Central role of soluble adenylyl cyclase and cAMP in sperm physiology

Cyclic adenosine 3′,5′-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cyclases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.Fil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Wertheimer Hermitte, Eva Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Visconti, Pablo E.. University Of Massachussets; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario; Argentin

Male Decapacitation Factor SPINK3 Blocks Membrane Hyperpolarization and Calcium Entry in Mouse Sperm

Mammalian sperm acquire ability to fertilize through a process called capacitation, occurring after ejaculation and regulated by both female molecules and male decapacitation factors. Bicarbonate and calcium present in the female reproductive tract trigger capacitation in sperm, leading to acrosomal responsiveness and hyperactivated motility. Male decapacitating factors present in the semen avert premature capacitation, until detached from the sperm surface. However, their mechanism of action remains elusive. Here we describe for the first time the molecular basis for the decapacitating action of the seminal protein SPINK3 in mouse sperm. When present in the capacitating medium, SPINK3 inhibited Src kinase, a modulator of the potassium channel responsible for plasma membrane hyperpolarization. Lack of hyperpolarization affected calcium channels activity, impairing the acquisition of acrosomal responsiveness and blocking hyperactivation. Interestingly, SPINK3 acted only on non-capacitated sperm, as it did not bind to capacitated cells. Binding selectivity allows its decapacitating action only in non-capacitated sperm, without affecting capacitated cells.Fil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Stival, Cintia Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Nicolli, Anabella Rita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: de Blas, Gerardo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Escuela Superior de Medicina.; Argentin

Determination of a Robust Assay for Human Sperm Membrane Potential Analysis

Mammalian sperm must undergo a complex process called capacitation in order to fertilize the egg. During this process, hyperpolarization of the sperm plasma membrane has been mostly studied in mouse, and associated to its importance in the preparation to undergo the acrosome reaction (AR). However, despite the increasing evidence of membrane hyperpolarization in human sperm capacitation, no reliable techniques have been set up for its determination. In this report we describe human sperm membrane potential (Em) measurements by a fluorimetric population assay, establishing optimal conditions for Em determination. In addition, we have conducted parallel measurements of the same human sperm samples by flow cytometry and population fluorimetry, before and after capacitation, to conclusively address their reliability. This integrative analysis sets the basis for the study of Em in human sperm allowing future work aiming to understand its role in human sperm capacitation.Fil: Baró Graf, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Ritagliati, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Stival, Cintia Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Balestrini, Paula Ania. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentin

Determination of a Robust Assay for Human Sperm Membrane Potential Analysis

Mammalian sperm must undergo a complex process called capacitation in order to fertilize the egg. During this process, hyperpolarization of the sperm plasma membrane has been mostly studied in mouse, and associated to its importance in the preparation to undergo the acrosome reaction (AR). However, despite the increasing evidence of membrane hyperpolarization in human sperm capacitation, no reliable techniques have been set up for its determination. In this report we describe human sperm membrane potential (Em) measurements by a fluorimetric population assay, establishing optimal conditions for Em determination. In addition, we have conducted parallel measurements of the same human sperm samples by flow cytometry and population fluorimetry, before and after capacitation, to conclusively address their reliability. This integrative analysis sets the basis for the study of Em in human sperm allowing future work aiming to understand its role in human sperm capacitation.Fil: Baró Graf, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; ArgentinaFil: Ritagliati, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Stival, Cintia Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Balestrini, Paula Ania. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentin

