146 EFFECT OF PROTEIN SYNTHESIS INHIBITOR ON BOAR SPERM CAPACITATION AND FERTILIZATION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 186
Author(s):  
Y. Okudaira ◽  
H. Funahashi
Keyword(s):  
Protein Synthesis ◽  
Acrosome Reaction ◽  
Specific Inhibitor ◽  
Protein Synthesis Inhibitor ◽  
Sperm Viability ◽  
Sperm Capacitation ◽  
Synthesis Inhibitor ◽  
Mitochondrial Ribosome ◽  
Cytoplasmic Ribosome

In human, bovine, mouse, and rat sperm, translation of RNA to proteins in the mitochondrial ribosome during capacitation has been reported to be important for fertilization. The objective of this study was to examine effect of protein synthesis inhibitor (ribosome inhibitor) on boar sperm capacitation and IVF. Sperm from an ejaculated sperm-rich fraction of Berkshire boars were washed by centrifugation (1500 rpm for 35 min) in a Percoll gradient (45/90%) and then incubated in modified Medium-199 containing 0.4% BSA and 5 mM caffeine sodium benzoate, supplemented with or without a mitochondrial ribosome-specific (55S ribosome) inhibitor, chloramphenicol (CP; 0.3 mM), or a cytoplasmic ribosome-specific (80S ribosome) inhibitor, cyclohexide (CH; 3.6 mM), in an atmosphere of 5% CO2 in air at 39°C for 45 or 90 min. At 45 and 90 min after culture, sperm viability, motility, and chlortetracyclin-stained patterns (to assess the sperm functional status, capacitation, and acrosome reaction) were examined. Porcine oocytes were matured in vitro for 44 h in porcine oocyte medium supplemented with eCG, hCG, and dibutyryl cyclic adenosine monophosphate for the first 20 h. Matured oocytes after the removal of cumulus cells were co-cultured with sperm (final conc.: 2.5 × 105 cells mL–1) in the absence or presence of CP or CH for 8 h. Sperm penetrability was also determined. Statistical analyses of data from 4 replicated trials were performed by ANOVA. After 45 and 90 min of culture, neither CP nor CH affected sperm viability and motility (P > 0.05). The addition of CP after 45 and 90 min of culture significantly (P < 0.05) decreased capacitated and acrosome-reacted sperm rates, as detected by chlortetracyclin fluorescence assay (capacitated: control 9.6 v. CP 5.6%, control 17.8 v. CP 10.2%; acrosome reacted: control 4.6 v. CP 2.2%, control 9.2 v. CP 4.8%, respectively; P < 0.05). In the presence of CH, IVF rate and number of sperm per penetrated egg were decreased (control 80.8 v. CH 46.8%, 2.2 v. 1.4, respectively; P < 0.05). In the presence of CH, however, the percentage of metaphase II oocytes after co-culture with sperm for 8 h was lower than other 2 groups (control 87.6 v. CP 85.5 v. CH 74.0%; P < 0.05), and the percentage of A/T-II oocytes was higher than in the other 2 groups (control 1.1 v. CP 0 v. CH 9.4%; P < 0.05). From these results, we conclude that mitochondrial ribosome-specific inhibitor, chloramphenicol, affects capacitation and acrosome reaction but not penetration, whereas cytoplasmic ribosome-specific inhibitor, cyclohexide, decreases the number of oocytes that reach metaphase II stage and are penetrated.

Evidence for a role of phospholipase A2 in the mechanism of LHRH priming in rat anterior pituitary tissue

1994 ◽  
Vol 141 (1) ◽  
pp. 15-31 ◽  
Author(s):  
F J Thomson ◽  
M S Johnson ◽  
R Mitchell ◽  
B Wolbers
Keyword(s):  
Protein Synthesis ◽  
Anterior Pituitary ◽  
Priming Effect ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Pituitary Tissue ◽  
Gonadotrophin Secretion ◽  
Lh Release ◽  
Functional Relevance

