Protective effects of L-carnitine on reproductive capacity in rats with diabetes.

To explore the protective effects of L-carnitine on erectile function and reproductive function in rats with diabetes. A total of 60 male diabetes mellitus induced-erectile dysfunction (DMED) rats were randomly divided into three groups, 20 rats in each group. The blank group was fed normally, the control group was fed with 0.9% sodium chloride solution 5 ml/kg/day, and the experimental group was given L-carnitine 300 mg/kg/day. After six weeks, the Corpus cavernosum penis pressure (ICP) and mean arterial pressure (MAP) were measured. The sperm of epididymis were taken to detect the parameters of sperm. After six weeks of treatment, ICP and MAP in the experimental group were significantly higher than those in the control group and blank group (p < 0.05), and sperm density and PR in the experimental group were significantly higher than those in the control group and the blank group (p < 0.05). Superoxide dismutase (SOD) in the experimental group was significantly higher than that in the control group and blank group (p < 0.05). Malondialdehyde (MDA) in the experimental group was significantly lower than that in the control group and blank group (p < 0.05). The follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone in the experimental group were significantly higher than those in the control group and blank group (p < 0.05). We conclude that L-carnitine can significantly improve erectile function and reproductive function in rats with diabetes and it has great potential in the treatment of systemic organ damage in DMED rats.

Effect of vitrification on the mRNA transcriptome of bovine oocytes.

Vitrification has been shown to decrease the developmental capacity of mammalian oocytes, and this is closely associated with the abnormal mRNA expressions of vitrified oocytes. However, the effect of vitrification on transcriptional machinery of oocytes examined by RNA sequencing (RNA-seq) has yet to be defined. In the present study, the mRNA transcriptomes of fresh and vitrified bovine oocytes were analysed by Smart-seq2 with the differently expressed genes determined by DEseq2 (an adjusted p-value of .05 and a minimum fold change of 2). The differentially expressed mRNAs were then searched against the Gene Ontology (GO) and Genomes (KEGG) database. Finally, the mRNA expressions of 10 candidate genes were validated using quantitative real-time PCR (qRT-PCR). Approximately 12,000 genes were detected in each sample of fresh or vitrified oocytes. Of these, the expression levels of 102 genes differed significantly in vitrified groups: 12 genes mainly involved in cell cycle, fertilization and glucose metabolism were upregulated, and 90 genes mainly involved in mitochondria, ribosomal protein, cytoskeleton, transmembrane protein, cell cycle and calcium ions were downregulated. GO analysis showed that these genes were mainly enriched in terms of membrane-bounded organelles, macromolecular complex, and intracellular part. The mRNA expression levels of 10 candidate genes selected randomly were in agreement with the results of the RNA-seq. In conclusion, our results showed that vitrification affected the mRNA transcriptome of bovine oocytes by downregulating genes, which contributed to the decreased developmental capacity of vitrified oocytes. Our findings will be useful in determining approaches to improve the efficiency of vitrified oocytes.

Composition of Mineral Produced by Dental Mesenchymal Stem Cells.

Mesenchymal stem cells isolated from different dental tissues have been described to have osteogenic/odontogenic-like differentiation capacity, but little attention has been paid to the biochemical composition of the material that each produces. Here, we used Raman spectroscopy to analyze the mineralized materials produced in vitro by different dental cell populations, and we compared them with the biochemical composition of native dental tissues. We show that different dental stem cell populations produce materials that differ in their mineral and matrix composition and that these differ from those of native dental tissues. In vitro, BCMP (bone chip mass population), SCAP (stem cells from apical papilla), and SHED (stem cells from human-exfoliated deciduous teeth) cells produce a more highly mineralized matrix when compared with that produced by PDL (periodontal ligament), DPA (dental pulp adult), and GF (gingival fibroblast) cells. Principal component analyses of Raman spectra further demonstrated that the crystallinity and carbonate substitution environments in the material produced by each cell type varied, with DPA cells, for example, producing a more carbonate-substituted mineral and with SCAP, SHED, and GF cells creating a less crystalline material when compared with other dental stem cells and native tissues. These variations in mineral composition reveal intrinsic differences in the various cell populations, which may in turn affect their specific clinical applications.

Effects of different activation regimens on pronuclear formation and developmental competence of in vitro-matured porcine oocytes after intracytoplasmic sperm injection.

To improve the activation protocol for in vitro-maturated porcine oocytes after intracytoplasmic sperm injection (ICSI), we examined the combined effect of U0126, a specific inhibitor of mitogen-activated protein kinase kinases, and an electrical pulse on pronuclear formation and developmental competence. Two approaches were tested: (i) 6-h treatment of ICSI oocytes with U0126 applied at different intervals (0, 2, 3 or 4 h) after the electrical pulse and (ii) treatment of ICSI oocytes with U0126 applied 4 h after the electrical pulse over an additional 4, 6 or 8 h. Another protein kinase inhibitor, 6-dimethylaminopurine, was used as a chemical activator in control experiments. The highest rates of diploid embryo formation, normal fertilization and blastocyst formation were observed after 6 h of exposure to U0126 starting 4 h after the electrical pulse. Therefore, U0126 can be used as an activating agent for porcine oocytes fertilized by ICSI.

