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Marcelo O. Cabada - One of the best experts on this subject based on the ideXlab platform.
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Vitelline envelope of Bufo Arenarum: biochemical and biological characterization.
Biology of reproduction, 2002Co-Authors: Gustavo A. Barisone, Jerry L. Hedrick, Marcelo O. CabadaAbstract:Abstract 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-m...
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Identification of mRNA-binding proteins during development: characterization of Bufo Arenarum cellular nucleic acid binding protein.
Development growth & differentiation, 1999Co-Authors: Nora B. Calcaterra, Javier F. Palatnik, Diego Martin Bustos, Silvia E. Arranz, Marcelo O. CabadaAbstract:Ultraviolet irradiation was used to covalently cross-link poly(A)+RNA and associated proteins in eggs and embryos of the toad Bufo Arenarum. Four major proteins with apparent sizes of 60, 57, 45 and 30–24 kDa were identified. It was observed that the same mRNA-binding proteins were isolated from eggs to gastrula and neural stages of development. The 30 kDa polypeptide, p30, appeared as the main ultraviolet (UV) cross-linked protein in the developmental stages analyzed. By means of polyclonal antibodies, it was determined that this polypeptide has a cytoplasmic localization and it was detected in liver, eggs and embryos. The presence of p30 was also analyzed by western blot during oogenesis and development. The 30 kDa polypeptide was present in all stages analyzed but it could not be detected in stages I–II of oogenesis. At the neural stage, the relative amount of p30 began to decrease, reaching its lowest levels after stages 26–30 (tail-bud in Bufo Arenarum). On the basis of purification, immunoprecipitation and western blot assays the 30 kDa protein was identified as the Bufo Arenarum cellular nucleic acid binding protein.
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Immunohistochemical localisation of a galectin from Bufo Arenarum ovary.
Zygote (Cambridge England), 1998Co-Authors: María T. Elola, Gustavo A. Barisone, Marcelo O. Cabada, Nilda E FinkAbstract:Galectins are a group of soluble animal lectins that exhibit specificity for beta-galactosides and conserve sequence homology in the carbohydrate-recognition domain. The galectin from Bufo Arenarum ovary showed a strong cross-reaction with the lectin of 14.5 kDa purified from embryos at early blastula stage. In this paper, we studied the immunohistochemical localisation of the galectin of 14.5 kDa from ovary of the toad B. Arenarum in adult ovary sections. We also analysed the immunohistochemical localisation of the embryonic lectin during early development using the antiserum anti-ovary galectin. In the ovary, oocytes in the previtellogenic stage showed strong reactivity in the nucleus and the cortex but not in the cytoplasm. Oocytes in the stage of primary vitellogenesis exhibited a similar pattern in the nuclear and cortical areas but showed immunostaining in the cytoplasm. Intense nuclear staining was detected in oocytes in the stage of late vitellogenesis and in mature oocytes, which also presented strong reactions in the yolk platelets that completely covered the cytoplasm. In blastula embryos the staining was found in the blastomeres, the yolk platelets and the blastocoele. Each lectin localisation is discussed in relation to potential biological roles in the corresponding tissues.
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Vitelline envelope formation during oogenesis in Bufo Arenarum.
Biocell : official journal of the Sociedades Latinoamericanas de Microscopia Electronica ... et. al, 1996Co-Authors: Marcelo O. Cabada, Gustavo A. BarisoneAbstract:The formation of vitelline envelope (VE) during the oogenesis of Bufo Arenarum (Amphibia Anura) is described. At the stage of early vitellogenesis, the first structures appear: the number of oocyte microvilli increases, and many cross sections of them are observed between the follicle cells and the oocyte. A filamentous material is observed inside the follicle cells and between the follicle cells and the oocyte. Multivesicular bodies are also found in the follicle cells, and in the perivitelline space. The micrographs also suggest the participation of the oocyte in the process of VE formation: large vesicles are present in the cortex of the oocyte, filled with an amorphous material of low and uniform electron density. Some of them are in the process of releasing their content to the perivitelline space. Many vesicles (probably resulting from microvilli fragmentation) are also observed in the perivitelline space. During late vitellogenesis the VE is a continuous structure between the layer of follicle cells and the oocyte. The filamentous material is aggregated in bundles, forming a net, and the spherical components are now either included in the orifices of the net, or free near the oocyte's surface. At the end of oogenesis, when the VE is completely formed, it is difficult to distinguish its components independently. Immunolocalization with antibodies against VE, show a positive reaction in follicle cells and oocytes in previtellogenic and full grown ovarian follicles. This analysis suggests that both the oocyte and the follicle cells are directly involved in the synthesis and secretion of the components of the vitelline envelope in Bufo Arenarum.
