RESUMO
The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.
Assuntos
Acrossomo/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Exocitose/fisiologia , Proteínas rab3 de Ligação ao GTP/metabolismo , Acrossomo/fisiologia , Acrossomo/ultraestrutura , Análise de Variância , Western Blotting , Cálcio/farmacologia , Ciclodextrinas/farmacologia , Exocitose/efeitos dos fármacos , Humanos , Masculino , Microscopia ImunoeletrônicaRESUMO
The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that alpha-soluble NSF-attachment protein (alpha-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for alpha-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.
Assuntos
Reação Acrossômica/fisiologia , Espermatozoides/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Acrossomo/química , Acrossomo/metabolismo , Anticorpos/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Humanos , Masculino , Proteínas SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Espermatozoides/química , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteína rab3A de Ligação ao GTP/metabolismo , Proteína rab3A de Ligação ao GTP/farmacologiaRESUMO
The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.
Assuntos
Reação Acrossômica/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Espermatozoides/enzimologia , Acrossomo/metabolismo , Animais , Cálcio/metabolismo , Humanos , Masculino , Camundongos , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Espermatozoides/efeitos dos fármacos , Vanadatos/farmacologia , Proteína rab3A de Ligação ao GTP/metabolismoRESUMO
The interaction between Rab3A and calmodulin is necessary for the inhibitory effect of Rab3A in neuroendocrine cells. Contrastingly, Rab3A triggers the exocytosis known as acrosome reaction in permeabilized spermatozoa. Here we show that a Rab3A mutant that cannot bind calmodulin was fully capable of triggering acrosomal exocytosis. Additionally, calmodulin by itself abrogated the exocytosis triggered by Rab3A. The effect was observed with both the wild type protein and the calmodulin binding deficient mutant. Our results indicate that the inhibitory and stimulatory effects of Rab3A in different exocytic processes are mediated by different effectors.
Assuntos
Reação Acrossômica/fisiologia , Calmodulina/metabolismo , Exocitose/fisiologia , Proteína rab3A de Ligação ao GTP/metabolismo , Reação Acrossômica/efeitos dos fármacos , Calcimicina/farmacologia , Calmodulina/antagonistas & inibidores , Calmodulina/farmacologia , Permeabilidade da Membrana Celular , Clorpromazina/farmacologia , Antagonistas de Dopamina/farmacologia , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Corantes Fluorescentes , Humanos , Ionóforos/farmacologia , Masculino , Mutação , Progesterona/farmacologia , Ligação Proteica/fisiologia , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Proteína rab3A de Ligação ao GTP/genética , Proteína rab3A de Ligação ao GTP/farmacologiaRESUMO
Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis.
Assuntos
Reação Acrossômica , Proteínas de Ligação ao Cálcio , Glicoproteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Espermatozoides/metabolismo , Western Blotting , Cálcio/metabolismo , Citosol/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Microscopia de Fluorescência , Ésteres de Forbol/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , SinaptotagminasRESUMO
The acrosome reaction of spermatozoa is a complex, calcium-dependent, regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. However, very little is known about the molecules that mediate and regulate this unique fusion process. Here, we show that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa. Moreover, we demonstrate that calcium-dependent exocytosis of permeabilized sperm requires active NSF. Previously, we have shown that the addition of the active (GTP-bound) form of the small GTPase Rab3A triggers exocytosis in permeabilized spermatozoa. In the present report we show that Rab3A is necessary for calcium-dependent exocytosis. The activation of Rab3A protects NSF from N-ethylmaleimide inhibition and precludes the exchange of the endogenous protein with recombinant dominant negative mutants of NSF. Furthermore, Rab3A activation of acrosomal exocytosis requires active NSF. Our results suggest that, upon calcium stimulation, Rab3A switches to its active GTP-bound form, triggering the formation of a protein complex in which NSF is protected. This process is suggested to be an essential part of the molecular mechanism of membrane fusion leading to the release of the acrosomal contents.
Assuntos
Reação Acrossômica , Acrossomo/fisiologia , Adenosina Trifosfatases/metabolismo , Cálcio/fisiologia , Proteínas de Transporte/metabolismo , Exocitose/fisiologia , Proteínas de Transporte Vesicular , Proteína rab3A de Ligação ao GTP/metabolismo , Acrossomo/efeitos dos fármacos , Acrossomo/ultraestrutura , Proteínas de Bactérias , Cálcio/farmacologia , Permeabilidade da Membrana Celular , Exocitose/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Cinética , Masculino , Proteínas Sensíveis a N-Etilmaleimida , Proteínas Recombinantes/metabolismo , EstreptolisinasRESUMO
The acrosome reaction is a regulated exocytotic process leading to a massive fusion between the outer acrosomal membrane and the cell membrane. In spite of the great amount of information available related to the acrosome reaction in several species, there is a remarkable paucity about the role of monomeric guanosine triphosphatases (GTPases) of the Rab family-well-established participants in exocytosis in other cell types-in the acrosome reaction. Western blot and immunofluorescence analysis indicate that Rab3A is present in human spermatozoa and localizes to the acrosomal region in the sperm head. One difficulty in studying the role of proteins in intact cells is the fact that they are unable to cross the cell membrane. Therefore, we established a working model of streptolysin O-permeabilized human spermatozoa. Permeabilized spermatozoa were able to respond in a regulated way to different stimuli, such as G protein activators and calcium. An acrosomal reaction was also triggered by a Rab3A peptide corresponding to the effector region. More important, recombinant Rab3A protein in the GTP-bound form caused acrosome exocytosis. The same protein loaded with GDP or Rab11 in the GTP-bound form was inactive. Also, recombinant GDI (GDP dissociation inhibitor)-a protein that releases Rab proteins from membrane-inhibited a GTPgammaS-stimulated acrosome reaction. Our results indicate that 1) permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, 2) Rab3A is present in human spermatozoa, and 3) Rab3A or another Rab3 isoform is involved in the exocytosis of the acrosomal granule in human spermatozoa.
