RESUMO
Glycosidases are present both in sperm and eggs in vertebrates and have been associated with different fertilization steps as gamete binding, egg coat penetration, and polyspermy prevention. In this manuscript, we have analyzed the activity of different glycosidases of Xenopus laevis eggs. The main activity corresponded to N-acetyl-ß-D-glucosaminidase (Hex), which was reported to participate both in gamete binding and polyspermy prevention among phylogenetically distant animals. We have raised homologous antibodies against a recombinant N-terminal fragment of a X. laevis Hex, and characterized egg's Hex both by Western blot and immunohistochemical assays. Noteworthy, Hex was mainly localized to the cortex of animal hemisphere of full-grown oocytes and oviposited eggs, and remained unaltered after fertilization. Hex is constituted by different pair arrangements of two subunits (α and ß), giving rise to three possible Hex isoforms: A (αß), B (ßß), and S (αα). However, no information was available regarding molecular identity of Hex in amphibians. We present for the first time the primary sequences of two isoforms of X. laevis Hex. Interestingly, our results suggest that α- and ß-like subunits that constitute Hex isoforms could be synthesized from a same gene in Xenopus, by alternative exon use. This finding denotes an evolutionary divergence with mammals, where α and ß Hex subunits are synthesized from different genes on different chromosomes.
Assuntos
Acetilglucosaminidase/metabolismo , Imuno-Histoquímica/métodos , Oócitos/enzimologia , Óvulo/enzimologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Acetilglucosaminidase/genética , Acetilglucosaminidase/isolamento & purificação , Sequência de Aminoácidos , Animais , Western Blotting , Domínio Catalítico , Clonagem Molecular , Sequência Conservada , DNA Complementar/genética , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Ensaios Enzimáticos , Éxons , Feminino , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Xenopus laevis/genéticaRESUMO
Integrins are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction. Nevertheless, their function in mammalian fertilization is still controversial, as different species behave differently. In amphibians, their role is mainly supported by Xenopus laevis studies, where RGDS peptide induces oocyte activation. We recently provided evidence suggesting the presence and involvement of integrins in the interaction of the oocyte plasma membrane (PM) with sperm in the amphibian Bufo arenarum. In order to understand the role of integrin homologs in oocytes and their possible contribution to egg activation mechanisms, we examined the presence of integrin subunits and the effect of RGDS peptide on oocytes and during fertilization. Western blot studies detected integrin subunits α5, αV and ß1 in oocytes. In sperm, we could detect only the αV integrin subunit. We found that RGDS peptide was unable to elicit egg activation or MAPK dephosphorylation, but can induce reversible inhibition of fertilization. A similar partial inhibition was produced by an anti-ß1 integrin antibody. Using an anti-phosphotyrosine antibody we found major changes in phosphotyrosine-containing proteins in egg extracts minutes after fertilization. Cytosol and PMs isolated from oocytes and fertilized eggs showed additional fertilization-induced phosphorylated proteins. Some of these were also present in cytosol and PMs from RGDS-treated oocytes (partially mimicking fertilization). These findings suggest that B. arenarum fertilization involves integrins (e.g. ß1 subunit) as adhesion proteins. Our data support the view that RGDS-binding receptors may function as signaling receptors in B. arenarum oocytes, but integrin engagement by RGDS is not sufficient for oocyte activation.
Assuntos
Proteínas de Anfíbios/metabolismo , Bufo arenarum/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fertilização/efeitos dos fármacos , Integrinas/metabolismo , Oligopeptídeos/farmacologia , Oócitos/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Animais , Western Blotting , Ativação Enzimática , Feminino , Masculino , Microscopia de Fluorescência , Oócitos/enzimologia , Fosforilação , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Fatores de Tempo , TirosinaRESUMO
Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation.
