Función de la proteína MARCKS en la exocitosis acrosomal de espermatozoides humanos

Acrosomal exocytosis is an absolute requirement for physiological fertilization. In this thesis, we report that MARCKS is expressed in human spermatozoa. \nCalcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies, indicating t...

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Autor principal: Rodriguez Peña, Marcelo Javier
Otros Autores: Mayorga, Luis
Formato: Tesis doctoral acceptedVersion
Lenguaje:Español
Publicado: Facultad de Farmacia y Bioquímica 2014
Materias:
MARCKS
Pip2
Exotosis Acrosomal
Espermatozoides
Ciencia de la vida
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_1340
http://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_1340.dir/1340.PDF
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spelling I28-R145-HWA_13402019-09-25 Acrosomal exocytosis is an absolute requirement for physiological fertilization. In this thesis, we report that MARCKS is expressed in human spermatozoa. \nCalcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2. \nIn non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. \nIn addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering PIP2, and that it is phosphorylated during acrosomal exocytosis.\n Fil: Rodriguez Peña, Marcelo Javier. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Mayorga, Luis Palaoro, Luis Facultad de Farmacia y Bioquímica Michaut, Marcela Rodriguez Peña, Marcelo Javier 2014-09-11 La exocitosis acrosomal (EA) es un proceso secretorio dependiente de Ca2+ fundamental para que el espermatozoide fecunde el ovocito. Durante este trabajo de tesis describimos la presencia y localización de MARCKS en espermatozoides humanos.\nEn un modelo de espermatozoides permeabilizado, anticuerpos que reconocen diferentes dominios de MARCKS inhibieron la EA. A su vez se demostró que el dominio efector (ED) de MARCKS es capaz de inhibir la EA y que el estado de fosforilación del ED regula su participación durante la EA. Asimismo la adición de PIP2 o adenofostina, un potente agonista de los receptores de IP3 revirtió el efecto inhibitorio del ED de MARCKS sobre la EA, sugiriendo que MARCKS regula la disponibilidad de PIP2 durante la EA. \nAdemás se demostró que un péptido que posee la secuencia del ED de MARCKS es permeable a la membrana plasmática y es capaz de inhibir los movimientos de Ca2+ que ocurren durante la EA.\nEn esta tesis demostramos que una vez que se estimula la EA se produce la fosforilación y translocación de MARCKS desde las membranas al citosol celular. Estos resultados nos permiten plantear que MARCKS es un componente importante en las vías de transducción de señales durante la exocitosis acrosomal.\n application/pdf Vaccaro, María Inés Vázquez-Levin, Mónica Buffone, Mariano MARCKS Pip2 Exotosis Acrosomal Espermatozoides spa Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencia de la vida Función de la proteína MARCKS en la exocitosis acrosomal de espermatozoides humanos info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_1340 http://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_1340.dir/1340.PDF
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-145
collection Repositorio Digital de la Universidad de Buenos Aires (UBA)
language Español
orig_language_str_mv spa
topic MARCKS
Pip2
Exotosis Acrosomal
Espermatozoides
Ciencia de la vida
spellingShingle MARCKS
Pip2
Exotosis Acrosomal
Espermatozoides
Ciencia de la vida
Rodriguez Peña, Marcelo Javier
Función de la proteína MARCKS en la exocitosis acrosomal de espermatozoides humanos
topic_facet MARCKS
Pip2
Exotosis Acrosomal
Espermatozoides
Ciencia de la vida
description Acrosomal exocytosis is an absolute requirement for physiological fertilization. In this thesis, we report that MARCKS is expressed in human spermatozoa. \nCalcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2. \nIn non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. \nIn addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering PIP2, and that it is phosphorylated during acrosomal exocytosis.\n
author2 Mayorga, Luis
author_facet Mayorga, Luis
Rodriguez Peña, Marcelo Javier
format Tesis doctoral
Tesis doctoral
acceptedVersion
author Rodriguez Peña, Marcelo Javier
author_sort Rodriguez Peña, Marcelo Javier
title Función de la proteína MARCKS en la exocitosis acrosomal de espermatozoides humanos
title_short Función de la proteína MARCKS en la exocitosis acrosomal de espermatozoides humanos
title_full Función de la proteína MARCKS en la exocitosis acrosomal de espermatozoides humanos
title_fullStr Función de la proteína MARCKS en la exocitosis acrosomal de espermatozoides humanos
title_full_unstemmed Función de la proteína MARCKS en la exocitosis acrosomal de espermatozoides humanos
title_sort función de la proteína marcks en la exocitosis acrosomal de espermatozoides humanos
publisher Facultad de Farmacia y Bioquímica
publishDate 2014
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_1340
http://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_1340.dir/1340.PDF
work_keys_str_mv AT rodriguezpenamarcelojavier funciondelaproteinamarcksenlaexocitosisacrosomaldeespermatozoideshumanos
_version_ 1766017466263142400

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