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1.
Combined effects of calcium and dibutyryl cyclic AMP on germinal vesicle breakdown in the mouse oocyte 总被引:1,自引:0,他引:1
Mouse oocytes were cultured in the presence of dibutyryl cyclic AMP (dbcAMP) and various agents that affect cytoplasmic calcium concentrations. Treatment that inhibited calcium uptake potentiated the inhibitory effect of dbcAMP and treatments which stimulated cellular calcium uptake overcame the effect of dbcAMP. Elevated extracellular calcium (greater than 10 mM) significantly decreased the inhibitory effect of concentrations of dbcAMP up to 150 microM when compared to control levels of calcium (1.7 mM). In addition, the calcium ionophore A23187 (greater than 1 microM) significantly overcame the effect of dbcAMP in media that contained 1.7 or 20 mM calcium. In the presence of 41 microM-dbcAMP the calcium antagonist verapamil increased (in a dose-dependent fashion) the percentage of oocytes blocked at the germinal vesicle stage, from 21% with 10 microM-verapamil to 99% with 200 microM. A similar dose-dependent, reversible potentiation of the effect of dbcAMP was found with tetracaine, which also lowers cytoplasmic calcium concentrations. These results suggest that a minimum level of cytoplasm calcium is required for the initiation of germinal vesicle breakdown and that the action of dbcAMP is mediated by its effect upon this calcium. 相似文献
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Lambert CC 《Development, growth & differentiation》2008,50(3):181-188
Many ascidian oocytes undergo 'spontaneous' germinal vesicle breakdown (GVBD) when transferred from the ovary to normal pH 8.2 sea water (SW); however, low pH inhibits GVBD, which can then be stimulated while remaining in the low pH SW. Oocytes of Boltenia villosa blocked from GVBD by pH 4 SW undergo GVBD in response to permeant cyclic AMP (8-bromo-cyclic AMP), phosphodiesterase inhibitors (isobutylmethylxanthine and theophylline) or the adenylyl cyclase activator forskolin. This suggests that cAMP increases during GVBD. Removal of the follicle cells or addition of a protease inhibitor inhibits GVBD in response to raised pH but not to forskolin, theophylline or 8 bromo-cAMP. Isolated follicle cells in low pH SW release protease activity in response to an increase in pH. These studies imply that the follicle cells release protease activity, which either itself stimulates an increase in oocyte cAMP level or reacts with other molecules to stimulate this process. Studies with the mitogen-activated protein (MAP) kinase inhibitors U0126 and CI 1040 suggest that MAP kinase is not involved in GVBD. The Cdc25 inhibitor NSC 95397 inhibits GVBD at 200 n m in a reversible manner. 相似文献
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Lambert CC 《Development, growth & differentiation》2005,47(4):265-272
The Ascidiacea, the invertebrate chordates, includes three orders; the Stolidobranchia is the most complex. Until the present study, the onset of oocyte maturation (germinal vesicle breakdown) had been investigated in only a single pyurid (Halocynthia roretzi), in which germinal vesicle breakdown (GVBD) begins when the oocyte contacts seawater (SW); nothing was known about internal events. This study strongly suggests the importance of protein phosphorylation in this process. Herdmania pallida (Pyuridae) functions like H. roretzi; GVBD occurs in SW. Oocytes of Cnemidocarpa irene (Styelidae) do not spontaneously undergo GVBD in SW but must be activated. Herdmania oocytes are inhibited from GVBD by pH 4 SW and subsequently activated by mastoparan (G-protein activator), A23187 (Ca2+ ionophore) or dimethylbenzanthracene (tyrosine kinase activator). This requires maturation promoting factor (MPF) activity; cyclin-dependent kinase inhibitors roscovitine and olomoucine are inhibitory. It also entails dephosphorylation as demonstrated by the ability of the phosphatase inhibitor vitamin K3 to inhibit GVBD. GVBD is also inhibited by the tyrosine kinase inhibitors tyrphostin A23 and genistein, and LY-294002, a phosphatidylinositol-3-kinase inhibitor previously shown to inhibit starfish GVBD. LY-294002 inhibits strongly when activation is by mastoparan or ionophore but not when activated by dimethylbenzanthracene (DMBA). The DMBA is hypothesized to phosphorylate a phosphatase directly or indirectly causing secondary activation, bypassing inhibition. 相似文献
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Hiradate Y Ohtake J Hoshino Y Tanemura K Sato E 《Biochemical and biophysical research communications》2011,(4):691-695
Adrenomedullin (ADM) is a multifunctional hormone that regulates processes as diverse as blood pressure and cell growth. Although expressed in the ovary, the role of ADM in this organ is not clear. In the present study, we found the expression of ADM receptor and receptor activity-modifying proteins in mouse cumulus cells but not in the oocytes. We report that germinal vesicle breakdown (GVBD), which is required for oocyte maturation, is not inhibited by ADM alone. However, ADM in the presence of the nitric oxide donor sodium nitroprusside (SNP) significantly inhibited GVBD. Furthermore, the ADM- and SNP-dependent inhibition of GVBD was abrogated by Akt blockade. Additionally, Akt expression and phosphorylation was exhibited by ADM, suggesting that Akt signaling upstream in cumulus cells is responsible. Additionally, immunohistochemical analysis revealed that ADM was localized in the granulosa cells of developed follicles, implying the possibility that ADM physiologically affects oocyte maturation in vivo. Our results provide the evidence that ADM can act as a GVBD regulator. 相似文献
