scholarly journals The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 118
Author(s):  
Tamara Muñoz-Caro ◽  
Amanda J. Gibson ◽  
Iván Conejeros ◽  
Dirk Werling ◽  
Anja Taubert ◽  
...  
Keyword(s):  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Polymorphonuclear Neutrophils ◽  
Neutrophil Extracellular Trap ◽  
Luciferase Reporter ◽  
Tlr4 Expression ◽  
Immunofluorescence Analysis ◽  
Hek Cells ◽  
Eimeria Bovis

Background: Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like receptors (TLRs) TLR2 and TLR4 and have been shown to generate Neutrophil extracellular traps (NETs) upon exposure to Eimeria bovis. The present work investigated the role of TLR2 and TLR4 in the recognition and uptake of E. bovis sporozoites, IL-8 production and neutrophil extracellular trap (NET) formation. Methods: TLR expression was performed by flow cytometric analysis on PMN exposed to live carboxyfluorescein succinimidyl ester (CFSE)-stained sporozoites. Supernatants of PMN exposed to different E. bovis sporozoite preparations and antigens in the absence or presence of TLR antibodies were assessed for IL-8 secretion. Cells were exposed to sporozoite preparations and assessed for the activation of transcription factor NF-κB using a luciferase reporter assay. Immunofluorescence analysis was done to investigate TLR2 and TLR4 surface expression and NET formation on bovine PMN exposed to vital sporozoites. Results: we observed significantly increased TLR2 and TLR4 expression with a mean increase in expression that was greater for TLR2 than TLR4. This upregulation neither inhibited nor promoted sporozoite phagocytosis by bovine PMN. Live sporozoites together with anti-TLR2 mAb resulted in a significant enhancement of IL-8 production. NF-κB activation was more strongly induced in TLR2-HEK cells than in TLR4/MD2-HEK cells exposed to heat-killed sporozoites and antigens. Immunofluorescence analysis showed TLR-positive signals on the surface of PMN and concomitant NET formation. Conclusions: This is the first report on E. bovis-induced concomitant TLR2 and TLR4 expression during bovine PMN-derived NETosis.

Autocrine regulation of interleukin-8 production in human monocytes

2000 ◽  
Vol 279 (6) ◽  
pp. L1129-L1136 ◽  
Author(s):  
Darren D. Browning ◽  
Wade C. Diehl ◽  
Matthew H. Hsu ◽  
Ingrid U. Schraufstatter ◽  
Richard D. Ye
Keyword(s):  
Mononuclear Cells ◽  
De Novo ◽  
Human Peripheral Blood ◽  
Surface Expression ◽  
Polymorphonuclear Neutrophils ◽  
Mitogen Activated Protein Kinase ◽  
Cell Surface Expression ◽  
Peripheral Blood Mononuclear ◽  
Mitogen Activated Protein

Interleukin (IL)-8 is a C-X-C chemokine that plays an important role in acute inflammation through its G protein-coupled receptors CXCR1 and CXCR2. In this study, we investigated the role of IL-8 as an autocrine regulator of IL-8 production and the signaling mechanisms involved in human peripheral blood mononuclear cells (MNCs). Sepharose-immobilized IL-8 stimulated a sevenfold increase in IL-8 production within 2 h. IL-8 induced the expression of its own message, and IL-8 biosynthesis was inhibited by cycloheximide and actinomycin D, indicating de novo RNA and protein synthesis. In contrast to MNCs, polymorphonuclear neutrophils did not respond to the immobilized IL-8 with IL-8 production despite cell surface expression of CXCR1 and CXCR2. Melanoma growth-stimulatory activity/growth-related protein-α (MGSA/GROα), which binds CXCR2 but not CXCR1, was unable to either stimulate IL-8 secretion in MNCs or desensitize these cells to respond to immobilized IL-8. The involvement of mitogen-activated protein kinase (MAPK) in IL-8-induced IL-8 biosynthesis was suggested by the ability of PD-98059, an inhibitor of MAPK kinase, to block this function. Furthermore, IL-8 induced a significant increase in extracellular signal-regulated kinase 2 phosphorylation, whereas MGSA/GROα was much less effective. These findings support the role of IL-8 as an autocrine regulator of IL-8 production and suggest that this function is mediated by CXCR1 through activation of MAPK.

