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    ABSTRACT: In vitro fertilization (IVF) in horses is rarely successful. One reason for this could be failure of sperm to fully capacitate or exhibit hyper-active motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm, and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVI) with equine sperm capacitated with dilauroyl phosphatidylcholine (PC12) would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In Experiment 1, bovine oocytes were used for: 1) IVF with bovine sperm, 2) IVF with equine sperm, and 3) intracytoplasmic sperm injections (ICSI) with equine sperm. Presumptive zygotes were either stained with 4',6-diamidino-2-phenylindole (DAPI) from 18 to 26 h at 2 h intervals or evaluated for cleavage 56 h following addition of sperm. Equine sperm fertilized bovine oocytes; however, pro-nuclei formation was delayed compared to bovine sperm after IVF. The delayed pro-nuclear formation was not seen following ICSI. In Experiment 2, bovine oocytes were assigned to five groups: 1) cumulus oocyte complexes (COC) co-incubated with bovine sperm, 2) COC exposed to sucrose then co-incubated with bovine sperm, 3) COC co-incubated with equine sperm, 4) COC exposed to sucrose and co-incubated with equine sperm, and 5) oocytes exposed to sucrose and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P=0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In Experiment 3, oocytes were assigned to four groups: 1) IVF, equine and bovine COC co-incubated with equine sperm, 2) PVI of equine and bovine oocytes, 3) PVI with equine oocytes pretreated with sucrose, and 4) ICSI of equine oocytes. Oocytes were examined at 24h for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with PC12 resulted in 85% of the oocytes cleaving. Sperm injected into the PV space of equine oocytes did not appear to enter the ooplasm. This study validated the use of bovine oocytes for equine sperm studies and indicates that failure of equine IVF is more than an inability of equine sperm to penetrate the ZP.
    Theriogenology 01/2014;
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    ABSTRACT: In 2050, beef likely will be produced much as occurs currently, as (1) a by-product of dairying-cull cows and calves not needed as replacements; (2) intensively managed cattle in environments rich in feed resources; or (3) extensively managed cattle grazing land unsuitable for tillage, with calves often moving to richer feed environments. Genetic progress will continue to depend on information such as weaning weights, but in addition, genetic, epigenetic, and phenotypic information will be obtained from blood, hair, semen, and/or biopsies of embryos.Most cattle will be genetically modified for efficient growth, desirable carcass traits, and management traits such as disease resistance. Some strains of cattle will have Y-chromosome-dependent terminal cross traits; sexed semen thus will automatically result in males with terminal cross characteristics and females with maternally desirable traits. In most cases, mother cows will have shorter gestations and smaller frame sizes than currently to decrease nutrient requirements for maintenance. The cow herd may disappear with some intensively managed systems; with sexed semen, each female can replace herself with a female calf and then be fattened for slaughter. The flexibility of being a ruminant will continue to be exploited by using a variety of feedstuffs, some of which are otherwise of little value.
    Advances in experimental medicine and biology 01/2014; 752:239-44.
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    ABSTRACT: To investigate the distribution of retinal ganglion cells (RGCs) and visual acuity in alpacas (Vicugna pacos) through Brn-3a immunofluorescent labeling. Five eyes from four healthy alpacas with normal ophthalmic examination findings were included in the study. The axial length of the globes was measured before fixation. All five retinas were treated with Brn-3a antibodies to label RGCs. Images taken with a fluorescent microscope were used for RGC counting. RGC density maps were reconstructed by computer software. Visual acuity was estimated based on the results of peak RGC density and ocular anatomical parameters. The reconstructed retinal maps from Brn-3a labeling showed a horizontal streak across the retinal meridian superior to the optic nerve head with a temporal, upward extension. The highest RGC densities were in the temporal retinas. The maximal visual acuity was located in the temporal retina and was estimated to range between 12.5 and 13.4 cycles per degree. Alpacas have a horizontal streak across the retinal meridian superior to the optic disk with a temporal, upward extension based on the Brn-3a labeling of RGCs. The maximal visual acuity was located in the temporal retina. The reconstructed retinal maps indicate the RGC topography of alpacas is similar to that of other herbivores, but is different from that of dromedary camels.
