Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†

2021 ◽  
Author(s):  
Cecilia Valencia ◽  
Felipe Alonso Pérez ◽  
Carola Matus ◽  
Ricardo Felmer ◽  
María Elena Arias
Keyword(s):  
Protein Synthesis ◽  
Kinase Activity ◽  
Cyclin B1 ◽  
Protein Synthesis Inhibitors ◽  
Vitro Fertilization ◽  
New Findings ◽  
Bovine Oocytes ◽  
Dependent Pathway

Abstract The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro

1997 ◽  
Vol 9 (6) ◽  
pp. 603 ◽  
Author(s):  
J. C. Bell ◽  
L. C. Smith ◽  
R. Rumpf ◽  
A. K. Goff
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Germinal Vesicle ◽  
Uv Exposure ◽  
One Dimensional ◽  
Repair Mechanisms ◽  
Bovine Oocytes

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulusoocyte complexes (COCs) were denuded before or after maturationin vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high-possibly due to nuclear repair mechanisms. Protein proles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specic proteins are synthesized in the nuclear region.

scholarly journals Effect of Ethanol on Parthenogenetic Activation and α-Tocopherol Supplementation during In Vitro Maturation on Developmental Competence of Summer-Collected Bovine Oocytes

2021 ◽  
Vol 43 (3) ◽  
pp. 2253-2265
Author(s):  
Francisco Báez ◽  
Belén Gómez ◽  
Victoria de Brun ◽  
Nélida Rodríguez-Osorio ◽  
Carolina Viñoles
Keyword(s):  
In Vitro Maturation ◽  
Apoptotic Index ◽  
Developmental Competence ◽  
Vitro Fertilization ◽  
Parthenogenetic Activation ◽  
Oocyte Competence ◽  
Maturation Medium ◽  
Bovine Oocytes

The use of α-tocopherol during in vitro maturation (IVM) is an alternative to minimize the adverse effects of heat stress on oocyte competence. However, α-tocopherol is diluted in ethanol, which can induce oocyte parthenogenetic activation (PA). This study aimed to evaluate the role of ethanol concentration on PA and the effect of α-tocopherol supplementation during IVM on the developmental competence and the expression of key genes in blastocysts derived from summer-collected oocytes. All in vitro embryo production was conducted at 5% O2, 5% CO2 at 38.5 °C. Experiment 1: oocytes were cultured with or without 0.05% ethanol. As positive PA control matured oocytes were subjected to 3% or 7% ethanol for 7 min. Oocytes from all groups were placed in fertilization medium (22 h) and culture medium (9 days). Ethanol at 0.05% during IVM did not induce oocyte PA, however, 3% and 7% ethanol were effective parthenogenetic inductors. Experiment 2: oocytes were cultured in maturation medium supplemented with 0, 50, 100 and 200 μM α-tocopherol, diluted in 0.05% ethanol. After in vitro fertilization and embryo culture, we assessed blastocyst apoptotic index and the transcription of a panel of genes. The results showed that supplementation with 100 μM α-tocopherol reduced apoptotic index and increased the expression of SOD2. In conclusion, 100 μM α-tocopherol, diluted in 0.05% ethanol, can be used during IVM to embryonic quality.

Role of different proteolytic pathways in degradation of muscle protein from streptozotocin-diabetic rats

1996 ◽  
Vol 271 (2) ◽  
pp. E340-E347 ◽  
Author(s):  
M. T. Pepato ◽  
R. H. Migliorini ◽  
A. L. Goldberg ◽  
I. C. Kettelhut
Keyword(s):  
Protein Synthesis ◽  
Muscle Protein ◽  
Phase 1 ◽  
Diabetic Rats ◽  
Time Interval ◽  
Streptozotocin Diabetic Rats ◽  
Dependent Pathway ◽  
Proteolytic Pathways

In vitro rats of overall proteolysis and the activities of four different proteolytic pathways (lysosomal, Ca2+ dependent, ATP dependent, and ATP independent), as well as rates of protein synthesis, were measured in soleus and extensor digitorum longus (EDL) muscles from streptozotocin-diabetic rats. In the acute phase (1-3 days) of diabetes, there was an increase in overall proteolysis that coincided with an increased activity of the Ca(2+)-dependent pathway in both soleus and EDL and of the ATP-dependent pathway in EDL. After longer periods (5-10 days) of diabetes, the overall rate of protein degradation decreased and reached values similar to or even lower than those of controls as a result of a reduction in the activities of Ca(2+)-dependent and ATP-dependent pathways. No change was detected at any time interval in the activity of the intralysosomal proteolytic system in muscles from diabetic animals. Rates of protein synthesis were already reduced 24 h after diabetes induction and decreased further thereafter. Insulin treatment restored to normal the activities of the proteolytic pathways and rates of protein synthesis.