Oral and Blood Neutrophil Activation States during Experimental Gingivitis

2017 ◽  
Vol 3 (1) ◽  
pp. 65-75 ◽  
Author(s):  
N.C. Wellappuli ◽  
N. Fine ◽  
H.P. Lawrence ◽  
M. Goldberg ◽  
H.C. Tenenbaum ◽  
...  
Keyword(s):  
At Risk ◽  
Early Stage ◽  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Polymorphonuclear Neutrophils ◽  
Induction Phase ◽  
Clinical Parameters ◽  
Activation Markers ◽  
Hygiene Practices ◽  
Experimental Gingivitis

Polymorphonuclear neutrophils (PMNs) are the primary leukocytes present in the healthy and inflamed oral cavity. While unique PMN activation states have been shown to differentiate health and periodontitis, little is known about the changes in PMN activation states that occur during the transition from periodontal health to gingivitis. The objective of this study was to characterize oral and circulatory PMNs during induction and resolution of experimental gingivitis. Healthy volunteers were recruited to undergo experimental gingivitis. Clinical assessment of pocket depths, bleeding on probing, gingival index, and plaque index, as well as flow cytometric analysis of CD (cluster of differentiation) activation markers on blood and oral PMNs, was performed weekly. All clinical parameters increased significantly during the induction period and returned to baseline levels during the resolution phase. During the induction phase, while oral PMN counts increased, oral PMN activation state based on surface expression of CD63, CD11b, CD16, and CD14 was diminished compared to those seen in health and during the resolution phase. PMNs in circulation during onset showed increased activation based on CD55, CD63, CD11b, and CD66a. Using clinical parameters and oral PMN counts assessed at day 21, we noted 2 unique disease patterns where one-third of subjects displayed an exaggerated influx of oral PMNs with severe inflammation compared to the majority of the population who experienced a moderate level of inflammation and PMN influx. This supports the notion that PMN influx and severe inflammatory changes during gingivitis could identify subjects at risk for the development of severe gingival inflammation and progression toward destructive periodontitis. This study demonstrates that oral PMN activation states are reduced in gingivitis and suggest that only in periodontitis do PMNs become hyperactivated and tissue damaging. Knowledge Transfer Statement: Our article creates a paradigm for future studies of the evolution of essential oral and circulatory biomarkers to identify individuals at risk to develop periodontitis at an early stage of periodontal disease, which is reversible upon proper oral hygiene practices and dental treatments.

Impaired TLR4 and HIF expression in cystic fibrosis bronchial epithelial cells downregulates hemeoxygenase-1 and alters iron homeostasis in vitro

2014 ◽  
Vol 307 (10) ◽  
pp. L791-L799 ◽  
Author(s):  
Shashi Chillappagari ◽  
Shalini Venkatesan ◽  
Virajith Garapati ◽  
Poornima Mahavadi ◽  
Antje Munder ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Iron Homeostasis ◽  
Hypoxia Inducible Factor ◽  
Immunohistochemical Analysis ◽  
Surface Expression ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Intracellular Iron

Hemeoxygenase-1 (HO-1), an inducible heat shock protein, is upregulated in response to multiple cellular insults via oxidative stress, lipopolysaccharides (LPS), and hypoxia. In this study, we investigated in vitro the role of Toll-like receptor 4 (TLR4), hypoxia-inducible factor 1α (HIF-1α), and iron on HO-1 expression in cystic fibrosis (CF). Immunohistochemical analysis of TLR4, HO-1, ferritin, and HIF-1α were performed on lung sections of CFTR−/− and wild-type mice. CFBE41o- and 16HBE14o- cell lines were employed for in vitro analysis via immunoblotting, immunofluorescence, real-time PCR, luciferase reporter gene analysis, and iron quantification. We observed a reduced TLR4, HIF-1α, HO-1, and ferritin in CFBE41o- cell line and CF mice. Knockdown studies using TLR4-siRNA in 16HBE14o- revealed significant decrease of HO-1, confirming the role of TLR4 in HO-1 downregulation. Inhibition of HO-1 using tin protoporphyrin in 16HBE14o- cells resulted in increased iron levels, suggesting a probable role of HO-1 in iron accumulation. Additionally, sequestration of excess iron using iron chelators resulted in increased hypoxia response element response in CFBE41o- and 16HBE14o-, implicating a role of iron in HIF-1α stabilization and HO-1. To conclude, our in vitro results demonstrate that multiple regulatory factors, such as impaired TLR4 surface expression, increased intracellular iron, and decreased HIF-1α, downregulate HO-1 expression in CFBE41o- cells.