Assuntos
Adenilil Ciclases/metabolismo , Bufo arenarum/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/fisiologia , Inibidores de Adenilil Ciclases , Animais , Membrana Celular/enzimologia , AMP Cíclico/metabolismo , Ativação Enzimática , Soluções Hipotônicas/farmacologia , Masculino , Fosforilação , Espermatozoides/efeitos dos fármacos , Espermatozoides/enzimologiaRESUMO
Vitellogenin (Vtg), a large lipoglycophosphoprotein, is the most important precursor of the yolk proteins, and the major source of nutrients for the developing embryo in oviparous species. After its uptake by the oocytes, Vtg is converted into lipovitellins (high and light) and phosvitin, which are deposited into crystalline yolk platelets. We describe here the presence of two high molecular mass lipovitellin isoforms in Bufo arenarum mature oocytes with masses of 113 and 100 kDa, respectively. The amino acid sequence analysis of p113 and p100 peptides showed a high sequence homology between both polypeptides and the complete reported sequences of Xenopus laevis vitellogenin. Using specific antibodies, we determined that the Vtg uptake begins early during oogenesis, at the previtellogenic stage, and continues until oocytes have reached their mature status. In addition, we found that large endocytic vesicles mediate Vtg uptake in stage I oocytes, and that the size of the endocytic vesicles declines with oogenesis progression. In terms of the Vtg protein trafficking, we detected the Vtg precursor (190 kDa) in the liver of estradiol-injected females. Finally, we propose a subclassification of B. arenarum stage II oocytes into three physiologically and morphologically distinct periods (early, mid and late).
Assuntos
Bufo arenarum/metabolismo , Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Vitelogênese , Sequência de Aminoácidos , Animais , Bufo arenarum/crescimento & desenvolvimento , Bufo arenarum/fisiologia , Proteínas do Ovo/isolamento & purificação , Feminino , Imuno-Histoquímica , Dados de Sequência Molecular , Peso Molecular , Oócitos/ultraestrutura , Transporte Proteico , Análise de Sequência de DNA , Fatores de TempoRESUMO
The presence, topology and dynamics of heparin-binding proteins (HBP) on boar sperm were evaluated. HBP distribution was analyzed by subcellular parting, using biotinylated heparin followed by colorimetric detection. HBP were detected as peripherical and integral periacrosomal membrane proteins. Indirect fluorescence microscopy of sperm incubated with biotinylated heparin was used to evidence heparin binding on sperm at different physiological stages. Two different fluorescent patterns (A and B) were found, which probably correspond to non-capacitated and capacitated sperm as assessed by the ability to undergo acrosome reaction with calcium ionophore A23187 and by the increase of p32 phosphorylated protein. In A pattern, corresponding to untreated sperm, fluorescence located mostly on the post-acrosomal region; in B pattern, corresponding to incubated sperm, on the acrosomal region. Upon incubation under capacitating conditions (TALP), sperm having the B pattern was augmented compared with non-incubated sperm (p<0.001). Differences in the HBP patterns (p<0.0001) were observed in sperm incubated under non-capacitating conditions in relation to sperm incubated in TALP, indicating that the modification of HBP patterns is probably related to capacitation. No difference was observed when untreated sperm were permeabilized prior to staining, suggesting that HBP are present on the sperm surface. The effect of heparin on capacitation dependent protein tyrosine phosphorylation was also analyzed, finding a decrease in p32 phosphorylation in the presence of heparin. This suggests that the capacitation enhancement mediated by this glycosaminoglycan involves an alternative intracellular pathway. The finding that heparin binds to sperm differently according to its physiological state, is a new evidence of the remodelling of sperm membrane surface upon capacitation and may provide a useful and relatively simple method to evaluate in vitro modification of boar sperm physiological state.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/metabolismo , Heparina/farmacocinética , Espermatozoides/metabolismo , Sus scrofa/metabolismo , Reação Acrossômica/efeitos dos fármacos , Reação Acrossômica/fisiologia , Algoritmos , Animais , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Proteínas Sanguíneas/isolamento & purificação , Proteínas de Transporte/isolamento & purificação , Extratos Celulares/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Masculino , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/fisiologia , Espermatozoides/química , Espermatozoides/efeitos dos fármacos , Tirosina/metabolismoRESUMO