Ca2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways

Ca2+ ionophore A23187 is known to induce the acrosome reaction of mammalian spermatozoa, but it also quickly immobilizes them. Although mouse spermatozoa were immobilized by this ionophore, they initiated vigorous motility (hyperactivation) soon after this reagent was washed away by centrifugation. About half of live spermatozoa were acrosome-reacted at the end of 10 min of ionophore treatment; fertilization of cumulus-intact oocytes began as soon as spermatozoa recovered their motility and before the increase in protein tyrosine phosphorylation, which started 30-45 min after washing out the ionophore. When spermatozoa were treated with A23187, more than 95% of oocytes were fertilized in the constant presence of the protein kinase A inhibitor, H89. Ionophore-treated spermatozoa also fertilized 80% of oocytes, even in the absence of HCO3-, a component essential for cAMP synthesis under normal in vitro conditions. Under these conditions, fertilized oocytes developed into normal offspring. These data indicate that mouse spermatozoa treated with ionophore are able to fertilize without activation of the cAMP/PKA signaling pathway. Furthermore, they suggest that the cAMP/PKA pathway is upstream of an intracellular Ca2+ increase required for the acrosome reaction and hyperactivation of spermatozoa under normal in vitro conditions.Fil: Tateno, Hiroyuki. Asahikawa Medical University. Department of Biological Sciences; JapónFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Hino,Toshiaki. Asahikawa Medical University. Department of Biological Sciences; JapónFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autonoma de Mexico. Instituto de Biotecnologia; MéxicoFil: Darszon, Alberto. Universidad Nacional Autonoma de Mexico. Instituto de Biotecnologia; MéxicoFil: Yanagimachi, Ryuzo. University of Hawaii Medical School. Institute for Biogenesis Research; Estados UnidosFil: Visconti, Pablo E.. University Of Massachussets; Estados Unido

Functional human sperm capacitation requires both bicarbonate dependent-PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases

In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/PKA pathway. Recent studies in mice revealed however that a Src Family Kinase (SFK) induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (p<0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6 h-incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster eggs. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analogue and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (p<0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm were exposed to SKI606 and OA. Interestingly, different concentrations of inhibitors were required to modulate human and mouse capacitation revealing the species-specificity of the molecular mechanisms underlying this process. In conclusion, our results describe for the first time the involvement of both PKA activation and Ser/Thr phosphatase down-regulation in functional human sperm capacitation and provide convincing evidence that early PKA-dependent phosphorylation is the convergent regulatory point between these two signaling pathways.Fil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas -conicet. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas -conicet. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Salicioni, A.. University Of Massachussets; Estados UnidosFil: Navarrete, F.. University Of Massachussets; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario; ArgentinaFil: Visconti, P. E.. University Of Massachussets;Fil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas -conicet. Instituto de Biología y Medicina Experimental (i); Argentin

Lysophosphatidylcholine Drives Neuroblast Cell Fate

Neuronal differentiation plays a key role during embryogenesis. However, based on the capacity of neuronal stem cells to either generate or regenerate neurons and because differentiation stops aberrant neuroblasts proliferation, neuronal differentiation is crucial during neuropathological conditions. Although phosphatidylcholine (PtdCho) has been proposed as an important molecule for neurite growth and neuronal regeneration, the identity of the molecular target has remained elusive. This study originally describes that lysophosphatidylcholine (LPtdCho), either exogenously supplied or generated by the imbalance of PtdCho metabolism through the enzymatic action of cytosolic phospholipase A2, acts as a neurotrophic-like factor. We demonstrated that LPtdCho induces neuronal differentiation by activation of the small G protein Ras followed by the Raf/MEK/ERK signaling pathway. Accordingly, LPtdCho redirects neuroblasts gene expression leading to the generation of functional mature neurons expressing βIII-tubulin and having increased acetylcholinesterase activity and membrane biosynthesis required for neuritogenesis. These findings provide mechanistic details of the role of cytidine-5-diphosphocholine (CDP-choline) and PtdCho as neuroprotectors. Furthermore, as LPtdCho recapitulates the effect of the therapeutic agent retinoic acid, these results open new avenues for drug discovery for the treatment of neuropathological conditions.Fil: Paoletti, Luciana Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Domizi, Pablo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Marcucci, Hebe Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Montaner, Aneley Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Salvador, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Banchio, Claudia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Cdc42 localized in the CatSper signaling complex regulates cAMP‐dependent pathways in mouse sperm

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+-dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Xu, Xinran. State University of Colorado - Fort Collins; Estados UnidosFil: Romarowski, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. State University of Colorado - Fort Collins; Estados UnidosFil: Gervasi, María G.. University of Massachussets; Estados UnidosFil: Orta, Gerardo. Universidad Autonoma de México. Instituto de Biotecnología; MéxicoFil: De la Vega Beltrán, José L.. Universidad Autonoma de México. Instituto de Biotecnología; MéxicoFil: Stival, Cintia Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gilio, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: D'alotto Moreno, Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Visconti, Pablo E.. University of Massachussets; Estados UnidosFil: Krapf, Diego. State University of Colorado - Fort Collins; Estados UnidosFil: Darszon, Alberto. Universidad Autonoma de México. Instituto de Biotecnología; MéxicoFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Biphasic Role of Calcium in Mouse Sperm Capacitation Signaling Pathways

