Boar spermadhesin AWN: Novel insights in its binding behavior and localization on sperm

2022 ◽  
Author(s):  
Pascal D Kroh ◽  
Beate C Braun ◽  
Fan Lui ◽  
Peter Müller ◽  
Karin Müller
Keyword(s):  
Electron Microscopy ◽  
Lipid Binding ◽  
Epididymal Sperm ◽  
Sperm Analysis ◽  
Binding Behavior ◽  
Binding Partners ◽  
Custom Made ◽  
Boar Sperm ◽  
Specific Association ◽  
Cytoplasmic Side

Abstract As a major spermadhesin first found in the seminal plasma of boars, AWN is described to fulfil a variety of reproduction related tasks. Although being the best investigated boar spermadhesin, information about its interaction with membranes is inconsistent. In this regard, previous reports locate AWN either inside or on the surface of sperm cells and at different regions, depending on the method and antibody used. Here, we localize native AWN in/on epididymal, ejaculated, capacitated and acrosome-reacted boar sperm using epifluorescence and electron microscopy, as well as an analysis of potential lipid binding partners of native and recombinant AWN. By applying a custom-made anti-AWN antibody, localization of AWN in the equatorial segment of ejaculated, capacitated and acrosome-reacted boar sperm was discovered. Electron microscopy showed that AWN is localized both on the sperm surface and on the cytoplasmic side of the plasma membrane, and in close vicinity to the nuclear and both acrosomal membranes of sperm. Analysis of epididymal sperm indicated migration of AWN from the retral postacrosomal part to the equatorial segment during the epididymal passage. In contrast to hypotheses claiming a specific association of AWN to phosphatidylethanolamine and in line with our previous study describing an interaction with phosphatidic acid, the current results show a rather electrostatically-driven binding mechanism of AWN to negative lipids. In conclusion, this work provides new insights into the arrangement of AWN in the equatorial segment that suggest a possible role in sperm-oocyte fusion.

scholarly journals Cholesterol Increases Lipid Binding Rate and Changes Binding Behavior of Bacillus thuringiensis Cytolytic Protein

2018 ◽  
Vol 19 (12) ◽  
pp. 3819 ◽  
Author(s):  
Sudarat Tharad ◽  
Öykü Üzülmez ◽  
Boonhiang Promdonkoy ◽  
José Toca-Herrera
Keyword(s):  
Bacillus Thuringiensis ◽  
Lipid Bilayers ◽  
Mammalian Cells ◽  
Layer Structure ◽  
Cholesterol Content ◽  
Lipid Binding ◽  
Lipid Layer ◽  
Binding Behavior ◽  
Binding Rate ◽  
Ring Shape

Cytolytic protein (Cyt) is a member of insecticidal proteins produced by Bacillus thuringiensis. Cyt protein has activity against insect cells and mammalian cells, which differ in lipid and cholesterol composition. This study presents the lipid binding behavior of Cyt2Aa2 protein on model membranes containing different levels of cholesterol content by combining Quartz Crystal Microbalance with Dissipation (QCM-D) and Atomic Force Microscopy (AFM). QCM-D results revealed that cholesterol enhances the binding rate of Cyt2Aa2 protein onto lipid bilayers. In addition, the thicker lipid bilayer was observed for the highest cholesterol content. These results were confirmed by AFM. The analysis of protein surface coverage as a function of time showed a slower process for 5:0 and 5:0.2 (POPC:Chol) ratios than for 5:1 and 5:2 (POPC:Chol) ratios. Significantly, the Cyt2Aa2-lipid binding behavior and the protein–lipid layer were different for the 5:3 (POPC:Chol) ratio. Furthermore, AFM images revealed a transformation of Cyt2Aa2/lipid layer structure from strip pattern to ring shape structures (which showed a strong repulsion with AFM tip). In summary, cholesterol increases the binding rate and alters the lipid binding behavior of Cyt2Aa2 protein, although it is not required for Cyt2Aa2 protein binding onto lipid bilayers.

scholarly journals Effects of Photoperiod on Epididymal and Sperm Morphology in a Wild Rodent, the Viscacha (Lagostomus maximus maximus)

ISRN Anatomy ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
A. M. Cruceño ◽  
J. C. de Rosas ◽  
M. Fóscolo ◽  
E. M. Chaves ◽  
L. Scardapane ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Reproductive Cycle ◽  
Sperm Morphology ◽  
Structural Parameters ◽  
Light And Electron Microscopy ◽  
Morphological Characteristics ◽  
Activity Period ◽  
Epididymal Sperm ◽  
Wild Rodent ◽  
Lagostomus Maximus

