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Scientific Reports Oct 2015For the past 30 years, oocytes from Xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment. Here we... (Comparative Study)
Comparative Study
For the past 30 years, oocytes from Xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment. Here we report the first use of oocytes from the closely related species Xenopus borealis as an alternative expression system for neuronal ion channels. Using the two-electrode voltage-clamp technique, we show that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either X. laevis or X. borealis oocytes. Potential advantages of the X. borealis oocytes include a smaller endogenous chloride current and the ability to produce more intense fluorescence signals when studied with voltage-clamp fluorometry. Scanning electron microscopy revealed a difference in vitelline membrane structure between the two species, which may be related to the discrepancy in fluorescence signals observed. We demonstrate that X. borealis oocytes are a viable heterologous system for expression of neuronal ion channels with some potential advantages over X. laevis oocytes for certain applications.
Topics: Acid Sensing Ion Channel Blockers; Acid Sensing Ion Channels; Animals; Female; Ion Channels; Microscopy, Electron, Scanning; Neurons; Oocytes; Patch-Clamp Techniques; Vitelline Membrane; Xenopus; Xenopus laevis
PubMed: 26440210
DOI: 10.1038/srep14763 -
Development (Cambridge, England) Apr 2015The eggshell serves as a depository for proteins that play an important role in early embryonic development. In particular, the Drosophila eggshell is responsible for...
The eggshell serves as a depository for proteins that play an important role in early embryonic development. In particular, the Drosophila eggshell is responsible for transferring asymmetries from the egg chamber to specify the regions at both ends of the embryo through the uneven activation of the Torso (Tor) receptor in its membrane. This process relies on the restricted expression of the gene torso-like (tsl) in subpopulations of follicle cells during oogenesis and its protein accumulation at both poles of the eggshell, but it is not known how this signal is transmitted to the embryo. Here, we show that Tsl accumulates at the embryonic plasma membrane, even in the absence of the Tor receptor. However, during oogenesis, we detected Tsl accumulation only at the eggshell. These results suggest that there is a two-step mechanism to transfer the asymmetric positional cues from the egg chamber into the early embryo: initial anchoring of Tsl at the eggshell as it is secreted, followed by its later translocation to the egg plasma membrane, where it enables Tor receptor activation. Translocation of anchored determinants from the eggshell might then regulate the spatial and temporal control of early embryonic developmental processes.
Topics: Animals; Blastoderm; Cell Membrane; Drosophila Proteins; Drosophila melanogaster; Egg Shell; Embryo, Nonmammalian; Female; Models, Biological; Ovarian Follicle; Protein Transport
PubMed: 25758463
DOI: 10.1242/dev.117630 -
Poultry Science Mar 2015The US egg industry is exploring alternative housing systems for laying hens. However, limited published research related to cage-free aviary systems and enriched colony... (Comparative Study)
Comparative Study
The US egg industry is exploring alternative housing systems for laying hens. However, limited published research related to cage-free aviary systems and enriched colony cages exists related to production, egg quality, and hen nutrition. The laying hen's nutritional requirements and resulting productivity are well established with the conventional cage system, but diminutive research is available in regards to alternative housing systems. The restrictions exist with limited availability of alternative housing systems in research settings and the considerable expense for increased bird numbers in a replicate due to alternative housing system design. Therefore, the objective of the current study was to evaluate the impact of nutrient and energy intake on production and egg quality parameters from laying hens housed at a commercial facility. Lohmann LSL laying hens were housed in three systems: enriched colony cage, cage-free aviary, and conventional cage at a single commercial facility. Daily production records were collected along with dietary changes during 15 production periods (28-d each). Eggs were analyzed for shell strength, shell thickness, Haugh unit, vitelline membrane properties, and egg solids each period. An analysis of covariance (ANCOVA) coupled with a principal components analysis (PCA) approach was utilized to assess the impact of nutritional changes on production parameters and monitored egg quality factors. The traits of hen-day production and mortality had a response only in the PCA 2 direction. This finds that as house temperature and Met intake increases, there is an inflection point at which hen-day egg production is negatively effected. Dietary changes more directly influenced shell parameters, vitelline membrane parameters, and egg total solids as opposed to laying hen housing system. Therefore, further research needs to be conducted in controlled research settings on laying hen nutrient and energy intake in the alternative housing systems and resulting impact on egg quality measures.