    Veterinary Ophthalmology 12/2013;
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    ABSTRACT: Foetal exposure to phthalates is known to adversely impact male reproductive development and function. Developmental anomalies of reproductive tract have been attributed to impaired testosterone synthesis. However, species differences in the ability to produce testosterone have been noted; e.g., following foetal exposure, abnormal clustering of Leydig cells or decreased production of testosterone that manifests in rats does not occur in mice or humans. Nonetheless, other facets of testicular dysgenesis do occur in both rats and mice as well as in some other species tested. We recently published a comprehensive evaluation of the foetal rat testis proteome following in utero exposure to diethylhexyl phthalate (DEHP), which revealed changes in individual proteins that are known to be factors in cellular differentiation and migration or related to the capacity of the foetal Leydig cell to produce T and fit a pathway network in which each is regulated directly or indirectly by oestradiol. Plasma oestradiol indeed was elevated ~two-fold in 19-day-old DEHP-exposed foetal male rats. In this brief review, we discuss our new findings vis-à-vis "oestrogen hypothesis" as a cause for testicular dysgenesis syndrome.
    Reproduction 11/2013;
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    ABSTRACT: The (pro)renin receptor (PRR), which binds both renin and prorenin, is a newly discovered component of the renin-angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, nonproteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate-salt-induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. PRR expression, detected by immunostaining and reverse transcription-polymerase chain reaction, was significantly decreased in the brains of knockout mice compared with wild-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild-type mice. This hypertensive response was abolished in PRR-knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate-salt increased PRR expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in PRR-knockout mice. PRR knockout in neurons prevented the development of deoxycorticosterone acetate-salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, nonproteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate-salt-induced hypertension, possibly through diminished angiotensin II formation.
    Hypertension 11/2013;
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    ABSTRACT: The only practicable method for sexing mammalian sperm is by measuring DNA content with a flow cytometer/cell sorter. Although many other methods have been tried and patented, they either are inaccurate, damage sperm severely, or otherwise are unsuitable for practical application. Procedures for sexing sperm damage them slightly, and fewer sperm are packaged per straw than with unsexed semen. These 2 characteristics result in lower fertility with sexed than unsexed semen. Incremental improvements of current sexing procedures are being developed constantly.
    Veterinary Clinics of North America Food Animal Practice 11/2013; 29(3):619-25.
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    ABSTRACT: Endocan is a secreted proteoglycan that has been shown to indicate angiogenic activity: remodeling in several tumor types in humans and mice. Serum endocan levels also indicate prognosis and has been proposed as a biomarker for certain cancers. Recently, monoclonal antibodies directed against mouse endocan have been developed allowing for further characterization of endocan function and potentially as a marker for angiogenesis through immunoreactivity in endothelial tip cells. The results of the current study show that endocan immunoreactivity in the mouse brain is present in blood vascular networks including but not limited to the cortex, hippocampus and paraventricular nucleus of the hypothalamus in C57BL/6J and FVB/N mice. Endocan immunoreactivity did not vary during postnatal development or by sex. Interestingly, after vascular perfusion with fluorescein isothiocyanate (FITC), endothelial cells positive for FITC were immunonegative for endocan suggesting FITC interference with the immunohistochemistry. A small number of FITC-negative blood vessels were endocan immunoreactive suggesting the identification of new blood vessels that are not yet functional. The current study shows that endocan is normally present in the mouse brain and prior vascular perfusion with FITC may provide a useful tool for identify newly forming blood vessels.