Expression of vesicular glutamate transporters in peripheral vestibular structures and vestibular nuclear complex of rat.

Glutamate transmission from vestibular end organs to central vestibular nuclear complex (VNC) plays important role in transferring sensory information about head position and movements. Three isoforms of vesicular glutamate transporters (VGLUTs) have been considered so far the most specific markers for glutamatergic neurons/cells. In this study, VGLUT1 and VGLUT2 were immunohistochemically localized to axon terminals in VNC and somata of vestibular primary afferents in association with their central and peripheral axon endings, and VGLUT1 and VGLUT3 were co-localized to hair cells of otolith maculae and cristae ampullaris. VGLUT1 and VGLUT2 defined three subsets of Scarpa's neurons (vestibular ganglionic neurons): those co-expressing VGLUT1 and VGLUT2 or expressing only VGLUT2, and those expressing neither. In addition, many neurons located in all vestibular subnuclei were observed to contain hybridized signals for VGLUT2 mRNA and a few VNC neurons, mostly scattered in medial vestibular nucleus (MVe), displayed VGLUT1 mRNA labelling. Following unilateral ganglionectomy, asymmetries of VGLUT1-immunoreactivity (ir) and VGLUT2-ir occurred between two VNCs, indicating that the VNC terminals containing VGLUT1 and/or VGLUT2 are partly of peripheral origin. The present data indicate that the constituent cells/neurons along the vestibular pathway selectively apply VGLUT isoforms to transport glutamate into synaptic vesicles for glutamate transmission.

Unusually mild phenotype of AADC deficiency in 2 siblings.

Aromatic L-amino acid decarboxylase deficiency is a rare neurotransmitter defect leading to serotonin, dopamine and norepinephrine deficiency. Affected individuals usually present in infancy with severe developmental delay, oculogyric crises and extrapyramidal movements. We present the clinical, molecular and biochemical features of a pair of siblings who presented with fatigability, hypersomnolence and dystonia and who showed excellent response to treatment. Analysis of CSF biogenic amines, plasma AADC levels and direct sequencing of the DDC gene was performed. CSF catecholamine metabolites were reduced, with elevation of 3-O-methyldopa. Plasma AADC activity was undetectable in both siblings, and decreased in their carrier parents. One missense mutation (853C>T) was found in exon 8, and a donor splice site mutation was found in the intron after exon 6 (IVS6+4A>T). Both siblings showed excellent response to MAO inhibitor and dopamine agonist treatment. This report expands the clinical spectrum of AADC deficiency and contributes to the knowledge of the genotype and phenotype correlation for the DDC gene. It is important to recognize the milder phenotypes of the disease as these patients might respond well to therapy.

The effects of nickel and cobalt ions on the spontaneous electrical activity, slow wave, in the circular muscle of the guinea-pig gastric antrum.

1. Circular muscle strips of the guinea-pig gastric muscle produced spontaneous electrical activity in the form of slow waves. The slow wave amplitude, maximum rate of rise, duration, and frequency were 31 mV, 60 mV sec-1, 4.3 sec, and 4.3 min-1 on average, respectively. These parameters were not appreciably affected by 3 microM nifedipine or nicardipine, even following membrane depolarization with 60 mM K+. 2. Ni2+ (1-100 microM) increased slow wave amplitude and frequency, but reduced the rate of rise, accompanied by membrane depolarization. The rate of rise and depolarization slowly recovered to the control values in the continuous presence of Ni2+, but slow wave frequency remained high. The recovery after wash-out was very poor particularly when a high concentration of Ni2+ was applied. 3. The effects of Co2+ were fundamentally the same as those of Ni2+. 4. Removal of external Ca2+ slowly reduced the rate of rise and amplitude of the slow waves in the absence and the presence of Ni2+ and Co2+, although the effects were reduced in the presence of these metal ions. 5. Concentrations of Ni2+ and Co2+ greater than 1 mM suppressed the slow waves. However, when the external Na+ was replaced with N-methyl-D-glucamine during the suppression, nearly normal electrical activity was resumed. 6. Since slow waves were not significantly affected by nifedipine (3 microM) and Ni2+ (100 microM), the inward currents generating slow waves do not seem to flow through L-type Ca2+ channels or typical T-type Ca2+ channels. Slow waves are probably potentiated by Ni2+ and Co2+ acting intracellularly. These ions at higher concentrations seem to inhibit the pacemaker activity more powerfully than they do the inward currents responsible for slow wave generation.

