Sperm function in vitro and fertility after antibiotic-free, hypothermic storage of liquid preserved boar semen

Abstract The role of antibiotics (AB) in semen extenders as a potential contribution to the global antimicrobial resistance threat is emerging. Here, we establish an AB-free hypothermic preservation strategy for boar semen and investigate its impact on sperm function, microbial load and fertility after artificial insemination (AI). Spermatozoa (12 boars) preserved in AB-free AndroStar Premium extender at 5 °C maintained high motility, membrane integrity, and a low DNA-fragmentation index throughout 72 h storage and results did not significantly differ from controls stored at 17 °C in extender containing AB (p = 0.072). Likewise, kinetic response of spermatoza to the capacitation stimulus bicarbonate during 180 min incubation in Tyrode’s medium did not differ from 17 °C-controls. In a competitive sperm oviduct binding assay, binding indices did not differ between semen stored for 72 h AB-free at 5 °C and 17 °C-controls (n = 6 boars). Bacterial load < 103 CFU/ml after 72 h was measured in 88.9% of samples stored at 5 °C AB-free compared to 97.2% in 17 °C-controls (n = 36 semen pools, 23 boars). Fertility traits of 817 females did not differ significantly between the two semen groups (p > 0.05). In conclusion, a hypothermic semen preservation strategy is presented which offers antibiotic-free storage of boar semen doses.

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Abstract WMP31: Dedifferentiation of Smooth Muscle Cells and its Potential Contribution to Pathogenesis of Intracranial Aneurysms

Stroke ◽

10.1161/str.51.suppl_1.wmp31 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Mieko Oka ◽

Nobuhiko Ohno ◽

Takakazu Kawamata ◽

Tomohiro Aoki

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Inflammatory Responses ◽

Muscle Cells ◽

Inflammatory Factors ◽

Potential Contribution ◽

Electron Microscopic

Introduction: Intracranial aneurysm (IA) affects 1 to 5 % in general public and becomes the primary cause of subarachnoid hemorrhage, the most severe form of stroke. However, currently, no drug therapy is available for IAs to prevent progression and rupture of lesions. Elucidation of mechanisms underlying the disease is thus mandatory. Considering the important role of vascular smooth muscle cells (SMCs) in the maintenance of stiffness of arterial walls and also in the pathogenesis of atherosclerosis via mediating inflammatory responses, we in the present study analyzed morphological or phenotypical changes of SMCs during the disease development in the lesions. Methods: We subjected rats to an IA model in which lesions are induced by increase of hemodynamic force loading on intracranial arterial bifurcations and performed histopathological analyses of induced lesions including the electron microscopic examination. We then immunostained specimens from induced lesions to explore factors responsible for dedifferentiation or migration of SMCs. In vitro study was also done to examine effect of some candidate factors on dedifferentiation or migration of cultured SMCs. Results: We first found the accumulation of SMCs underneath the endothelial cell layer mainly at the neck portion of the lesion. These cells was positive for the embryonic form of myosin heavy chain, a marker for the dedifferentiated SMCs, and the expression of pro-inflammatory factors like TNF-α. In immunostaining to explore the potential factor regulating the dedifferentiation of SMCs, we found that Platelet-derived growth factor-BB (PDGF-BB) was expressed in endothelial cells at the neck portion of IA walls. Consistently, recombinant PDGF-BB could promote the dedifferentiate of SMCs and chemo-attracted them in in vitro. Finally, in the stenosis model of the carotid artery, PDGF-BB expression was induced in endothelial cells in which high wall shear stress was loaded and the dedifferentiation of SMCs occurred there. Conclusions: The findings from the present study imply the role of dedifferentiated SMCs partially recruited by PDGF-BB from endothelial cells in the formation of inflammatory microenvironment at the neck portion of IA walls, leading to the progression of the disease.