    Journal of immunological methods 09/2013;
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    ABSTRACT: Important functions of the vascular endothelium, including permeability, production of antithrombotic factors, and control of vascular tone, are regulated by changes in intracellular Ca(2+). The molecular identities and regulation of Ca(2+) influx channels in the endothelium are incompletely understood, in part because of experimental difficulties associated with application of patch-clamp electrophysiology to native endothelial cells. However, advances in confocal and total internal reflection fluorescence microscopy and the development of fast, high affinity Ca(2+)-binding fluorophores have recently allowed for direct visualization and characterization of single channel transient receptor potential (TRP) channel Ca(2+) influx events in endothelial cells. These events, called "TRP channel Ca(2+) sparklets," have been optically recorded from primary endothelial cells and the intact endothelium, and the biophysical properties and fundamental significance of these Ca(2+) signals in vasomotor regulation have been characterized. This review will first briefly discuss the role of endothelial cell TRP channel Ca(2+) influx in endothelium-dependent vasodilation, will describe improved methods for recording unitary TRP channel activity using optical methods, and will highlight discoveries regarding the regulation and physiological significance of TRPV4 Ca(2+) sparklets in the vascular endothelium enabled by this new technology. Perspectives on the potential use of these techniques to evaluate changes in TRP channel Ca(2+) influx activity associated with endothelial dysfunction are offered.
    AJP Cell Physiology 09/2013;
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    ABSTRACT: Electrical synapses are abundant in the vertebrate brain, but their functional and molecular complexities are still poorly understood. We report here that electrical synapses between auditory afferents and goldfish Mauthner cells are constructed by apposition of hemichannels formed by two homologs of mammalian connexin 36 (Cx36) and that, while Cx35 is restricted to presynaptic hemiplaques, Cx34.7 is restricted to postsynaptic hemiplaques, forming heterotypic junctions. This molecular asymmetry is associated with rectification of electrical transmission that may act to promote cooperativity between auditory afferents. Our data suggest that, in similarity to pre- and postsynaptic sites at chemical synapses, one side in electrical synapses should not necessarily be considered the mirror image of the other. While asymmetry based on the presence of two Cx36 homologs is restricted to teleost fish, it might also be based on differences in posttranslational modifications of individual connexins or in the complement of gap junction-associated proteins.
    Neuron 09/2013; 79(5):957-69.
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    ABSTRACT: We examined the involvement of membrane microdomains during human luteinizing hormone (LH) receptor recovery from receptor desensitization after removal of bound hormone. Lateral motions of individual desensitized LH receptors expressed on the surface of Chinese hamster ovary cells and transient association of these receptors with detergent-resistant membrane (DRM) microdomains isolated using isopycnic sucrose gradient ultracentrifugation were assessed. Single particle tracking experiments showed untreated individual LH receptors to be confined within cell-surface membrane compartments with an average diameter of 199 ± 17 nm and associated with membrane fractions characteristic of bulk plasma membrane. After brief exposure to human chorionic gonadotropin (hCG), LH receptors remained for several hours desensitized to hCG challenge. Throughout this period, significantly increased numbers of LH receptors were confined within smaller diameter (<120 nm) membrane compartments and associated with DRM fragments of characteristically low density. By 5 h, when cells again produced cAMP in response to hCG, unoccupied LH receptors were found in larger 169 ± 22 nm diameter cell-surface membrane compartments and >90 % of LH receptors were again found in high-density membrane fragments characteristic of bulk plasma membrane. Taken together, these results suggest that, during recovery from LH receptor desensitization, LH receptors are both located with DRM lipid environments and confined within small, mesoscale (80-160 nm) cell-surface compartments. This may reflect hormone-driven translocation of receptors into DRM and formation there of protein aggregates too large or too rigid to permit effective signaling. Once bound hormone is removed, receptor structures would have to dissociate before receptors can again signal effectively in response to hormone challenge. Moreover, such larger protein complexes would be more easily constrained laterally by membrane structural elements and so appear resident in smaller cell-surface compartments.
    Cell biochemistry and biophysics 08/2013;
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