Slo3 K+ channel blocker clofilium extends bull and mouse sperm-fertilizing competence

Reproduction ◽  
2018 ◽  
Vol 156 (6) ◽  
pp. 463-476 ◽  
Author(s):  
Roland Abi Nahed ◽  
Guillaume Martinez ◽  
Jean Pascal Hograindleur ◽  
Emilie Le Blévec ◽  
Sabine Camugli ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Acrosome Reaction ◽  
Channel Blocker ◽  
Specific Inhibitor ◽  
K Channel ◽  
Sperm Dna ◽  
The Impact ◽  
First Time ◽  
Bovine Species

For artificial insemination (AI) to be successful, it is essential that sperm delivery be perfectly timed relative to ovulation, as sperm lifespan is limited due to oxidative metabolism induced by capacitation. Extending the window of sperm capacitation could therefore increase sperm lifespan, prolong sperm-fertilizing competence and increase AI efficiency. Hyperpolarization of sperm is a crucial step in capacitation and is induced by activation of the potassium calcium-activated channel subfamily U member 1 (KCNU1, also named Slo3 or KSper). Given the essential role played by KCNU1 in capacitation, this study assessed the impact of its pharmacological inhibition on sperm lifespan. We showed that treatment of murine sperm with sub-micromolar concentrations of clofilium, a specific inhibitor of KCNU1, slowed down capacitation, decreased the rate of acrosome reaction and extended the fertilizing competence of capacitated sperm for 12 h. Clofilium also extended fertilizing competence and motility of bovine-capacitated sperm, and increased the rate of fertilization with sperm capacitated for 24 h by 100%, and the rate of blastocyst formation by 150%. Finally, toxicity experiments showed clofilium to have no impact on sperm DNA and no embryotoxicity at the concentration used to extend sperm lifespan. Our results demonstrate that clofilium prolongs fertilizing competence of aging capacitated sperm in vitro in both rodent and bovine species. To our knowledge, this is the first time the duration of sperm-fertilizing competence is shown to be extended by potassium channels blockers.

Application of reproductive technology to the Australian livestock industries

1991 ◽  
Vol 3 (6) ◽  
pp. 627 ◽  
Author(s):  
G Evans
Keyword(s):  
Artificial Insemination ◽  
Management Practices ◽  
Significant Degree ◽  
Reproductive Technology ◽  
Commercial Application ◽  
Embryo Freezing ◽  
Commercial Practice ◽  
Breeding Programmes ◽  
The Impact

Current use of reproductive technology in the Australian livestock industries is limited, though it increased in line with higher prices for beef and wool through the 1980s. The required techniques, many of which were developed in Australia, are available and the level of expertise is comparable to the best in the world. However, the extensive pastoral industries do not readily lend themselves to these procedures. Only in the dairy industry is artificial insemination used to a significant degree. On the other hand, application of the technology in the pastoral industries is confined largely to studs and breeding cooperatives which provide breeding animals for producer flocks and herds. Hence the impact of applied technology may be more widespread than first appears. Until recently, little regard was paid to application of the technology along sound breeding principles. Artificial insemination and multiple ovulation and embryo transfer (MOET) have not been used so much in planned breeding programmes aimed at local improvement of stock, but more to proliferate genes of reputedly superior stock, imported either from overseas or elsewhere in Australia. This is particularly true of MOET, where the incentive to use it is commonly a short term cash gain made from proliferating breeding stock of a particularly valuable and usually novel strain or breed. Recent technological improvements which render the use of reproductive technology cheaper and more effective will lead to its more widespread use in commercial practice. Techniques for embryo freezing and splitting have been greatly simplified and quickly put into practice. The novel livestock technologies of in vitro oocyte maturation and fertilization have already found commercial application overseas. Fecundity-enhancing products have also been adopted by the livestock industries. There is potential value for greater use of reproductive technology in the livestock industries provided it is implemented according to sound breeding principles and provided associated management practices are applied simultaneously.

scholarly journals Phenotypic Susceptibility to Bevirimat in Isolates from HIV-1-Infected Patients without Prior Exposure to Bevirimat

