scholarly journals Using Fluorescence Recovery After Photobleaching to Study Gap Junctional Communication In Vitro

2016 ◽  
pp. 171-179 ◽  
Author(s):  
Maria Kuzma-Kuzniarska ◽  
Clarence Yapp ◽  
Philippa A. Hulley
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Fluorescence Recovery ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional

76 Effects of serum type in maturation medium on in vitro development of bovine embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 163
Author(s):  
A. Mesalam ◽  
S. Zhang ◽  
K.-L. Lee ◽  
S.-H. Song ◽  
L. Xu ◽  
...  
Keyword(s):  
Lipid Content ◽  
Cumulus Cell ◽  
Cell Number ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Maturation Medium ◽  
Gap Junctional

This study investigated the effect of bovine serum albumin (BSA), charcoal:dextran stripped fetal bovine serum (CDS FBS), and heat-inactivated FBS (HI FBS) in maturation medium on their ability to support in vitro oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos. Charcoal:dextran treatment of FBS removes lipophilic chemicals, certain steroid hormones, and certain growth factors; however, HI FBS have a lot-to-lot variation in steroid hormones level that can affect the reproducibility of experimental findings. Oocytes were cultured in TCM-199 supplemented with either 8% (w/v) BSA, 10% (v/v) CDS FBS, or 10% (v/v) HI FBS and 1µg mL−1 oestradiol-17β, 10µg mL−1 FSH, 10ng mL−1 epidermal growth factor, 0.6mM cysteine, 0.2mM sodium pyruvate, and followed by IVF, and the zygotes were cultured in SOF-BE1 medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, immunocytochemistry, and cryo-tolerance. The differences in embryo development between experimental groups were analysed by 1-way ANOVA. The Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P<0.05. We have shown that CDS FBS significantly improved (P<0.05) the percentage of MII oocytes compared with that in the BSA supplemented group (77.2±1.0% v. 69.3%±2.3%, respectively). Moreover, CDS FBS had a higher significant (P<0.05) effect on the rate of blastocyst formation compared with HI FBS and BSA (45.2±0.7% v. 37.4±1.5% and 31.1±3.9%, respectively; 6 replicates were performed). Culture of oocytes with CDS FBS increased (P<0.05) the expression of gap junction proteins, CX37 and CX43, at both transcriptional and translation levels as determined by quantitative RT-PCR and immunofluorescence analysis, respectively. We also found that CDS FBS significantly increased total cell number and decreased the apoptotic index in Day-8 blastocysts compared with the BSA group. Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocysts as identified by Nile red and MitoTracker Green staining, respectively. Taken together, these data suggest that supplementation of maturation medium with CDS FBS, as an alternative to HI FBS, affected cumulus cell-oocyte gap junctional communication, and subsequently improved in vitro developmental competence of bovine oocytes and embryos. Research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant numbers: 117029-3 and 315017-5).

scholarly journals Interaction between Connexin50 and Mitogen-activated Protein Kinase Signaling in Lens Homeostasis

2009 ◽  
Vol 20 (10) ◽  
pp. 2582-2592 ◽  
Author(s):  
Teresa I. Shakespeare ◽  
Caterina Sellitto ◽  
Leping Li ◽  
Clio Rubinos ◽  
Xiaohua Gong ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Signal Transduction Pathways ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Mitogen Activated Protein ◽  
Gap Junctional

Both connexins and signal transduction pathways have been independently shown to play critical roles in lens homeostasis, but little is known about potential cooperation between these two intercellular communication systems. To investigate whether growth factor signaling and gap junctional communication interact during the development of lens homeostasis, we examined the effect of mitogen-activated protein kinase (MAPK) signaling on coupling mediated by specific lens connexins by using a combination of in vitro and in vivo assays. Activation of MAPK signaling pathways significantly increased coupling provided by Cx50, but not Cx46, in paired Xenopus laevis oocytes in vitro, as well as between freshly isolated lens cells in vivo. Constitutively active MAPK signaling caused macrophthalmia, cataract, glucose accumulation, vacuole formation in differentiating fibers, and lens rupture in vivo. The specific removal or replacement of Cx50, but not Cx46, ameliorated all five pathological conditions in transgenic mice. These results indicate that MAPK signaling specifically modulates coupling mediated by Cx50 and that gap junctional communication and signal transduction pathways may interact in osmotic regulation during postnatal fiber development.

scholarly journals Regulation of Gap Junctional Communication by Astrocytic Mitochondrial KATP Channels following Neurotoxin Administration in in vitro and in vivo Models

