scholarly journals Fluorescent labelling of boar spermatozoa for quantitative studies on competitive sperm–oviduct binding

2019 ◽  
Vol 31 (9) ◽  
pp. 1520 ◽  
Author(s):  
Heiko H. W. Henning ◽  
Julia Batz-Schott ◽  
Benita Grünther ◽  
Xuyen Le Thi ◽  
Dagmar Waberski
Keyword(s):  
Storage Temperature ◽  
Gradient Centrifugation ◽  
Fluorescent Labelling ◽  
Binding Assays ◽  
Binding Studies ◽  
Fluorescent Markers ◽  
Semen Storage ◽  
Labelling Procedure ◽  
Mitotracker Red ◽  
Boar Spermatozoa

Invitro sperm–oviduct binding assays enable assessment of the capacity of spermatozoa to form a ‘reservoir’ in the oviduct. Competitive approaches, such as experimental set-ups that test multiple males or semen samples simultaneously on the same tissue explants, are desirable because they reduce the likelihood of bias when using material from different females. Therefore, we established a fluorescent labelling technique that allows tagging and storage of spermatozoa before competitive studies of sperm–oviduct binding invitro. Fluorescent markers were tested for reliability and compatibility with parameters of boar spermatozoa viability. The addition of seminal plasma after density gradient centrifugation was essential to counteract centrifugation stress during the labelling procedure. It was demonstrated that sperm tagged with MitoTracker Green FM or MitoTracker Red FM can be successfully used in competitive sperm–oviduct binding studies. The assay was sensitive enough to indicate subtle effects of semen storage temperature on the ability of the spermatozoa to contribute to the female sperm reservoir.

scholarly journals Metabolic activity of boar semen stored in different extenders supplemented with ostrich egg yolk lipoproteins

2017 ◽  
Vol 61 (1) ◽  
pp. 127-133 ◽  
Author(s):  
Anna Dziekońska ◽  
Marek Kinder ◽  
Leyland Fraser ◽  
Jerzy Strzeżek ◽  
Władysław Kordan
Keyword(s):  
Oxygen Consumption ◽  
Metabolic Activity ◽  
Storage Temperature ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Atp Production ◽  
Beneficial Effects ◽  
Semen Storage ◽  
Boar Semen ◽  
Boar Spermatozoa

AbstractIntroduction:The aim of this study was to evaluate the effect of lipoprotein fraction isolated from ostrich egg yolk (LPFo) on the metabolic activity of boar spermatozoa following liquid semen storage in different extenders and temperatures.Material and Methods:Boar ejaculates were extended in Androhep, Beltsville thawing solution (BTS), and Martín-Rillo and Alias (MR-A) without (control) or with the addition of LPFo and stored for three days at either 5°C or 16°C. The analysed sperm parameters included total motility (TMOT), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), oxygen consumption, and adenosine triphosphate (ATP) production.Results:The sperm metabolic activity seemed to be higher in the LPFo-based extenders following storage for three days, irrespective of the storage temperature. Compared with the LPFo-free extenders, significantly higher (P < 0.05) sperm PMI and MMP were observed in BTS and MR-A extenders supplemented with LPFo during storage for three days at 5°C. Spermatozoa stored in the BTS-LPFo extender exhibited higher (P < 0.05) TMOT and oxygen consumption, whereas higher (P < 0.05) PMI was observed in spermatozoa stored in Androhep-LPFo and MR-A-LPFo for three days at 16°C. No significant differences (P > 0.05) in ATP content were observed between the LPFo-free and LPFo-based extenders during storage.Conclusions:Supplementation of LPFo to semen extenders had varying effects on the metabolic activity of boar spermatozoa stored at different temperatures. It can be suggested that the interactions of various components of the extenders and seminal plasma with LPFo exert beneficial effects on the sperm metabolic activity during liquid storage of boar semen.

scholarly journals Binding of boar spermatozoa to oviductal epithelium in vitro in relation to sperm morphology and storage time