2010 ◽  
Vol 54 (6) ◽  
pp. 2345-2353 ◽  
Author(s):  
Nicolas A. Margot ◽  
Craig S. Gibbs ◽  
Michael D. Miller
Keyword(s):  
Recombinant Viruses ◽  
Gag Polyprotein ◽  
New Class ◽  
Major Mutation ◽  
Hiv Drugs ◽  
The Impact ◽  
First Time ◽  
Hiv 1

ABSTRACT Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conducting in vitro phenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entire gag and protease genes from 31 plasma samples from HIV-1-infected patients with (n = 21) or without (n = 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient's ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selected in vitro (H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.

scholarly journals Effects of calpain and Rho-kinase inhibitors on the acrosome reaction and motility of fowl spermatozoa in vitro

Reproduction ◽  
2006 ◽  
Vol 131 (1) ◽  
pp. 71-79 ◽  
Author(s):  
K Ashizawa ◽  
G J Wishart ◽  
S Katayama ◽  
D Takano ◽  
M Maeda ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Acrosome Reaction ◽  
Kinase Inhibitors ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Immunoblot Analysis ◽  
Calpain Inhibitor ◽  
Cytoplasmic Matrix ◽  
Sperm Extract

At the avian body temperature of 40 °C, intact fowl spermatozoa require Ca2+for the initiation of motility and a combination of both Ca2+and homogenized inner perivitelline layer (IPVL) together to induce the acrosome reaction. Within the range of 1–100 μmol/l, neither PD 150606 (a Ca2+-dependent calpain inhibitor) nor Y-27632 (an inhibitor of Ca2+-dependent Rho-kinase) were able to inhibit the acrosome reaction induced by the presence of Ca2+and IPVL. However, PD 150606, although not Y-27632, was able to inhibit sperm motility initiated by Ca2+, as well as motility initiated by calyculin A – a specific inhibitor of protein phosphatases, which also initiates sperm motility at 40 °C. The addition of PD 150606 did not reduce the ATP concentrations of intact spermatozoa, nor the motility of demembranated spermatozoa. Immunoblot analysis of sperm extract using a polyclonal antibody against calpain 12 revealed a cross-reacting protein of approximately 80 kDa. These results suggest that Rho-kinase is not involved in the regulation of the acrosome reaction or of motility in fowl spermatozoa. In contrast, calpain appears to be involved in the regulation of flagellar movement, but not izn that of the acrosome reaction. Furthermore, it seems that endogenous calpain is present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.

Comparison of acetylcholine-dependent relaxation in large and small arteries of rat mesenteric vascular bed

1994 ◽  
Vol 266 (3) ◽  
pp. H952-H958 ◽  
Author(s):  
J. J. Hwa ◽  
L. Ghibaudi ◽  
P. Williams ◽  
M. Chatterjee
Keyword(s):  
Methylene Blue ◽  
Channel Blocker ◽  
Mesenteric Arteries ◽  
K Channel ◽  
Resistance Arteries ◽  
Vascular Bed ◽  
Mesenteric Vascular Bed ◽  
Relaxation Responses ◽  
Endothelium Dependent Relaxation

The relative contributions of nitric oxide (NO) to in vitro relaxation responses elicited by acetylcholine (ACh) were compared in vessels of different sizes from the rat mesenteric vascular bed. ACh elicited an endothelium-dependent relaxation in phenylephrine-contracted superior mesenteric arteries (SMA, unstretched luminal diam 650 microns), which was blocked by compounds that inhibited NO, such as hemoglobin (10 microM), methylene blue (10 microM), and NG-monomethyl-L-arginine (1 mM). In contrast, the endothelium-dependent relaxation induced by ACh in phenylephrine-contracted mesenteric resistance arteries (MRA, unstretched luminal diam 200 microns) was not blocked by hemoglobin, methylene blue, or NG-monomethyl-L-arginine. KCl (25 mM) partially inhibited the ACh-dependent relaxation in MRA. Furthermore, the ACh-dependent relaxation in MRA was selectively inhibited by the Ca(2+)-activated K+ channel blocker charybdotoxin (0.1 microM). In contrast, the ATP-sensitive K+ channel blocker glibenclamide (50 microM) did not block the ACh-dependent relaxation in MRA. We conclude that 1) NO is a major component of the ACh-dependent relaxation in SMA and 2) the ACh-dependent relaxation of MRA is resistant to NO inhibitors but sensitive to a Ca(2+)-activated K+ channel blocker. This suggests that an endothelium-derived hyperpolarization factor may be involved in the relaxation of MRA.

