Extracellular Vesicles from Follicular and Ampullary Fluid Isolated by Density Gradient Ultracentrifugation Improve Bovine Embryo Development and Quality.

Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality.

Forskolin and protein kinase inhibitors differentially affect hair cell potassium currents and transmitter release at the cytoneural junction in the isolated frog labyrinth.

The post-transductional elaboration of sensory input at the frog semicircular canal has been studied by correlating the effects of drugs that interfere with phosphorylation processes on: (i) potassium conductances in isolated hair cell and (ii) transmitter release at the cytoneural junction in the intact labyrinth. At hair cells, delayed potassium currents (IKD) undergo voltage- and time-dependent inactivation; inactivation removal requires ATP, is sensitive to kinase blockade, but is unaffected by exogenous application of cyclic nucleotides. We report here that forskolin, an activator of endogenous adenylyl cyclase, enhances IKD inactivation removal in isolated hair cells, but produces an overall decrease in IKD amplitude consistent with the direct blocking action of the drug on several families of K channels. In the intact labyrinth, forskolin enhances transmitter release, consistent with such depression of K conductances. Kinase blockers - H-89 and KT5823 - have been shown to reduce IKD inactivation removal and IKD amplitude at isolated hair cells. In the labyrinth, the effects of these drugs on junctional activity are quite variable, with predominant inhibition of transmitter release, rather than the enhancement expected from the impairment of K currents. The overall action of forskolin and kinase inhibitors on K conductances is similar (depression), but they have opposite effects on transmitter release: this indicates that some intermediate steps between the bioelectric control of hair cell membrane potential and transmitter release are affected in opposite ways and therefore are presumably regulated by protein phosphorylation.

Role of saposin C and D in auditory and vestibular function.

OBJECTIVES/HYPOTHESIS: Saposins are small proteins derived from a precursor protein, prosaposin. Each of the four saposins (A-D) is necessary for the activity of lysosomal glycosphingolipid hydrolases. Individual saposin mutations lead to lysosomal storage diseases, some of which are associated with hearing loss. Here we evaluate the effects of the loss of saposins C and D on auditory and vestibular function in transgenic mice. METHODS: Transgenic mice with either loss of saposin C function or a combined loss of saposin C + D function were studied. Light microscopy and immunofluorescence were used to evaluate histologic and morphologic changes in the auditory and vestibular organs. Acoustic brainstem response thresholds and distortion product otoacoustic emissions were used to study the auditory phenotype. RESULTS: A null mutation of saposin C did not result in any identifiable histologic changes or loss of hearing through postnatal day 55. Combined losses of saposins C and D similarly did not result in any changes in organ of Corti histology or loss of hearing. However, inclusions within the vestibular end organs was noted, consistent with afferent and efferent neuronal sprouting, although to a much milder degree than seen in the previously studied prosaposin knockout mouse. CONCLUSIONS: Loss of saposin C and D function, although causing mild phenotypic changes in the vestibular end organs, otherwise results in minimal functional impairment and no changes in the auditory system. It is more likely that the auditory and vestibular effects of the loss of prosaposin are mediated through the actions of saposin A and/or B. LEVEL OF EVIDENCE: NA.

Uptake of fluorescent gentamicin by peripheral vestibular cells after systemic administration.

OBJECTIVE: In addition to cochleotoxicity, systemic aminoglycoside pharmacotherapy causes vestibulotoxicity resulting in imbalance and visual dysfunction. The underlying trafficking routes of systemically-administered aminoglycosides from the vasculature to the vestibular sensory hair cells are largely unknown. We investigated the trafficking of systemically-administered gentamicin into the peripheral vestibular system in C56Bl/6 mice using fluorescence-tagged gentamicin (gentamicin-Texas-Red, GTTR) imaged by scanning laser confocal microscopy to determine the cellular distribution and intensity of GTTR fluorescence in the three semicircular canal cristae, utricular, and saccular maculae at 5 time points over 4 hours. RESULTS: Low intensity GTTR fluorescence was detected at 0.5 hours as both discrete puncta and diffuse cytoplasmic fluorescence. The intensity of cytoplasmic fluorescence peaked at 3 hours, while punctate fluorescence was plateaued after 3 hours. At 0.5 and 1 hour, higher levels of diffuse GTTR fluorescence were present in transitional cells compared to hair cells and supporting cells. Sensory hair cells typically exhibited only diffuse cytoplasmic fluorescence at all time-points up to 4 hours in this study. In contrast, non-sensory cells rapidly exhibited both intense fluorescent puncta and weaker, diffuse fluorescence throughout the cytosol. The numbers and size of fluorescent puncta in dark cells and transitional cells increased over time. There is no preferential GTTR uptake by the five peripheral vestibular organs' sensory cells. Control vestibular tissues exposed to Dulbecco's phosphate-buffered saline or hydrolyzed Texas Red had negligible fluorescence. CONCLUSIONS: All peripheral vestibular cells rapidly take up systemically-administered GTTR, reaching peak intensity 3 hours after injection. Sensory hair cells exhibited only diffuse fluorescence, while non-sensory cells displayed both diffuse and punctate fluorescence. Transitional cells may act as a primary pathway for trafficking of systemic GTTR from the vasculature to endolymph prior to entering hair cells.

Improved biolistic transfection of hair cells.

Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca(2+)-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells.

Evolution of electrosensory ampullary organs: conservation of Eya4 expression during lateral line development in jawed vertebrates.

