scholarly journals Assessment of Sperm Binding Capacity in the Tubal Reservoir Using a Bovine Ex Vivo Oviduct Culture and Fluorescence Microscopy

2021 ◽  
Vol 4 (4) ◽  
pp. 67
Author(s):  
Miguel Camara Pirez ◽  
Simeng Li ◽  
Sabine Koelle
Keyword(s):  
Fluorescence Microscopy ◽  
Ex Vivo ◽  
Binding Capacity ◽  
Sperm Binding ◽  
Bovine Spermatozoa ◽  
Sperm Reservoir ◽  
Multiple Species ◽  
The Impact ◽  
Molecular Nature

Sperm binding within the oviductal sperm reservoir plays an important role for reproductive success by enabling sperm survival and maintaining fertilizing capacity. To date, numerous in vitro technologies have been established to measure sperm binding capacity to cultured oviductal cells or oviductal explants. However, these methods do not accurately represent the microenvironment and complex multi-molecular nature of the oviduct. In this paper, we describe a novel protocol for assessing sperm binding capacity in the tubal sperm reservoir using an ex vivo oviduct culture in the bovine model. This protocol includes the staining of frozen-thawed bovine spermatozoa with the DNA-binding dye Hoechst 33342, the co-incubation of stained sperm in closed segments of the oviduct and the visualization and quantification of bound spermatozoa by fluorescence microscopy. By generating overlays of multiple Z-stacks of randomly selected regions of interest (ROIs), spermatozoa bound in the sperm reservoir can be visualized and quantified within the 3D arrangement of the oviductal folds. This method, which is applicable to multiple species, can be used to assess individual sperm binding capacity in males for prognostic purposes as well as to assess the impact of diseases and medications on the formation of the sperm reservoir in the oviduct in humans and animals.

scholarly journals Bovine sperm-oviduct interactions are characterized by specific sperm behaviour, ultrastructure and tubal reactions which are impacted by sex sorting

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Miguel Camara Pirez ◽  
Heather Steele ◽  
Sven Reese ◽  
Sabine Kölle
Keyword(s):  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Sperm Binding ◽  
Tail Movement ◽  
Bovine Spermatozoa ◽  
Sperm Reservoir ◽  
And Function ◽  
The Impact

Abstract To date sperm-oviduct interactions have largely been investigated under in vitro conditions. Therefore we set out to characterize the behaviour of bovine spermatozoa within the sperm reservoir under near in vivo conditions and in real-time using a novel live cell imaging technology and a newly established fluorescent sperm binding assay. Sperm structure and tubal reactions after sperm binding were analysed using scanning and transmission electron microscopy and histochemistry. As a model to specify the impact of stress on sperm-oviduct interactions, frozen-thawed conventional and sex-sorted spermatozoa from the same bulls (n = 7) were co-incubated with oviducts obtained from cows immediately after slaughter. Our studies revealed that within the oviductal sperm reservoir agile (bound at a tangential angle of about 30°, actively beating undulating tail), lagging (bound at a lower angle, reduced tail movement), immotile (absence of tail movement) and hyperactivated (whip-like movement of tail) spermatozoa occur, the prevalence of which changes in a time-dependent pattern. After formation of the sperm reservoir, tubal ciliary beat frequency is significantly increased (p = 0.022) and the epithelial cells show increased activity of endoplasmic reticula. After sex sorting, spermatozoa occasionally display abnormal movement patterns characterized by a 360° rotating head and tail. Sperm binding in the oviduct is significantly reduced (p = 0.008) following sexing. Sex-sorted spermatozoa reveal deformations in the head, sharp bends in the tail and a significantly increased prevalence of damaged mitochondria (p < 0.001). Our results imply that the oviductal cells specifically react to the binding of spermatozoa, maintaining sperm survival within the tubal reservoir. The sex-sorting process, which is associated with mechanical, chemical and time stress, impacts sperm binding to the oviduct and mitochondrial integrity affecting sperm motility and function.

scholarly journals Selective sperm binding to pig oviductal epithelium in vitro

Reproduction ◽  
2001 ◽  
pp. 889-896 ◽  
Author(s):  
AM Petrunkina ◽  
R Gehlhaar ◽  
W Drommer ◽  
D Waberski ◽  
E Topfer-Petersen
Keyword(s):  
Seminal Fluid ◽  
Binding Capacity ◽  
Sperm Binding ◽  
Oviductal Epithelium ◽  
Female Donor ◽  
Sperm Reservoir ◽  
The Individual ◽  
Boar Spermatozoa ◽  
Initial Binding