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Effect of egg water from Bufo Arenarum on the fertilizing capacity of homologous spermatozoa
Journal of Experimental Zoology, 1991Co-Authors: Marina F. Diaz Fontdevila, Bernabé Bloj, Marcelo O. CabadaAbstract:The effect of egg water (EW) from oocytes' string of Bufo Arenarum on the fertilizing capacity of homologous spermatozoa was studied. When Bufo Arenarum spermatozoa were incubated in EW, a decrease in the fertilizing capacity of spermatozoa was observed. This effect depended on the osmolarity of the incubation medium. When the osmolarity was between 35 and 40 mOsm/kg water, inhibition of fertility was observed even in the presence of only 10% EW. When osmolarity was greater than 60 mOsm/kg water, even 90% EW had no effect. Control spermatozoa incubated in saline medium with osmolarity similar to that of EW did not show any decrease in fertility. These effects seem to be related to a component of the EW that can be extracted with agents used to extract lipids. The effect of such lipidic extracts of egg water (LEEW) in reducing the fertilizing capacity of a sperm suspension depends on the concentration of LEEW and the duration of incubation.
Dora C. Miceli - One of the best experts on this subject based on the ideXlab platform.
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Characterization and properties of steroid binding protein in Bufo Arenarum serum
Molecular reproduction and development, 1994Co-Authors: Silvia N. Fernández, Zulema C. Mansilla-whitacre, Dora C. MiceliAbstract:Serum steroid binding properties of mature Bufo Arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo Arenarum sex binding protein (Ba SBP), which binds 5 α-dihydrotestosterone (DHT), testosterone (T), and estradiol-17β (E2) with high affinity (107 M−1 – 108 M−1) and fair capacity (10−6 M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady-state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E2. Estriol and estrone competed poorly, while diethylstilbestrol and C21 steroids did not compete. The binding capacity of this protein is under estrogenic control. © 1994 Wiley-Liss, Inc.
Nora R. Ceballos - One of the best experts on this subject based on the ideXlab platform.
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Effects of mGnRH on testicular steroidogenesis in the toad Bufo Arenarum
General and Comparative Endocrinology, 2002Co-Authors: Luis Fabián Canosa, Andrea G. Pozzi, Gustavo Manuel Somoza, Nora R. CeballosAbstract:Abstract GnRH controls vertebrate reproduction in several ways. This hormone not only affects the secretion of gonadotropins from the pituitary gland but also has a direct influence on several gonadal functions such as steroidogenesis, spermatogenesis, and spermiation. In the present paper we have studied the in vitro effects of GnRH on the testicular steroidogenesis of Bufo Arenarum to ascertain the role of this peptide in the control of the steroidogenic pathway previously described in this species. It was found that GnRH is able to reduce basal as well as hCG-stimulated testosterone release, having an inhibitory effect on P450 c17 activity. Thus, GnRH could be involved in the mechanism that regulates the metabolic change in the testicular steroidogenesis. Additionally, testicular GnRH binding site has been characterised, showing a K d of 34 nM and a maximum binding of 4.7 pmol/mg protein.
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Pregnenolone and progesterone metabolism by the testes of Bufo Arenarum
Journal of comparative physiology. B Biochemical systemic and environmental physiology, 1998Co-Authors: Luis Fabián Canosa, Andrea G. Pozzi, Nora R. CeballosAbstract:Sliced testis tissue from Bufo Arenarum was incubated in the presence of [3H]pregnenolone. Testis fragments were also used for double isotope experiments using [3H]pregnenolone and [14C]progesterone. Specific activities were equated with the addition of radioinert pregnenolone. When yields of radiometabolites were analysed, pregnenolone was found to be a good precursor for C19 steroids such as dehydroepiandrosterone, 5-androsten-3 beta, 17 beta diol, testosterone, 5 alpha-dihydrotestosterone and a C21 steroid, 5 alpha-pregnan-3,20 dione. Progesterone mainly converts to 5 alpha-pregnan-3,20 dione, a steroid with unknown function in amphibians. The 5-ene pathway, including 5-androsten-3 beta, 17 beta diol as intermediate, could be predominant for androgen biosynthesis. Testes bypass not only progesterone but also androstenedione for testosterone biosynthesis.