OBJECTIVE: To study the effect of the human tubal tissue conditioned medium (CM) on sperm parameters related to sperm-zona pellucida interaction. DESIGN: Controlled experimental laboratory study. SETTING: Research laboratory. SUBJECT(S): Semen samples from donors with normozoospermia. Human tubal tissue obtained from women undergoing hysterectomies. Human follicular fluids (hFF) and oocytes collected from patients undergoing IVF-ET. INTERVENTION(S): Incubation of spermatozoa with CM proteins obtained from human tubal tissue culture; sperm binding to the zona pellucida assessment. MAIN OUTCOME MEASURE(S): Explants' viability was assessed by tissue DNA analysis. Sperm ability to interact with zona was tested with use of the whole oocyte test. Expression of d-mannose binding sites was assessed with use of a fluorescent probe on mannose coupled to bovine serum albumin. Human FF-induced acrosome reaction was assessed by the Pisum sativum technique. RESULT(S): Although treatment with 0.8 microg/microL of CM allowed sperm binding to the zona and the expression of d-mannose binding sites comparable with sperm in control medium, with 3.2 microg/mL of CM resulted in a significant decrease of both parameters. No effect of CM on spontaneous or hFF-induced acrosome reaction or in sperm viability was observed. CONCLUSION(S): The results indicate that the incubation of spermatozoa in the presence of CM reduces sperm affinity for the zona pellucida. This effect can be partly explained by the decreased expression of d-mannose binding sites on the sperm surface.
Assuntos
Reação Acrossômica , Tubas Uterinas/metabolismo , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Adulto , Sítios de Ligação , Sobrevivência Celular , Meios de Cultivo Condicionados/metabolismo , Dano ao DNA , Feminino , Líquido Folicular/metabolismo , Humanos , Masculino , Manose/metabolismo , Pessoa de Meia-Idade , Técnicas de Cultura de TecidosRESUMO
Sperm from the toad Bufo arenarum must penetrate the egg jelly before reaching the vitelline envelope (VE), where the acrosome reaction is triggered. When the jelly coat is removed, sperm still bind to the VE, but acrosomal exocytosis is not promoted. Our previous work demonstrated that diffusible substances of the jelly coat, termed "egg water" (EW), triggered capacitation-like changes in B. arenarum sperm, promoting the acquisition of a transient fertilizing capacity. In the present work, we correlated this fertilizing capacity with the ability of the sperm to undergo the acrosome reaction, further substantiating the role of the jelly coat in fertilization. When sperm were exposed to the VE, only those preincubated in EW for 5 or 8 min underwent an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which led to acrosomal exocytosis. Responsiveness to the VE was not acquired on preincubation in EW for 2 or 15 min or in Ringer solution regardless of the preincubation time. In contrast, depletion of intracellular Ca(2+) stores (induced by thapsigargin) promoted [Ca(2+)](i) rise and the acrosome reaction even in sperm that were not exposed to EW. Acrosomal exocytosis was blocked by the presence of Ca(2+) chelators independent of whether a physiological or pharmacological stimulus was used. However, Ni(2+) and mibefradil prevented [Ca(2+)](i) rise and the acrosome reaction of sperm exposed to the VE but not of sperm exposed to thapsigargin. These data suggest that the acrosomal responsiveness of B. arenarum sperm, present during a narrow period, is acquired during EW incubation and involves the modulation of a voltage-dependent Ca(2+) channel.
