RESUMEN
Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.
Asunto(s)
AMP Cíclico , Capacitación Espermática , Espermatozoides , Proteína que Contiene Valosina , Animales , Masculino , Capacitación Espermática/efectos de los fármacos , Proteína que Contiene Valosina/metabolismo , Proteína que Contiene Valosina/genética , Espermatozoides/metabolismo , Ratones , AMP Cíclico/metabolismo , Fosforilación , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMEN
The actin cytoskeleton reorganization during sperm capacitation is essential for the occurrence of acrosomal exocytosis (AR) in several mammalian species. Here, we demonstrate that in mouse sperm, within the first minutes of exposure upon capacitating conditions, the activity of RHOA/C and RAC1 is essential for LIMK1 and COFILIN phosphorylation. However, we observed that the signaling pathway involving RAC1 and PAK4 is the main player in controlling actin polymerization in the sperm head necessary for the occurrence of AR. Moreover, we show that the transient phosphorylation of COFILIN is also influenced by the Slingshot family of protein phosphatases (SSH1). The activity of SSH1 is regulated by the dual action of two pathways. On one hand, RHOA/C and RAC1 activity promotes SSH1 phosphorylation (inactivation). On the other hand, the activating dephosphorylation is driven by okadaic acid-sensitive phosphatases. This regulatory mechanism is independent of the commonly observed activating mechanisms involving PP2B and emerges as a new finely tuned modulation that is, so far, exclusively observed in mouse sperm. However, persistent phosphorylation of COFILIN by SSH1 inhibition or okadaic acid did not altered actin polymerization and the AR. Altogether, our results highlight the role of small GTPases in modulating actin dynamics required for AR.
Asunto(s)
Factores Despolimerizantes de la Actina , Capacitación Espermática , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Animales , Cofilina 1/metabolismo , Exocitosis , Masculino , Mamíferos/metabolismo , Ratones , Ácido Ocadaico/metabolismo , Ácido Ocadaico/farmacología , Fosforilación , Semen/metabolismoRESUMEN
Sperm capacitation is essential to gain fertilizing capacity. During this process, a series of biochemical and physiological modifications occur that allow sperm to undergo acrosomal exocytosis (AE). At the molecular level, hyperpolarization of the sperm membrane potential (Em) takes place during capacitation. This study shows that human sperm incubated under conditions that do not support capacitation (NC) can become ready for an agonist stimulated AE by pharmacologically inducing Em hyperpolarization with Valinomycin or Amiloride. To investigate how Em hyperpolarization promotes human sperm's ability to undergo AE, live single-cell imaging experiments were performed to simultaneously monitor changes in [Ca2+ ]i and the occurrence of AE. Em hyperpolarization turned [Ca2+ ]i dynamics in NC sperm from spontaneously oscillating into a sustained slow [Ca2+ ]i increase. The addition of progesterone (P4) or K+ to Valinomycin-treated sperm promoted that a significant number of cells displayed a transitory rise in [Ca2+ ]i which then underwent AE. Altogether, our results demonstrate that Em hyperpolarization is necessary and sufficient to prepare human sperm for the AE. Furthermore, this Em change decreased Ca2+ oscillations that block the occurrence of AE, providing strong experimental evidence of the molecular mechanism that drives the acquisition of acrosomal responsiveness.
Asunto(s)
Reacción Acrosómica , Señalización del Calcio , Exocitosis , Potenciales de la Membrana , Capacitación Espermática , Espermatozoides/fisiología , Humanos , Masculino , FosforilaciónRESUMEN
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.
Asunto(s)
Canales de Calcio/metabolismo , Espermatozoides/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/deficiencia , Canales de Calcio/genética , Señalización del Calcio , AMP Cíclico/metabolismo , Femenino , Fertilización In Vitro , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Transducción de Señal , Capacitación Espermática/fisiología , Motilidad Espermática/fisiología , Cola del Espermatozoide/metabolismo , Espermatozoides/efectos de los fármacos , Espermatozoides/ultraestructura , Proteína de Unión al GTP cdc42/antagonistas & inhibidoresRESUMEN
Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkalinization, beat frequency and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and human sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in women.
