The Experts below are selected from a list of 141 Experts worldwide ranked by ideXlab platform
Noritaka Hirohashi - One of the best experts on this subject based on the ideXlab platform.
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Lactic acid is a sperm motility inactivation factor in the sperm storage tubules
Scientific Reports, 2015Co-Authors: Mei Matsuzaki, Kazuo Inaba, Shusei Mizushima, Kogiku Shiba, Noritaka Hirohashi, Gen Hiyama, Tomohiro Suzuki, Hideo Dohra, Toshiyuki Ohnishi, Yoshikatsu SatoAbstract:Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41 °C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar Dynein ATPase inactivation following cytoplasmic acidification (
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Sperm–Egg Binding in the Sea Urchin: A High Level of Intracellular ATP Stabilizes Sperm Attachment to the Egg Receptor☆
Developmental biology, 1998Co-Authors: Noritaka Hirohashi, William J. LennarzAbstract:Previous studies have established that a recombinant protein fragment (45A) of the egg receptor for sperm of the sea urchinStrongylocentrotus purpuratusexhibits several characteristics that are consistent with that expected of a receptor. Using a quantitative sperm binding assay with glutathioneS-transferase fused to a recombinant protein containing the C-terminal half of the 45A construct immobilized on glutathione beads, it was found that the interaction between sperm and this protein is a kinetically transient event. Sperm binding to the receptor fragment reached a maximum at 20 s after adding sperm in the presence of egg jelly to beads coated with recombinant receptor. In the next 20–120 s, approximately 50–70% of the sperm detached from the beads. Similar phenomena were observed when the kinetics of sperm binding to dejellied, glutaraldehyde-fixed eggs were studied. Because the acrosome reaction, a prelude to binding, is known to be accompanied by a decrease in the ATP level of sperm, we studied the effect of various inhibitors on both sperm detachment and the level of ATP. It was found that the detachment rate increased slightly when respiration inhibitors that blocked ATP production in mitochondria were added. In contrast, the Dynein ATPase inhibitor,erythro-9-[3-hydroxynonyl]adenine, which is known to inhibit flagellum motility by blocking ATP utilization, stabilized the binding of sperm to the receptor and allowed maintenance of a high internal ATP level. Immotile, tailless sperm that physically lacked Dynein ATPase, and therefore sustained their internal ATP levels, also exhibited stable binding provided that the sperm and beads were physically mixed. These results suggest that the internal ATP level of the sperm controls the stability of its binding to the receptor. The possible mechanism of the detachment and its significance with respect to the overall process of fertilization are discussed.
Michael Plamann - One of the best experts on this subject based on the ideXlab platform.
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null mutants of the neurospora actin related protein 1 pointed end complex show distinct phenotypes
Molecular Biology of the Cell, 2001Co-Authors: Santosh Kumar, Michael PlamannAbstract:Dynactin is a multisubunit complex that regulates the activities of cytoplasmic Dynein, a microtubule-associated motor. Actin-related protein 1 (Arp1) is the most abundant subunit of dynactin, and it forms a short filament to which additional subunits associate. An Arp1 filament pointed-end–binding subcomplex has been identified that consists of p62, p25, p27, and Arp11 subunits. The functional roles of these subunits have not been determined. Recently, we reported the cloning of an apparent homologue of mammalian Arp11 from the filamentous fungus Neurospora crassa. Here, we report that N. crassa ro-2 and ro-12 genes encode the respective p62 and p25 subunits of the pointed-end complex. Characterization of Δro-2, Δro-7, and Δro-12 mutants reveals that each has a distinct phenotype. All three mutants have reduced in vivo vesicle trafficking and have defects in vacuole distribution. We showed previously that in vivo dynactin function is required for high-level Dynein ATPase activity, and we find that all three mutants have low Dynein ATPase activity. Surprisingly, Δro-12 differs from Δro-2 and Δro-7 and other previously characterized Dynein/dynactin mutants in that it has normal nuclear distribution. Each of the mutants shows a distinct Dynein/dynactin localization pattern. All three mutants also show stronger Dynein/dynactin-membrane interaction relative to wild type, suggesting that the Arp1 pointed-end complex may regulate interaction of dynactin with membranous cargoes.
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Cytoplasmic Dynein ATPase activity is regulated by dynactin-dependent phosphorylation.