Mammalian sperm acquire fertilizing ability in the female tract in a process known as capacitation. At the molecular level, capacitation is associated with up-regulation of a cAMP-dependent pathway, changes in intracellular pH, intracellular Ca2+, and an increase in tyrosine phosphorylation. How these signaling systems interact during capacitation is not well understood. Results presented in this study indicate that Ca2+ ions have a biphasic role in the regulation of cAMP-dependent signaling. Media without added Ca2+ salts (nominal zero Ca2+) still contain micromolar concentrations of this ion. Sperm incubated in this medium did not undergo PKA activation or the increase in tyrosine phosphorylation suggesting that these phosphorylation pathways require Ca2+. However, chelation of the extracellular Ca2+ traces by EGTA induced both cAMP-dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca2+ media was mimicked by two calmodulin antagonists, W7 and calmidazolium, and by the calcineurin inhibitor cyclosporine A. These results suggest that Ca2+ ions regulate sperm cAMP and tyrosine phosphorylation pathways in a biphasic manner and that some of its effects are mediated by calmodulin. Interestingly, contrary to wild-type mouse sperm, sperm from CatSper1 KO mice underwent PKA activation and an increase in tyrosine phosphorylation upon incubation in nominal zero Ca2+ media. Therefore, sperm lacking Catsper Ca2+ channels behave as wild-type sperm incubated in the presence of EGTA. This latter result suggests that Catsper transports the Ca2+ involved in the regulation of cAMP-dependent and tyrosine phosphorylation pathways required for sperm capacitation.Fil: Navarrete, Felipe A.. University of Massachusetts; Estados UnidosFil: García Vázquez, Francisco A.. University of Massachusetts; Estados Unidos. Universidad de Murcia; EspañaFil: Alvau, Antonio. University of Massachusetts; Estados UnidosFil: Escoffier, Jessica. University of Massachusetts; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autónoma de México; MéxicoFil: Salicioni, Ana M.. University of Massachusetts; Estados UnidosFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Visconti, Pablo E.. University of Massachusetts; Estados Unido

Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis

Filamentous actin (F-actin) is a key factor in exocytosis in many cell types. In mammalian sperm, acrosomal exocytosis (denoted the acrosome reaction or AR), a special type of controlled secretion, is regulated by multiple signaling pathways and the actin cytoskeleton. However, the dynamic changes of the actin cytoskeleton in live sperm are largely not understood. Here, we used the powerful properties of SiR-actin to examine actin dynamics in live mouse sperm at the onset of the AR. By using a combination of super-resolution microscopy techniques to image sperm loaded with SiR-actin or sperm from transgenic mice containing Lifeact-EGFP, six regions containing F-actin within the sperm head were revealed. The proportion of sperm possessing these structures changed upon capacitation. By performing live-cell imaging experiments, we report that dynamic changes of F-actin during the AR occur in specific regions of the sperm head. While certain F-actin regions undergo depolymerization prior to the initiation of the AR, others remain unaltered or are lost after exocytosis occurs. Our work emphasizes the utility of live-cell nanoscopy, which will undoubtedly impact the search for mechanisms that underlie basic sperm functions.Fil: Romarowski, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Velasco Félix, Ángel G.. Universidad Nacional Autónoma de México; MéxicoFil: Rodriguez, Paulina Torres. Universidad Nacional Autónoma de México; MéxicoFil: Gervasi, Mar?á G.. University Of Massachusetts Amherst;Fil: Xu, Xinran. School Of Biomedical Engineering;Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Contreras-Jiménez, Gastón. Universidad Nacional Autónoma de México; MéxicoFil: Sánchez-Cárdenas, Claudia. Universidad Nacional Autónoma de México; MéxicoFil: Ramírez-Gómez, Héctor V.. Universidad Nacional Autónoma de México; MéxicoFil: Krapf, Diego. School Of Biomedical Engineering;Fil: Visconti, Pablo E.. University Of Massachusetts Amherst;Fil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Guerrero, Adán. Universidad Nacional Autónoma de México; MéxicoFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin