RESUMEN
Sperm chemotaxis may facilitate the finding of the oocyte. Only capacitated spermatozoa can orient their movement by chemotaxis, which as well as capacitation, is regulated in part by the cAMP-PKA pathway. Reactive oxygen species (ROS) are produced during sperm capacitation which is closely related to chemotaxis. Then, the ROS participation in the chemotactic signaling can be expected. Here we studied the role of ROS in the chemotaxis signaling of equine spermatozoa which produce high quantities of ROS because of their energy metabolism. The level of capacitated and chemotactic spermatozoa was increased with 0.1 and 0.2 mM hydrogen peroxide (H2O2), which was involved in the chemotactic signaling. By combining a concentration gradient of H2O2 with inhibitors/chelators of some of the signaling pathway elements, we showed that the activation of NOX (membrane NADPH oxidase) increases the intracellular ROS which activate the chemotaxis AMPc-PKA pathway. Our results provide evidence about the participation of ROS in the chemotactic signaling mediated by progesterone (P).
Asunto(s)
Quimiotaxis , Caballos/metabolismo , Especies Reactivas de Oxígeno , Capacitación Espermática , Espermatozoides/metabolismo , Animales , MasculinoRESUMEN
The acrosome reaction is the regulated exocytosis of mammalian sperm's single secretory granule, essential for fertilization. It relies on small GTPases, the cAMP binding protein Epac, and the SNARE complex, among other components. Here, we describe a novel tool to investigate Rab27-related signaling pathways: a hybrid recombinant protein consisting of human Rab27A fused to TAT, a cell penetrating peptide. With this tool, we aimed to unravel the connection between Rab3, Rab27 and Rap1 in sperm exocytosis and to deepen our understanding about how isoprenylation and guanine nucleotides influence the behaviour of Rab27 in exocytosis. Our results show that TAT-Rab27A-GTP-γ-S permeated into live sperm and triggered acrosomal exocytosis per se when geraylgeranylated but inhibited it when not lipid-modified. Likewise, an impermeant version of Rab27A elicited exocytosis in streptolysin O-permeabilized - but not in non-permeabilized - cells when geranylgeranylated and active. When GDP-ß-S substituted for GTP-γ-S, isoprenylated TAT-Rab27A inhibited the acrosome reaction triggered by progesterone and an Epac-selective cAMP analogue, whereas the non-isoprenylated protein did not. Geranylgeranylated TAT-Rab27A-GTP-γ-S promoted the exchange of GDP for GTP on Rab3 and Rap1 detected by far-immunofluorescence with Rab3-GTP and Rap1-GTP binding cassettes. In contrast, TAT-Rab27A lacking isoprenylation or loaded with GDP-ß-S prevented the activation of Rab3 and Rap1 elicited by progesterone. Challenging streptolysin O-permeabilized human sperm with calcium increased the population of sperm with Rap1-GTP, Rab3-GTP and Rab27-GTP in the acrosomal region; pretreatment with anti-Rab27 antibodies prevented the activation of all three. The novel findings reported here include: the description of membrane permeant TAT-Rab27A as a trustworthy tool to unveil the regulation of the human sperm acrosome reaction by Rab27 under physiological conditions; that the activation of endogenous Rab27 is required for that of Rab3 and Rap1; and the connection between Epac and Rab27 and between Rab27 and the configuration of the SNARE complex. Moreover, we present direct evidence that Rab27A's lipid modification, and activation/inactivation status correlate with its stimulatory or inhibitory roles in exocytosis.
Asunto(s)
Reacción Acrosómica , Exocitosis , Nucleótidos de Guanina/metabolismo , Prenilación , Proteínas de Unión a Telómeros/metabolismo , Proteínas rab27 de Unión a GTP/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/metabolismo , Factores de Intercambio de Guanina Nucleótido , Humanos , Masculino , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Prenilación de Proteína , Proteínas Recombinantes/genética , Proteínas SNARE/metabolismo , Complejo Shelterina , Transducción de Señal , Proteínas rab27 de Unión a GTP/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
At the final stage of exocytotis, a fusion pore opens between the plasma and a secretory vesicle membranes; typically, when the pore dilates the vesicle releases its cargo. Sperm contain a large dense-core secretory granule (the acrosome) whose contents are secreted by regulated exocytosis at fertilization. Minutes after the arrival of the triggering signal, the acrosomal and plasma membranes dock at multiple sites and fusion pores open at the contact points. It is believed that immediately afterward, fusion pores dilate spontaneously. Rab3A is an essential component of human sperm exocytotic machinery. Yet, recombinant, persistently active Rab3A halts calcium-triggered secretion when introduced after docking into streptolysin O-permeabilized cells; so does a Rab3A-22A chimera. Here, we applied functional assays, electron and confocal microscopy to show that the secretion blockage is due to the stabilization of open fusion pores. Other novel findings are that sperm SNAREs engage in α-SNAP/NSF-sensitive complexes at a post-fusion stage. Complexes are disentangled by these chaperons to achieve vesiculation and acrosomal contents release. Thus, post-fusion regulation of the pores determines their expansion and the success of the acrosome reaction.
