Involvement of actin cytoskeleton and intracellular calcium in the control of sperm acrosomal exocytosis

Los espermatozoides de mamífero adquieren su capacidad fecundante después de una serie de modificaciones bioquímicas en el tracto reproductor femenino, colectivamente llamadas capacitación. Estos cambios son esenciales para que los espermatozoides puedan realizarla exocitosis acrosomal (EA), un proceso que es fundamental para la fecundación. En este trabajo se estudiaron los cambios en el citoesqueleto de actina en la preparación para la ocurrencia de la EA así como también, los cambios en los niveles de calcio intracelular como evento fundamental para las etapas finales de la fusión de membranas. La dinámica del citoesqueleto de actina juega un rol central en controlar el proceso de exocitosis en células somáticas así como en los espermatozoides de varias especies demamíferos. A pesar de que en células somáticas, laspequeñas GTPasas de la familia Rho son ampliamente conocidas como reguladores principales de la dinámica dela actina, su función en espermatozoides es desconocida. En el presente trabajo se caracterizó la participación de las pequeñas GTPasas de la familia Rho en la vía de señalización que conduce a la polimerización de actina durante la capacitación del espermatozoide de ratón. Se observó que la mayoría de las proteínas de esta cascada de señalización y sus proteínas efectoras se expresan en el espermatozoide de ratón. La activación de las vías de señalización de AMPc/PKA, RhoA/C y Rac1 es esencial para la activación por fosforilación de LIMK1 en Treonina 508. Cofilin es fosforilada en Serina 3porLIMK1 durante la capacitación de manera transitoria. La inhibición de LIMK1 por un inhibidor específico (BMS-3) resultó en menores niveles de polimerización de actina durante la capacitación y una marcada disminución en el porcentaje de espermatozoides que realizan EA. Asimismo se sabe que es necesario un aumento en el Ca2+ intracelular ([Ca2+]i) para que la EA se produzca. La progesterona producida por las células del cúmulus ha sido asociada con diversos procesos fisiológicos en los espermatozoides, incluyendo la estimulación de la EA. En este trabajo, investigamos la correlación espacio temporal entre los cambios en el [Ca2+]iy la EA en espermatozoidesindividuales de ratón en respuesta a la progesterona. Se encontró que la progesterona estimula un incremento en el [Ca2+]i encinco patrones diferentes: gradual,oscilatorio, transitorio tardío, transitorioinmediato, y sostenido.Tambiénse observó que el aumento en el [Ca2+]ipromovido por la progesterona puedecomenzar tanto en el flagelo como en la cabeza del espermatozoide. Se validó la utilización de FM4-64 como un indicador de la ocurrencia de la EA mediante la detección simultánea del aumento de su fluorescencia y la pérdida del EGFP en espermatozoides transgénicos EGFPAcr. Por primera vez, se logró visualizar simultáneamente el aumento en el [Ca2+]i y el proceso de exocitosis en respuesta a la progesterona, observándose que sólo un aumento transitorio específico en el [Ca2+]i originado en la cabeza del espermatozoide promueve la iniciación de la EA. En conclusión, en esta tesis se logró evidenciar la importanciade las pequeñas GTPasas de la familia Rho y sus efectores principales LIMK1 y Cofilin en la regulación de la dinámica del citoesqueleto de actina en la preparación del espermatozoide para poder realizar la EA. A su vez, se identificó el tipo de aumento de [Ca2+]i específico que es necesario para iniciar los eventos finales de la EA estimulada por progesterona.Mammalia sperm must acquire their fertilizing ability after a series of biochemical modifications in the female reproductive tract collectively called capacitation to undergo acrosomal exocytosis (AE), a process that is essential for fertilization. In this work, the changes in the actin cytoskeleton in preparation for the occurrence of the AE as well as the changes in intracellular calcium levels as a crucial event to the final stages of membrane fusion, were studied. 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 the role of the small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during mouse sperm capacitation was studied. It was observed that most of the proteins of this signaling cascade and their downstream effector proteins are expressed in mouse sperm. The activation of the cAMP/PKA, RhoA/C and Rac1 signaling pathways are 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. In addition, it is well known that an increase in intracellular Ca2+ ([Ca2+]i) is necessary for AE to occur. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AE. In this study, the spatio-temporal correlation between the changes in [Ca2+]i and AEin single mouse spermatozoa in response to progesterone was investigated. Progesterone stimulated an [Ca2+]i increase in five different patterns: gradual, oscillatory, late transitory, immediate transitory and sustained. It was also observed that the [Ca2+]i increase promoted by progesterone started at either the flagellum or the head. The use of FM4-64 as an indicator for the occurrence of the AE was validated by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, it was simultaneously visualized the rise in [Ca2+]i at the onset of AE in response to progesterone. Only a specific transitory increase in [Ca2+]i originated in the sperm head promoted the initiation of AE. In conclusion, in this thesis it was demonstrated the importance of the small GTPases of the Rho family and its main effector proteins LIMK1 and Cofilin in the regulation of the actin cytoskeleton in preparation for AE. Moreover, it was identified the type of [Ca2 +]i increase necessary to trigger the final events of AE induced by progesterone.Fil: Romarowski, Ana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Mouse sperm energy restriction and recovery (SER) revealed novel metabolic pathways