The sperm reservoir in the caudal isthmus of the oviduct of a number of species is created by binding of spermatozoa to oviductal epithelium. The sperm reservoir fulfills a number of functions such as control of sperm transport, maintenance of sperm viability and modulation of capacitation. The initial capacities of ejaculated and epididymal boar spermatozoa to bind to oviductal epithelium were investigated using a modified pig oviductal explant assay. The number of spermatozoa that bound to 0.01 mm(2) of explant surface was used as the parameter of binding capacity. Binding of spermatozoa to oviductal epithelial explants was dependent in a linear manner on the number of spermatozoa added (P < or = 0.05). No difference was found in initial sperm binding between isthmic and ampullar explants. There was no effect of the stage of the oestrous cycle or the reproductive status of the female donor. There was a significant effect (P < or = 0.05) of the individual boar on the binding index. The binding index correlated negatively with the percentage of spermatozoa with cytoplasmic droplets and the percentage of morphologically abnormal spermatozoa (P < or = 0.05). Epididymal spermatozoa showed significantly lower initial binding capability than did ejaculated spermatozoa from the same boars (P < or = 0.05); therefore, components of seminal plasma may play a role in the binding process. The individual differences revealed by this study and their relation to morphology and contact of spermatozoa with seminal fluid indicate a selective function of sperm-oviduct binding.

scholarly journals Cell–cell coupling and DNA methylation abnormal phenotypes in the after-hours mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Federico Tinarelli ◽  
Elena Ivanova ◽  
Ilaria Colombi ◽  
Erica Barini ◽  
Edoardo Balzani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Neuronal Networks ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Neuronal Cultures ◽  
Functional Impairments ◽  
After Hours ◽  
The Impact

Abstract Background DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. Methods In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. Results We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. Conclusions The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.

scholarly journals Allergenic Characterization of New Mutant Forms of Pru p 3 as New Immunotherapy Vaccines

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
C. Gómez-Casado ◽  
M. Garrido-Arandia ◽  
P. Gamboa ◽  
N. Blanca-López ◽  
G. Canto ◽  
...  
Keyword(s):  
Food Allergy ◽  
Ex Vivo ◽  
Binding Capacity ◽  
Cross Reactivity ◽  
T Cell Epitopes ◽  
Native Form ◽  
Ige Binding ◽  
Pru P 3

Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named asPru p 3.01, Pru p 3.02, andPru p 3.03.Pru p 3.01showed very similar allergenic activity as the wild type byin vitroassays. However,Pru p 3.02andPru p 3.03presented reduced IgE binding with respect to the native form, byin vitro,ex vivo,and in vivo assays. In addition,Pru p 3.03had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, bothPru p 3.02andPru p 3.03maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus,Pru p 3.02andPru p 3.03could be good candidates for potential immunotherapy in peach-allergic patients.

Corticosteroids alter alveolar macrophage control of Lichtheimia corymbifera spores in an ex vivo mouse model

2020 ◽  
Author(s):  
Kévin Brunet ◽  
François Arrivé ◽  
Jean-Philippe Martellosio ◽  
Isabelle Lamarche ◽  
Sandrine Marchand ◽  
...  
Keyword(s):  
Mouse Model ◽  
Alveolar Macrophage ◽  
Oxidative Burst ◽  
Ex Vivo ◽  
Fungal Growth ◽  
Invasive Infection ◽  
Ex Vivo Model ◽  
Lichtheimia Corymbifera ◽  
The Impact

Abstract Alveolar macrophages (AM) are the first-line lung defense against Mucorales in pulmonary mucormycosis. Since corticosteroid use is a known risk factor for mucormycosis, the aim of this study was to describe the role of corticosteroids on AM capacities to control Lichtheimia corymbifera spore growth using a new ex vivo model. An in vivo mouse model was developed to determine the acetate cortisone dose able to trigger pulmonary invasive infection. Then, in the ex vivo model, male BALB/c mice were pretreated with the corticosteroid regimen triggering invasive infection, before AM collection through bronchoalveolar lavage. AMs from corticosteroid-treated mice and untreated control AMs were then exposed to L. corymbifera spores in vitro (ratio 1:5). AM control of fungal growth, adherence/phagocytosis, and oxidative burst were assessed using optical densities by spectrophotometer, flow cytometry, and 2', 7'-dichlorofluoresceine diacetate fluorescence, respectively. Cortisone acetate at 500 mg/kg, at D-3 and at D0, led to pulmonary invasive infection at D3. Co-incubated spores and AMs from corticosteroid-treated mice had significantly higher absorbance (fungal growth) than co-incubated spores and control AMs, at 24 h (P = .025), 36 h (P = .004), and 48 h (P = .001). Colocalization of spores with AMs from corticosteroid-treated mice was significantly lower than for control AMs (7.6 ± 1.9% vs 22.3 ± 5.8%; P = .003), reflecting spore adherence and phagocytosis inhibition. Finally, oxidative burst was significantly increased when control AMs were incubated with spores (P = 0.029), while corticosteroids hampered oxidative burst from treated AMs (P = 0.321). Corticosteroids enhanced fungal growth of L. corymbifera through AM phagocytosis inhibition and burst oxidative decrease in our ex vivo model. Lay Summary The aim of this study was to describe the impact of corticosteroids on alveolar macrophage (AM) capacities to control Mucorales growth in a new murine ex vivo model. Corticosteroids enhanced fungal growth of L. corymbifera through AM phagocytosis inhibition and burst oxidative decrease.