Abstract The phospholipase A2 (PLA2) inhibitors, quinacrine, p-bromophenacyl bromide, ONO-RS-082, aristolochic acid and chloracysine blocked the priming effect of LHRH, but not acute LHRH-induced gonadotrophin release measured in anterior pituitary pieces in pro-oestrous rats in vitro. These results suggest that the intracellular mechanisms underlying LHRH priming are distinct from those which mediate LH release in the present circumstances in that they involve PLA2. Furthermore, neither LHRH-induced LH release from preprimed tissue nor Ca2+-induced LH release were attenuated by quinacrine, indicating that this inhibitor does not interfere with the general Ca2+-dependent secretory apparatus of the gonadotroph and that the critical period for its action is in the induction of priming. LHRH induced the release of [3H]arachidonic acid ([3H]AA) from [3H]AA-prelabelled anterior pituitary tissue from pro-oestrous rats; a response which was sensitive to inhibitors of PLA2, of protein kinase C (PKC) and of protein synthesis. Activation of PKC also resulted in [3H]AA release which was inhibited with exactly the same pharmacological profile as the response to LHRH. Both gonadotrophin secretion and [3H]AA release responses to LHRH and to phorbol ester varied in parallel during the oestrous cycle and in ovariectomized/oestradiol-17β-replaced animals, as did their sensitivity to quinacrine and the protein synthesis inhibitor cycloheximide. These results indicate that LHRH priming is dependent on a hormonally regulated cascade involving a distinct form of PKC acting through a protein synthesis-dependent step to release AA by means of PLA2 activity. The priming effect was mimicked (at least in part) by conditioning preincubation with AA, confirming the functional relevance of this signalling cascade. Results using standard inhibitors of lipoxygenase/epoxygenase pathways were equivocal as to whether these pathways were critically involved, whilst cyclo-oxygenase inhibitors were completely without effect. The steps downstream from AA (and its possible metabolites) by which stimulus–secretion coupling is up-regulated in priming remain to be clarified. Journal of Endocrinology (1994) 141, 15–31

scholarly journals Long-Lasting Effects of GABA Infusion Into the Cerebral Cortex of the Rat

2000 ◽  
Vol 7 (1-2) ◽  
pp. 1-8 ◽  
Author(s):  
Teresa Montiel ◽  
Daniel Almeida ◽  
Iván Arango ◽  
Eduardo Calixto ◽  
César Casasola ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Long Term Potentiation ◽  
Information Storage ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Receptor Stimulation ◽  
Paroxysmal Activity ◽  
The Brain

In electrophysiological terms, experimental models of durable information storage in the brain include long-term potentiation (LTP), long-term depression, and kindling. Protein synthesis correlates with these enduring processes. We propose a fourth example of long-lasting information storage in the brain, which we call the GABA-withdrawal syndrome (GWS). In rats, withdrawal of a chronic intracortical infusion of GABA, a ubiquitous inhibitory neurotransmitter, induced epileptogenesis at the infusion site. This overt GWS lasted for days. Anisomycin, a protein synthesis inhibitor, prevented the appearance of GWSin vivo. Hippocampal and neocortical slices showed a similar post-GABA hyperexcitabilityin vitroand an enhanced susceptibility to LTP induction. One to four months after the epileptic behavior disappeared, systemic administration of a subconvulsant dose of pentylenetetrazol produced the reappearance of paroxysmal activity. The long-lasting effects of tonicGABAAreceptor stimulation may be involved in long-term information storage processes at the cortical level, whereas the cessation ofGABAAreceptor stimulation may be involved in chronic pathological conditions, such as epilepsy. Furthermore, we propose that GWS may represent a common key factor in the addiction to GABAergic agents (for example, barbiturates, benzodiazepines, and ethanol). GWS represents a novel form of neurono-glial plasticity. The mechanisms of this phenomenon remain to be understood.

Role of corticosterone in TNF and IL-6 production in isolated perfused rat liver

1995 ◽  
Vol 268 (3) ◽  
pp. R699-R706 ◽  
Author(s):  
J. Liao ◽  
J. A. Keiser ◽  
W. E. Scales ◽  
S. L. Kunkel ◽  
M. J. Kluger
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Intravenous Infusion ◽  
Isolated Perfused Rat Liver ◽  
Protein Synthesis Inhibitor ◽  
Perfused Rat Liver ◽  
Synthesis Inhibitor ◽  
In Situ Data ◽  
Data Support