Reproduction ◽  
2006 ◽  
Vol 131 (2) ◽  
pp. 311-318 ◽  
Author(s):  
D Waberski ◽  
F Magnus ◽  
F Ardón ◽  
A M Petrunkina ◽  
K F Weitze ◽  
...  
Keyword(s):  
Semen Quality ◽  
Binding Capacity ◽  
Storage Period ◽  
Sperm Function ◽  
Sperm Binding ◽  
Oviductal Epithelium ◽  
Semen Storage ◽  
Boar Semen ◽  
Boar Spermatozoa

In vitro short-term storage of boar semen for up to 72 h before insemination negatively affects fertility, but this often remains undetected during semen quality assessment. One important sperm function is the ability to form the functional sperm reservoir in the oviduct. In the present study, we used the modified oviductal explant assay to study sperm binding to oviductal epithelium in vitro in diluted boar semen stored for 24 or 72 h. First, we determined the kinetics of in vitro sperm binding to oviductal epithelium in relation to co-incubation time of sperm and oviductal tissue pieces. Then, we studied how the binding of sperm to oviductal epithelium was affected by in vitro semen storage and by differences among individual boars. Sperm binding after different incubation times was significantly higher when semen was stored 24 h than after 72-h storage (P < 0.05), and peaked at 30–90 min of incubation. Sperm binding differed between boars (n = 44), and was negatively correlated to the percentage of sperm with cytoplasmic droplets (R = −0.51, P < 0.001). There were no significant changes in motility, acrosome integrity and propidium iodide stainability during the 72-h storage period. However, sperm-binding indices were significantly lower after 72 h in vitro storage than after 24-h storage in sperm from boars with normal semen quality (P < 0.05); in contrast, the binding capacity of sperm from boars with higher percentages of morphologically altered sperm remained at a low level. The sperm-binding capacity of sperm from four of the five boars with known subfertility was lower than the mean binding index minus one standard deviation of the boar population studied here. It is concluded that changes in the plasma membrane associated with in vitro ageing reduce the ability of stored boar sperm to bind to the oviductal epithelium. This study shows the potential of sperm–oviduct binding as a tool to assess both male fertility and changes in sperm function associated with in vitro ageing.

scholarly journals Contribution of factor VIII light-chain residues 2007–2016 to an activated protein C-interactive site

2013 ◽  
Vol 109 (02) ◽  
pp. 187-198 ◽  
Author(s):  
Masahiro Takeyama ◽  
Hironao Wakabayashi ◽  
Philip Fay
Keyword(s):  
Light Chain ◽  
Protein C ◽  
Activated Protein C ◽  
Fluid Phase ◽  
Fold Increase ◽  
Binding Assays ◽  
Dot Blot ◽  
Binding Studies ◽  
Dot Blot Assay ◽  
Dot Blotting

SummaryAlthough factor (F) VIIIa is inactivated by activated protein C (APC) through cleavages in the FVIII heavy chain-derived A1 (Arg336) and A2 subunits (Arg562), the FVIII light chain (LC) contributes to catalysis by binding the enzyme. ELISA-based binding assays showed that FVIII and FVIII LC bound to immobilised active site-modified APC (DEGRAPC) (apparent K d ~270 nM and 1.0 μM, respectively). Fluid-phase binding studies using fluorescence indicated an estimated K d of ~590 nM for acrylodan-labelled LC binding to DEGR-APC. Furthermore, FVIII LC effectively competed with FVIIIa in blocking APC-catalysed cleavage at Arg336 (K i = 709 nM). A binding site previously identified near the C-terminal end of the A3 domain (residues 2007–2016) of FVIII LC was subjected to Ala-scanning mutagenesis. FXa generation assays and western and dot blotting were employed to assess the contribution of these residues to FVIIIa interactions with APC. Virtually all variants tested showed reductions in the rates of APC-catalysed inactivation of the cofactor and cleavage at the primary inactivation site (Arg336), with maximal reductions in inactivation rates (~3-fold relative to WT) and cleavage rates (~3 to ~9-fold relative to WT) observed for the Met2010Ala, Ser2011Ala, and Leu2013Ala variants. Titration of FVIIIa substrate concentration monitoring cleavage by a dot blot assay indicated that these variants also showed ~3-fold increases relative to WT while a double mutant (Met2010Ala/Ser2011Ala) showed a >4-fold increase in K m. These results show a contribution of a number of residues within the 2007–2016 sequence, and in particular residues Met2010, Ser2011, and Leu2013 to an APC-interactive site.