542 Objective Sleep Duration, Sleep Timing and Completion of In Vitro Fertilization Cycles; a Prospective Cohort Study

SLEEP ◽  
2021 ◽  
Vol 44 (Supplement_2) ◽  
pp. A214-A214
Author(s):  
Chawanont Pimolsri ◽  
Xiru Lyu ◽  
Cathy Goldstein ◽  
Chelsea Fortin ◽  
Sunni Mumford ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Sleep Duration ◽  
Medical Center ◽  
Working Women ◽  
Vitro Fertilization ◽  
Circadian Misalignment ◽  
Logistic Regression Models ◽  
Sleep Timing

Abstract Introduction Sleep duration and circadian misalignment have been linked to fertility and fecundability. However, sleep in women undergoing IVF has rarely been examined. This study investigated the role of sleep duration and timing with completion of an IVF cycle. Methods Prospective study of women undergoing IVF at a tertiary medical center between 2015 and 2017. Sleep was assessed by wrist-worn actigraphy 1–2 weeks prior to the initiation of their IVF cycle. Reproductive profile, IVF cycle details, demographic and health information were obtained from medical charts. Sleep duration, midpoint and bedtime were examined in relation to IVF cycle completion using logistic regression models, adjusted for age and anti-Müllerian hormone levels. A sub-analysis excluded women who worked non-day shifts to control for circadian misalignment. Results A total of 48 women were studied. Median age was 33y (range 25–42), with 29% of women older than 35 years. Ten women had an IVF cycle cancellation prior to embryo transfer. These women had shorter sleep duration, more nocturnal awakenings, lower sleep efficiency, and later sleep timing in comparison to those who completed their cycle. Twenty-minute increases in sleep duration were associated with lower odds of an uncompleted IVF cycle (OR = 0.88; 95% CI 0.78, 1.00). Women with later sleep midpoints and later bedtime had higher odds of an uncompleted cycle relative to those with earlier midpoints and earlier bedtime; OR=1.24; 95% CI 1.09, 1.40 and OR=1.33; 95% CI 1.17, 1.53 respectively, per 20-minute increments. These results were independent of age, levels of anti-Müllerian hormone, or sleep duration, and remained unchanged after exclusion of shift-working women. Conclusion This study demonstrated the influence of sleep duration and sleep timing on the odds of an uncompleted IVF cycle prior to embryo transfer. Sleep is a modifiable behavior that may contribute to IVF cycle success. Support (if any):

scholarly journals 1202. Subinhibitory Concentrations of Omadacycline Inhibit Staphylococcus aureus Hemolytic Activity in Vitro

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S622-S623
Author(s):  
Alisa W Serio ◽  
S Ken Tanaka ◽  
Kelly Wright ◽  
Lynne Garrity-Ryan
Keyword(s):  
Staphylococcus Aureus ◽  
Protein Synthesis ◽  
Virulence Factors ◽  
Hemolytic Activity ◽  
Protein Biosynthesis ◽  
The United States ◽  
Aureus Strain ◽  
Protein Synthesis Inhibitors ◽  
Subinhibitory Concentrations

