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Introduction: Cortical reaction is a secretory process that occurs after a spermatozoon fuses with the oocyte, avoiding the fusion of additional sperm. During this exocytic event, the cortical granule membrane fuses with the oocyte plasma membrane. We have identified several molecular components involved in this process and confirmed that SNARE proteins regulate membrane fusion during cortical reaction in mouse oocytes. In those studies, we microinjected different nonpermeable reagents to demonstrate the participation of a specific protein in the cortical reaction. However, the microinjection technique has several limitations. In this work, we aimed to assess the potential of cell-penetrating peptides (CPP) as biotechnological tools for delivering molecules into oocytes, and to evaluate the functionality of the permeable tetanus toxin (bound to CPP sequence) during cortical reaction. Methods: Arginine-rich cell-penetrating peptides have demonstrated the optimal internalization of small molecules in mammalian cells. Two arginine-rich CPP were used in the present study. One, labeled with 5-carboxyfluorescein, to characterize the factors that can modulate its internalization, and the other, the permeable light chain of tetanus toxin, that cleaves the SNAREs VAMP1 and VAMP3 expressed in mouse oocytes. Results: Results showed that fluorescent CPP was internalized into the oocyte cytoplasm and that internalization was dependent on the concentration, time, temperature, and maturation stage of the oocyte. Using our functional assay to study cortical reaction, the light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibited cortical granules exocytosis. Discussion: Results obtained from the use of permeable peptides demonstrate that this CPP is a promising biotechnological tool to study functional macromolecules in mouse oocytes.
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Before fertilization, spermatozoa must undergo calcium-regulated acrosome exocytosis in response to physiological stimuli such as progesterone and zona pellucida. Our laboratory has elucidated the signaling cascades accomplished by different sphingolipids during human sperm acrosomal exocytosis. Recently, we established that ceramide increases intracellular calcium by activating various channels and stimulating the acrosome reaction. However, whether ceramide induces exocytosis on its own, activation of the ceramide kinase/ceramide 1-phosphate (CERK/C1P) pathway or both is still an unsolved issue. Here, we demonstrate that C1P addition induces exocytosis in intact, capacitated human sperm. Real-time imaging in single-cell and calcium measurements in sperm population showed that C1P needs extracellular calcium to induce [Ca2+]i increase. The sphingolipid triggered the cation influx through voltage-operated calcium (VOC) and store-operated calcium (SOC) channels. However, it requires calcium efflux from internal stores through inositol 3-phosphate receptors (IP3R) and ryanodine receptors (RyR) to achieve calcium rise and the acrosome reaction. We report the presence of the CERK in human spermatozoa, the enzyme that catalyzes C1P synthesis. Furthermore, CERK exhibited calcium-stimulated enzymatic activity during the acrosome reaction. Exocytosis assays using a CERK inhibitor demonstrated that ceramide induces acrosomal exocytosis, mainly due to C1P synthesis. Strikingly, progesterone required CERK activity to induce intracellular calcium increase and acrosome exocytosis. This is the first report, implicating the bioactive sphingolipid C1P in the physiological progesterone pathway leading to the sperm acrosome reaction.
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Human voltage-gated proton channels (hHv1) extrude protons from cells to compensate for charge and osmotic imbalances due metabolism, normalizing intracellular pH and regulating protein function. Human albumin (Alb), present at various levels throughout the body, regulates oncotic pressure and transports ligands. Here, we report Alb is required to activate hHv1 in sperm and neutrophils. Dose-response studies reveal the concentration of Alb in semen is too low to activate hHv1 in sperm whereas the higher level in uterine fluid yields proton efflux, allowing capacitation, the acrosomal reaction, and oocyte fertilization. Likewise, Alb activation of hHv1 in neutrophils is required to sustain production and release of reactive oxygen species during the immune respiratory burst. One Alb binds to both voltage sensor domains (VSDs) in hHv1, enhancing open probability and increasing proton current. A computational model of the Alb-hHv1 complex, validated by experiments, identifies two sites in Alb domain II that interact with the VSDs, suggesting an electrostatic gating modification mechanism favoring the active "up" sensor conformation. This report shows how sperm are triggered to fertilize, resolving how hHv1 opens at negative membrane potentials in sperm, and describes a role for Alb in physiology that will operate in the many tissues expressing hHv1.
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Albuminas/metabolismo , Mediadores da Inflamação/metabolismo , Canais Iônicos/metabolismo , Neutrófilos/metabolismo , Capacitação Espermática/fisiologia , Reação Acrossômica/fisiologia , Albuminas/química , Sequência de Aminoácidos , Fertilização/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/química , Canais Iônicos/genética , Masculino , Potenciais da Membrana/fisiologia , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Prótons , Sêmen/citologia , Sêmen/metabolismo , Homologia de Sequência de Aminoácidos , Espermatozoides/fisiologia , Eletricidade EstáticaRESUMO
Using a de novo peptide inhibitor, Corza6 (C6), we demonstrate that the human voltage-gated proton channel (hHv1) is the main pathway for H+ efflux that allows capacitation in sperm and permits sustained reactive oxygen species (ROS) production in white blood cells (WBCs). C6 was identified by a phage-display strategy whereby â¼1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) scaffold and sorting on purified hHv1 protein. Two C6 peptides bind to each dimeric channel, one on the S3-S4 loop of each voltage sensor domain (VSD). Binding is cooperative with an equilibrium affinity (Kd) of â¼1 nM at -50 mV. As expected for a VSD-directed toxin, C6 inhibits by shifting hHv1 activation to more positive voltages, slowing opening and speeding closure, effects that diminish with membrane depolarization.
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Canais Iônicos/fisiologia , Leucócitos/metabolismo , Capacitação Espermática/fisiologia , Reação Acrossômica/efeitos dos fármacos , Reação Acrossômica/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Células HEK293 , Humanos , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/genética , Masculino , Potenciais da Membrana , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Capacitação Espermática/efeitos dos fármacos , Toxinas Biológicas/química , Toxinas Biológicas/farmacologiaRESUMO
OBJECTIVE: To develop and evaluate a method to detect acrosome reaction (AR) in live human sperm. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Human semen samples with normal parameters obtained from healthy donors. INTERVENTION(S): Acrosome reaction assays. MAIN OUTCOME MEASURE(S): Fluorescence assessment of AR. RESULT(S): Evaluating acrosomal exocytosis in live human sperm is challenging. In this study, we report that in reacting sperm, Pisum sativum agglutinin conjugated to fluorescein isothiocyanate rapidly permeates into the acrosome when fusion pores open and stabilizes the acrosomal matrix, preventing the dispersal of the granule contents. CONCLUSION(S): Fluorescent Pisum sativum agglutinin can be used to visualize AR in real time, to determine the percentage of sperm undergoing exocytosis upon stimulation, and to separate the population of reacting sperm by flow cytometry.