Abstract 11607: Novel Assay for Detection of LDL Transcytosis Across Coronary Endothelium Reveals an Unexpected Role for SR-B1

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Susan M Armstrong ◽  
Michael G Sugiyama ◽  
Andrew Levy ◽  
Dante Neculai ◽  
Mark Roufaiel ◽  
...  
Keyword(s):  
Coronary Artery ◽  
Fluorescence Microscopy ◽  
Ex Vivo ◽  
Scavenger Receptor ◽  
Intercellular Junctions ◽  
Live Cells ◽  
Human Coronary Artery ◽  
Class B ◽  
Major Route

Introduction: Retention of LDL beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. How LDL crosses the endothelium to enter the arterial wall remains unknown. While LDL could conceivably pass between endothelial cells (paracellularly) or through them (transcytosis), electron microscopy studies in animals revealed LDL in intracellular vesicles and none at intercellular junctions. This, combined with the absence of endothelial injury or intercellular gaps in early atherosclerosis, suggests that transcytosis is the major route. However, technical challenges with studying transcytosis have made confirming and extending these findings difficult. We developed and validated a novel assay for measuring the transcytosis of native LDL across live human coronary artery endothelium in vitro. Using this assay, we propose to elucidate the regulation of LDL transcytosis and have identified a novel role for SR-B1. Methods and Results: Experiments were performed using primary human coronary artery endothelial monolayers. Transcytosis was quantified in single live cells in real time using total internal reflectance fluorescence microscopy. Transcytosis of LDL was saturable and inhibited by excess unlabeled LDL. By fluorescence microscopy we found that DiI-LDL colocalized significantly with scavenger receptor, class B, type 1 (SR-B1). Unexpectedly, overexpression of SR-BI resulted in increased LDL transcytosis, while knockdown of SR-BI by siRNA inhibited transcytosis. Excess HDL, the canonical SR-B1 ligand, also decreased LDL transcytosis. To confirm the occurrence of transcytosis in an intact vessel, we perfused murine aortas ex vivo with both LDL and dextran of a smaller molecular radius. We observed the accumulation of subendothelial LDL without dextran, indicating that transcytosis of LDL occurs in intact vessels. Conclusions: The accumulation of LDL in the subendothelial intima is the first step of atherosclerosis yet little is known about how it occurs. Our data suggests that transcytosis of LDL is an important contributor, particularly in the early stages of the disease. By identifying the mechanisms of transcytosis, our work could have important implications for its pathogenesis and therapy.

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 Emulsion of Chloramphenicol: an Overwhelming Approach for Ocular Delivery

Folia Medica ◽  
2017 ◽  
Vol 59 (1) ◽  
pp. 23-30 ◽  
Author(s):  
Kalpesh C. Ashara ◽  
Ketan V. Shah
Keyword(s):  
Ternary Phase Diagram ◽  
Ex Vivo ◽  
Sterility Testing ◽  
Ocular Irritation ◽  
Peg 400 ◽  
Significant Difference ◽  
Oil Mixtures ◽  
Student’S T ◽  
The Impact

Abstract Background: Ophthalmic formulations of chloramphenicol have poor bioavailability of chloramphenicol in the ocular cavity. Aim: The present study aimed at exploring the impact of different oil mixtures in the form of emulsion on the permeability of chloramphenicol after ocular application. Materials and methods: Selection of oil mixture and ratio of the components was made by an equilibrium solubility method. An emulsifier was chosen according to its emulsification properties. A constrained simplex centroid design was used for the assessment of the emulsion development. Emulsions were evaluated for physicochemical properties; zone of inhibition, in-vitro diffusion and ex-vivo local accumulation of chloramphenicol. Validation of the design using check-point batch and reduced polynomial equations were also developed. Optimization of the emulsion was developed by software Design® expert 6.0.8. Assessment of the osmolarity, ocular irritation, sterility testing and isotonicity of optimized batch were also made. Results: Parker Neem®, olive and peppermint oils were selected as an oil phase in the ratio 63.64:20.2:16.16. PEG-400 was selected as an emulsifier according to a pseudo-ternary phase diagram. Constrained simplex-centroid design was applied in the range of 25-39% water, 55-69% PEG-400, 5-19% optimized oil mixture, and 1% chloramphenicol. Unpaired Student’s t-test showed for in-vitro and ex-vivo studies that there was a significant difference between the optimized batch of emulsion and Chloramphenicol eye caps (a commercial product) according to both were equally safe. Conclusion: The optimized batch of an emulsion of chloramphenicol was found to be as safe as and more effective than Chloramphenicol eye caps.