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Regulation of mouse oocyte maturation: involvement of cyclic AMP phosphodiesterase and calmodulin 总被引:2,自引:0,他引:2
A decrease in mouse oocyte cAMP occurs during commitment to resume meiosis (R. M. Schultz, R. R. Montgomery, and J. R. Belanoff, 1983, Dev. Biol. 97, 264-273). Experiments described in this report were performed to ascertain if oocyte cyclic nucleotide phosphodiesterase (PDE) is involved in this decrease. PDE activity was found in extracts of mouse oocytes. The activity appeared soluble and not membrane bound. For each of three different PDE inhibitors, a positive correlation was found between the ability of increasing concentrations of each compound to inhibit PDE in oocyte extracts and to inhibit germinal vesicle breakdown (GVBD). Moreover, the more potent the PDE inhibitor, the more effectively it inhibited GVBD. The possibility that calmodulin (CaM) plays a role in maturation was examined since CaM modulates PDE activity in other systems. About 0.3% of total oocyte protein is CaM as determined by radioimmunoassay and activation of exogenous PDE. A CaM-dependent step in maturation was suggested since the CaM inhibitors trifluoperazine and calmidizolium inhibited GVBD in a dose-dependent manner. In addition, the CaM inhibitors W7 and W13 inhibited GVBD at lower concentrations than the less-active corresponding congeners W5 and W12. Oocyte extracts contained a CaM-modulated PDE. Activity was inhibited about 50% by addition of EGTA, and fully restored by addition of exogenous CaM and excess calcium. cAMP hydrolysis was inhibited in a dose-dependent manner by either trifluoperazine, calmidizolium, or W7; maximal inhibition was also about 50%. CaM-modulated PDE, however, did not appear to be the target for the effects of CaM inhibitors on GVBD, since concentrations of W7 that inhibited maturation did not inhibit cAMP hydrolysis in the oocyte. Results from these studies suggest that oocyte PDE is involved in the decrease in cAMP associated with resumption of meiosis, but that the CaM-dependent step occurs subsequent to or concurrently with the drop in cAMP. 相似文献
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We have examined the activity of cyclic AMP phosphodiesterase, cyclic GMP phosphodiesterase and the protein activator of cyclic AMP phosphodiesterase in various anatomic and subcellular fractions of the bovine eye. Cyclic GMP hydrolysis was 1.6--12 times faster than hydrolysis of cyclic AMP in the subcellular fractions of the retina and in the precipitate of the rod outer segment. An opposite pattern was seen in the bovine lens, where the hyrolysis of cyclic AMP occurred 17 and 169 times faster than that of cyclic GMP in the supernatant and precipitate of lens, respectively. The activity of cyclic AMP phosphodiesterase was not affected by ethylene-glycol bis(beta-aminoethylether)-N,N'-tetraacetic acid in any fractions except in the retinal supernatant, suggesting that the phosphodiesterase exists primarily as a Ca2+-independent, activator-independent form. However, the protein activator of cyclic AMP phosphodiesterase existed in all fractions examine. A complex kinetic patternwas observed for both cyclic AMP and cyllic GMP hydrolysis by the 105000 times g lens supernatant. The Michaelis constants for both cyclic AMP (1.3-10(-6) and 9.I-10(-6) M) and cyclic GMP (1.04-10(6) AND 1.22 10(-5) M) appeared to be similar. 相似文献
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This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes. 相似文献
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Testosterone, progesterone, and estradiol-17 beta each inhibit cAMP phosphodiesterase activity of mouse oocyte extracts in a concentration-dependent manner. This finding provides an explanation for the inhibitory effect of steroid hormones on germinal vesicle breakdown (GVBD) of mouse oocytes in vitro. Furthermore, it raises the possibility that steroid hormones present in follicular fluid participate in maintaining meiotic arrest in vivo by acting in a nonclassical manner. 相似文献