The lateral line system of fishes and amphibians comprises two ancient sensory systems: mechanoreception and electroreception. Electroreception is found in all major vertebrate groups (i.e. jawless fishes, cartilaginous fishes, and bony fishes); however, it was lost in several groups including anuran amphibians (frogs) and amniotes (reptiles, birds, and mammals), as well as in the lineage leading to the neopterygian clade of bony fishes (bowfins, gars, and teleosts). Electroreception is mediated by modified "hair cells," which are collected in ampullary organs that flank lines of mechanosensory hair cell containing neuromasts. In the axolotl (a urodele amphibian), grafting and ablation studies have shown a lateral line placode origin for both mechanosensory neuromasts and electrosensory ampullary organs (and the neurons that innervate them). However, little is known at the molecular level about the development of the amphibian lateral line system in general and electrosensory ampullary organs in particular. Previously, we identified Eya4 as a marker for lateral line (and otic) placodes, neuromasts, and ampullary organs in a shark (a cartilaginous fish) and a paddlefish (a basal ray-finned fish). Here, we show that Eya4 is similarly expressed during otic and lateral line placode development in the axolotl (a representative of the lobe-finned fish clade). Furthermore, Eya4 expression is specifically restricted to hair cells in both neuromasts and ampullary organs, as identified by coexpression with the calcium-buffering protein Parvalbumin3. As well as identifying new molecular markers for amphibian mechanosensory and electrosensory hair cells, these data demonstrate that Eya4 is a conserved marker for lateral line placodes and their derivatives in all jawed vertebrates.

Computational model of a circulation current that controls electrochemical properties in the mammalian cochlea.

Sound-evoked mechanical stimuli permit endolymphatic K(+) to enter sensory hair cells. This transduction is sensitized by an endocochlear potential (EP) of +80 mV in endolymph. After depolarizing the cells, K(+) leaves hair cells in perilymph, and it is then circulated back to endolymph across the lateral cochlear wall. In theory, this process entails a continuous and unidirectional current carried by apical K(+) channels and basolateral K(+) uptake transporters in both the marginal cell and syncytial layers of the lateral wall. The transporters regulate intracellular and extracellular [K(+)], allowing the channels to form K(+) diffusion potentials across each of the two layers. These diffusion potentials govern the EP. What remains uncertain is whether these transport mechanisms accumulating across diverse cell layers make up a continuous circulation current in the lateral wall and how this current might affect the characteristics of the endolymph. To address this question, we developed an electrophysiological model that incorporates channels and transporters of the lateral wall and channels of hair cells that derive a circulation current. The simulation replicated normal experimental EP values and reproduced experimentally measured changes in the EP and intra- and extracellular [K(+)] in the lateral wall when different transporters and channels were blocked. The model predicts that, under these different conditions, the circulation current's contribution to the EP arises from different sources. Finally, our model also accurately simulated EP loss in a mouse model of a chloride channelopathy associated with deafness.

K+ currents in isolated vestibular afferent calyx terminals.

Vestibular hair cells transduce mechanical displacements of their hair bundles into an electrical receptor potential which modulates transmitter release and subsequent action potential firing in afferent neurons. To probe ionic mechanisms underlying sensory coding in vestibular calyces, we used the whole-cell patch-clamp technique to record action potentials and K(+) currents from afferent calyx terminals isolated from the semicircular canals of Mongolian gerbils. Calyx terminals showed minimal current at the mean zero-current potential (-60 mV), but two types of outward K(+) currents were identified at potentials above -50 mV. The first current was a rapidly activating and inactivating K(+) current that was blocked by 4-aminopyridine (4-AP, 2.5 mM) and BDS-I (up to 250 nM). The time constant for activation of this current decreased with membrane depolarization to a minimum value of approximately 1 ms. The 4-AP-sensitive current showed steady-state inactivation with a half-inactivation of approximately -70 mV. A second, more slowly activating current (activation time constant was 8.5 +/- 0.7 ms at -8 mV) was sensitive to TEA (30 mM). The TEA-sensitive current also showed steady-state inactivation with a half-inactivation of -95.4 +/- 1.4 mV, following 500-ms duration conditioning pulses. A combination of 4-AP and TEA blocked approximately 90% of the total outward current. In current clamp, single Na(+)-dependent action potentials were evoked following hyperpolarization to potentials more negative than the resting potential. 4-AP application increased action potential width, whereas TEA both increased the width and greatly reduced repolarization of the action potential.

Ultrastructural co-localization of cochlin and type II collagen in the rat semicircular canal.

Cochlin and type II collagen are major constituents of the inner ear extracellular matrix. To investigate the morphological relation of cochlin and type II collagen in the rat semicircular canal, immuno-electronmicroscopic analysis was performed using the post-embedding immunogold method. Immunolabeling for cochlin was detected in the fibrillar substance underlying the supporting epithelium of the sensory cells and beneath the epithelial cells facing the endolymph in the semicircular canals. Immunolabeling for type II collagen was observed in the same fibrillar substance in the subepithelial area. The co-localization of cochlin and type II collagen in the fibrillar substance in the subepithelial area indicate that cochlin may play a role in the structural homeostasis of the vestibule acting in concert with the fibrillar type II collagen bundles.

Histamine H1 receptors are expressed in mouse and frog semicircular canal sensory epithelia.

Histamine-related drugs are commonly used in the treatment of vertigo and related vestibular disorders. Their site and mechanism of action, however, are still poorly understood. To increase our knowledge of the histaminergic system in the vestibular organs, we have investigated the expression of H1 and H3 histamine receptors in the frog and mouse semicircular canal sensory epithelia. Analysis was performed by mRNA reverse transcriptase-PCR, immunoblotting and immunocytochemistry experiments. Our data show that both frog and mouse vestibular epithelia express H1 receptors. Conversely no clear evidence for H3 receptors expression was found.