scholarly journals Neuroinflammation in Aged Brain: Impact of the Oral Administration of Ellagic Acid Microdispersion

2020 ◽  
Vol 21 (10) ◽  
pp. 3631 ◽  
Author(s):  
Raffaella Boggia ◽  
Federica Turrini ◽  
Alessandra Roggeri ◽  
Guendalina Olivero ◽  
Francesca Cisani ◽  
...  
Keyword(s):  
Oral Administration ◽  
Ellagic Acid ◽  
Ex Vivo ◽  
Behavioral Skills ◽  
Aged Mice ◽  
Age Related ◽  
The Central Nervous System ◽  
The Impact ◽  
Old Mice

The immune system and the central nervous system message each other to preserving central homeostasis. Both systems undergo changes during aging that determine central age-related defects. Ellagic acid (EA) is a natural product which is beneficial in both peripheral and central diseases, including aging. We analyzed the impact of the oral administration of a new oral ellagic acid micro-dispersion (EAm), that largely increased the EA solubility, in young and old mice. Oral EAm did not modify animal weight and behavioral skills in young and old mice, but significantly recovered changes in “ex-vivo, in vitro” parameters in old animals. Cortical noradrenaline exocytosis decreased in aged mice. EAm administration did not modify noradrenaline overflow in young animals, but recovered it in old mice. Furthermore, GFAP staining was increased in the cortex of aged mice, while IBA-1 and CD45 immunopositivities were unchanged when compared to young ones. EAm treatment significantly reduced CD45 signal in both young and old cortical lysates; it diminished GFAP immunopositivity in young mice, but failed to affect IBA-1 expression in both young and old animals. Finally, EAm treatment significantly reduced IL1beta expression in old mice. These results suggest that EAm is beneficial to aging and represents a nutraceutical ingredient for elders.

scholarly journals Kinetic Cytokine Secretion Profile of LPS-Induced Inflammation in the Human Skin Organ Culture

Pharmaceutics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 299 ◽  
Author(s):  
Raanan Gvirtz ◽  
Navit Ogen-Shtern ◽  
Guy Cohen
Keyword(s):  
Organ Culture ◽  
Human Skin ◽  
Comprehensive Evaluation ◽  
Ex Vivo ◽  
Cytokine Secretion ◽  
Peak Time ◽  
Local Skin ◽  
The Impact ◽  
Skin Organ Culture

Several in vitro models that mimic different aspects of local skin inflammation exist. The use of ex vivo human skin organ culture (HSOC) has been reported previously. However, comprehensive evaluation of the cytokine secretory capacity of the system and its kinetics has not been performed. Objective: the aim of the current study was to investigate the levels and secretion pattern of key cytokine from human skin tissue upon lipopolysaccharide (LPS) stimulation. HSOC maintained in an air–liquid interface was used. Epidermal and tissue viability was monitored by MTT and Lactate Dehydrogenase (LDH) activity assay, respectively. Cytokine levels were examined by ELISA and multiplex array. HSOCs were treated without or with three different LPS subtypes and the impact on IL-6 and IL-8 secretion was evaluated. The compounds enhanced the secreted levels of both cytokines. However, differences were observed in their efficacy and potency. Next, a kinetic multiplex analysis was performed on LPS-stimulated explants taken from three different donors to evaluate the cytokine secretion pattern during 0–72 h post-induction. The results revealed that the pro-inflammatory cytokines IL-6, IL-8, TNFα and IL-1β were up-regulated by LPS stimuli. IL-10, an anti-inflammatory cytokine, was also induced by LPS, but exhibited a different secretion pattern, peak time and maximal stimulation values. IL-1α and IL-15 showed donor-specific changes. Lastly, dexamethasone attenuated cytokine secretion in five independent repetitions, supporting the ability of the system to be used for drug screening. The collective results demonstrate that several cytokines can be used as valid inflammatory markers, regardless of changes in the secretion levels due to donor’s specific alterations.

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