Topics: Animal Husbandry; Animal Welfare; Animals; Chickens; Eating; Eggs; Energy Intake; Housing, Animal; Ovum
PubMed: 25630672
DOI: 10.3382/ps/peu078 -
PloS One 2014Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their...
Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.
Topics: Amino Acids; Animals; Embryonic Development; Glutathione; Lipids; Ovum; Spectrometry, Mass, Electrospray Ionization; Subcellular Fractions; Xenopus laevis; gamma-Aminobutyric Acid
PubMed: 25506922
DOI: 10.1371/journal.pone.0115173 -
Mechanisms of Development Nov 2014Monospermic fertilization in the frog, Xenopus laevis, is ensured by a fast-rising, positive fertilization potential to prevent polyspermy on the fertilized egg,...
Monospermic fertilization in the frog, Xenopus laevis, is ensured by a fast-rising, positive fertilization potential to prevent polyspermy on the fertilized egg, followed by a slow block with the formation of a fertilization envelope over the egg surface. In this paper, we found that not only the enzymatic activity of sperm matrix metalloproteinase-2 (MMP-2) was necessary for a sperm to bind and/or pass through the extracellular coat of vitelline envelope, but also the hemopexin (HPX) domain of MMP-2 on the sperm surface was involved in binding and membrane fusion between the sperm and eggs. A peptide with a partial amino acid sequence of the HPX domain caused egg activation accompanied by an increase in [Ca(2+)]i in a voltage-dependent manner, similar to that in fertilization. The membrane microdomain (MD) of unfertilized eggs bound the HPX peptide, and this was inhibited by ganglioside GM1 distributed in the MD. The treatment of sperm with GM1 or anti-MMP-2 HPX antibody allows the sperm to fertilize an egg clamped at 0 mV, which untreated sperm cannot achieve. We propose a model accounting for the mechanism of voltage-dependent fertilization based on an interaction between the positively charged HPX domain in the sperm membrane and negatively-charged GM1 in the egg plasma membrane.
Topics: Animals; Calcium; Cell Membrane; Fertilization; Hemopexin; Male; Matrix Metalloproteinase 2; Membrane Potentials; Ovum; Spermatozoa; Xenopus laevis
PubMed: 25296387
DOI: 10.1016/j.mod.2014.09.005 -
Wiley Interdisciplinary Reviews.... 2014The pathway that generates the dorsal-ventral (DV) axis of the Drosophila embryo has been the subject of intense investigation over the previous three decades. The... (Review)
Review
UNLABELLED
The pathway that generates the dorsal-ventral (DV) axis of the Drosophila embryo has been the subject of intense investigation over the previous three decades. The initial asymmetric signal originates during oogenesis by the movement of the oocyte nucleus to an anterior corner of the oocyte, which establishes DV polarity within the follicle through signaling between Gurken, the Drosophila Transforming Growth Factor (TGF)-α homologue secreted from the oocyte, and the Drosophila Epidermal Growth Factor Receptor (EGFR) that is expressed by the follicular epithelium cells that envelop the oocyte. Follicle cells that are not exposed to Gurken follow a ventral fate and express Pipe, a sulfotransferase that enzymatically modifies components of the inner vitelline membrane layer of the eggshell, thereby transferring DV spatial information from the follicle to the egg. These ventrally sulfated eggshell proteins comprise a localized cue that directs the ventrally restricted formation of the active Spätzle ligand within the perivitelline space between the eggshell and the embryonic membrane. Spätzle activates Toll, a transmembrane receptor in the embryonic membrane. Transmission of the Toll signal into the embryo leads to the formation of a ventral-to-dorsal gradient of the transcription factor Dorsal within the nuclei of the syncytial blastoderm stage embryo. Dorsal controls the spatially specific expression of a large constellation of zygotic target genes, the Dorsal gene regulatory network, along the embryonic DV circumference. This article reviews classic studies and integrates them with the details of more recent work that has advanced our understanding of the complex pathway that establishes Drosophila embryo DV polarity. For further resources related to this article, please visit the WIREs website.
CONFLICT OF INTEREST
The authors have declared no conflicts of interest for this article.