The Journal of biological chemistry, 2000Co-Authors: Santosh Kumar, In Hyung Lee, Michael PlamannAbstract:Abstract Cytoplasmic Dynein is a microtubule-associated motor that utilizes ATP hydrolysis to conduct minus-end directed transport of various organelles. Dynactin is a multisubunit complex that has been proposed to both link Dynein with cargo and activate Dynein motor function. The mechanisms by which dynactin regulates Dynein activity are not clear. In this study, we examine the role of dynactin in regulating Dynein ATPase activity. We show that Dynein-microtubule binding and ATP-dependent release of Dynein from microtubules are reduced in dynactin null mutants, Δro-3 (p150Glued) and Δro-4(Arp1), relative to wild-type. The Dynein-microtubule binding activity, but not the ATP-dependent release of Dynein from microtubules, is restored by in vitro mixing of extracts from Dynein and dynactin mutants. Dynein produced in a Δro-3 mutant has ∼8-fold reduced ATPase activity relative to Dynein isolated from wild-type. However, Dynein ATPase activity from wild-type is not reduced when dynactin is separated from Dynein, suggesting that Dynein produced in a dynactin mutant is inactivated. Treatment of Dynein isolated from the Δro-3mutant with λ protein phosphatase restores the ATPase activity to near wild-type levels. The reduced Dynein ATPase activity observed in dynactin null mutants is mainly due to altered affinity for ATP. Radiolabeling experiments revealed that low molecular mass proteins, particularly 20- and 8-kDa proteins, that immunoprecipitate with Dynein heavy chain are hyperphosphorylated in the dynactin mutant and dephosphorylated upon λ protein phosphatase treatment. The results suggest that cytoplasmic Dynein ATPase activity is regulated by dynactin-dependent phosphorylation of Dynein light chains.
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Two approaches to isolate cytoplasmic Dynein ATPase from Neurospora crassa
Biochimie, 2000Co-Authors: Santosh Kumar, In Hyung Lee, Michael PlamannAbstract:Abstract Cytoplasmic Dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic Dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal Dynein, we isolated/partially purified cytoplasmic Dynein ATPase from the filamentous fungus Neurospora crassa . N. crassa Dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic Dynein that uses Dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate Dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K m , ATP obtained by isolating Dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the Dynein ATPase activity. Like vertebrate Dynein, N. crassa Dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K m , ATP for N. crassa cytoplasmic Dynein is 10- to 15-fold higher than that of the vertebrate enzyme.
William J. Lennarz - One of the best experts on this subject based on the ideXlab platform.
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Sperm–Egg Binding in the Sea Urchin: A High Level of Intracellular ATP Stabilizes Sperm Attachment to the Egg Receptor☆
Developmental biology, 1998Co-Authors: Noritaka Hirohashi, William J. LennarzAbstract:Previous studies have established that a recombinant protein fragment (45A) of the egg receptor for sperm of the sea urchinStrongylocentrotus purpuratusexhibits several characteristics that are consistent with that expected of a receptor. Using a quantitative sperm binding assay with glutathioneS-transferase fused to a recombinant protein containing the C-terminal half of the 45A construct immobilized on glutathione beads, it was found that the interaction between sperm and this protein is a kinetically transient event. Sperm binding to the receptor fragment reached a maximum at 20 s after adding sperm in the presence of egg jelly to beads coated with recombinant receptor. In the next 20–120 s, approximately 50–70% of the sperm detached from the beads. Similar phenomena were observed when the kinetics of sperm binding to dejellied, glutaraldehyde-fixed eggs were studied. Because the acrosome reaction, a prelude to binding, is known to be accompanied by a decrease in the ATP level of sperm, we studied the effect of various inhibitors on both sperm detachment and the level of ATP. It was found that the detachment rate increased slightly when respiration inhibitors that blocked ATP production in mitochondria were added. In contrast, the Dynein ATPase inhibitor,erythro-9-[3-hydroxynonyl]adenine, which is known to inhibit flagellum motility by blocking ATP utilization, stabilized the binding of sperm to the receptor and allowed maintenance of a high internal ATP level. Immotile, tailless sperm that physically lacked Dynein ATPase, and therefore sustained their internal ATP levels, also exhibited stable binding provided that the sperm and beads were physically mixed. These results suggest that the internal ATP level of the sperm controls the stability of its binding to the receptor. The possible mechanism of the detachment and its significance with respect to the overall process of fertilization are discussed.
H.s. Juneja - One of the best experts on this subject based on the ideXlab platform.
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Estrogen receptor, calcium mobilization and rat sperm motility
Molecular and Cellular Biochemistry, 2002Co-Authors: G. Sethi Saberwal, M.k. Sharma, N. Balasinor, J. Choudhary, H.s. JunejaAbstract:Oral treatment with 0.4 mg/kg/day of tamoxifen citrate, an antiestrogen, has been reported to reduce the fertility of adult male rat, presumably through estrogen receptors expressed throughout the male reproductive tract. During the course of these studies, tamoxifen was observed to gradually alter the pattern of sperm motility in the cauda epididymides without reducing sperm counts. Studies were carried out to understand the mechanism involved in tamoxifen induced change in the sperm motility pattern. In order to study the direct effects of tamoxifen on motility, biochemical levels/activities of sperm calcium, cAMP, phosphodiesterase and Dynein ATPase, normally implicated in sperm motility were studied In view of the fact that tamoxifen is a ligand of estrogen receptor, estrogen receptor alpha protein and transcript were localized on rat sperm membrane and the effect of tamoxifen studied. The present study demonstrated presence of estrogen receptor protein and mRNA in the rat sperm by immunofluorescence, western blotting and in situ hybridization respectively. Specificity of sperm estrogen receptors was confirmed by conventional binding studies using [^3H]-estradiol. There was no effect of tamoxifen treatment on estrogen receptors in rat sperms. Biochemical analysis of the sperms from tamoxifen treated cauda epididymides revealed a significant increase in the levels of calcium and cAMP. A significant reduction was also apparent in the activity of Dynein ATPase. Tamoxifen treatment did not alter phosphodiesterase activity. Estrogen receptors could be identified both in the control as well as tamoxifen treated rat sperms. It was concluded that tamoxifen treatment mobilized calcium from the intra- or extra-cellular pools with a concomitant increase in cAMP and presumably activation of PKA (protein kinase A). Tamoxifen altered the pattern of sperm motility through a calcium induced block in the activity of Dynein ATPase, presumably through the activation of sperm phosphatase. The putative estrogen receptor mediated signal transduction pathway appears to be directly affected in the tamoxifen treated, sub-motile rat sperm.