The viscacha (Lagostomus maximus maximus) is a seasonal South American wild rodent. The adult males exhibit an annual reproductive cycle with periods of maximum and minimum gonadal activity. Four segments have been identified in the epididymis of this species: initial, caput, corpus, and cauda. The main objective of this work was to relate the seasonal morphological changes observed in the epididymal duct with the data from epididymal sperm during periods of activity and gonadal regression using light and scanning electron microscopy (SEM). Under light and electron microscopy, epididymal corpus and cauda showed marked seasonal variations in structural parameters and in the distribution of different cellular populations of epithelium. Initial and caput segments showed mild morphological variations between the two periods. Changes in epididymal sperm morphology were observed in the periods analyzed and an increased number of abnormal gametes were found during the regression period. During this period, anomalies were found mainly in the head, midpiece, and neck, while in the activity period, defects were found only in the head. Our results confirm that the morphological characteristics of the epididymal segments, as well as sperm morphology, undergo significant changes during the reproductive cycle of Lagostomus.

scholarly journals Exosomes derived from HEK293T cells interact in an efficient and noninvasive manner with mammalian sperm in vitro

Nanomedicine ◽  
2020 ◽  
Vol 15 (20) ◽  
pp. 1965-1980
Author(s):  
Teresa Vilanova-Perez ◽  
Celine Jones ◽  
Stefan Balint ◽  
Rebecca Dragovic ◽  
Michael L Dustin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Sperm Function ◽  
Sperm Analysis ◽  
Mammalian Sperm ◽  
Boar Sperm ◽  
Computer Assisted Sperm Analysis

Aim: To investigate exosomes as a noninvasive delivery tool for mammalian sperm. Materials & Methods: Exosomes were isolated from HEK293T cells and co-incubated with boar sperm in vitro. Results: Internalized exosomes were detected within 10 min of co-incubation. Computer-assisted sperm analysis and flow cytometry demonstrated that even after 5-h of exposure to exosomes, there were no significant deleterious effects with regard to sperm motility, viability, membrane integrity and mitochondrial membrane potential (p > 0.05), thus indicating that exosomes did not interfere with basic sperm function. Conclusion: HEK293T-derived exosomes interacted with boar sperm without affecting sperm function. Exosomes represent a versatile and promising research tool for studying sperm biology and provide new options for the diagnosis and treatment of male infertility.

scholarly journals Effects of Astaxanthin on Miniature Pig Sperm Cryopreservation

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Eunjoo Lee ◽  
Daeyoung Kim
Keyword(s):  
Sperm Motility ◽  
Semen Parameters ◽  
Control Group ◽  
Computer Assisted ◽  
Miniature Pig ◽  
Oxidation Stress ◽  
Sperm Analysis ◽  
Semen Parameter ◽  
Boar Sperm ◽  
Sperm Plasma Membrane

The purpose of this study is to evaluate the effects of astaxanthin added to freezing buffer on semen parameters, total sperm oxidation stress after postthawing of boar sperm, and lipid peroxidation (LPO) which is caused by reactive oxygen species (ROS) in sperm membrane. Varying concentrations of astaxanthin (0, 10, 50, 100, and 500 μM) were used in the freezing buffer during cryopreservation to protect the DNA of thawed miniature pig sperm. Semen parameter was measured using computer-assisted sperm analysis (CASA) for sperm motility, and then ROS rate and oxidative stress of boar sperm were determined using fluorescence-activated cell sorting (FACS). Sperm motility was higher (p<0.05) in the astaxanthin group than in the control group. Sperm motility and the number of progressive motile sperm were higher (p<0.05) in the astaxanthin 500 μM group than in the control group. In ROS evaluation, the astaxanthin group had lower intracellular O2 and H2O2 in viable sperm. Yo-Pro-I/HE and PI/H2DCFDA staining as revealed using flow cytometry was lower in astaxanthin groups than in the other groups. As a result, we found that astaxanthin could protect the sperm plasma membrane from free radicals and LPO during boar sperm postthawing.

scholarly journals Native Tandem and Ion Mobility Mass Spectrometry Highlight Structural and Modular Similarities in Clustered-Regularly-Interspaced Shot-Palindromic-Repeats (CRISPR)-associated Protein Complexes From Escherichia coli and Pseudomonas aeruginosa

2012 ◽  
Vol 11 (11) ◽  
pp. 1430-1441 ◽  
Author(s):  
Esther van Duijn ◽  
Ioana M. Barbu ◽  
Arjan Barendregt ◽  
Matthijs M. Jore ◽  
Blake Wiedenheft ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Ion Mobility ◽  
Protein Complexes ◽  
Acquired Resistance ◽  
Native Mass Spectrometry ◽  
Ion Mobility Mass Spectrometry ◽  
E Coli ◽  
Ribonucleoprotein Complexes ◽  
Specific Association

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.