Mammalian sperm must undergo capacitation to become fertilization-competent. While working on mice, we recently developed a new methodology for treating sperm in vitro, which results in higher rates of fertilization and embryo development after in vitro fertilization. Sperm incubated in media devoid of nutrients lose motility, although they remain viable. Upon re-adding energy substrates, sperm resume motility and become capacitated with improved functionality. Here, we explore how sperm energy restriction and recovery (SER) treatment affects sperm metabolism and capacitation-associated signaling. Using extracellular flux analysis and metabolite profiling and tracing via nuclear magnetic resonance (NMR) and mass spectrometry (MS), we found that the levels of many metabolites were altered during the starvation phase of SER. Of particular interest, two metabolites, AMP and L-carnitine, were significantly increased in energy-restricted sperm. Upon re-addition of glucose and initiation of capacitation, most metabolite levels recovered and closely mimic the levels observed in capacitating sperm that have not undergone starvation. In both control and SER-treated sperm, incubation under capacitating conditions upregulated glycolysis and oxidative phosphorylation. However, ATP levels were diminished, presumably reflecting the increased energy consumption during capacitation. Flux data following the fate of 13C glucose indicate that, similar to other cells with high glucose consumption rates, pyruvate is converted into 13C-lactate and, with lower efficiency, into 13C-acetate, which are then released into the incubation media. Furthermore, our metabolic flux data show that exogenously supplied glucose is converted into citrate, providing evidence that in sperm cells, as in somatic cells, glycolytic products can be converted into Krebs cycle metabolites

CFTR/ENaC-dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC

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.Fil: Puga Molina, Lis del Carmen. 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: Pinto, Nicolás Alejandro. 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: Torres, Nicolás I.. 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: González Cota, Ana, L.. University of Washington; Estados UnidosFil: 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: 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: 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; 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: Santi, Celia M.. University of Washington; Estados UnidosFil: Treviño, Claudia L.. Universidad Nacional Autónoma de México; MéxicoFil: Darszon, Alberto. Universidad Nacional Autónoma de México; 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

Molecular Basis of Human Sperm Capacitation

In the early 1950s, Austin and Chang independently described the changes that are required for the sperm to fertilize oocytes in vivo. These changes were originally grouped under name of “capacitation” and were the first step in the development of in vitro fertilization (IVF) in humans. Following these initial and fundamental findings, a remarkable number of observations led to characterization of the molecular steps behind this process. The discovery of certain sperm-specific molecules and the possibility to record ion currents through patch-clamp approaches helped to integrate the initial biochemical observation with the activity of ion channels. This is of particular importance in the male gamete due to the fact that sperm are transcriptionally inactive. Therefore, sperm must control all these changes that occur during their transit through the male and female reproductive tracts by complex signaling cascades that include post-translational modifications. This review is focused on the principal molecular mechanisms that govern human sperm capacitation with particular emphasis on comparing all the reported pieces of evidence with the mouse model

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

Capacitation induces changes in metabolic pathways supporting motility of epididymal and ejaculated sperm

Mammalian sperm require sufficient energy to support motility and capacitation for successful fertilization. Previous studies cataloging the changes to metabolism in sperm explored ejaculated human sperm or dormant mouse sperm surgically extracted from the cauda epididymis. Due to the differences in methods of collection, it remains unclear whether any observed differences between mouse and human sperm represent species differences or reflect the distinct maturation states of the sperm under study. Here we compare the metabolic changes during capacitation of epididymal versus ejaculated mouse sperm and relate these changes to ejaculated human sperm. Using extracellular flux analysis and targeted metabolic profiling, we show that capacitation-induced changes lead to increased flux through both glycolysis and oxidative phosphorylation in mouse and human sperm. Ejaculation leads to greater flexibility in the ability to use different carbon sources. While epididymal sperm are dependent upon glucose, ejaculated mouse and human sperm gain the ability to also leverage non-glycolytic energy sources such as pyruvate and citrate

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

Redistribution of the intra-acrosomal EGFP before acrosomal exocytosis in mouse spermatozoa

Mammalian spermatozoa must undergo complex physiological and morphological alterations within the female reproductive tract before they become fertilization competent. Two important alterations are capacitation and the acrosome reaction (AR), by which spermatozoa become capable of penetrating the zona pellucida (ZP) of the oocyte. Although various biochemical stimulants have been reported to induce the AR, the true physiological inducer in vivo remains to be identified. Previously, it has been reported that most fertilizing spermatozoa undergo the AR before contacting the ZP and that only a small fraction of in vitro-capacitated spermatozoa can penetrate the ZP. Therefore, it is important to identify which capacitating spermatozoa undergo the AR in response to potential AR inducers such as progesterone. Here we show that spermatozoa undergo a dynamic rearrangement of the acrosome during in vitro capacitation. This involves the rapid movement of an artificially introduced soluble component of the acrosome, enhanced green fluorescent protein (EGFP), from the acrosomal cap region to the equatorial segment (EQ) of the sperm head. Spermatozoa exhibiting the EQ pattern were more sensitive to progesterone than were those without it. We suggest that spermatozoa that are ready to undergo acrosomal exocytosis can be detected by real-time EGFP imaging. This offers a promising new method for identifying where spermatozoa undergo the AR in the female reproductive tract in vivo.Fil: Hirohashi, Noritaki. Shimane University; Japón. Ochanomizu University; JapónFil: la Spina, Florenza Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Romarowski, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Role of actin cytoskeleton during mammalian sperm acrosomal exocytosis

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.Fil: 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; ArgentinaFil: 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: la Spina, Florenza Antonella. 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: 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