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The Periplasmic α-Carbonic Anhydrase Activity of Helicobacter pylori Is Essential for Acid Acclimation

Journal of Bacteriology ◽

10.1128/jb.187.2.729-738.2005 ◽

2005 ◽

Vol 187 (2) ◽

pp. 729-738 ◽

Cited By ~ 124

Author(s):

Elizabeth A. Marcus ◽

Amiel P. Moshfegh ◽

George Sachs ◽

David R. Scott

Keyword(s):

Helicobacter Pylori ◽

Membrane Potential ◽

Carbonic Anhydrase ◽

Membrane Integrity ◽

Carbonic Anhydrase Activity ◽

Knockout Mutant ◽

Inner Membrane ◽

Acid Acclimation

ABSTRACT The role of the periplasmic α-carbonic anhydrase (α-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since α-CA catalyzes the conversion of CO2 to HCO3 −, the role of CO2 in periplasmic buffering was studied using an α-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that α-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the α-CA knockout. In the mutant or in the presence of acetazolamide, there was an ∼3 log10 decrease in acid survival. In acid, absence of α-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the α-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of α-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH3 efflux from the cytoplasm but also on the conversion of CO2, produced by urease, to HCO3 − by the periplasmic α-CA.

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

Reproduction ◽

10.1530/rep.1.00814 ◽

2006 ◽

Vol 131 (2) ◽

pp. 311-318 ◽

Cited By ~ 28

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.

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Liver X Receptor Activation Impairs Neutrophil Functions and Aggravates Sepsis

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz635 ◽

2019 ◽

Vol 221 (9) ◽

pp. 1542-1553 ◽

Cited By ~ 4

Author(s):

Fabrício O Souto ◽

Fernanda V S Castanheira ◽

Silvia C Trevelin ◽

Braulio H F Lima ◽

Guilherme Cesar Martelossi Cebinelli ◽

...

Keyword(s):

Multiorgan Failure ◽

Bacterial Load ◽

Cecal Ligation ◽

Neutrophil Infiltration ◽

Receptor Activation ◽

Messenger Ribonucleic Acid ◽

X Receptors ◽

Lxr Agonists

Abstract Background Liver X receptors (LXRs) are nuclear receptors activated by oxidized lipids and were previously implicated in several metabolic development and inflammatory disorders. Although neutrophils express both LXR-α and LXR-β, the consequences of their activation, particularly during sepsis, remain unknown. Methods We used the model of cecal ligation and puncture (CLP) to investigate the role of LXR activation during sepsis. Results In this study, we verified that LXR activation reduces neutrophil chemotactic and killing abilities in vitro. Mice treated with LXR agonists showed higher sepsis-induced mortality, which could be associated with reduced neutrophil infiltration at the infectious foci, increased bacteremia, systemic inflammatory response, and multiorgan failure. In contrast, septic mice treated with LXR antagonist showed increased number of neutrophils in the peritoneal cavity, reduced bacterial load, and multiorgan dysfunction. More important, neutrophils from septic patients showed increased ABCA1 messenger ribonucleic acid levels (a marker of LXR activation) and impaired chemotactic response toward CXCL8 compared with cells from healthy individuals. Conclusions Therefore, our findings suggest that LXR activation impairs neutrophil functions, which might contribute to poor sepsis outcome.

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Role of ascorbic acid in promoting follicle integrity and survival in intact mouse ovarian follicles in vitro

Reproduction ◽

10.1530/rep.0.1210089 ◽

2001 ◽

pp. 89-96 ◽

Cited By ~ 61

Author(s):

AA Murray ◽

MD Molinek ◽

SJ Baker ◽

FN Kojima ◽

MF Smith ◽

...

Keyword(s):