Asunto(s)
Inhibidores de Adenilato Ciclasa/farmacología , Fertilización/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Adenilil Ciclasas/genética , Adenilil Ciclasas/fisiología , Animales , Células Cultivadas , Femenino , Fertilización/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Embarazo , Espermatozoides/fisiologíaRESUMEN
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.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Acrosoma/metabolismo , Citoesqueleto de Actina/metabolismo , Espermatozoides/metabolismo , Animales , Exocitosis , Masculino , Ratones , Imagen MolecularRESUMEN
Acrosomal exocytosis (AR) is a critical process that sperm need to undergo to fertilize an egg. The evaluation of the presence or absence of the acrosome is usually performed by using lectins or dyes in fixed cells. With this approach, it is neither possible to monitor the dynamic process of exocytosis and related molecular events while discriminating between live and dead cells, nor to evaluate the acrosomal status while sperm reside in the female reproductive tract. However, over the last two decades, several new methodologies have been used to assess the occurrence of AR in living cells allowing different groups to obtain information that was not possible in the past. These techniques have revolutionized the whole study of this process. This review summarizes current methods available to analyze AR in living cells as well as the important information that emerged from studies using these approaches.
Asunto(s)
Reacción Acrosómica/fisiología , Acrosoma/metabolismo , Exocitosis/fisiología , Fertilización In Vitro/métodos , Capacitación Espermática/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Calcio/metabolismo , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Zona Pelúcida/metabolismoRESUMEN
Serratia marcescens is an opportunistic pathogen with increasing incidence in clinical settings. This is mainly attributed to the timely expression of a wide diversity of virulence factors and intrinsic and acquired resistance to antibiotics, including ß-lactams, aminoglycosides, quinolones, and polypeptides. For these reasons, S. marcescens has been recently categorised by the World Health Organization as one priority to strengthen efforts directed to develop new antibacterial agents. Therefore, it becomes critical to understand the underlying mechanisms that allow Serratia to succeed within the host. S. marcescens ShlA pore-forming toxin mediates phenotypes that alter homeostatic and signal transduction pathways of host cells. It has been previously demonstrated that ShlA provokes cytotoxicity, haemolysis and autophagy and also directs Serratia egress and dissemination from invaded nonphagocytic cells. However, molecular details of ShlA mechanism of action are still not fully elucidated. In this work, we demonstrate that Ni2+ selectively and reversibly blocks ShlA action, turning wild-type S. marcescens into a shlA mutant strain phenocopy. Combined use of Ni2+ and calcium chelators allow to discern ShlA-triggered phenotypes that require intracellular calcium mobilisation and reveal ShlA function as a calcium channel, providing new insights into ShlA mode of action on target cells.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Canales de Calcio/metabolismo , Proteínas Hemolisinas/antagonistas & inhibidores , Níquel/farmacología , Serratia marcescens/efectos de los fármacos , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/metabolismo , Células CHO , Calcio/metabolismo , Cricetulus , Eritrocitos/microbiología , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidad , Hemólisis/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Fenotipo , Serratia marcescens/metabolismo , Serratia marcescens/patogenicidadRESUMEN
Protein kinase A (PKA) is a broad-spectrum Ser/Thr kinase involved in the regulation of several cellular activities. Thus, its precise activation relies on being localized at specific subcellular places known as discrete PKA signalosomes. A-Kinase anchoring proteins (AKAPs) form scaffolding assemblies that play a pivotal role in PKA regulation by restricting its activity to specific microdomains. Because one of the first signaling events observed during mammalian sperm capacitation is PKA activation, understanding how PKA activity is restricted in space and time is crucial to decipher the critical steps of sperm capacitation. Here, we demonstrate that the anchoring of PKA to AKAP is not only necessary but also actively regulated during sperm capacitation. However, we find that once capacitated, the release of PKA from AKAP promotes a sudden Ca2+ influx through the sperm-specific Ca2+ channel CatSper, starting a tail-to-head Ca2+ propagation that triggers the acrosome reaction. Three-dimensional super-resolution imaging confirmed a redistribution of PKA within the flagellar structure throughout the capacitation process, which depends on anchoring to AKAP. These results represent a new signaling event that involves CatSper Ca2+ channels in the acrosome reaction, sensitive to PKA stimulation upon release from AKAP.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Reacción Acrosómica , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Mapas de Interacción de Proteínas , Capacitación Espermática , Espermatozoides/citología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Exocitosis , Fertilización , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Espermatozoides/metabolismoRESUMEN
To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3--dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (cystic fibrosis transmembrane conductance regulator channel) activity and for the modulation of membrane potential (Em). However, the main HCO3- transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na+/HCO3- cotransporter (NBC) and epithelial Na+ channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3- influx, resulting in lower PKA activity, and that events downstream of the cAMP activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3- also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation.