scholarly journals miR-27a-3p attenuates induce autophagy-related cell death by suppressing Inhibitor of growth family 5 in cholangiocarcinoma

2020 ◽  
Author(s):  
Ming WAN ◽  
Fu-min Zhang ◽  
Peng-cheng Kang ◽  
Xing-ming Jiang ◽  
yunfu cui
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Immunofluorescence Analysis ◽  
Ki 67 ◽  
Western Blot Assay ◽  
Qrt Pcr ◽  
Related Cell ◽  
The Relationship

Abstract Background MicroRNAs (miRNAs) are abnormally expressed in human tumors, including cholangiocarcinoma (CCA). miR-27a-3p was observed up-regulated in CCA, but its functions in CCA are largely unknown.Methods CCK8 assay, Colony formation assays and Ki-67 staining was employed to detect the cell growth. The autophagy and proliferation relative-protein analyzed by western blot. The immunofluorescence staining was applied to analyze the expression level of LC3 I/II. Tumor xenografts was used to test the role of miR-27a-3p. Luciferase reporter assay, western bolt and qRT-PCR showed the relationship between miR-27a-3p and ING5.Results miR-27a-3p expression was increased in human CCA tissues. Inhibition of miR-27a-3p suppressed the proliferative capacity of CCA cells, silencing of miR-27a-3p dramatically induced cell death and suppressed tumor growth in vivo. The proteins, such as Beclin-1, p62, p21, p-p53, CDK4 and CDK6, were decreased upon miR-27a-3p inhibitor transfection. Western blot assay and immunofluorescence analysis were showed the induced-autophagy after transfecting with miR-27a-3p or inhibitor of growth family 5 (ING5) in RBE. ING5 as a direct miR-27a-3p target in CCA. Co-transfect of miR-27a-3p and ING5 can reverse CCA cell death which induced by miR-27a-3p inhibitor alone.Conclusions miR-27a-3p promotes oncogenesis of CCA by triggering autophagy-related cell death by interacting with ING5 directly.

Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange

Blood ◽  
2002 ◽  
Vol 100 (7) ◽  
pp. 2472-2478 ◽  
Author(s):  
Judith Lahav ◽  
Kerstin Jurk ◽  
Oded Hess ◽  
Michael J. Barnes ◽  
Richard W. Farndale ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Disulfide Exchange ◽  
Fibrinogen Receptor ◽  
Flow Cytometric ◽  
Fibrinogen Binding ◽  
Protein Disulfide ◽  
Mediated Reduction

Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor αIIbβ3 is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified αIIbβ3, specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in αIIbβ3 ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to αIIbβ3 as well as ligation-induced allosteric changes in the conformation of αIIbβ3. We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiol-independent initial ligation followed by a thiol-dependent stabilization of binding. Flow cytometric analysis showed that sustained binding of fibrinogen, as well as expression of ligand-induced binding site epitopes and ligand-bound conformation, depended on free thiols and disulfide exchange. Expression of P-selectin was minimally affected, even with complete inhibition of αIIbβ3function. These data indicate that although agonist-induced platelet stimulation is independent of ecto-sulfhydryls, engagement of integrin αIIbβ3 on the intact platelet depends totally on their enzymatically catalyzed surface expression.

scholarly journals Estrogen Promotes Pituitary Prolactinoma by Upregulating TLR4 / NF-κB/p38MAPK Pathway

2021 ◽  
Author(s):  
Yu Zhang ◽  
Qiaoyan Ding ◽  
Shuman Wang ◽  
Qi Wu ◽  
Ping Ni ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
In Vitro Study ◽  
Tlr4 Expression ◽  
Immunofluorescence Analysis ◽  
Immunohistochemistry Analysis ◽  
P38mapk Pathway ◽  
Interfering Rna ◽  
Harmful Effects