Topics: Animals; Body Patterning; Drosophila; Epistasis, Genetic; Gene Expression Regulation, Developmental; Gene Regulatory Networks
PubMed: 25124754
DOI: 10.1002/wdev.138 -
Poultry Science Sep 2014This study was conducted to assess the effect of dietary supplementation of meat-type quail breeders with guanidinoacetic acid (GAA) on their reproductive parameters and...
This study was conducted to assess the effect of dietary supplementation of meat-type quail breeders with guanidinoacetic acid (GAA) on their reproductive parameters and progeny performance. Two hundred forty meat-type quails at 25 wk of age were distributed in a completely randomized design with 5 treatments and 8 replicates of 6 birds each. The treatments consisted of 5 dietary levels of GAA (0.00, 0.06, 0.12, 0.18, and 0.24%). The progenies from quail breeders were housed according to breeder treatments and fed a conventional diet based on corn and soybean meal without GAA supplementation. Dietary GAA levels did not affect (P > 0.05) the productivity of meat-type quail breeders, although the concentration of guanidinic compounds (creatine, GAA, and creatinine) in the eggs from the breeders increased linearly (P < 0.05) according to the increase in dietary GAA levels. The number of spermatozoa present in the vitelline membrane was not affected (P > 0.05) by the treatments, but there was a quadratic effect (P < 0.05) of the levels of GAA on fertility, embryonic mortality, and egg hatchability, with the best results estimated at 0.13, 0.15, and 0.14% GAA, respectively. The creatine levels of the pectoral muscle in newborn quails showed a quadratic effect (P ≤ 0.07), and the dietary GAA level of 0.11% was estimated to maximize the muscular creatine level in the progeny. There was a quadratic effect (P < 0.05) of GAA levels on weight gain and feed conversion of progeny at 35 d of age with an optimization point of 0.14% GAA for these variables. Dietary GAA supplementation of meat-type quail breeders increases the availability of creatine in eggs and muscle of progeny, which results in better reproductive parameters and better postnatal progeny performance.
Topics: Animal Feed; Animals; Chromatography, Ion Exchange; Coturnix; Diet; Dietary Supplements; Female; Glycine; Male; Random Allocation; Reproduction
PubMed: 24974392
DOI: 10.3382/ps.2014-03894 -
American Journal of Physiology. Cell... Nov 2014Exposing an oocyte to CO2/HCO3 (-) causes intracellular pH (pHi) to decline and extracellular-surface pH (pHS) to rise to a peak and decay. The two companion papers...
Exposing an oocyte to CO2/HCO3 (-) causes intracellular pH (pHi) to decline and extracellular-surface pH (pHS) to rise to a peak and decay. The two companion papers showed that oocytes injected with cytosolic carbonic anhydrase II (CA II) or expressing surface CA IV exhibit increased maximal rate of pHi change (dpHi/dt)max, increased maximal pHS changes (ΔpHS), and decreased time constants for pHi decline and pHS decay. Here we investigate these results using refinements of an earlier mathematical model of CO2 influx into a spherical cell. Refinements include 1) reduced cytosolic water content, 2) reduced cytosolic diffusion constants, 3) refined CA II activity, 4) layer of intracellular vesicles, 5) reduced membrane CO2 permeability, 6) microvilli, 7) refined CA IV activity, 8) a vitelline membrane, and 9) a new simulation protocol for delivering and removing the bulk extracellular CO2/HCO3 (-) solution. We show how these features affect the simulated pHi and pHS transients and use the refined model with the experimental data for 1.5% CO2/10 mM HCO3 (-) (pHo = 7.5) to find parameter values that approximate ΔpHS, the time to peak pHS, the time delay to the start of the pHi change, (dpHi/dt)max, and the change in steady-state pHi. We validate the revised model against data collected as we vary levels of CO2/HCO3 (-) or of extracellular HEPES buffer. The model confirms the hypothesis that CA II and CA IV enhance transmembrane CO2 fluxes by maximizing CO2 gradients across the plasma membrane, and it predicts that the pH effects of simultaneously implementing intracellular and extracellular-surface CA are supra-additive.
Topics: Animals; Bicarbonates; Biological Transport; Carbon Dioxide; Carbonic Anhydrase II; Carbonic Anhydrase IV; Cell Membrane; Humans; Hydrogen-Ion Concentration; Models, Biological; Oocytes; Vitelline Membrane; Xenopus laevis
PubMed: 24965589
DOI: 10.1152/ajpcell.00049.2014