Ascorbic Acid ◽

Basement Membrane ◽

Growth And Development ◽

Culture Media ◽

Membrane Integrity ◽

Tissue Inhibitor Of Metalloproteinase ◽

Follicular Growth ◽

Intact Mouse

Ascorbic acid has three known functions: it is necessary for collagen synthesis, promotes steroidogenesis and acts as an antioxidant. Within the ovary, most studies have concentrated on the role of ascorbic acid in luteal formation and regression and little is known about the function of this vitamin in follicular growth and development. Follicular growth and development were investigated in this study using an individual follicle culture system that allows the growth of follicles from the late preantral stage to Graafian morphology. Follicles were isolated from prepubertal mice and cultured for 6 days. Control media contained serum and human recombinant FSH. Further groups of follicles were cultured in the same media but with the addition of ascorbic acid at concentrations of either 28 or 280 micromol l(-1). Addition of ascorbic acid at the higher concentration significantly increased the percentage of follicles that maintained basement membrane integrity throughout culture (P < 0.001). Ascorbic acid had no effect on the growth of the follicles or on oestradiol production. Metalloproteinase 2 activity tended to increase at the higher concentration of ascorbic acid and there was a significant concomitant increase in the activity of tissue inhibitor of metalloproteinase 1 (P < 0.01). Follicles cultured without the addition of serum but with FSH and selenium in the culture media underwent apoptosis. Addition of ascorbic acid to follicles cultured under serum-free conditions significantly reduced apoptosis (P < 0.05). From these data it is concluded that ascorbic acid is necessary for remodelling the basement membrane during follicular growth and that the ability of follicles to uptake ascorbic acid confers an advantage in terms of granulosa cell survival.

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Membrane lysis during biological membrane fusion: collateral damage by misregulated fusion machines

The Journal of Cell Biology ◽

10.1083/jcb.200805182 ◽

2008 ◽

Vol 183 (2) ◽

pp. 181-186 ◽

Cited By ~ 34

Author(s):

Alex Engel ◽

Peter Walter

Keyword(s):

Membrane Fusion ◽

Biological Membrane ◽

Membrane Integrity ◽

Fusion Pore ◽

Mechanistic Models ◽

Collateral Damage ◽

Vacuole Fusion ◽

Membrane Lysis

In the canonical model of membrane fusion, the integrity of the fusing membranes is never compromised, preserving the identity of fusing compartments. However, recent molecular simulations provided evidence for a pathway to fusion in which holes in the membrane evolve into a fusion pore. Additionally, two biological membrane fusion models—yeast cell mating and in vitro vacuole fusion—have shown that modifying the composition or altering the relative expression levels of membrane fusion complexes can result in membrane lysis. The convergence of these findings showing membrane integrity loss during biological membrane fusion suggests new mechanistic models for membrane fusion and the role of membrane fusion complexes.

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

Nanomedicine ◽

10.2217/nnm-2020-0056 ◽

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.

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Otitis Media and Nasopharyngeal Colonization in ccl3−/− Mice

Infection and Immunity ◽

10.1128/iai.00148-17 ◽

2017 ◽

Vol 85 (11) ◽

Cited By ~ 6

Author(s):

Dominik Deniffel ◽

Brian Nuyen ◽

Kwang Pak ◽

Keigo Suzukawa ◽

Jun Hung ◽

...

Keyword(s):

Otitis Media ◽

Respiratory Infections ◽

Expression Profiles ◽

Bacterial Load ◽

Content Type ◽

Cell Recruitment ◽

Chemokine Ligand ◽

Infection And Inflammation

ABSTRACT We previously found CC chemokine ligand 3 (CCL3) to be a potent effector of inflammation during otitis media (OM): exogenous CCL3 rescues the OM phenotype of tumor necrosis factor-deficient mice and the function of macrophages deficient in several innate immune molecules. To further delineate the role of CCL3 in OM, we evaluated middle ear (ME) responses of ccl3 −/−mice to nontypeable Haemophilus influenzae (NTHi). CCL chemokine gene expression was evaluated in wild-type (WT) mice during the complete course of acute OM. OM was induced in ccl3 −/− and WT mice, and infection and inflammation were monitored for 21 days. Phagocytosis and killing of NTHi by macrophages were evaluated by an in vitro assay. The nasopharyngeal bacterial load was assessed in naive animals of both strains. Many CCL genes showed increased expression levels during acute OM, with CCL3 being the most upregulated, at levels 600-fold higher than the baseline. ccl3 −/− deletion compromised ME bacterial clearance and prolonged mucosal hyperplasia. ME recruitment of leukocytes was delayed but persisted far longer than in WT mice. These events were linked to a decrease in the macrophage capacity for NTHi phagocytosis and increased nasopharyngeal bacterial loads in ccl3 −/− mice. The generalized impairment in inflammatory cell recruitment was associated with compensatory changes in the expression profiles of CCL2, CCL7, and CCL12. CCL3 plays a significant role in the clearance of infection and resolution of inflammation and contributes to mucosal host defense of the nasopharyngeal niche, a reservoir for ME and upper respiratory infections. Therapies based on CCL3 could prove useful in treating or preventing persistent disease.