Assuntos
Reação Acrossômica/fisiologia , Bufo arenarum/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Membrana Vitelina/fisiologia , Zona Pelúcida/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Canais de Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Feminino , Masculino , Modelos Biológicos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Tapsigargina/farmacologia , Membrana Vitelina/efeitos dos fármacos , Membrana Vitelina/metabolismoRESUMO
PURPOSE: In the female genital tract spermatozoa interact with the oviductal secretion. The aim of the study was to evaluate the effect of conditioned media (CM) from cultures of human oviductal tissue, on sperm DNA integrity. The effect of H(2)O(2) on sperm DNA integrity, before and after incubation under capacitating conditions, was also investigated. METHODS: Motile sperm obtained from normozoospermic semen samples were incubated (4 h or 22 h) in the presence or absence of CM and further exposed to H(2)O(2). DNA damage was detected by the comet assay. RESULTS: The CM significantly reduced the DNA damage associated with sperm incubation, and also decreased the effect of H(2)O(2) after 4 h incubation, compared to controls. The H(2)O(2) caused a dose-dependent deleterious effect on sperm DNA integrity both before and following 22 h of capacitation. CONCLUSION: The oviductal tissue CM increased the stabilization of the sperm DNA structure under culture conditions.
Assuntos
Líquidos Corporais/fisiologia , Dano ao DNA , Tubas Uterinas/metabolismo , Espermatozoides/metabolismo , Adulto , Algoritmos , Líquidos Corporais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , DNA/efeitos dos fármacos , DNA/metabolismo , DNA/fisiologia , Dano ao DNA/fisiologia , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Masculino , Pessoa de Meia-Idade , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologiaRESUMO
The oviduct is a dynamic organ which modulates gamete physiology. Two subpopulations of sperm have been described in the oviduct of sows, a majority with normal appearance in the deep furrows and a minority, centrally located, and showing damaged membranes. Sperm-oviduct interaction provides the formation of a sperm storage and allows the selection of sperm with certain qualities. Pig (Sus scrofa) oviductal sperm binding glycoprotein (SBG) binds to sperm and exposes Gal beta1-3GalNAc. This disaccharide may be recognized by boar spermadhesin AQN1, which seems to be involved in sperm interaction with the oviduct. SBG is present at the apical surface of the epithelial cells that surround the lumen of the oviduct rather than at the bottom of the crypts. These characteristics imply it could be involved in sperm interaction with this organ. In this study, we evaluate the effect of SBG over boar sperm. We show that the presence of SBG produces alterations of the acrosome morphology of sperm only when they are incubated in capacitating conditions. SBG binds to the periacrosomal region of sperm undergoing capacitation. Its presence induces an increase on the tyrosine-phosphorylation of a polypeptide of apparent molecular mass 97 kDa, as occurs with a 95 kDa protein in other mammalian sperm upon acrosomic reaction. Altogether, these results suggest that SBG might be involved in sperm selection by alteration of the acrosome of sperm that have already begun the capacitation process when they arrive to the oviduct.
Assuntos
Acrossomo/metabolismo , Glicoproteínas/metabolismo , Oviductos/metabolismo , Proteínas de Plasma Seminal/metabolismo , Capacitação Espermática/fisiologia , Sus scrofa/metabolismo , Animais , Dissacarídeos/metabolismo , Feminino , Masculino , Fosforilação/efeitos dos fármacos , Proteínas de Plasma Seminal/farmacologia , Capacitação Espermática/efeitos dos fármacosRESUMO
Mammalian sperm acquire fertilizing capacity after residing in the female tract, where physiological changes named capacitation take place. In animals with external fertilization as amphibians, gamete interactions are first established between sperm and molecules of the egg jelly coat released into the medium. Since dejellied oocytes are not normally fertilized, the aim of this study was to determine if the jelly coat of the toad Bufo arenarum promotes a "capacitating" activity on homologous sperm. We found that sperm incubation in diffusible substances of the jelly coat (egg water) for 90-180 s is sufficient to render sperm transiently capable of fertilizing dejellied oocytes. The fertilizing state was correlated with an increase of protein tyrosine phosphorylation and a decrease of sperm cholesterol content. Inhibition of either the increase in tyrosine phosphorylation or cholesterol efflux affected the acquisition of fertilizing capacity. Phosphorylation and fertilization could be promoted with NaHCO(3) and also by addition of beta cyclodextrin. Moreover, sperm could gain the ability to fertilize dejellied oocytes in the presence of these compounds. These data indicate that sperm should undergo a series of molecular changes to gain fertilizing capacity; these changes are reminiscent of mammalian sperm capacitation and take place before the acrosome reaction.
