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Involvement of actin cytoskeleton and intracellular calcium in the control of sperm acrosomal exocytosis
Full text linkLos 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
Involvement of actin cytoskeleton and intracellular calcium in the control of sperm acrosomal exocytosis
No full textLos 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
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Role of actin cytoskeleton during mammalian sperm acrosomal exocytosis
No full textMammalian 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
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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