Abstract Background In animal models of Staphylococcus aureus infection, α-hemolysin has been shown to be a key virulence factor. Treatment of S. aureus with subinhibitory levels of protein synthesis inhibitors can decrease α-hemolysin expression. Omadacycline, a novel aminomethylcycline antibiotic in the tetracycline class of bacterial protein biosynthesis inhibitors, is approved in the United States for treatment of community-acquired bacterial pneumonia (CABP) and acute bacterial skin and skin structure infections (ABSSSI) in adults. This study was performed to determine the durability of inhibition and effect of subinhibitory concentrations of omadacycline on S. aureus hemolytic activity. Methods All experiments used the methicillin-sensitive S. aureus strain Wood 46 (ATCC 10832), a laboratory strain known to secrete high levels of α-hemolysin. Minimum inhibitory concentrations (MICs) of omadacycline and comparator antibiotics (tetracycline, cephalothin, clindamycin, vancomycin, linezolid) were determined. Growth of S. aureus with all antibiotics was determined and the percentage of hemolysis assayed. “Washout” experiments were performed with omadacycline only. Results S. aureus cultures treated with 1/2 or 1/4 the MIC of omadacycline for 4 hours showed hemolysis units/108 CFU of 47% and 59% of vehicle-treated cultures, respectively (Fig. 1A, 1B). In washout experiments, treatment with as little as 1/4 the MIC of omadacycline for 1 hour decreased the hemolysis units/108 CFU by 60% for 4 hours following removal of the drug (Table 1). Figure 1 Table 1 Conclusion Omadacycline inhibited S. aureus hemolytic activity in vitro at subinhibitory concentrations and inhibition was maintained for ≥ 4 hours after removal of extracellular drug (Fig. 2). The suppression of virulence factors throughout the approved omadacycline dosing interval, in addition to the in vitro potency of omadacycline, may contribute to the efficacy of omadacycline for ABSSSI and CABP due to virulent S. aureus. This finding may apply to other organisms and other virulence factors that require new protein synthesis to establish disease. Figure 2 Disclosures Alisa W. Serio, PhD, Paratek Pharmaceuticals, Inc. (Employee, Shareholder) S. Ken Tanaka, PhD, Paratek Pharmaceuticals, Inc. (Employee, Shareholder) Kelly Wright, PharmD, Paratek Pharmaceuticals, Inc. (Employee, Shareholder) Lynne Garrity-Ryan, PhD, Paratek Pharmaceuticals, Inc. (Employee, Shareholder)

scholarly journals Novel Insights on the Role of Nitric Oxide in the Ovary: A Review of the Literature

2021 ◽  
Vol 18 (3) ◽  
pp. 980
Author(s):  
Maria Cristina Budani ◽  
Gian Mario Tiboni
Keyword(s):  
Nitric Oxide ◽  
In Vivo Studies ◽  
Published Data ◽  
Vitro Fertilization ◽  
Peripheral Neurons ◽  
Relevant Role ◽  
Oocyte Meiotic Maturation

Nitric oxide (NO) is formed during the oxidation of L-arginine to L-citrulline by the action of multiple isoenzymes of NO synthase (NOS): neuronal NOS (nNOS), endotelial NOS (eNOS), and inducible NOS (iNOS). NO plays a relevant role in the vascular endothelium, in central and peripheral neurons, and in immunity and inflammatory systems. In addition, several authors showed a consistent contribution of NO to different aspects of the reproductive physiology. The aim of the present review is to analyse the published data on the role of NO within the ovary. It has been demonstrated that the multiple isoenzymes of NOS are expressed and localized in the ovary of different species. More to the point, a consistent role was ascribed to NO in the processes of steroidogenesis, folliculogenesis, and oocyte meiotic maturation in in vitro and in vivo studies using animal models. Unfortunately, there are few nitric oxide data for humans; there are preliminary data on the implication of nitric oxide for oocyte/embryo quality and in-vitro fertilization/embryo transfer (IVF/ET) parameters. NO plays a remarkable role in the ovary, but more investigation is needed, in particular in the context of human ovarian physiology.

Inhibition of atrial peptide secretion at different stages of the secretory process: Ca2+ dependence

1991 ◽  
Vol 261 (6) ◽  
pp. C1162-C1172 ◽  
Author(s):  
E. Page ◽  
J. Upshaw-Earley ◽  
G. E. Goings ◽  
D. A. Hanck
Keyword(s):  
Protein Synthesis ◽  
Brefeldin A ◽  
Atp Depletion ◽  
Secretory Process ◽  
Protein Synthesis Inhibitors ◽  
Large Reservoir ◽  
Secretory Rate ◽  
Vesicular Traffic ◽  
Dependent Inactivation