Asunto(s)
Bicarbonatos/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Canales Epiteliales de Sodio/metabolismo , Potenciales de la Membrana , Capacitación Espermática , Espermatozoides/fisiología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Masculino , Fosforilación , Transducción de Señal , Sodio/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismoRESUMEN
Mammalian sperm must undergo a functionally defined process called capacitation to be able to fertilize oocytes. They become capacitated in vivo by interacting with the female reproductive tract or in vitro in a defined capacitation medium that contains bovine serum albumin, calcium (Ca2+ ), and bicarbonate (HCO3- ). In this work, sperm were double stained with propidium iodide and the Ca2+ dye Fluo-4 AM and analyzed by flow cytometry to determine changes in intracellular Ca2+ concentration ([Ca2+ ]i ) in individual live sperm. An increase in [Ca2+ ]i was observed in a subpopulation of capacitated live sperm when compared with noncapacitated ones. Sperm exposed to the capacitating medium displayed a rapid increase in [Ca2+ ]i within 1 min of incubation, which remained sustained for 90 min. These rise in [Ca2+ ]i after 90 min of incubation in the capacitating medium was evidenced by an increase in the normalized median fluorescence intensity. This increase was dependent on the presence of extracellular Ca2+ and, at least in part, reflected the contribution of a new subpopulation of sperm with higher [Ca2+ ]i . In addition, it was determined that the capacitation-associated [Ca2+ ]i increase was dependent of CatSper channels, as sperm derived from CatSper knockout (CatSper KO) or incubated in the presence of CatSper inhibitors failed to increase [Ca2+ ]i . Surprisingly, a minimum increase in [Ca2+ ]i was also observed in CatSper KO sperm suggesting the existence of other Ca2+ transport systems. Altogether, these results indicate that a subpopulation of sperm increases [Ca2+ ]i very rapidly during capacitation mainly due to a CatSper-mediated influx of extracellular Ca2+ .
Asunto(s)
Canales de Calcio/genética , Calcio/farmacología , Capacitación Espermática/genética , Espermatozoides/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Femenino , Citometría de Flujo , Técnicas de Inactivación de Genes , Genitales Femeninos/metabolismo , Genitales Femeninos/fisiología , Humanos , Masculino , Ratones , Ratones Noqueados , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/crecimiento & desarrolloRESUMEN
Several pathogens co-opt host intracellular compartments to survive and replicate, and they thereafter disperse progeny to prosper in a new niche. Little is known about strategies displayed by Serratia marcescens to defeat immune responses and disseminate afterwards. Upon invasion of nonphagocytic cells, Serratia multiplies within autophagosome-like vacuoles. These Serratia-containing vacuoles (SeCV) circumvent progression into acidic/degradative compartments, avoiding elimination. In this work, we show that ShlA pore-forming toxin (PFT) commands Serratia escape from invaded cells. While ShlA-dependent, Ca2+ local increase was shown in SeCVs tight proximity, intracellular Ca2+ sequestration prevented Serratia exit. Accordingly, a Ca2+ surge rescued a ShlA-deficient strain exit capacity, demonstrating that Ca2+ mobilization is essential for egress. As opposed to wild-type-SeCV, the mutant strain-vacuole was wrapped by actin filaments, showing that ShlA expression rearranges host actin. Moreover, alteration of actin polymerization hindered wild-type Serratia escape, while increased intracellular Ca2+ reorganized the mutant strain-SeCV actin distribution, restoring wild-type-SeCV phenotype. Our results demonstrate that, by ShlA expression, Serratia triggers a Ca2+ signal that reshapes cytoskeleton dynamics and ends up pushing the SeCV load out of the cell, in an exocytic-like process. These results disclose that PFTs can be engaged in allowing bacteria to exit without compromising host cell integrity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Exocitosis , Proteínas Hemolisinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Serratia marcescens/fisiología , Vacuolas/microbiología , Animales , Células CHO , Calcio/metabolismo , Señalización del Calcio , Cationes Bivalentes/metabolismo , Cricetinae , Cricetulus , Citoesqueleto/metabolismo , Serratia marcescens/metabolismoRESUMEN
Recent evidence demonstrated that most fertilizing mouse sperm undergo acrosomal exocytosis (AE) before binding to the zona pellucida of the eggs. However, the sites where fertilizing sperm could initiate AE and what stimuli trigger it remain unknown. Therefore, the aim of this study was to determine physiological sites of AE by using double transgenic mouse sperm, which carried EGFP in the acrosome and DsRed2 fluorescence in mitochondria. Using live imaging of sperm during in vitro fertilization of cumulus-oocyte complexes, it was observed that most sperm did not undergo AE. Thus, the occurrence of AE within the female reproductive tract was evaluated in the physiological context where this process occurs. Most sperm in the lower segments of the oviduct were acrosome-intact; however, a significant number of sperm that reached the upper isthmus had undergone AE. In the ampulla, only 5% of the sperm were acrosome-intact. These results support our previous observations that most of mouse sperm do not initiate AE close to or on the ZP, and further demonstrate that a significant proportion of sperm initiate AE in the upper segments of the oviductal isthmus.