The impact of the Maillard reaction on the in vitro proteolytic breakdown of bovine lactoferrin in adults and infants

2014 ◽  
Vol 5 (8) ◽  
pp. 1898-1908 ◽  
Author(s):  
Alice M. Moscovici ◽  
Yousef Joubran ◽  
Valerie Briard-Bion ◽  
Alan Mackie ◽  
Didier Dupont ◽  
...  
Keyword(s):  
Maillard Reaction ◽  
Bioactive Peptide ◽  
Bovine Lactoferrin ◽  
Peptide Formation ◽  
The Impact ◽  
First Time

The impact of the Maillard reaction on proteolysis of the bioactive bovine lactoferrin is comparedin vitrobetween adults and infants for the first time, coupling proteomics to elucidate bioactive peptide formation.

Paternal Diet and Obesity: Effects on Reproduction

2017 ◽  
Vol 35 (04) ◽  
pp. 313-317 ◽  
Author(s):  
Alex Polotsky ◽  
Jasmine Aly
Keyword(s):  
Maternal Obesity ◽  
Absolute Risk ◽  
Human Studies ◽  
Semen Parameters ◽  
Sperm Dna Fragmentation ◽  
Vitro Fertilization ◽  
Sperm Dna Damage ◽  
Sperm Dna ◽  
The Impact

AbstractAlthough most research has focused on maternal obesity, there is growing data to indicate that obesity in the father can affect reproduction. Supporting data come from both mouse and human studies. Murine studies found that obese male mice exhibited decreased motility and reduced hyperactivated progression versus lean mice. Obese mice also exhibited sperm with increased levels of intracellular and mitochondrial levels of reactive oxygen species, increased sperm damage, and lower levels of capacitation, which has been shown to be associated with poor fertilization rates following in vitro fertilization, defective preimplantation embryonic development, and high rates of miscarriage and morbidity in the offspring. Furthermore, diet-induced paternal obesity was found to initiate intergenerational transmission of obesity and insulin resistance in two generations of murine offspring. Meta-analysis from human studies found obese males were more likely to demonstrate sperm DNA fragmentation, infertility, decreased live birth per cycle of assisted reproduction technology, and increased absolute risk of pregnancy nonviability, with no consistent effect on conventional semen parameters. There is a need for future studies to expound on the mechanisms of sperm DNA damage and the impact of weight loss in reversing this damage.

Semen collection, characterisation and artificial insemination in the beluga (Delphinapterus leucas) using liquid-stored spermatozoa

2008 ◽  
Vol 20 (7) ◽  
pp. 770 ◽  
Author(s):  
J. K. O'Brien ◽  
K. J. Steinman ◽  
T. Schmitt ◽  
T. R. Robeck
Keyword(s):  
Artificial Insemination ◽  
Storage Temperature ◽  
Sperm Concentration ◽  
Serum Testosterone ◽  
Delphinapterus Leucas ◽  
Short Term ◽  
Preservation Method ◽  
First Time ◽  
Pregnancy And Birth

Ejaculates were collected from a beluga (Delphinapterus leucas) to gain an understanding of sperm biology and develop a short-term sperm preservation method for use in artificial insemination (AI). Ejaculate parameters and biochemistry, semen production and serum testosterone concentrations of an adult male were characterised for 21 months. Sperm viability, acrosome integrity and morphology did not change (P > 0.05) but ejaculate volume, sperm concentration and total spermatozoa per ejaculate were higher (P < 0.05) from January to June than from July to December. Peak testosterone concentrations (P < 0.05) were observed from October to April (8.0 ± 1.6 ng mL–1). The effects of hyaluronic acid (HA), antioxidants, storage temperature and time on in vitro sperm characteristics were examined. Motility parameters and viability were improved (P < 0.05) when semen was stored at 5°C compared with 21°C. During the first 24 h of storage sperm agglutination was absent only at 5°C in the presence of HA. A nulliparous 28-year-old female was inseminated endoscopically with liquid-stored semen. A pregnancy and birth of a calf was achieved following AI for the first time in this species, thereby validating both the AI technique and the fertility of beluga spermatozoa after chilled storage in a specialised diluent.