Assuntos
Reação Acrossômica/fisiologia , Espermatozoides/fisiologia , Proteína rab3A de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Western Blotting , Separação Celular , Eletroforese em Gel de Poliacrilamida , Exocitose/fisiologia , Imunofluorescência , Técnica Indireta de Fluorescência para Anticorpo , GTP Fosfo-Hidrolases/metabolismo , Glutationa Transferase/metabolismo , Humanos , Técnicas In Vitro , Indicadores e Reagentes , Masculino , Dados de Sequência Molecular , Permeabilidade , Prenilação de Proteína , Proteína rab3A de Ligação ao GTP/metabolismoRESUMO
The effect of 17 inhibitors of cyclic nucleotide phosphodiesterases (PDEs) was assayed on cAMP binding activity of Mucor rouxii protein kinase A (PKA), on PKA activity in the absence of cAMP and on free catalytic subunit (C) activity. Isobutylmethylxanthine (IBMX), SQ 20,009 and cilostamide, at 0.2 mM, behaved as partial agonists of cAMP since they inhibited binding of 0.15 microM [3H]cAMP to the regulatory subunit (R), stimulated slightly PKA activity in the absence of cAMP and did not modify C activity. Amrinone at 0.2 mM inhibited C activity competitively towards ATP. These four compounds displayed the same effects when assayed on eukaryotic protein kinase A types I (PKI) and II (PKII). The combined effect of IBMX and cAMP was analysed on Mucor PKA. Under dissociating conditions (+ 0.5 M NaCl) IBMX antagonized activation by low concentrations of cAMP, while in the absence of NaCl, IBMX potentiated the stimulating activity of cAMP.
Assuntos
1-Metil-3-Isobutilxantina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Animais , AMP Cíclico/metabolismo , Etazolato/farmacologia , Técnicas In Vitro , Mucor/enzimologia , Miocárdio/enzimologia , Quinolonas/farmacologia , Ratos , Transdução de SinaisRESUMO
The antimicrobial activity of extracts and constituents of Gomphrena martiana and Gomphrena boliviana (Amaranthaceae) were determined in order to identify the compounds responsible for the folk-medicinal use of these plants. Each extract was evaluated against 20 microorganisms, including Gram-positive and Gram-negative bacteria, spore-forming Gram-positive bacteria, an acid-fast bacterium, a fungus and two yeasts. Fractionation of each petroleum ether (PE) extract yielded five 5,6,7-trisubstituted flavones that were separately tested showing high activity against M. phlei (minimum inhibitory concentration (MIC) 15, 20 and 75 micrograms/ml) approaching that of commercial bactericides. Other natural and synthetic flavonoids with diverse structures were also tested to define structure-activity relationships. Each EtOH extract was subsequently fractionated and monitored by bioassays leading to isorhamnetin 3-O-beta-robinobioside (MIC 50 micrograms/ml) in both instances. This glycoside is reported here for the first time in G. boliviana.
Assuntos
Fungos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Plantas Medicinais , Flavonoides/farmacologia , Testes de Sensibilidade Microbiana , Plantas Medicinais/químicaRESUMO
A partially purified preparation (200-fold) of cAMP phosphodiesterase (PDE) was obtained from Mucor rouxii grown and extracted under conditions minimizing endogenous proteolysis. Four purification steps were applied: batch DEAE-Sepharose, DEAE-Sepharose chromatography, Sephadex G-150 super-fine gel filtration and sucrose gradient centrifugation. The final PDE preparation was activatable by cAMP-dependent phosphorylation and controlled trypsin treatment. A careful correlation of protein patterns with PDE activity was done throughout the whole procedure by analyzing the active fractions of each step by mini-polyacrylamide non-denaturing gel electrophoresis. The final preparation displayed four major protein bands, none of which corresponded to PDE, although PDE activity comigrated with two of them. Some properties of this preparation were studied. Vmax increased around 10-15 fold by activation of PDE by phosphorylation or proteolysis; Km values were unaffected. PDE had Stokes radius of 3.5 nm, sedimentation coefficient of 4.3 S and molecular weight of 70,000 daltons. The treatment of sucrose gradient fractions with [gamma-32P] ATP and cAMP-dependent protein kinase catalytic subunit and further analysis through minigels showed that none of the visible bands was phosphorylated, and that among the four phosphorylated bands there was one that cosedimented and comigrated with PDE activity. Trypsin treatment of the phosphorylated samples removed the label but did not modify the staining pattern.