Asunto(s)
Reacción Acrosómica , Células del Cúmulo/citología , Exocitosis , Oviductos/fisiología , Espermatozoides/fisiología , Acrosoma/metabolismo , Animales , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oocitos/citología , Capacitación Espermática/fisiología , Interacciones Espermatozoide-Óvulo , Zona Pelúcida/metabolismoRESUMEN
Mammalian sperm must acquire their fertilizing ability after a series of biochemical modifications in the female reproductive tract collectively called capacitation to undergo acrosomal exocytosis, a process that is essential for fertilization. Actin dynamics play a central role in controlling the process of exocytosis in somatic cells as well as in sperm from several mammalian species. In somatic cells, small GTPases of the Rho family are widely known as master regulators of actin dynamics. However, the role of these proteins in sperm has not been studied in detail. In the present work we characterized the participation of small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during mouse sperm capacitation. We observed that most of the proteins of this signaling cascade and their effector proteins are expressed in mouse sperm. The activation of the signaling pathways of cAMP/PKA, RhoA/C and Rac1 is essential for LIMK1 activation by phosphorylation on Threonine 508. Serine 3 of Cofilin is phosphorylated by LIMK1 during capacitation in a transiently manner. Inhibition of LIMK1 by specific inhibitors (BMS-3) resulted in lower levels of actin polymerization during capacitation and a dramatic decrease in the percentage of sperm that undergo acrosomal exocytosis. Thus, we demonstrated for the first time that the master regulators of actin dynamics in somatic cells are present and active in mouse sperm. Combining the results of our present study with other results from the literature, we have proposed a working model regarding how LIMK1 and Cofilin control acrosomal exocytosis in mouse sperm.
Asunto(s)
Reacción Acrosómica/fisiología , Cofilina 1/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Exocitosis , Quinasas Lim/metabolismo , Capacitación Espermática/fisiología , Actinas/metabolismo , Animales , Cruzamientos Genéticos , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Fosforilación , Transducción de Señal , Espermatozoides/metabolismoRESUMEN
Plasma membrane hyperpolarization is crucial for mammalian sperm to acquire acrosomal responsiveness during capacitation. Among the signaling events leading to mammalian sperm capacitation, the immediate activation of protein kinase A plays a pivotal role, promoting the subsequent stimulation of protein tyrosine phosphorylation that associates with fertilizing capacity. We have shown previously that mice deficient in the tyrosine kinase cSrc are infertile and exhibit improper cauda epididymis development. It is therefore not clear whether lack of sperm functionality is due to problems in epididymal maturation or to the absence of cSrc in sperm. To further address this problem, we investigated the kinetics of cSrc activation using anti-Tyr(P)-416-cSrc antibodies that only recognize active cSrc. Our results provide evidence that cSrc is activated downstream of PKA and that inhibition of its activity blocks the capacitation-induced hyperpolarization of the sperm plasma membrane without blocking the increase in tyrosine phosphorylation that accompanies capacitation. In addition, we show that cSrc inhibition also blocks the agonist-induced acrosome reaction and that this inhibition is overcome by pharmacological hyperpolarization. Considering that capacitation-induced hyperpolarization is mediated by SLO3, we evaluated the action of cSrc inhibitors on the heterologously expressed SLO3 channel. Our results indicate that, similar to SLO1 K(+) channels, cSrc blockers significantly decreased SLO3-mediated currents. Together, these results are consistent with findings showing that hyperpolarization of the sperm plasma membrane is necessary and sufficient to prepare the sperm for the acrosome reaction and suggest that changes in sperm membrane potential are mediated by cSrc activation.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Potenciales de la Membrana/genética , Familia-src Quinasas/metabolismo , Acrosoma/metabolismo , Animales , Membrana Celular/genética , Polaridad Celular/genética , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Masculino , Ratones , Transducción de Señal/genética , Capacitación Espermática/genética , Espermatozoides/metabolismo , Familia-src Quinasas/genéticaRESUMEN
Mammalian sperm require to undergo an exocytotic process called acrosomal exocytosis in order to be able to fuse with the oocyte. This ability is acquired during the course of sperm capacitation. This review is focused on one aspect related to this acquisition: the role of the actin cytoskeleton. Evidence from different laboratories indicates that actin polymerization occurs during capacitation, and the detection of several actin-related proteins suggests that the cytoskeleton is involved in important sperm functions. In other mammalian cells, the cortical actin network acts as a dominant negative clamp which blocks constitutive exocytosis but, at the same time, is necessary to prepare the cell to undergo regulated exocytosis. Thus, F-actin stabilizes structures generated by exocytosis and supports the physiological progression of this process. Is this also the case in mammalian sperm? This review summarizes what is currently known about actin and its related proteins in the male gamete, with particular emphasis on their role in acrosomal exocytosis.