We have used a noncontracting in vitro preparation of stretched and unstretched rat atria to estimate contributions of constitutive and regulated pathways to the rates of stretch-augmented and basal secretion of immunoreactive atrial natriuretic peptide (ANP) and to examine effects of inhibition of the secretory sequence by 1) protein synthesis inhibitors, 2) disruption of forward vesicular traffic between endoplasmic reticulum and Golgi with brefeldin A (BFA, and 3) cellular ATP depletion. Protein synthesis inhibition with cycloheximide for 44 min slowed neither basal nor stretch-augmented ANP secretion but instead accelerated stretch-augmented secretion at low (but not at physiological) external Ca2+ concentration, suggesting that the constitutive component does not contribute substantially to either basal or stretch-augmented secretion. BFA, which disassembled Golgi cisternae, increased the stretch-augmented secretory rate via the regulated pathway and prevented Ca(2+)-dependent inactivation with time. Cellular ATP depletion rapidly and completely inhibited stretch-augmented secretion. We conclude that both basal and stretch-augmented utilize the energy-dependent regulated pathway, drawing on a large reservoir of concentrated prohormone stored in granules that is not detectably depleted during 44 min of stretch-augmented secretion at 37 degrees C.

scholarly journals Protein Synthesis in Human Leukocytes and Lymphocytes: 1. Effect of Steroids and Sterols

Blood ◽  
1969 ◽  
Vol 34 (3) ◽  
pp. 348-356 ◽  
Author(s):  
SEYMOUR WERTHAMER ◽  
CARL HICKS ◽  
LEONARD AMARAL
Keyword(s):  
Protein Synthesis ◽  
Steroid Hormones ◽  
Plasma Protein ◽  
Human Lymphocytes ◽  
Amino Acid Incorporation ◽  
Binding Factor ◽  
In Vitro Effects ◽  
Physiologic Role

Abstract The in vitro effects of sterols, cholesterol and 3-methyl cholanthrene and steroids, cortisol, prednisolone and testosterone on protein synthesis in separate popultions of human lymphocytes and leukocytes has been investigated. It has been shown that all agents used result in the inhibition of protein synthesis under these conditions. It has also been shown that the inhibitory mechanism of the steroid hormones requires the presence of plasma, presumably as a protein binding factor in order to achieve its effect. The sterol, cholesterol and 3-methyl cholanthrene, in the absence of plasma, still inhibit amino acid incorporation. However, in the case of cholesterol, the magnitude of inhibition is lower than that observed in the presence of plasma, perhaps indicating a partial plasma dependence. The results presented therefore support the hypothesis that the inhibition of lymphocyte protein synthesis by steroid hormones occurs only when the steroid is bound to a plasma protein. The physiologic role of the plasma protein-cortisol complex and its relation to the condition of lymphopenia in man is discussed.

Role of iPLA2 and store-operated channels in agonist-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries

2008 ◽  
Vol 294 (3) ◽  
pp. H1183-H1187 ◽  
Author(s):  
Kristen M. Park ◽  
Mario Trucillo ◽  
Nicolas Serban ◽  
Richard A. Cohen ◽  
Victoria M. Bolotina
Keyword(s):  
Carotid Arteries ◽  
Cerebral Arteries ◽  
Present Evidence ◽  
Voltage Gated ◽  
Functional Inhibition ◽  
Intracellular Ca ◽  
Dependent Pathway ◽  
Secondary Activation

Store-operated channels (SOC) and store-operated Ca2+ entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type Ca2+ (CaL2+) channels are thought to be fully responsible for agonist-induced Ca2+ influx and vasoconstriction. We present evidence that SOC and their activation via a Ca2+-independent phospholipase A2 (iPLA2)-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular Ca2+ in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) Ca2+ and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a CaL2+ inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and CaL2+ channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of iPLA2β totally inhibited PE-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries, whereas K+-induced Ca2+ influx and vasoconstriction mediated by CaL2+ channels were not affected. Thus iPLA2-dependent activation of SOC is crucial for agonist-induced Ca2+ influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of Ca2+ stores, nonselective SOC are activated via an iPLA2-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of CaL2+ channels and lead to Ca2+ entry and vasoconstriction.

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