Neurosignals ◽  
2011 ◽  
Vol 19 (2) ◽  
pp. 63-74 ◽  
Author(s):  
Kewen Jiang ◽  
Jiangping Wang ◽  
Congying Zhao ◽  
Mei Feng ◽  
Zheng Shen ◽  
...  
Keyword(s):  
In Vivo Models ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional

scholarly journals Blockade of Brain Stem Gap Junctions Increases Phrenic Burst Frequency and Reduces Phrenic Burst Synchronization in Adult Rat

2003 ◽  
Vol 89 (1) ◽  
pp. 135-149 ◽  
Author(s):  
Irene C. Solomon ◽  
Ki H. Chon ◽  
Melissa N. Rodriguez
Keyword(s):  
Gap Junctions ◽  
Brain Stem ◽  
Gap Junction ◽  
Phrenic Nerve ◽  
Respiratory Rhythm ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Nerve Discharge

Recent investigations have examined the influence of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in vitro using transverse medullary slice and en bloc brain stem-spinal cord preparations obtained from neonatal (1–5 days postnatal) mice. Gap junction proteins, however, have been identified in both neurons and glia in brain stem regions implicated in respiratory control in both neonatal and adult rodents. Here, we used an in vitro arterially perfused rat preparation to examine the role of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in adult rodents. We recorded rhythmic inspiratory motor activity from one or both phrenic nerves before and during pharmacological blockade (i.e., uncoupling) of brain stem gap junctions using carbenoxolone (100 μM), 18α-glycyrrhetinic acid (25–100 μM), 18β-glycyrrhetinic acid (25–100 μM), octanol (200–300 μM), or heptanol (200 μM). During perfusion with a gap junction uncoupling agent, we observed an increase in the frequency of phrenic bursts (∼95% above baseline frequency; P < 0.001) and a decrease in peak amplitude of integrated phrenic nerve discharge ( P < 0.001). The increase in frequency of phrenic bursts resulted from a decrease in both T I ( P < 0.01) and T E ( P < 0.01). In addition, the pattern of phrenic nerve discharge shifted from an augmenting discharge pattern to a “bell-shaped” or square-wave discharge pattern in most experiments. Spectral analyses using a fast Fourier transform (FFT) algorithm revealed a reduction in the peak power of both the 40- to 50-Hz peak (corresponding to the MFO) and 90- to 110-Hz peak (corresponding to the HFO) although spurious higher frequency activity (≥130 Hz) was observed, suggesting an overall loss or reduction in inspiratory-phase synchronization. Although additional experiments are required to identify the specific brain stem regions and cell types (i.e., neurons, glia) mediating the observed modulations in phrenic motor output, these findings suggest that gap junction communication modulates generation of respiratory rhythm and inspiratory motoneuron synchronization in adult rodents in vitro.

scholarly journals Anticonvulsant Actions of Gap Junctional Blockers in an In Vitro Seizure Model

2002 ◽  
Vol 88 (4) ◽  
pp. 1893-1902 ◽  
Author(s):  
Shokrollah S. Jahromi ◽  
Kirsten Wentlandt ◽  
Sanaz Piran ◽  
Peter L. Carlen
Keyword(s):  
Neuronal Excitability ◽  
Intracellular Acidosis ◽  
Sodium Propionate ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Cellular Excitability ◽  
Different Types ◽  
Gap Junctional ◽  
Whole Cell Recordings

Gap junctions (gjs) are increasingly recognized as playing a significant role in seizures. We demonstrate that different types of gap junctional blocking agents reduce the duration of evoked seizure-like primary afterdischarges (PADs) in the rat in vitro CA1 hippocampal pyramidal region, following repetitive tetanization of the Schaffer collaterals. Intracellular acidosis, which is known to block gap junctional communication, decreased the PADs, whereas alkalinization increased the PADs. Cellular excitability was not significantly depressed as determined by input/output relations recorded before and during perfusion of the gj blockers blockers carbenoxolone and sodium propionate. There was a small decrease following 1-octanol perfusion and a large decrease following NH4Cl application. Carbenoxolone diminished PAD duration, but increased neuronal excitability in whole-cell recordings. After robust PADs were established, the expression of several gj proteins including connexins (Cxs) 26, 32, 36, and 43, as measured by Western blotting, was unchanged, although the level of nonphosphorylated Cx43 was decreased. Our data support the concept that blocking gap junctional communication is an anticonvulsant mechanism.

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