Assuntos
Bufo arenarum/metabolismo , Espermatozoides/metabolismo , Reação Acrossômica , Animais , Colesterol/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Fertilização , Masculino , Oócitos/metabolismo , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Capacitação Espermática , Interações Espermatozoide-Óvulo , beta-Ciclodextrinas/metabolismoRESUMO
The vitelline envelope (VE) participates in sperm-egg interactions during the first steps of fertilization. In Bufo arenarum, this envelope is composed of at least four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa and molar ratio of 1:1.3:7.4:4.8, respectively. These components were isolated and covalently coupled to silanized glass slides in order to study their sperm-binding capacity. When considering the molar ratio of the glycoproteins in the egg-envelope and assuming that each protein is monovalent for sperm, the assay showed that gp41 and gp38 possess 55 and 25% of total sperm-binding activity. We obtained a full-length cDNA of gp41 (ZPC), comprising a sequence for 486 amino acids, with 43.3% homology with Xenopus laevis ZPC. As in the case of mammalian ZP3 and Xenopus ZPC, Bufo ZPC presented a furin-like (convertase) and a C-terminal transmembrane domain (TMD) reflecting common biosynthetic and secretory pathways. As it was reported for some fishes, we obtained evidence that suggests the presence of more than one zpc gene in Bufo genome, based on different partial cDNA sequences of zpc, Southern blots and two-dimensional SDS-PAGE of deglycosylated egg-envelope components. As far as we are aware, this is the first observation of the presence of different zpc genes in an Amphibian species.
Assuntos
Bufo arenarum/fisiologia , Proteínas do Ovo/metabolismo , Glicoproteínas de Membrana/metabolismo , Oócitos/fisiologia , Receptores de Superfície Celular/metabolismo , Interações Espermatozoide-Óvulo/fisiologia , Membrana Vitelina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bufo arenarum/metabolismo , Clonagem Molecular , Diploide , Proteínas do Ovo/genética , Proteínas do Ovo/isolamento & purificação , Feminino , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Oócitos/metabolismo , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificação , Espermatozoides/fisiologia , Xenopus laevis/genética , Glicoproteínas da Zona PelúcidaRESUMO
In several mammals a sperm reservoir is formed at the isthmus of the Fallopian tube, providing viable, potentially fertile sperm for an extensive period. In pig (Sus scrofa) the spermadhesin AQN-1 seems to be involved in the establishment of the sperm reservoir. The pig oviductal protein, sperm binding glycoprotein (SBG), binds to sperm and exposes carbohydrate groups that can be recognized by AQN-1. In this study we obtain anti-SBG polyclonal antibodies and use them to localize SBG in the oviduct. Immunohistochemical analysis shows that SBG is present at the apical surface of isthmic and ampullar epithelial cells. The presence of SBG is limited to the upper two-thirds of the crypts of the isthmus and to cells located near the oviductal lumen in the ampulla. The ratio of the amount of SBG detected by western blot is 1:3 (ampulla:isthmus). Sperm entering the Fallopian tube probably contact the epithelial cells at the lumen before they reach the cells at the bottom of the folds. In vitro sperm can bind to isthmus and, at less extent, to ampulla. Thus, the localization and the relative amount of SBG in the isthmus and ampulla of pig's oviduct are compatible with its possible function in sperm binding to oviductal epithelial cells.