Abstract Background: Prolactinomas have harmful effects on human health, and the pathogenesis is still unknown. Furthermore, the morbidity of women is much more than man, maybe related with estradiol level. Thus, it is important to reveal the pathogenesis and develop new therapeutic methods for prolactinomas.Methods: Immunofluorescence analysis or Immunohistochemistry analysis were performed on the ERβ, TLR4 and prolactin (PRL) expressions of pituitary gland in C57BL/6 mice and human prolactinoma specimen. In the present study, the role of TLR4 in prolactinoma was determined using estradiol-induced mice models in C57BL/6 wild-type (WT) and TLR4−/− mice. MMQ cells were treated with estradiol, fulvestrant, LPS or transfected with different TLR4 small interfering RNA, which to study ERβ, TLR4 and PRL expression in MMQ cells. Co‑immunoprecipitates analysis was used to investigate the interaction between ERβ and TLR4.Results: Immunofluorescence analysis or Immunohistochemistry analysis showed that PRL and TLR4 expression were co-located and increased in the pituitary gland of mice and human prolactinoma specimen compared with the control specimen. It was shown that PRL and TLR4 expression was co-located and increased significantly in the pituitary gland of estradiol-injected prolactinoma mice compared with the control mice. Knockout of TLR4 significantly inhibited tumor overgrowth, and PRL expression was decreased in estradiol-induced mice through regulating TLR4/NF‑κB/p38MAPK pathway. Estradiol promoted PRL expression through regulating TLR4/NF‑κB/p38MAPK pathway in vitro study, and pre-Inhibiting ERβ or TLR4 reverse the effect, while simultaneously activating ERβ and TLR4 enhanced PRL expression than activating single ERβ or TLR4. Furthermore, ERβ co-immunoprecipitates with endogenous TLR4 was assessed by co-immunoprecipitation analysis.Conclusions: These results suggest that estradiol promoted prolactinoma development by activating the TLR4/NF‑κB/ p38MAPK pathway through Erβ and TLR4 knockout inhibited the proliferation and secretion of prolactin in prolactinoma.

Role of BAFF in Adhesion and Growth of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 627-627
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Rory Coffey ◽  
Iris Breitkreutz ◽  
Laurence Catley Laurence Catley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Luciferase Activity ◽  
Flow Cytometric Analysis ◽  
Gene Promoter ◽  
Bone Marrow Microenvironment ◽  
Luciferase Reporter ◽  
Dependent Manner

Abstract Recent studies have underscored the role of B-cell activating factor (BAFF), a member of the tumor necrosis factor superfamily, in promoting the survival of malignant B cells, including human MM. Since the tumor bone marrow microenvironment plays a crucial role in MM cell growth and survival, we here characterize the functional significance of BAFF in the interaction between MM and bone marrow stromal cells (BMSCs), and further defined molecular mechanisms regulating these processes. We first confirmed the expression of BAFF and its receptors (BCMA, TACI, BAFF-R) on majority of MM cell lines and CD138-purified patient MM cells. Significantly, gene expression profiling revealed increased BCMA expression on newly diagnosed and relapsed MM versus normal plasma cells (p<0.0001, student T test, Abstract # 552872). Although BAFF and its 3 receptors are expressed on CD138+patient MM cells (n=10) by RT-PCR analysis, the pattern of expression of TACI and BAFF-R receptors was heterogeneous, assessed by flow cytometric analysis. We next examined BAFF expression in BMSC lines (n=4) and BMSCs derived from patient BM (n=5). Baff expression is easily detected in BMSCs, and Baff levels are approximately 3–10-fold higher in supernatants of BMSCs than equivalent numbers of MM cells. Thus BMSCs are the main source of BAFF in MM patients. We then asked whether MM adhesion to BMSCs further upregulates BAFF secretion from BMSCs. Adhesion of 5 MM cell lines to BMSCs augments BAFF secretion by 2–5 fold, using BAFF ELISA. Immunoblotting using anti-BAFF Ab confirmed markedly higher expression of BAFF in BMSCs than MCCAR MM cells, as well as greater BAFF upregulation induced by MM adhesion. Since NF-kappaB (NF-κB) is crucial for MM adhesion-induced cytokine secretion from BMSCs and the BAFF gene promoter contains at least six NF-κB-binding sites, we next transfected KM104 BMSC line with a luciferase reporter vector carrying the BAFF gene promoter (BAFF-LUC) and then allowed MM cells to adhere to BMSCs for 24 hr, followed by measurement of luciferase activity. NF-κB-LUC reporter was used as positive and pGL3 plasmid as a negative control. Adhesion of MCCAR and MM1S MM lines to KM103 BMSC line with BAFF-LUC and NF-κB-LUC, but not control reporters, significantly enhanced luciferase activity, suggesting that NF-κB activation induced by MM adhesion to BMSCs mediates BAFF upregulation. In parallel, we also asked whether BAFF induces MM adhesion to BMSCs. BAFF (0–100 ng/ml) increases adhesion of 5 MM lines to BMSCs in a dose-dependent manner; conversely, TACI-Ig or blocking anti-BCMA Ab inhibited BAFF stimulation, indicating increased adhesion specific triggered by BAFF. Using adenoviruses expressing dominant-negative and constitutively expressed AKT as well as IkappaB kinase (IKK) inhibitor (PS-1145), we further showed that BAFF-induced MM cell adhesion is primarily mediated via activation of AKT and NF-κB signaling. Finally, BAFF significantly increased adhesion of CD138-expressing patient MM cells to BMSCs. These studies suggest a role for BAFF in localization and growth of MM cells in the BM microenvironment and strongly support novel important therapeutics targeting the interaction between BAFF and its receptors in human MM.