Effects of phosphodiesterase inhibitors on spontaneous electrical activity (slow waves) in the guinea-pig gastric muscle.

1. The effects of the phosphodiesterase inhibitors caffeine, theophylline, isobutylmethylxanthine (IBMX) and rolipram on spontaneous electrical activity (slow waves) were studied in the circular muscle of the guinea-pig gastric antrum. 2. All the inhibitors reduced slow wave frequency without changing the membrane potential and the slow wave configuration, but at higher concentrations they blocked the slow waves and caused membrane hyperpolarization. In the presence of the inhibitors a low level of irregular electrical activity could be observed in many preparations. 3. Isoprenaline, forskolin, dibutyryl cAMP and 8-bromo-cAMP all produced effects essentially similar to those of phosphodiesterase inhibitors. K+ (12 mM) and removal of K+ both depolarized the membrane and these were not affected by IBMX (1-3 microM). A decrease in frequency caused by IBMX was also not significantly affected by 12 mM K+ or K+ removal and only partially antagonized by TEA or 4-aminopyridine. 4. These results suggest that an increase in intracellular cAMP inhibits pacemaker activity of slow waves. An increase in K+ conductance does not seem to be a major factor in this inhibition. Slow waves appear to be a compound electrical activity in a group of muscle cells and are likely to be disintegrated by xanthine derivatives.

Sustained contraction produced by caffeine after ryanodine treatment in the circular muscle of the guinea-pig gastric antrum and rabbit portal vein.

1. Caffeine inhibited spontaneous mechanical activity at 0.3-1 mM, but produced a tonic contraction at concentrations higher than 3 mM in the circular muscle of the guinea-pig gastric antrum. In the circular muscle of the rabbit portal vein, caffeine at concentrations higher than 1 mM produced an early phasic contraction followed by a small tonic component. The caffeine-induced contraction was abolished by removal of the external Ca2+ more rapidly in the gastric antrum than the portal vein. 2. When the preparations were pretreated with ryanodine (1 microM) a sustained contraction developed on wash-out of caffeine (10 mM) both in the gastric antrum and portal vein. This contraction was not affected by nicardipine (3 microM) or verapamil (3 microM), but was readily abolished by removal of the external Ca2+ or by addition of cobalt (1 mM). Spontaneous electrical activity, the slow wave, in gastric muscles was blocked in the presence of 10 mM caffeine, but reappeared during the sustained contraction. 3. Both the contractions induced directly by caffeine and those produced following caffeine wash-out after ryanodine treatment were accompanied by a maintained increase in intracellular Ca2+ concentration measured with fura-2. 4. The presence or absence of Ca2+ during the application of ryanodine did not affect the ability of caffeine to initiate sustained contractions, provided Ca2+ was present during the exposure to caffeine. 5. It is concluded that caffeine can induce a sustained contraction after ryanodine treatment both in the guinea-pig gastric antrum and rabbit portal vein, by activating a Ca2+ influx pathway insensitive to organic Ca2+ channel blockers. No clear evidence was obtained for involvement of the Ca2+ influx pathway activated through depletion of intracellular Ca2+ stores. A hypothesis is proposed that the plasma membrane of these preparations is similar to the sarcoplasmic reticulum membrane in that Ca2+permeability can be increased almost irreversibly by a combination of caffeine and ryanodine in the presence of the external Ca2+.

The inner sublayer of the circular muscle coat in the canine proximal colon: origins of spontaneous electrical and mechanical activity.

In the canine proximal colon, tissue near the submucosal surface of the circular muscle layer produces spontaneous mechanical contractions, synchronized with electrical slow waves. Comparative physiological examination of tissue strips from various regions of the submucosa and circular muscle coat revealed that the characteristic smooth muscle tissue of the innermost sublayer of the circular muscle is required for this rhythmical phenomenon. Histological examination showed that tissues containing special smooth muscle cells form an inner sublayer of the circular muscle coat. These innermost muscle cells were distinguishable from the bulk circular muscle cells by the following features: 1) flattened and shorter shapes of the cell and nucleus, 2) numerous caveolae on the cell surface, 3) abundant mitochondria, and 4) frequent gap-junction formations. Neither slow waves nor spontaneous mechanical rhythmicities were recorded from the submucosal connective tissue or from the bulk circular muscle tissue without the inner sublayer. The thicker smooth muscle cells found in the submucosal border specimens were identical in histological features to the bulk circular muscles which produced no slow waves and no spontaneous contractions. Cellular elements in the interstitium, such as fibroblasts, mast cells and macrophages, were found in all tissue strips that were physiologically examined. Nerve elements were found in all the specimens; however, there was a unique nerve network probably corresponding to the plexus entericus (submucosus) extremus described by Stach (1972). It was concluded, therefore, that the inner sublayer characterized by special smooth muscle cells with a delicate nerve plexus is essential for producing spontaneous activities of the circular muscle coat in the canine proximal colon.