M.i. Bühler - One of the best experts on this subject based on the ideXlab platform.
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Role of phospholipase A2 pathway in regulating activation of Bufo Arenarum oocytes.
Zygote (Cambridge England), 2012Co-Authors: M.t. Ajmat, L. Zelarayán, F. Bonilla, P.c. Hermosilla, M.i. BühlerAbstract:Transient increases in the concentration of cytosolic Ca(2+) are essential for triggering egg activation events. Increased Ca(2+) results from its rapid release from intracellular stores, mainly mediated by one or both intracellular calcium channels: the inositol trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). Several regulatory pathways that tailor the response of these channels to the specific cell type have been proposed. Among its many modulatory actions, calcium can serve as an activator of a cytosolic phospholipase A(2) (cPLA2), which releases arachidonic acid from phospholipids of the endoplasmic reticulum as well as from the nuclear envelope. Previous studies have suggested that arachidonic acid and/or its metabolites were able to modulate the activity of several ion channels. Based on these findings, we have studied the participation of the phospholipase A(2) (PLA(2)) pathway in the process of Bufo Arenarum oocyte activation and the interrelation between any of its metabolites and the ion channels involved in the calcium release from the intracellular reservoirs at fertilization. We found that addition of both melittin, a potent PLA(2) activator, and arachidonic acid, the main PLA(2) reaction metabolite, was able to induce activation events in a bell-shaped manner. Differential regulation of IP3Rs and RyRs by arachidonic acid and its products could explain melittin and arachidonic acid behaviour in Bufo Arenarum egg activation. The concerted action of arachidonic acid and/or its metabolites could provide controlled mobilization of calcium from intracellular reservoirs and useful tools for understanding calcium homeostasis in eggs that express both types of receptors.
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Spontaneous and LH-induced maturation in Bufo Arenarum oocytes: importance of gap junctions.
Zygote (Cambridge England), 2007Co-Authors: G. Sánchez Toranzo, L. Zelarayán, J. Oterino, F. Bonilla, M.i. BühlerAbstract:It has been demonstrated in Bufo Arenarum that fully grown oocytes are capable of meiotic resumption in the absence of a hormonal stimulus if they are deprived of their follicular envelopes. This event, called spontaneous maturation, only takes place in oocytes collected during the reproductive period, which have a metabolically mature cytoplasm. In Bufo Arenarum , progesterone acts on the oocyte surface and causes modifications in the activities of important enzymes, such as a decrease in the activity of adenylate cyclase (AC) and the activation of phospholipase C (PLC). PLC activation leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP 3 ), second messengers that activate protein kinase C (PKC) and cause an increase in intracellular Ca 2+ . Recent data obtained from Bufo Arenarum show that progesterone-induced maturation causes significant modifications in the level and composition of neutral lipids and phospholipids of whole fully grown ovarian oocytes and of enriched fractions in the plasma membrane. In amphibians, the luteinizing hormone (LH) is responsible for meiosis resumption through the induction of progesterone production by follicular cells. The aim of this work was to study the importance of gap junctions in the spontaneous and LH-induced maturation in Bufo Arenarum oocytes. During the reproductive period, Bufo Arenarum oocytes are capable of undergoing spontaneous maturation in a similar way to mammalian oocytes while, during the non-reproductive period, they exhibit the behaviour that is characteristic of amphibian oocytes, requiring progesterone stimulation for meiotic resumption (incapable oocytes). This different ability to mature spontaneously is coincident with differences in the amount and composition of the phospholipids in the oocyte membranes. Capable oocytes exhibit in their membranes higher quantities of phospholipids than incapable oocytes, especially of PC and PI, which are precursors of second messengers such as DAG and IP 3 . The uncoupling of the gap junctions with 1-octanol or halothane fails to induce maturation in follicles from the non-reproductive period, whose oocytes are incapable of maturing spontaneously. However, if the treatment is performed during the reproductive period, with oocytes capable of undergoing spontaneous maturation, meiosis resumption occurs in high percentages, similar to those obtained by manual defolliculation. Interestingly, results show that LH is capable of inducing GVBD in both incapable oocytes and in oocytes capable of maturing spontaneously as long as follicle cells are present, which would imply the need for a communication pathway between the oocyte and the follicle cells. This possibility was analysed by combining LH treatment with uncoupling agents such as 1-octanol or halothane. Results show that maturation induction with LH requires a cell–cell coupling, as the uncoupling of the gap junctions decreases GVBD percentages. Experiments with LH in the presence of heparin, BAPTA/AM and theophylline suggest that the hormone could induce GVBD by means of the passage of IP 3 or Ca 2+ through the gap junctions, which would increase the Ca 2+ level in the oocyte cytoplasm and activate phosphodiesterase (PDE), thus contributing to the decrease in cAMP levels and allowing meiosis resumption.