Using an isolated perfused rat liver (IPRL) preparation, we assessed whether corticosterone may contribute to the rise in tumor necrosis factor (TNF) and interleukin-6 (IL-6) in rats after injection with lipopolysaccharide (LPS) or exposure to psychological stress. Intravenous infusion of LPS into the IPRL led to dose-dependent increases in TNF and IL-6 concentrations in the effluent. Anisomycin, a protein synthesis inhibitor, completely blocked the rise in TNF and IL-6 concentration in the IPRL effluent, supporting the hypothesis that the synthesis (or release) of these cytokines was dependent on protein synthesis. Intravenous infusion of corticosterone at nonstressed (35 ng/ml) and stressed levels (350 ng/ml) increased TNF and/or IL-6 release. However, when LPS was combined with corticosterone, the lower dose of corticosterone facilitated the release of cytokines, whereas the higher dose of corticosterone suppressed the release of cytokines. We then showed that isolated Kupffer cells were capable of significant TNF and IL-6 production and that corticosterone decreased LPS-induced cytokine production in these cells. Our data support the hypothesis that the liver is an important source of circulating cytokines in response to LPS. In addition, although in vitro data generally support the hypothesis that corticosterone suppresses the production of cytokines, our in situ data support the hypothesis that physiological levels of corticosterone cause an increase in TNF and IL-6.

scholarly journals TePhe, a tellurium-containing phenylalanine mimic, allows monitoring of protein synthesis in vivo with mass cytometry

2019 ◽  
Vol 116 (17) ◽  
pp. 8155-8160 ◽  
Author(s):  
Jay Bassan ◽  
Lisa M. Willis ◽  
Ravi N. Vellanki ◽  
Alan Nguyen ◽  
Landon J. Edgar ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Direct Detection ◽  
Aromatic Amino Acids ◽  
Protein Synthesis Inhibitor ◽  
Mass Cytometry ◽  
Synthesis Inhibitor ◽  
Molecular Specificity ◽  
Noncanonical Amino Acid

Protein synthesis is central to maintaining cellular homeostasis and its study is critical to understanding the function and dysfunction of eukaryotic systems. Here we report L-2-tellurienylalanine (TePhe) as a noncanonical amino acid for direct measurement of protein synthesis. TePhe is synthetically accessible, nontoxic, stable under biological conditions, and the tellurium atom allows its direct detection with mass cytometry, without postexperiment labeling. TePhe labeling is competitive with phenylalanine but not other large and aromatic amino acids, demonstrating its molecular specificity as a phenylalanine mimic; labeling is also abrogated in vitro and in vivo by the protein synthesis inhibitor cycloheximide, validating TePhe as a translation reporter. In vivo, imaging mass cytometry with TePhe visualizes translation dynamics in the mouse gut, brain, and tumor. The strong performance of TePhe as a probe for protein synthesis, coupled with the operational simplicity of its use, suggests TePhe could become a broadly applied molecule for measuring translation in vitro and in vivo.

scholarly journals Role of lysosomal cathepsin activities in cell hypertrophy induced by NH4Cl in cultured renal proximal tubule cells.

1996 ◽  
Vol 7 (1) ◽  
pp. 73-80
Author(s):  
H Ling ◽  
S Vamvakas ◽  
M Gekle ◽  
L Schaefer ◽  
M Teschner ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cell Protein ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Renal Hypertrophy ◽  
Cytosolic Ph ◽  
Cell Hypertrophy ◽  
Renal Proximal Tubule Cells

An increase of renal ammoniagenesis has been implicated in renal hypertrophy associated with various clinical disorders such as metabolic acidosis, diabetic nephropathy, and renal insufficiency. In vivo and in vitro studies have shown that ammonia promotes hypertrophy in tubular epithelial cells. To elucidate its role on protein turnover, the effects of NH4Cl on the activities of cathepsins B, H, and L+B, as well as on protein synthesis and degradation in LLC-PK1 cells, were investigated. The results show that NH4Cl (20 mM) induced cell hypertrophy, as defined by an increase in both cell protein content and cell volume (+25.5 +/- 1.3 and +10.4 +/- 0.1% after 48 h). This hypertrophy was associated with the suppression of the activities of cathepsins B and L+B (-57.0 +/- 0.9 and -54.5 +/- 1.5% after 48 h) and a reduction of protein degradation rate (-59.7 +/- 4.1% after 48 h), but without enhanced protein synthesis. The findings were further supported with an additional experiment, showing that the protein synthesis inhibitor cycloheximide (10 microM) did not blunt NH4Cl-induced cell hypertrophy. Moreover, NH4Cl (20 mM) resulted in a persistent elevation of the lysosomal pH, whereas the rise in the cytosolic pH was only transient. This alkalinization in lysosomes may be causatively involved in the impairment of the activities of cathepsins B and L+B. In conclusion, the suppression of the activities of cathepsins B and L+B and the subsequent reduction of protein breakdown due to intralysosomal alkalinization contribute to NH4Cl-induced hypertrophy in LLC-PK1 cells.