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Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7

Reproduction ◽

10.1530/rep-13-0191 ◽

2014 ◽

Vol 148 (2) ◽

pp. 221-235 ◽

Cited By ~ 48

Author(s):

Juan M Gallardo Bolaños ◽

Carolina M Balao da Silva ◽

Patricia Martín Muñoz ◽

Antolín Morillo Rodríguez ◽

María Plaza Dávila ◽

...

Keyword(s):

Critical Role ◽

Membrane Integrity ◽

Specific Inhibitor ◽

Akt Inhibitor ◽

Akt Phosphorylation ◽

Mitochondrial Superoxide ◽

Phosphorylated Akt ◽

Sperm Survival

AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers–Whitten–Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, with inhibition reaching a maximum after 1 h of incubation. This decrease in phosphorylation was attributable to either dephosphorylation or suppression of the active phosphorylation pathway. Stallion spermatozoa spontaneously dephosphorylated during in vitro incubation, resulting in a lack of a difference in AKT phosphorylation between the SH5-treated sperm and the control after 4 h of incubation. AKT inhibition decreased the proportion of motile spermatozoa (total and progressive) and the sperm velocity. Similarly, AKT inhibition reduced membrane integrity, leading to increased membrane permeability and reduced the mitochondrial membrane potential concomitantly with activation of caspases 3 and 7. However, the percentage of spermatozoa exhibiting oxidative stress, the production of mitochondrial superoxide radicals, DNA oxidation and DNA fragmentation were not affected by AKT inhibition. It is concluded that AKT maintains the membrane integrity of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7, which prevents the progression of spermatozoa to an incomplete form of apoptosis.Free Spanish abstractA Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/148/2/221/suppl/DC1.

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Deactivation of the JNK Pathway by GSTP1 Is Essential to Maintain Sperm Functionality

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.627140 ◽

2021 ◽

Vol 9 ◽

Author(s):

Marc Llavanera ◽

Yentel Mateo-Otero ◽

Ariadna Delgado-Bermúdez ◽

Sandra Recuero ◽

Samuel Olives ◽

...

Keyword(s):

Membrane Integrity ◽

Mitochondrial Activity ◽

Computer Assisted ◽

Sperm Function ◽

Jnk Pathway ◽

Male Subfertility ◽

Mammalian Sperm ◽

Genetic Abnormalities ◽

Protein Dysfunction

Fifty percent of male subfertility diagnosis is idiopathic and is usually associated with genetic abnormalities or protein dysfunction, which are not detectable through the conventional spermiogram. Glutathione S-transferases (GSTs) are antioxidant enzymes essential for preserving sperm function and maintaining fertilizing ability. However, while the role of GSTP1 in cell signaling regulation via the inhibition of c-Jun N-terminal kinases (JNK) has been enlightened in somatic cells, it has never been investigated in mammalian spermatozoa. In this regard, a comprehensive approach through immunoblotting, immunofluorescence, computer-assisted sperm assessment (CASA), and flow cytometry analysis was used to characterize the molecular role of the GSTP1–JNK heterocomplex in sperm physiology, using the pig as a model. Immunological assessments confirmed the presence and localization of GSTP1 in sperm cells. The pharmacological dissociation of the GSTP1–JNK heterocomplex resulted in the activation of JNK, which led to a significant decrease in sperm viability, motility, mitochondrial activity, and plasma membrane stability, as well as to an increase of intracellular superoxides. No effects in intracellular calcium levels and acrosome membrane integrity were observed. In conclusion, the present work has demonstrated, for the first time, the essential role of GSTP1 in deactivating JNK, which is crucial to maintain sperm function and has also set the grounds to understand the relevance of the GSTP1–JNK heterocomplex for the regulation of mammalian sperm physiology.

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