Glycerol decreases the integrity of the perinuclear theca in boar sperm

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 172-177 ◽  
Author(s):  
Marco Antonio Arenas Núñez ◽  
María de Lourdes Juárez-Mosqueda ◽  
Oscar Gutiérrez-Pérez ◽  
Santiago René Anzaldúa Arce ◽  
Alejandro Córdova Izquierdo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Adverse Effect ◽  
Protease Inhibitors ◽  
Free Group ◽  
Glycerol Concentration ◽  
Boar Sperm ◽  
Perinuclear Theca ◽  
First Time ◽  
Control Samples

SummaryWe evaluated the effect of glycerol on the perinuclear theca (PT) of boar sperm. Samples from six ejaculates obtained from three different boars were incubated in the detergent Brij 36-T. Spermatozoa were treated with a glycerol concentration of either 2 or 4%, and incubated for 10 or 30 min; two other samples were treated with protease inhibitors (PI; leupeptin or an inhibitor commercial cocktail), mixed with 4% glycerol, and incubated for 30 min. A third glycerol-free group was used as the control. The samples were processed for electron microscopy evaluation. The PT remained intact in 78% of the control samples while, after addition of glycerol for 30 min, the proportion of spermatozoa with disrupted or absent PT increased (P < 0.05). PT was preserved in PI samples, but PT changes increased (P < 0.05). Differences due to treatment with glycerol (2 or 4%) at 10 or 30 min were not observed. These results show, to our knowledge for the first time, the adverse effect of glycerol on the integrity of the PT.

scholarly journals Taxol-stabilized microtubules promote the formation of filaments from unmodified full-length Tau in vitro

2012 ◽  
Vol 23 (24) ◽  
pp. 4796-4806 ◽  
Author(s):  
Aranda R. Duan ◽  
Holly V. Goodson
Keyword(s):  
Electron Microscopy ◽  
Experimental Design ◽  
Fluorescence Microscopy ◽  
Filament Formation ◽  
Binding Assays ◽  
Neuronal Protein ◽  
Binding Behavior ◽  
Health And Disease ◽  
Disease Understanding

Tau is a neuronal protein that stabilizes the microtubule (MT) network, but it also forms filaments associated with Alzheimer's disease. Understanding Tau–MT and Tau–Tau interactions would help to establish Tau function in health and disease. For many years, literature reports on Tau–MT binding behavior and affinity have remained surprisingly contradictory (e.g., 10-fold variation in Tau–MT affinity). Tau–Tau interactions have also been investigated, but whether MTs might affect Tau filament formation is unknown. We have addressed these issues through binding assays and microscopy. We assessed Tau–MT interactions via cosedimentation and found that the measured affinity of Tau varies greatly, depending on the experimental design and the protein concentrations used. To investigate this dependence, we used fluorescence microscopy to examine Tau–MT binding. Strikingly, we found that Taxol-stabilized MTs promote Tau filament formation without characterized Tau-filament inducers. We propose that these novel Tau filaments account for the incongruence in Tau–MT affinity measurements. Moreover, electron microscopy reveals that these filaments appear similar to the heparin-induced Alzheimer's model. These observations suggest that the MT-induced Tau filaments provide a new model for Alzheimer's studies and that MTs might play a role in the formation of Alzheimer's-associated neurofibrillary tangles.

scholarly journals Could night-guards be used as a simple method to detect leached-elements from dental restorations intra-orally? A study on amalgam restorations

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1786
Author(s):  
Rasha Mohamed Abdelraouf
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Elemental Analysis ◽  
Dental Amalgam ◽  
Simple Method ◽  
X Ray ◽  
Dental Restorations ◽  
Custom Made ◽  
Amalgam Restorations ◽  
Scanning Electron

Background: Detection of leached-elements from dental restorations intra-orally has been a subject of prime importance in dental research. However, this is challenging as most of the present techniques have some limitations. In this study, a new simple method was proposed via using night-guards. Thus, the aim of the study was to verify if night-guards could detect leached-elements from restorations as dental amalgam. Methods: Ten upper custom-made night-guards were fabricated for patients suffering from bruxism, who had amalgam-restorations in their upper molars. The night-guards were delivered to the patients and they were instructed to wear the night-guards during when they were asleep. After six months, the night-guards were taken from the patients to be analyzed. A new unused night-guard was fabricated from the same material to be used as a control. In the used night-guards, two areas were studied: the fitting surfaces contacting the amalgam restorations and the fitting surfaces not contacting amalgam restorations. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Analysis (EDXA) were used to examine the structural and elemental changes in the night-guards.   Results: SEM of the unused night-guard revealed a homogenous structure, and the composition was carbon and oxygen, as shown using EDXA (C=88.9wt% and O=11.1wt%). By contrast, the fitting surfaces of the night-guards contacting amalgam restorations showed numerous lustrous particles. Elemental analysis of these areas showed the presence of mercury and sulfur, in addition to carbon and oxygen (Hg=21.2wt%, S=2.5wt%, C=67.1wt% and O=9.2wt%). The night-guards’ fitting surfaces not contacting amalgam restorations showed slight cracking, and the composition was carbon and oxygen (C=88.3wt% and O=11.7 wt%).  Conclusions: Analyzing fitting surfaces of night-guards contacting dental restorations, such as amalgam, could aid in understanding the nature of leached-elements from these restorations intra-orally. However, further studies about its application upon dental-restorations other than amalgam are recommended.

Sign in / Sign up

Export Citation Format

Share Document