Santosh Kumar - One of the best experts on this subject based on the ideXlab platform.
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null mutants of the neurospora actin related protein 1 pointed end complex show distinct phenotypes
Molecular Biology of the Cell, 2001Co-Authors: Santosh Kumar, Michael PlamannAbstract:Dynactin is a multisubunit complex that regulates the activities of cytoplasmic Dynein, a microtubule-associated motor. Actin-related protein 1 (Arp1) is the most abundant subunit of dynactin, and it forms a short filament to which additional subunits associate. An Arp1 filament pointed-end–binding subcomplex has been identified that consists of p62, p25, p27, and Arp11 subunits. The functional roles of these subunits have not been determined. Recently, we reported the cloning of an apparent homologue of mammalian Arp11 from the filamentous fungus Neurospora crassa. Here, we report that N. crassa ro-2 and ro-12 genes encode the respective p62 and p25 subunits of the pointed-end complex. Characterization of Δro-2, Δro-7, and Δro-12 mutants reveals that each has a distinct phenotype. All three mutants have reduced in vivo vesicle trafficking and have defects in vacuole distribution. We showed previously that in vivo dynactin function is required for high-level Dynein ATPase activity, and we find that all three mutants have low Dynein ATPase activity. Surprisingly, Δro-12 differs from Δro-2 and Δro-7 and other previously characterized Dynein/dynactin mutants in that it has normal nuclear distribution. Each of the mutants shows a distinct Dynein/dynactin localization pattern. All three mutants also show stronger Dynein/dynactin-membrane interaction relative to wild type, suggesting that the Arp1 pointed-end complex may regulate interaction of dynactin with membranous cargoes.
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Cytoplasmic Dynein ATPase activity is regulated by dynactin-dependent phosphorylation.
The Journal of biological chemistry, 2000Co-Authors: Santosh Kumar, In Hyung Lee, Michael PlamannAbstract:Abstract Cytoplasmic Dynein is a microtubule-associated motor that utilizes ATP hydrolysis to conduct minus-end directed transport of various organelles. Dynactin is a multisubunit complex that has been proposed to both link Dynein with cargo and activate Dynein motor function. The mechanisms by which dynactin regulates Dynein activity are not clear. In this study, we examine the role of dynactin in regulating Dynein ATPase activity. We show that Dynein-microtubule binding and ATP-dependent release of Dynein from microtubules are reduced in dynactin null mutants, Δro-3 (p150Glued) and Δro-4(Arp1), relative to wild-type. The Dynein-microtubule binding activity, but not the ATP-dependent release of Dynein from microtubules, is restored by in vitro mixing of extracts from Dynein and dynactin mutants. Dynein produced in a Δro-3 mutant has ∼8-fold reduced ATPase activity relative to Dynein isolated from wild-type. However, Dynein ATPase activity from wild-type is not reduced when dynactin is separated from Dynein, suggesting that Dynein produced in a dynactin mutant is inactivated. Treatment of Dynein isolated from the Δro-3mutant with λ protein phosphatase restores the ATPase activity to near wild-type levels. The reduced Dynein ATPase activity observed in dynactin null mutants is mainly due to altered affinity for ATP. Radiolabeling experiments revealed that low molecular mass proteins, particularly 20- and 8-kDa proteins, that immunoprecipitate with Dynein heavy chain are hyperphosphorylated in the dynactin mutant and dephosphorylated upon λ protein phosphatase treatment. The results suggest that cytoplasmic Dynein ATPase activity is regulated by dynactin-dependent phosphorylation of Dynein light chains.
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Two approaches to isolate cytoplasmic Dynein ATPase from Neurospora crassa
Biochimie, 2000Co-Authors: Santosh Kumar, In Hyung Lee, Michael PlamannAbstract:Abstract Cytoplasmic Dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic Dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal Dynein, we isolated/partially purified cytoplasmic Dynein ATPase from the filamentous fungus Neurospora crassa . N. crassa Dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic Dynein that uses Dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate Dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K m , ATP obtained by isolating Dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the Dynein ATPase activity. Like vertebrate Dynein, N. crassa Dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K m , ATP for N. crassa cytoplasmic Dynein is 10- to 15-fold higher than that of the vertebrate enzyme.