10 research outputs found
Fibronectin modulates the endocannabinoid system through the cAMP/PKA pathway during human sperm capacitation
Fibronectin (Fn) enhances human sperm capacitation via the cAMP/PKA pathway, and the endocannabinoid system participates in this process. Moreover, Fn has been linked to endocannabinoid system components in different cellular models, even though no evidence of such interactions in human sperm is available. Normal semen samples were evaluated over a 4-year period. Our findings suggest that (a) the capacitating effects of Fn were reversed by preincubating the sperm with a cannabinoid receptor 1 (CB1) or transient receptor potential cation channel subfamily V member 1 (TRPV1) antagonist (p < 0.001 and p < 0.05, respectively); (b) cooperation between CB1 and TRPV1 may exist (p < 0.01); (c) the activity of specific fatty acid amide hydroxylase (FAAH) decreased after 1 min (p < 0.01) and increased after 60 min (p < 0.01) of capacitation in the presence of Fn; (d) the effects of Fn on FAAH activity were prevented by preincubating spermatozoa with a protein kinase A (PKA) inhibitor (p < 0.01); (e) Fn modulated both the cyclic adenosine monophosphate concentration and PKA activity (p < 0.05) during early capacitation; and (f) FAAH was a PKA substrate modulated by phosphorylation. These findings indicate that Fn stimulates human sperm capacitation via the cAMP/PKA pathway through modulation of the endocannabinoid system. Understanding the functional competence of human spermatozoa is essential for facilitating clinical advances in infertility treatment and for developing novel contraceptive strategies.Fil: Martínez León, Eduardo Antonio. Universidad de Antofagasta; Chile. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Osycka Salut, Claudia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Signorelli, Janetti. Universidad de Antofagasta; ChileFil: Kong, Milene. Universidad de Antofagasta; ChileFil: Morales, Patricio. Universidad de Antofagasta; ChileFil: Perez Martinez, Silvina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Díaz, Emilce Silvina. Universidad de Antofagasta; Chil
Contribution to van der Waerden's conjecture
AbstractIn this paper, we give two different elementary proofs for the inequality which states that the permanent of doubly stochastic matrices is greater than or equal to (n!/nn). This inequality was proved earlier by the author, and independently by Egorychev and Falikman
Protein Kinase A (PRKA) Activity Is Regulated by the Proteasome at the Onset of Human Sperm Capacitation
The proteasome increases its activity at the onset of sperm capacitation due to the action of the SACY/PRKACA pathway; this increase is required for capacitation to progress. PRKA activity also increases and remains high during capacitation. However, intracellular levels of cAMP decrease in this process. Our goal was to evaluate the role of the proteasome in regulating PRKA activity once capacitation has started. Viable human sperm were incubated in the presence and absence of epoxomicin or with 0.1% DMSO. The activity of PRKA; the phosphorylation pattern of PRKA substrates (pPRKAs); and the expression of PRKAR1, PRKAR2, and AKAP3 were evaluated by Western blot. The localization of pPRKAs, PRKAR1, PRKAR2, and AKAP3 was evaluated by immunofluorescence. Treatment with epoxomicin changed the localization and phosphorylation pattern and decreased the percentage of pPRKAs-positive sperm. PRKA activity significantly increased at 1 min of capacitation and remained high throughout the incubation. However, epoxomicin treatment significantly decreased PRKA activity after 30 min. In addition, PRKAR1 and AKAP3 were degraded by the proteasome but with a different temporal kinetic. Our results suggest that PRKAR1 is the target of PRKA regulation by the proteasome
Gonadotropin-Releasing Hormone-Stimulated Sperm Binding to the Human Zona Is Mediated by a Calcium Influx1
Thy-1-mediated cell-cell contact induces astrocyte migration through the engagement of αVβ3 integrin and syndecan-4.
Cell adhesion to the extracellular matrix proteins occurs through interactions with integrins that bind to Arg-Gly-Asp (RGD) tripeptides, and syndecan-4, which recognizes the heparin-binding domain of other proteins. Both receptors trigger signaling pathways, including those that activate RhoGTPases such as RhoA and Rac1. This sequence of events modulates cell adhesion to the ECM and cell migration. Using a neuron-astrocyte model, we have reported that the neuronal protein Thy-1 engages αVβ3 integrin and syndecan-4 to induce RhoA activation and strong astrocyte adhesion to their underlying substrate. Thus, because cell-cell interactions and strong cell attachment to the matrix are considered antagonistic to cell migration, we hypothesized that Thy-1 stimulation of astrocytes should preclude cell migration. Here, we studied the effect of Thy-1 expressing neurons on astrocyte polarization and migration using a wound-healing assay and immunofluorescence analysis. Signaling molecules involved were studied by affinity precipitation, western blotting and the usage of specific antibodies. Intriguingly, Thy-1 interaction with its two receptors was found to increase astrocyte polarization and migration. The latter events required interactions of these receptors with both the RGD-like sequence and the heparin-binding domain of Thy-1. Additionally, prolonged Thy-1-receptor interactions inhibited RhoA activation while activating FAK, PI3K and Rac1. Therefore, sustained engagement of integrin and syndecan-4 with the neuronal surface protein Thy-1 induces astrocyte migration. Interestingly we identify here, a cell-cell interaction that despite initially inducing strong cell attachment, favors cell migration upon persistent stimulation by engaging the same signaling receptors and molecules as those utilized by the extracellular matrix proteins to stimulate cell movement
Additional file 4: Figure S4. of αVβ3 Integrin regulates astrocyte reactivity
Representative western blot of the markers of astrocyte reactivity GFAP and iNOS from non-transgenic and hSOD1G93A-derived astrocytes. β-actin is a loading control. (PDF 166 kb
Additional file 2: Figure S2. of αVβ3 Integrin regulates astrocyte reactivity
Representative western blot of β3 Integrin from rat astrocytes treated with Il-6, IL+db cAMP, IL-1β, IL-1β+db cAMP, or IFN-γ. (PDF 77 kb
Additional file 3: Figure S3. of αVβ3 Integrin regulates astrocyte reactivity
β3 Integrin over expression permits Thy-1-induced signaling. A) Quantification of intracellular calcium levels in rat primary astrocytes transfected with pEGFP-β3 Integrin or pEGFP and stimulated with Thy-1-Fc. Trail-R2-Fc was used as a negative control. Cells were starved for 30 min. The arrow indicates the addition of Thy-1. B) Extracellular ATP measurement of primary astrocytes transfected with pEGFP-β3 Integrin or pEGFP, and treated with Trail-R2-Fc or Thy-1-Fc for 10 min. Cells were previously starved for 30 min. Values shown in the graph are the mean ± s.e.m. from three independent experiments. *p < 0.05. (PDF 174 kb