Asunto(s)
Reacción Acrosómica/genética , Acrosoma/metabolismo , Citoesqueleto de Actina/genética , Actinas/genética , Exocitosis/genética , Capacitación Espermática/genética , Acrosoma/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Actinas/química , Actinas/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Humanos , Quinasas Lim/genética , Quinasas Lim/metabolismo , Masculino , Ratones , Fosfolipasa D/genética , Fosfolipasa D/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Transducción de SeñalRESUMEN
Physiological changes that endow mammalian sperm with fertilizing capacity are known as sperm capacitation. As part of capacitation, sperm develop an asymmetrical flagellar beating known as hyperactivation and acquire the ability to undergo the acrosome reaction. Together, these processes promote fertilizing competence in sperm. At the molecular level, capacitation involves a series of signal transduction events which include activation of cAMP-dependent phosphorylation pathways, removal of cholesterol, hyperpolarization of the sperm plasma membrane, and changes in ion permeability. In recent years, new technologies have aided in the study of sperm signaling molecules with better resolution, at both spatial and temporal levels, unraveling how different cascades integrate and cooperate to render a fertilizing sperm. Despite this new information, the molecular mechanisms connecting capacitation with acrosomal exocytosis and hyperactivation are not well understood. This review brings together results obtained in mammalian species in the field of sperm capacitation with special focus on those pathways involved in the preparation to undergo the acrosomal reaction.
Asunto(s)
Reacción Acrosómica/fisiología , Membrana Celular/metabolismo , Capacitación Espermática/fisiología , Espermatozoides/fisiología , Animales , Permeabilidad de la Membrana Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Transporte Iónico , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Masculino , Mamíferos , Fosforilación , Transducción de Señal , Espermatozoides/citologíaRESUMEN
Ca(2+) 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 Ca(2+) increase required for the acrosome reaction and hyperactivation of spermatozoa under normal in vitro conditions.
Asunto(s)
Calcimicina/farmacología , Fertilización/efectos de los fármacos , Capacitación Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Animales , Western Blotting , Calcio/metabolismo , AMP Cíclico/metabolismo , Femenino , Masculino , Ratones , Fosforilación , Embarazo , Resultado del Embarazo , Transducción de Señal/fisiología , Espermatozoides/fisiologíaRESUMEN
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.
Asunto(s)
Adenilil Ciclasas/fisiología , AMP Cíclico/fisiología , Espermatozoides/fisiología , Adenilil Ciclasas/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Hidrólisis , Masculino , Transducción de Señal , Espermatozoides/enzimologíaRESUMEN
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 Ca(2+), and an increase in tyrosine phosphorylation. How these signaling systems interact during capacitation is not well understood. Results presented in this study indicate that Ca(2+) ions have a biphasic role in the regulation of cAMP-dependent signaling. Media without added Ca(2+) salts (nominal zero Ca(2+)) 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 Ca(2+). However, chelation of the extracellular Ca(2+) traces by EGTA induced both cAMP-dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca(2+) media was mimicked by two calmodulin antagonists, W7 and calmidazolium, and by the calcineurin inhibitor cyclosporine A. These results suggest that Ca(2+) 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 Ca(2+) media. Therefore, sperm lacking Catsper Ca(2+) channels behave as wild-type sperm incubated in the presence of EGTA. This latter result suggests that Catsper transports the Ca(2+) involved in the regulation of cAMP-dependent and tyrosine phosphorylation pathways required for sperm capacitation.