stimulation with tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate and teleocidin B, or with epidermal growth factor in a dose-and time-dependent manner. Interestingly, however, the tumor promoters did not exert a marked effect on the cellular binding of epidermal growth factor, indicating that the receptors for these reagents in T3M-3 cells are not shared by epidermal growth factor.

High pressure conditions promote the proliferation of rat cultured mesangial cells in vitro.

Glomerular capillary pressure is involved in the development of chronic renal failure and has at least two effects on mesangial cells: transmembrane hydrostatic pressure and stretch. To clarify whether pure hydrostatic pressure itself affects the proliferation of cultured rat mesangial cells, we compared the cell number under atmospheric pressure condition with high pressure condition. At 24 and 48 h with 0.5% serum, cell number was significantly higher under high pressure condition than under atmospheric pressure condition. At 48 h, cell number under high pressure condition was increased in a pressure-dependent manner. Furthermore, flow cytometric assay indicated that pressure-load could promote DNA synthesis rate at S phase and enhance G1/S progression induced by low concentration of serum (0.5%). These results suggest that pure hydrostatic pressure itself can promote the proliferation of cultured rat mesangial cells by advancing cell cycle progression in vitro.

Molecular cloning of rat transmembrane domain protein of 40 kDa regulated in adipocytes and its expression in H9c2 cells exposed to ischemic hypoxia and reoxygenation.

We clone a 1230 bp complementary DNA encoding rat transmembrane domain protein of 40 kDa regulated in adipocytes (TPRA40), an orphan receptor, by reverse transcription-polymerase chain reaction using H9c2 cells derived from embryonic rat heart. The deduced amino acid sequence of rat TPRA40 consists of 369 amino acids and has a longer carboxyl terminus than that of the mouse protein. The level of TPRA40 mRNA decreases significantly throughout ischemic hypoxia and reoxygenation.

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H(2)O(2) stimulation.

Cytokines and various cellular stresses are known to activate c-Jun N-terminal kinase-1 (JNK1), which is involved in physiological function. Here, we investigate the activation of JNK1 by oxidative stress in H9c2 cells derived from rat cardiomyocytes. H(2)O(2) (100 microM) significantly induces the tyrosine phosphorylation of JNK1 with a peak 25 min after the stimulation. The amount of JNK1 protein remains almost constant during stimulation. Immunocytochemical observation shows that JNK1 staining in the nucleus is enhanced after H(2)O(2) stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 microM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H(2)O(2)-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H(2)O(2) stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H(2)O(2). Under these conditions, the H(2)O(2)-induced phosphorylation of proteins with molecular masses of 55, 72, and 78 kDa is blocked by treatment with the antisense DNA for JNK1 as compared with the sense DNA for JNK1. These findings suggest that JNK1 induces apoptotic cell death in response to H(2)O(2), and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.

Detection of fibrous cap in atherosclerotic plaque by intravascular ultrasound by use of color mapping of angle-dependent echo-intensity variation.

BACKGROUND: The thickness of the fibrous cap is a major determinant in the vulnerability of atherosclerotic plaque to rupture. It has been demonstrated that intravascular ultrasound (IVUS) backscatter from fibrous tissue is strongly dependent on the ultrasound beam angle of incidence. This study investigated the feasibility of using a new IVUS color mapping technique representing the angle-dependent echo-intensity variation to determine the thickness of the fibrous cap in atherosclerotic plaque. METHODS AND RESULTS: Nineteen formalin-fixed noncalcified human atherosclerotic plaques from necropsy were imaged in vitro with a 30-MHz IVUS catheter. The IVUS catheter was moved coaxially relative to the plaque. The images showing maximum and minimum echo intensity of the plaque surface were selected to calculate the angle-dependent echo-intensity variation. A colorized representation of the echo-intensity variation in the plaque was obtained from the 2 IVUS images. A clearly bordered area with large variation in echo intensity was revealed for each plaque surface in the colorized IVUS image. The thickness (x, mm) of this area correlated significantly with that of fibrous cap (y, mm) measured from histologically prepared sections as y=1.05x-0.01 (r=0.81, P:<0.0001). Bland-Altman analysis also supported the reliability of this method (mean difference, 0.00+/-0.10 mm). CONCLUSIONS: This novel technique for color mapping the echo-intensity variation in IVUS provided an accurate representation of the thickness of the fibrous cap in atherosclerotic plaque. This method may be useful in assessing plaque vulnerability to rupture in atherosclerosis.