Asunto(s)
Exocitosis , Espermatozoides/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rab3A/metabolismo , Acrosoma/metabolismo , Calcio/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Masculino , Proteínas de Unión al GTP rab/genética , Proteína de Unión al GTP rab3A/genéticaRESUMEN
Exocytosis of the sperm's single secretory granule, or acrosome, is a regulated exocytosis triggered by components of the egg's investments. In addition to external calcium, sperm exocytosis (termed the acrosome reaction) requires cAMP synthesized endogenously and calcium mobilized from the acrosome through IP3-sensitive channels. The relevant cAMP target is Epac. In the first part of this paper, we present a novel tool (the TAT-cAMP sponge) to investigate cAMP-related signaling pathways in response to progesterone as acrosome reaction trigger. The TAT-cAMP sponge consists of the cAMP-binding sites of protein kinase A regulatory subunit RIß fused to the protein transduction domain TAT of the human immunodeficiency virus-1. The sponge permeated into sperm, sequestered endogenous cAMP, and blocked exocytosis. Progesterone increased the population of sperm with Rap1-GTP, Rab3-GTP, and Rab27-GTP in the acrosomal region; pretreatment with the TAT-cAMP sponge prevented the activation of all three GTPases. In the second part of this manuscript, we show that phospholipase Cε (PLCε) is required for the acrosome reaction downstream of Rap1 and upstream of intra-acrosomal calcium mobilization. Last, we present direct evidence that cAMP, Epac, Rap1, and PLCε are necessary for calcium mobilization from sperm's secretory granule. In summary, we describe here a pathway that connects cAMP to calcium mobilization from the acrosome during sperm exocytosis. Never before had direct evidence for each step of the cascade been put together in the same study.
Asunto(s)
Acrosoma/metabolismo , Calcio/metabolismo , AMP Cíclico/metabolismo , Espermatozoides/metabolismo , AMP Cíclico/fisiología , Exocitosis/genética , Exocitosis/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Humanos , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/fisiología , Masculino , Fosfoinositido Fosfolipasa C/metabolismo , Fosfoinositido Fosfolipasa C/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Transfección , Proteínas de Unión al GTP rap1/metabolismo , Proteínas de Unión al GTP rap1/fisiologíaRESUMEN
Regulated exocytosis employs a conserved molecular machinery in all secretory cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Rab superfamilies are members of this machinery. Rab proteins are small GTPases that organize membrane microdomains on organelles by recruiting specific effectors that strongly influence the movement, fusion and fission dynamics of intracellular compartments. Rab3 and Rab27 are the prevalent exocytotic isoforms. Many events occur in mammalian spermatozoa before they can fertilize the egg, one of them is the acrosome reaction (AR), a type of regulated exocytosis. The AR relies on the same fusion machinery as all other cell types, which includes members of the exocytotic SNARE and Rab superfamilies. Here, we describe in depth two protocols designed to determine the activation status of small G proteins. One of them also serves to determine the subcellular localization of active Rabs, something not achievable with other methods. By means of these techniques, we have reported that Rab27 and Rab3 act sequentially and are organized in a RabGEF cascade during the AR. Although we developed them to scrutinize the exocytosis of the acrosome in human sperm, the protocols can potentially be extended to study other Ras-related proteins in virtually any cellular model.
Asunto(s)
Acrosoma/metabolismo , Exocitosis , Proteínas de Unión al GTP Monoméricas/metabolismo , Acrosoma/efectos de los fármacos , Reacción Acrosómica/efectos de los fármacos , Calcimicina/farmacología , Precipitación Química , Enzimas Inmovilizadas/aislamiento & purificación , Enzimas Inmovilizadas/metabolismo , Exocitosis/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Factores de Intercambio de Guanina Nucleótido/metabolismo , Nucleótidos de Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Masculino , Proteínas de Unión al GTP Monoméricas/aislamiento & purificación , Permeabilidad/efectos de los fármacos , Prenilación de Proteína/efectos de los fármacosRESUMEN
Two so-called "secretory Rabs," Rab3 and Rab27, regulate late steps during dense-core vesicle exocytosis in neuroendocrine cells. Sperm contain a single large dense-core granule that is released by regulated exocytosis (termed the acrosome reaction) during fertilization or on exposure to inducers in vitro. Sperm exocytosis uses the same fusion machinery as neurons and neuroendocrine cells, with an additional requirement for active Rab3. Here we show that Rab27 is also required for the acrosome reaction, as demonstrated by the inability of inducers to elicit exocytosis when streptolysin O-permeabilized human sperm were loaded with inhibitory anti-Rab27 antibodies or the Rab27-GTP binding domain of the effector Slac2-b. The levels of GTP-bound Rab27 increased on initiation of exocytosis, as did the proportion of GTP-bound Rab3A. We have developed a fluorescence microscopy-based method for detecting endogenous Rab3A-GTP and Rab27-GTP in the acrosomal region of human sperm. Challenge with an inducer increased the population of cells exhibiting GTP-bound Rabs in this subcellular domain. Interestingly, introducing recombinant Rab27A loaded with GTP-γ-S into sperm elicited a remarkable increase in the number of cells evincing GTP-bound Rab3A. In the converse condition, recombinant Rab3A did not modify the percentage of Rab27-GTP-containing cells. Furthermore, Rab27A-GTP recruited a Rab3 GDP/GTP exchange factor (GEF) activity. Our findings suggest that Rab27/Rab3A constitutes a Rab-GEF cascade in dense-core vesicle exocytosis.