Assuntos
Tubas Uterinas/metabolismo , Proteínas de Plasma Seminal/metabolismo , Suínos/anatomia & histologia , Animais , Anticorpos/metabolismo , Western Blotting , Tubas Uterinas/anatomia & histologia , Feminino , Imuno-Histoquímica , Proteínas de Plasma Seminal/imunologia , Suínos/metabolismo , Distribuição TecidualRESUMO
Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.
Assuntos
Bufo arenarum/metabolismo , Proteínas de Choque Térmico HSP70/análise , Integrinas/análise , Proteínas de Membrana/análise , Oócitos/química , Interações Espermatozoide-Óvulo , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Integrinas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Oócitos/metabolismo , Espermatozoides/metabolismoRESUMO
BACKGROUND INFORMATION: The role of the jelly coat that surrounds the amphibian oocytes has been widely discussed, but is poorly understood. The presence of the jelly coat is essential for fertilization. However, the structure and function of the molecules that comprise the jelly coat have not been thoroughly documented. L-HGP (low-molecular-mass highly glycosylated protein) is a highly glycosylated protein that is present in the jelly coat of the toad, Bufo arenarum, oocytes and diffuses to the surrounding media. L-HGP, when purified from egg water, protects the sperm acrosome from breakdown induced by hypotonic solutions. RESULTS: L-HGP is an acidic glycoprotein, formed by two different subunits, linked by disulphide bonds. We raised polyclonal antibodies in rabbits against the deglycosylated protein. We determined that L-HGP is secreted along the oviduct, being hence present in all the jelly layers. The molecular mass of L-HGP is higher in the most cephalic region of the oviduct. The lower-M(r) L-HGP isoform, produced in the caudal regions of the oviduct, presents an acrosome-protecting property. L-HGP is produced by secretory cells in the oviduct and is deposited on the cilia at the oviduct lumen. CONCLUSIONS: Biochemical characterization of L-HGP has been carried out. It is synthesized by secretory cells in the oviduct and, when secreted, is deposited over the ciliated surface of the cells. The lower-M(r) isoform, secreted by the caudal region of the oviduct, protects acrosome integrity. This isoform diffuses into the medium. The role of the higher-M(r) L-HGP isoform in fertilization remains unknown.
Assuntos
Acrossomo/metabolismo , Bufo arenarum/metabolismo , Glicoproteínas/química , Oviductos/metabolismo , Reação Acrossômica/fisiologia , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Fertilização , Glicoproteínas/metabolismo , Masculino , Isoformas de ProteínasRESUMO
Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 microM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 microg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.
Assuntos
Heparina/farmacologia , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Clortetraciclina , Corantes , Masculino , Microscopia de Fluorescência , Espermatozoides/fisiologia , Sus scrofaRESUMO
Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 µM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 µg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.
Assuntos
Animais , Masculino , Heparina/farmacologia , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/fisiologia , Clortetraciclina , Corantes , Microscopia de Fluorescência , Sus scrofaRESUMO
In pigs the binding of sperm to oviductal epithelial cells to form a sperm reservoir involves carbohydrate interactions. In the present study, we purify a sperm binding glycoprotein (SBG) from cells from the isthmus of the oviduct using an affinity column. This protein conjugated with FITC is able to bind to the heads of pig sperm. SBG is shown to contain carbohydrates by PAS-silver staining and lectin binding assays. Enzymatic treatment and lectin affinity demonstrate that SBG exposes Galbeta1-3GalNAc disaccharide, which is bound to a serine or a threonin residue by an O-link. After enzymatic deglycosylation SBG shows an apparent molecular mass of 67.5 kDa, which changes to 85 kDa by reduction with 2-mercaptoethanol. Both SBG and enzymatically deglycosylated SBG show by isoelectrofocusing two forms of pI 3.6 and pI 3.8. SBG may be involved on sperm-oviduct interaction.
Assuntos
Proteínas de Transporte/isolamento & purificação , Células Epiteliais/química , Glicoproteínas/isolamento & purificação , Oviductos/química , Espermatozoides/metabolismo , Suínos , Animais , Metabolismo dos Carboidratos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cromatografia de Afinidade , Feminino , Fluoresceína-5-Isotiocianato , Glicoproteínas/química , Glicoproteínas/metabolismo , MasculinoRESUMO
A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.