scholarly journals Rapid Multiplex Detection and Differentiation of Listeria Cells by Use of Fluorescent Phage Endolysin Cell Wall Binding Domains

2010 ◽  
Vol 76 (17) ◽  
pp. 5745-5756 ◽  
Author(s):  
Mathias Schmelcher ◽  
Tatiana Shabarova ◽  
Marcel R. Eugster ◽  
Fritz Eichenseher ◽  
Vincent S. Tchang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Modular Organization ◽  
Binding Assays ◽  
Surface Plasmon Resonance Analysis ◽  
Fluorescent Marker ◽  
Binding Domains ◽  
Cell Wall Binding ◽  
Fluorescent Markers ◽  
Severe Infections ◽  
And Control

ABSTRACT The genus Listeria comprises food-borne pathogens associated with severe infections and a high mortality rate. Endolysins from bacteriophages infecting Listeria are promising tools for both their detection and control. These proteins feature a modular organization, consisting of an N-terminal enzymatically active domain (EAD), which contributes lytic activity, and a C-terminal cell wall binding domain (CBD), which targets the lysin to its substrate. Sequence comparison among 12 different endolysins revealed high diversity among the enzyme's functional domains and allowed classification of their CBDs into two major groups and five subclasses. This diversity is reflected in various binding properties, as determined by cell wall binding assays using CBDs fused to fluorescent marker proteins. Although some proteins exhibited a broad binding range and recognize Listeria strains representing all serovars, others target specific serovars only. The CBDs also differed with respect to the number and distribution of ligands recognized on the cells, as well as their binding affinities. Surface plasmon resonance analysis revealed equilibrium affinities in the pico- to nanomolar ranges for all proteins except CBD006, which is due to an internal truncation. Rapid multiplexed detection and differentiation of Listeria strains in mixed bacterial cultures was possible by combining CBDs of different binding specificities with fluorescent markers of various colors. In addition, cells of different Listeria strains could be recovered from artificially contaminated milk or cheese by CBD-based magnetic separation by using broad-range CBDP40 and subsequently identified after incubation with two differently colored CBD fusion proteins of higher specificity.

scholarly journals Quercetin and Naringenin Provide Functional and Antioxidant Protection to Stored Boar Semen

Animals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1930
Author(s):  
Eva Tvrdá ◽  
Mégane Debacker ◽  
Michal Ďuračka ◽  
Ján Kováč ◽  
Ondřej Bučko
Keyword(s):  
Mitochondrial Activity ◽  
Ros Generation ◽  
Dna Integrity ◽  
Membrane Stabilization ◽  
New Information ◽  
Semen Storage ◽  
Boar Semen ◽  
Boar Spermatozoa ◽  
The Impact ◽  
Sample Dilution

In this study, we evaluated the impact of 5–50 μM quercetin (QUE) and naringenin (NAR) on extended boar spermatozoa in the BTS (Beltsville Thawing Solution) medium for 72 h. Spermatozoa motion, membrane, acrosome, and DNA integrity were investigated immediately after sample dilution (0 h) as well as after 24 h, 48 h, and 72 h of semen storage. Furthermore, reactive oxygen species (ROS) and superoxide production, as well as the extent of oxidative damage to the sperm proteins and lipids, were assessed to determine the potential of QUE and NAR to prevent a potential loss of sperm vitality due to oxidative stress development. Our results indicate that the most notable parameter influenced by QUE was the mitochondrial activity, which remained significantly higher throughout the experiment (p < 0.001 and p < 0.0001; 10 μM), and which correlated with the most prominent maintenance of sperm motility (p < 0.01, 48 h; p < 0.05, 72 h). A significant membrane stabilization (p < 0.01, 24 h and 48 h; p < 0.0001, 72 h) and prevention of lipid peroxidation (p < 0.05, 24 h and 48 h; p < 0.01, 72 h) was primarily observed following administration of 10 and 25 μM NAR; respectively. Administration of 10 μM QUE led to a significant decrease of superoxide (p < 0.0001, 48 h and 72 h) while the most notable decline of ROS generation was recorded in the case of 10 and 25 μM NAR (p < 0.001). This study may provide new information on the specific mechanisms of action involved in the favorable effects of natural biomolecules on spermatozoa.

scholarly journals Neurofascin induces neurites by heterophilic interactions with axonal NrCAM while NrCAM requires F11 on the axonal surface to extend neurites.