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Activation of maturation promoting factor in Bufo Arenarum oocytes: injection of mature cytoplasm and germinal vesicle contents.
Zygote (Cambridge England), 2006Co-Authors: G. Sánchez Toranzo, L. Zelarayán, J. Oterino, F. Bonilla, M.i. BühlerAbstract:Although progesterone is the established maturation inducer in amphibians, Bufo Arenarum oocytes obtained during the reproductive period (spring–summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. In this species it is possible to obtain oocytes competent and incompetent to undergo spontaneous maturation according to the seasonal period in which animals are captured. Reinitiation of meiosis is regulated by maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34 cdc2 and cyclin B. Although the function and molecule of MPF are common among species, the formation and activation mechanisms of MPF differ according to species. This study was undertaken to evaluate the presence of pre-MPF in Bufo Arenarum oocytes incompetent to mature spontaneously and the effect of the injection of mature cytoplasm or germinal vesicle contents on the resumption of meiosis. The results of our treatment of Bufo Arenarum immature oocytes incompetent to mature spontaneously with sodium metavanadate (NaVO 3 ) and dexamethasone (DEX) indicates that these oocytes have a pre-MPF, which activates and induces germinal vesicle breakdown (GVBD) by dephosphorylation on Thr-14/Tyr-15 by cdc25 phosphatase and without cyclin B synthesis. The injection of cytoplasm containing active MPF is sufficient to activate an amplification loop that requires the activation of cdc25 and protein kinase C, the decrease in cAMP levels, and is independent of protein synthesis. However, the injection of germinal vesicle content also induces GVBD in the immature receptor oocyte, a process dependent on protein synthesis but not on cdc25 phosphatase or PKC activity.
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Effect of insulin on spontaneous and progesterone-induced GVBD on Bufo Arenarum denuded oocytes.
Zygote (Cambridge England), 2004Co-Authors: G. Sánchez Toranzo, L. Zelarayán, J. Oterino, F. Bonilla, M.i. BühlerAbstract:Progesterone is considered as the physiological steroid hormone that triggers meiosis reinitiation in amphibian oocytes. Nevertheless, isolated oocytes can be induced to undergo germinal vesicle breakdown (GVBD) in a saline medium by means of treatment with various hormones or inducing agents such as other steroid hormones, insulin or an insulin-like growth factor. It has been demonstrated that Bufo Arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. This study was undertaken to evaluate the participation of the purine and phosphoinositide pathway in the insulin-induced maturation of oocytes competent and incompetent to mature spontaneously, as well as to determine whether the activation of the maturation promoting factor (MPF) involved the activation of cdc25 phosphatase in Bufo Arenarum denuded oocytes. Our results indicate that insulin was able to induce GBVD in oocytes incompetent to mature spontaneously and to enhance spontaneous and progesterone-induced maturation. In addition, high intracellular levels of purines such as cAMP or guanosine can reversibly inhibit the progesterone and insulin-induced maturation process in Bufo Arenarum as well as spontaneous maturation. Assays of the inhibition of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis and its turnover by neomycin and lithium chloride respectively exhibited a different response in insulin- or progesterone-treated oocytes, suggesting that phosphoinositide turnover or hydrolysis of PIP2 is involved in progesterone- but not in insulin-induced maturation. In addition, the inhibitory effect of vanadate suggests that an inactive pre-maturation promoting factor (pre-MPF), activated by dephosphorylation of Thr-14 and Tyr-15 on p34cdc2, is present in Bufo Arenarum full-grown oocytes; this step would be common to both spontaneous and hormone-induced maturation. The data presented here strongly suggest that insulin initiates at the cell surface a chain of events leading to GVBD. However, our studies point to the existence of certain differences between the steroid and the peptide hormone pathways, although both involve the decrease in intracellular levels of cAMP, the activation of phosphodiesterase (PDE) and the activation of pre-MPF.