Assuntos
Bufo arenarum/fisiologia , Matriz Extracelular/química , Oócitos/citologia , Acetilglucosamina/análise , Animais , Blástula/química , Blástula/citologia , Proteínas do Ovo/análise , Eletroforese em Gel de Poliacrilamida , Matriz Extracelular/ultraestrutura , Proteínas da Matriz Extracelular/análise , Feminino , Fertilização , Fucose/análise , Galactose/análise , Glucosamina/análise , Glicoproteínas/análise , Lectinas/metabolismo , Oócitos/química , Inibidores de Proteases/farmacologia , Receptores Mitogênicos/metabolismo , Zigoto/química , Zigoto/citologiaRESUMO
Vitelline envelopes (VEs) of Bufo arenarum were isolated in order to study their composition and their role in fertilization. VEs are composed of four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa. To characterize its biological properties, we quantitatively determined sperm-VE binding and the induction of the acrosome reaction. Heterologous binding of B. arenarum sperm to Xenopus laevis VE components was observed with about one-third the efficiency of homologous binding. Equivalent binding of X. laevis sperm to the B. arenarum VE was observed. When B. arenarum sperm were incubated with fluorescein isothiocyanate-labeled VE, the labeled glycoproteins bound to the anterior end of the sperm head, showing a lateral distribution. Induction of the acrosome reaction was evaluated by incubating sperm in hypotonic saline media with VE glycoproteins. VEs induced the acrosome reaction in a time- and concentration-dependent manner. The acrosome reaction was maximal after 10 min. The half-maximal effect was obtained at a glycoprotein concentration of 1 microg/ml. Specificity was determined using fertilization envelope glycoproteins, which failed to induce the acrosome reaction. The B. arenarum VE is biochemically similar to other egg envelopes. It also seems that its biological properties are similar to other species in regard to sperm binding and induction of the acrosome reaction. However, as far as we are aware, this is the first observation of the VE inducing the sperm acrosome reaction in amphibians. The relatively small differences observed in heterologous sperm-VE binding in X. laevis and B. arenarum are inconsistent with the current paradigm that species specificity in fertilization is regulated at the sperm-VE binding step.
Assuntos
Bufo arenarum/anatomia & histologia , Fertilização , Membrana Vitelina/química , Membrana Vitelina/fisiologia , Reação Acrossômica , Animais , Eletroforese em Gel de Poliacrilamida , Feminino , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Glicoproteínas/análise , Glicoproteínas/química , Glicoproteínas/metabolismo , Masculino , Microscopia de Fluorescência , Peso Molecular , Espermatozoides/metabolismoRESUMO
Se determinó la captación de 3H adenosina en células foliculares y ovocitos ováricos totalmente crecidos de Bufo arenarum, y la incorporación del trazador radioactivo en ARN total y en la fracción reistente a ribonucleasas. Se incubaron 3H adenosina en folículos y envolturas foliculares (I-FE). En el primer caso, luego de la incubación las envolturas foliculares fueron manualmente separadas (O-FE). En el primer caso, luego de la incubación las envolturas foliculares fueron manualmente separadas (O-FE) y procesadas independiente de los ovocitos . I-FE incorporó 2,9 veces más 3H adenosina que O-FE. Los medios de incubación de I-FE y O-FE fueron analizados; en el primer caso se encontró 10.2 veces más radioactividad en macromoléculas que en el segundo caso. Se realizó una caracterización parcial de la ARN recientemente sintetizada, mediante electroforesis, encontrándose diferentes patrones para ARN de I-FE y O-FE. Una posible explicación para los resultados obtenidos es que las células foliculares transfieren ARN recientemente sintetizado al ovocito y, en ausencia de esto, al medio de incubación (AU)