scholarly journals Sperm chromatin structure assay versus sperm chromatin dispersion kits: Technical repeatability and choice of assisted reproductive technology procedure

2020 ◽  
Vol 47 (4) ◽  
pp. 277-283
Author(s):  
Vidya Laxme B ◽  
Silviya Stephen ◽  
Ramyashree Devaraj ◽  
Sridurga Mithraprabhu ◽  
Ricardo P. Bertolla ◽  
...  
Keyword(s):  
Assisted Reproductive Technology ◽  
Chromatin Structure ◽  
Reproductive Technology ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Vitro Fertilization ◽  
Sperm Chromatin Structure Assay ◽  
Sperm Dna ◽  
The Impact

Objective: The sperm DNA fragmentation index (DFI) guides the clinician’s choice of an appropriate assisted reproductive technology (ART) procedure. The DFI can be determined using commercially available methodologies, including sperm chromatin dispersion (SCD) kits and sperm chromatin structure assay (SCSA). Currently, when DFI is evaluated using SCD kits, the result is analyzed in reference to the SCSA-derived threshold for the choice of an ART procedure. In this study, we compared DFI values obtained using SCSA with those obtained using SCD and determined whether the difference affects the choice of ART procedure.Methods: We compared SCSA to two SCD kits, CANfrag (n=36) and Halosperm (n=31), to assess the DFI values obtained, the correlations between tests, the technical repeatability, and the impact of DFI on the choice of ART. Results: We obtained higher median DFI values using SCD kits than when using SCSA, and this difference was significant for the CANfrag kit (p<0.001). The SCD kits had significantly higher coefficients of variation than SCSA (p<0.0001). In vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) would be chosen for a significantly higher proportion of patients if a decision were made based on DFI derived from SCD rather than DFI determined using SCSA (p=0.003). Conclusion: Our results indicate that SCD kit-specific thresholds should be established in order to avoid the unnecessary use of IVF/ICSI based on sperm DNA damage for the management of infertility. Appropriate measures should be taken to mitigate the increased variability inherent to the methods used in these tests.

scholarly journals 14-3-3γ, a novel regulator of the large-conductance Ca2+-activated K+ channel

2020 ◽  
Vol 319 (1) ◽  
pp. F52-F62
Author(s):  
Shan Chen ◽  
Xiuyan Feng ◽  
Xinxin Chen ◽  
Zhizhi Zhuang ◽  
Jia Xiao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Specific Inhibitor ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Protein Ubiquitination ◽  
293 Cells

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.

scholarly journals 2′-O-Methylation of Ribosomal RNA: Towards an Epitranscriptomic Control of Translation?

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 106 ◽  
Author(s):  
Piero Monaco ◽  
Virginie Marcel ◽  
Jean-Jacques Diaz ◽  
Frédéric Catez
Keyword(s):  
Ribosomal Rna ◽  
Translational Control ◽  
Ribosome Biogenesis ◽  
In Vitro Translation ◽  
Ribosome Structure ◽  
Quantitative Mapping ◽  
And Function ◽  
The Impact ◽  
First Time

Ribosomal RNA (rRNA) undergoes post-transcriptional modification of over 200 nucleotides, predominantly 2′-O-methylation (2′-O-Me). 2′-O-Methylation protects RNA from hydrolysis and modifies RNA strand flexibility but does not contribute to Watson-Crick base pairing. The contribution of 2′-O-Me to the translational capacity of ribosomes has been established. Yet, how 2′-O-Me participates in ribosome biogenesis and ribosome functioning remains unclear. The development of 2′-O-Me quantitative mapping methods has contributed to the demonstration that these modifications are not constitutive but rather provide heterogeneity to the ribosomal population. Moreover, recent advances in ribosome structure analysis and in vitro translation assays have proven, for the first time, that 2′-O-Me contributes to regulating protein synthesis. This review highlights the recent data exploring the impact of 2′-O-Me on ribosome structure and function, and the emerging idea that the rRNA epitranscriptome is involved in translational control.

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