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The role of calcium in the nuclear maturation of Bufo Arenarum oocytes.
Zygote (Cambridge England), 2004Co-Authors: L. Zelarayán, Graciela Sánchez Toranzo, J. Oterino, M.i. BühlerAbstract:In Bufo Arenarum, progesterone is the physiological maturation inducer. However, in this species, oocytes reinitiate meiosis with no need of an exogenous hormonal stimulus when deprived of their enveloping cell, a phenomenon called spontaneous maturation. We demonstrated that in Bufo Arenarum spontaneous maturation occurs only in oocytes obtained during the reproductive period, which can be considered competent to mature spontaneously, in contrast to those in the non-reproductive period, which are incompetent. Interestingly, full-grown Bufo Arenarum oocytes always respond to progesterone regardless of the season in which they are obtained. There is a general consensus that both a transient increase in intracellular calcium and a decrease in cAMP-dependent protein kinase activity are the first steps in the mechanisms by which progesterone induces maturation in amphibians. In the present work we analysed the role of calcium in the spontaneous and progesterone-induced maturation of Bufo Arenarum oocytes. Results demonstrated that the absence of calcium in the incubation medium or the prevention of Ca(2+) influx by channel blockers such as CdCl2 or NiCl2 did not prevent meiosis reinitiation in either type of maturation. The inhibition of the Ca(2+)-calmodulin complex in no case affected the maturation of the treated oocytes. However, when the oocytes were deprived of calcium by incubation in Ca(2+)-free AR + A23187, meiosis resumption was inhibited. In brief, we demonstrated that in Bufo Arenarum the reinitiation of meiosis is a process independent of extracellular calcium at any period of the year and that oocytes require adequate levels of intracellular calcium for germinal vesicle breakdown to occur.
Alfredo Coviello - One of the best experts on this subject based on the ideXlab platform.
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Effects of atrial natriuretic peptide and toad heart extract on isolated toad Bufo Arenarum aortic rings
General and comparative endocrinology, 1992Co-Authors: Maráperal De Bruno, Alfredo CovielloAbstract:Abstract The vascular effects of synthetic atrial natriuretic peptide (ANP) (rANP-99-126), and toad heart extract (THE) were examined on isolated toad aortic rings from the toad Bufo Arenarum. ANP inhibited contraction produced by human angiotensin 11 (AT 11), norepinephrine (NE), and arginine vasopressin (AVP) in isolated toad aortic rings. The present data show that a relaxant effect of ANP could be obtained also in the noncontracted aortic smooth muscle of the toad if it had been previously challenged with AT 11 or NE and allowed to return to the original basal tension. Bufo Arenarum THE was able to relax the AT II-induced contraction in toad aortic rings. In toad arteries contracted with 10−6 M AT II, ANP produced a dose-dependent relaxation with a half-maximal inhibitory concentration IC50, of 1.2 × 10−8 M. ANP was not effective in relaxing contraction induced by high K+. The vasorelaxant effect of ANP on AT I1-induced contraction was significantly increased in Ca2+-free medium containing 3 mM ethylene glycol bis(β-aminoethyl ether) N,N,N′,N′tetraacetic acid (EGTA-Ringer) or by pretreatment with the calcium antagonist, diltiazem (DIL). The vasorelaxant effect of ANP on basal tension after treatment with AT II was also obtained in absence of extracellular calcium (EGTA-Ringer). These results show that Bufo Arenarum contains ANP-like material and that the ANP relaxant action in the toad aorta is similar to that in mammals.