scholarly journals A Dual-Specificity Phosphatase Cdc25B Is an Unstable Protein and Triggers p34cdc2/Cyclin B Activation in Hamster BHK21 Cells Arrested with Hydroxyurea

1997 ◽  
Vol 138 (5) ◽  
pp. 1105-1116 ◽  
Author(s):  
Hitoshi Nishijima ◽  
Hideo Nishitani ◽  
Takashi Seki ◽  
Takeharu Nishimoto
Keyword(s):  
Protein Synthesis ◽  
S Phase ◽  
Cyclin B ◽  
Protein Synthesis Inhibitor ◽  
Temperature Sensitive ◽  
Synthesis Inhibitor ◽  
Dual Specificity ◽  
Dual Specificity Phosphatases ◽  
Initial Activation

By incubating at 30°C in the presence of an energy source, p34cdc2/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40°C, the nonpermissive temperature (Nishimoto, T., E. Eilen, and C. Basilico. 1978. Cell. 15:475–483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34cdc2/cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34cdc2/cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40°C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34cdc2/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34cdc2/cyclin B. Cdc25B was found to be unstable (half-life &lt; 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34cdc2/cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34cdc2/cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34cdc2/cyclin B, which initiates mitosis through the activation of Cdc25C.

Tween-80 Alters Natural Resistance of Sarcoma-180 to Bouvardin (NSC 259968)

1984 ◽  
Vol 70 (1) ◽  
pp. 23-27 ◽  
Author(s):  
Ravi Menon ◽  
Manik Chitnis ◽  
Manohar Adwankar ◽  
Vathsala Basrur ◽  
Ashok Amonkar
Keyword(s):  
Protein Synthesis ◽  
Tween 80 ◽  
Natural Resistance ◽  
Protein Synthesis Inhibitor ◽  
Sarcoma 180 ◽  
Synthesis Inhibitor ◽  
Subsequent Increase ◽  
Rna And Dna Synthesis ◽  
Rna And Dna

Sarcoma-180 tumour (S-180) exhibits natural resistance to bouvardin (NSC 259968), a protein synthesis inhibitor that also inhibits RNA and DNA synthesis when administered over a range of non-toxic doses using 0.9 % NaCl as a vehicle. However, using Tween-80 as a vehicle, there is a substantial enhancement of cytotoxicity and a subsequent increase in the life span of animals bearing the S-180 tumour. This observation was substantiated in vitro by exposing S-180 cells to 10−6 M bouvardin in the presence and absence of Tween-80. Bouvardin, 10−6 M, in the absence of Tween-80, inhibited the incorporation of [3H]uridine by 46 %, whereas the presence of Tween-80 resulted in a 66 % inhibition of uridine incorporation.

scholarly journals Neuroprotection by Osteopontin in Stroke

2005 ◽  
Vol 25 (2) ◽  
pp. 217-225 ◽  
Author(s):  
Robert Meller ◽  
Susan L Stevens ◽  
Manabu Minami ◽  
Jennifer A Cameron ◽  
Sonya King ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Synthesis ◽  
Protective Effect ◽  
Cortical Neuron ◽  
Neuroprotective Effect ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
In Vivo Models

Osteopontin (OPN) is a secreted extracellular phosphoprotein involved in diverse biologic functions, including inflammation, cell migration, and antiapoptotic processes. Here we investigate the neuroprotective potential of OPN to reduce cell death using both in vitro and in vivo models of ischemia. We show that incubation of cortical neuron cultures with OPN protects against cell death from oxygen and glucose deprivation. The effect of OPN depends on the Arg–Gly–Asp (RGD)-containing motif as the protective effect of OPN in vitro was blocked by an RGD-containing hexapeptide, which prevents integrin receptors binding to their ligands. Osteopontin treatment of cortical neuron cultures caused an increase in Akt and p42/p44 MAPK phosphorylation, which is consistent with OPN-inducing neuroprotection via the activation of these protein kinases. Indeed, the protective effect of OPN was reduced by inhibiting the activation of Akt and p42/p44 MAPK using LY294002 and U0126, respectively. The protective effect of OPN was also blocked by the protein synthesis inhibitor cycloheximide, suggesting that the neuroprotective effect of OPN required new protein synthesis. Finally, intracerebral ventricular administration of OPN caused a marked reduction in infarct size after transient middle cerebral artery occlusion in a murine stroke model. These data suggest that OPN is a potent neuroprotectant against ischemic injury.

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