1996 ◽  
Vol 135 (4) ◽  
pp. 1059-1069 ◽  
Author(s):  
H Volkmer ◽  
R Leuschner ◽  
U Zacharias ◽  
F G Rathjen
Keyword(s):  
Direct Interaction ◽  
Binding Activity ◽  
Soluble Form ◽  
Binding Assays ◽  
Binding Studies ◽  
Experimental Conditions ◽  
Neurite Extension ◽  
Ig Superfamily ◽  
Inert Surface ◽  
Axonal Extension

Neurofascin and NrCAM are two axon-associated transmembrane glycoproteins belonging to the L1 subgroup of the Ig superfamily. In this study, we have analyzed the interaction of both proteins using neurite outgrowth and binding assays. A neurofascin-Fc chimera was found to stimulate the outgrowth of tectal cells when immobilized on an inert surface but not as a soluble form using polylysine as substrate. Antibody blocking experiments demonstrate that neurite extension on immobilized neurofascin is mediated by NrCAM on the axonal surface. Under the reverse experimental conditions where NrCAM induces neurite extension, F11, and not neurofascin, serves as axonal receptor. Binding studies using transfected COS7 cells and immunoprecipitations reveal a direct interaction between neurofascin and NrCAM. This binding activity was mapped to the Ig domains within neurofascin. The neurofascin-NrCAM binding can be modulated by alternative splicing of specific stretches within neurofascin. These studies indicate that heterophilic interactions between Ig-like proteins implicated in axonal extension underlie a regulation by the neuron.

A simple, inexpensive filtration device suitable for neurotransmitter receptor binding studies in brain tissue

1981 ◽  
Vol 59 (5) ◽  
pp. 504-506 ◽  
Author(s):  
M. Wilkinson ◽  
Dale Grovestine
Keyword(s):  
Rat Brain ◽  
Receptor Binding ◽  
Radioligand Binding ◽  
Benzodiazepine Receptors ◽  
Brain Homogenate ◽  
Binding Assays ◽  
Binding Studies ◽  
Neurotransmitter Receptor ◽  
Binding Data ◽  
Filtration Device

We describe the construction and use of an inexpensive filtration manifold suitable for radioligand binding assays. Experiments with [3H]flunitrazepam indicate that the binding data for benzodiazepine receptors in rat brain homogenate are almost indistinguishable from those obtained with a commercially available manifold.

scholarly journals Binding of immunoglobulin classes to subpopulations of human red blood cells separated by density-gradient centrifugation

Blood ◽  
1980 ◽  
Vol 55 (5) ◽  
pp. 817-822
Author(s):  
EM Alderman ◽  
HH Fudenberg ◽  
RE Lovins
Keyword(s):  
Plasma Membranes ◽  
Colloidal Silica ◽  
Gradient Centrifugation ◽  
Immunofluorescence Assay ◽  
Silica Matrix ◽  
Binding Studies ◽  
Dense Population ◽  
Specific Density ◽  
Human Red Blood Cells ◽  
Membrane Bound

Human erythrocytes (RBC) from whole blood were separated according to their specific densities by centrifugation on a polyvinyl-pyrrolidine- coated colloidal silica matrix (Percoll) into four major subpopulations. By indirect immunofluorescence assay, the most dense RBC subpopulation, with specific density greater than 1.110 g/ml (3%-5% of total RBC), was positive for membrane-bound immunoglobulin; the remaining, less dense subpopulations were negative. IgG was present on 85%-95%, IgM on 28%-32%, and IgA on 15%-20% of the RBC in the most dense population. When these immunoglobulins were eluted, radiolabeled, and used in binding studies with autologous RBC fractions subjected to thermal and/or enzymatic treatment, they reacted specifically with the less dense RBC subpopulations. These results suggest that previously cryptic antigens were revealed by the activity of neuraminidase on the plasma membranes of the treated RBC.

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