The Signaling Pathways in Nitric Oxide Production by Neutrophils Exposed to N-nitrosodimethylamine

2018 ◽  
Vol 16 (2) ◽  
pp. 194-199
Author(s):  
Wioletta Ratajczak-Wrona ◽  
Ewa Jablonska
Keyword(s):  
Nitric Oxide ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Polymorphonuclear Neutrophils ◽  
Microbial Pathogens ◽  
Human Neutrophils ◽  
No Production ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Background: Polymorphonuclear neutrophils (PMNs) play a crucial role in the innate immune system’s response to microbial pathogens through the release of reactive nitrogen species, including Nitric Oxide (NO). </P><P> Methods: In neutrophils, NO is produced by the inducible Nitric Oxide Synthase (iNOS), which is regulated by various signaling pathways and transcription factors. N-nitrosodimethylamine (NDMA), a potential human carcinogen, affects immune cells. NDMA plays a major part in the growing incidence of cancers. Thanks to the increasing knowledge on the toxicological role of NDMA, the environmental factors that condition the exposure to this compound, especially its precursors- nitrates arouse wide concern. Results: In this article, we present a detailed summary of the molecular mechanisms of NDMA’s effect on the iNOS-dependent NO production in human neutrophils. Conclusion: This research contributes to a more complete understanding of the mechanisms that explain the changes that occur during nonspecific cellular responses to NDMA toxicity.

Exosomal miR-214-5p Released from Glioblastoma Cells Modulates Inflammatory Response of Microglia after Lipopolysaccharide Stimulation through Targeting CXCR5

2019 ◽  
Vol 18 (1) ◽  
pp. 78-87 ◽  
Author(s):  
Jian-kai Yang ◽  
Hong-jiang Liu ◽  
Yuanyu Wang ◽  
Chen Li ◽  
Ji-peng Yang ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Critical Role ◽  
Luciferase Reporter ◽  
Clinical Prognosis ◽  
Direct Target ◽  
And Migration ◽  
High Level ◽  
Cxcr5 Expression ◽  
Proliferation And Migration

Background and Objective: Exosomes communicate inter-cellularly and miRNAs play critical roles in this scenario. MiR-214-5p was implicated in multiple tumors with diverse functions uncovered. However, whether miR-214-5p is mechanistically involved in glioblastoma, especially via exosomal pathway, is still elusive. Here we sought to comprehensively address the critical role of exosomal miR-214-5p in glioblastoma (GBM) microenvironment.Methods:The relative expression of miR-214-5p was determined by real-time PCR. Cell viability and migration were measured by MTT and transwell chamber assays, respectively. The secretory cytokines were measured with ELISA kits. The regulatory effect of miR-214-5p on CXCR5 expression was interrogated by luciferase reporter assay. Protein level was analyzed by Western blot.Results:We demonstrated that miR-214-5p was aberrantly overexpressed in GBM and associated with poorer clinical prognosis. High level of miR-214-5p significantly contributed to cell proliferation and migration. GBM-derived exosomal miR-214-5p promoted inflammatory response in primary microglia upon lipopolysaccharide challenge. We further identified CXCR5 as the direct target of miR-214- 5p in this setting.Conclusion:Overexpression of miR-214-5p in GBM modulated the inflammatory response in microglia via exosomal transfer.

scholarly journals Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species

Reproduction ◽  
2013 ◽  
Vol 146 (2) ◽  
pp. 119-133 ◽  
Author(s):  
Barbara Ambruosi ◽  
Gianluca Accogli ◽  
Cécile Douet ◽  
Sylvie Canepa ◽  
Géraldine Pascal ◽  
...  
Keyword(s):  
Brain Tumor ◽  
Western Blot ◽  
Immunohistochemical Analysis ◽  
Fertilization Rate ◽  
Malignant Brain Tumor ◽  
Estrus Cycle ◽  
Immunofluorescence Analysis ◽  
Oviductal Fluid ◽  
Porcine Spermatozoa

Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus–oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.

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