Ischemic preconditioning attenuates cardiac sympathetic nerve injury via ATP-sensitive potassium channels during myocardial ischemia.

BACKGROUND: During myocardial ischemia, massive norepinephrine (NE) is released from the cardiac sympathetic nerve terminals, reflecting the sympathetic nerve injury. A brief preceding ischemia can reduce infarct size; this is known as ischemic preconditioning (PC). The effect of PC on sympathetic nerves, however, including its underlying mechanisms in dog hearts, has remained unclear. Thus, this study was designed to elucidate whether the activation of ATP-sensitive potassium (K(ATP)) channels is involved in the mechanism of cardiac sympathetic nerve protection conferred by PC. METHODS AND RESULTS: Interstitial NE concentration was measured by the in situ cardiac microdialysis method in 45 anesthetized dogs. Five minutes of ischemia followed by 5 minutes of reperfusion was performed as PC. In the controls, the dialysate NE concentration (dNE) increased 15-fold after the 40-minute ischemia. PC decreased dNE at 40-minute ischemia by 59% (P<0.01), which was reversed by glibenclamide. A K(ATP) channel opener, nicorandil (25 microg. kg(-1). min(-1) IV), decreased dNE at 40 minutes of ischemia by 76% (P<0.01), which was also reversed by glibenclamide. During the PC procedure, no significant increase in dNE was detected, even with the uptake-1 inhibitor desipramine. CONCLUSIONS: Cardiac sympathetic nerve injury during myocardial ischemia was attenuated by PC via the activation of K(ATP) channels, but the trigger of the PC effect is unlikely to be NE release in dog hearts.

Altered stoichiometry of FKBP12.6 versus ryanodine receptor as a cause of abnormal Ca(2+) leak through ryanodine receptor in heart failure.

BACKGROUND: In the pathogenesis of cardiac dysfunction in heart failure, a decrease in the activity of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase is believed to be a major determinant. Here, we report a novel mechanism of cardiac dysfunction revealed by assessing the functional interaction of FK506-binding protein (FKBP12.6) with the cardiac ryanodine receptor (RyR) in a canine model of pacing-induced heart failure. METHODS AND RESULTS: SR vesicles were isolated from left ventricular muscles (normal and heart failure). The stoichiometry of FKBP12.6 per RyR was significantly decreased in failing SR, as assessed by the ratio of the B(max) values for [(3)H]dihydro-FK506 to those for [(3)H]ryanodine binding. In normal SR, the molar ratio was 3.6 ( approximately 1 FKBP12.6 for each RyR monomer), whereas it was 1.6 in failing SR. In normal SR, FK506 caused a dose-dependent Ca(2+) leak that showed a close parallelism with the conformational change in RyR. In failing SR, a prominent Ca(2+) leak was observed even in the absence of FK506, and FK506 produced little or no further increase in Ca(2+) leak and only a slight conformational change in RyR. The level of protein expression of FKBP12.6 was indeed found to be significantly decreased in failing SR. CONCLUSIONS: An abnormal Ca(2+) leak through the RyR is present in heart failure, and this leak is presumably caused by a partial loss of RyR-bound FKBP12.6 and the resultant conformational change in RyR. This abnormal Ca(2+) leak might possibly cause Ca(2+) overload and consequent diastolic dysfunction, as well as systolic dysfunction.