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The fatty-acid composition of follicular fluid from small and large developing follicles was analysed and the effects of saturated and unsaturated fatty acids on spontaneous breakdown of germinal vesicles were investigated. Fatty acids were bound to bovine serum albumin and cultured with oocytes at 100 mumol/l. Linoleic acid (18:2) was the only fatty acid tested that significantly inhibited breakdown of germinal vesicles (P less than 0.01). The effect was dose-dependent and was greatest at 50 mumol fatty acid/l (% breakdown of control, 81.1 +/- 6.8 vs. 50 mumol linoleic acid/l, 35.4 +/- 7.3; P less than 0.02). Linoleic acid was the major fatty acid, constituting about a third of the total fatty acid in the follicular fluid; followed by 18.9 +/- 1.0% and 16.9 +/- 1.3% oleic acid (18:1) in small and large follicles, respectively. Saturated fatty acids accounted for less than 30% of the total fatty acid composition. There was a marked absence of tetraenoic acids in small and large follicles. Proportions of linoleic acid were significantly lower in follicular fluid from large follicles (31.1 +/- 1.2% of total fatty acid) than from small follicles (34.8 +/- 0.7% of total fatty acid) (P less than 0.05) and there was a significant inverse correlation between follicle diameter and percentage of linoleic acid in the follicular fluid (r = -0.6966; P less than 0.05). There was no significant alteration in any other fatty acid during follicular development.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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T E Donnelly 《Biochimica et biophysica acta》1978,543(3):273-282
CuCl2 non-competitively inhibited the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent phosphodiesterase from bovine heart in the presence of 5 mM Mg2+, 10 muM Ca2+ and phosphodiesterase activator with Ki values of approximately 2 muM for both substrates. CuCl2 inhibition was also non-competitive with Mg2+, Ca2+ and phosphodiesterase activator. Dialysis demonstrated that CuCl2 inhibition is reversible. Treatment of the enzyme with p-hydroxymercuribenzoate resulted in the loss of enzyme activity, suggesting the presence of sulfhydryl groups essential for enzyme activity. The inhibitory activity of CuCl2 was not additive with that of p-hydroxymercuribenzoate, therefore CuCl2 may inhibit enzyme activity by binding to one or more essential sulfhydryl groups. CuCl2 also inhibited the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase from bovine heart with an I50 value of 18 muM. Several effects of Cu2+ are discussed which have been noted in other studies and might be due, in part, to changes in cyclic nucleotide levels following alterations in phosphodiesterase activity. 相似文献
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Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (Km 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (Km 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg2+. Mn2+ (3mm) was as effective as Mg2+ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca2+ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg2+ was 3mm. At concentrations of Mg2+ between 0.1 and 1mm, 1mm-Ca2+ was activatory, and at concentrations of Mg2+ below 0.1mm, 1mm-Ca2+ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling. 相似文献
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T E Donnelly 《Biochimica et biophysica acta》1978,522(1):151-160
In the presence of 10 micrometer Ca2+ and 5 mM Mg2+ (or 0.25 mM Mg2+), the addition of 100 micrometer Zn2+, Ni2+, Co2+, Fe2+, Cu2+ or 1 mM Mn2+ resulted in varying degrees of stimulation or inhibition of 10(-6) M cyclic GMP and cyclic AMP hydrolysis by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart in the absence or presence of phosphodiesterase activator. The substrate specificity of the enzyme was altered under several conditions. The addition of Zn2+ in the presence of 5 mM Mg2+ and the absence of activator resulted in the stimulation of cyclic GMP hydrolysis over a narrow substrate range while reducing the V 65% due to a shift in the kinetics from non-linear with Mg2+ alone to linear in the presence of Zn2+ and Mg2+. Zn2+ inhibited the hydrolysis of cyclic GMP and cyclic AMP in the presence of activator with Ki values of 70 and 100 micrometer, respectively. Zn2+ inhibition was non-competitive with substrate, activator and Ca2+ but was competitive with Mg2+. In the presence of 10 micrometer Ca2+ and activator, a Ki of 15 micrometer for Zn2+ vs. Mg2+ was noted in the hydrolysis of 10(-6) M cyclic GMP. Several effects of Zn2+ are discussed which have been noted in other studies and might be due in part to changes in cyclic nucleotide levels following phosphodiesterase inhibition. 相似文献
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Tyrosine, as well as small amounts of phenylalanine, were removed selectively and quantitatively from purified chick brain tubulin by enzymatic digestion with carboxypeptidase. The fraction of molecules containing hydrolyzable tyrosine changed with the stage of development and had the highest value (~0.5) around days 14–16 of the embryo. The increase in the fraction of tyrosinated molecules was found to be temporally correlated with an increased specific activity of the enzyme catalyzing the incorporation reaction. In addition, it was found that the availability of α-chain C-termini for tyrosination also reached a maximum during the same period. Changes in the extent of modification of the C-terminus of tubulin may be relevant for the participation of the resultant microbubules in different developmental events. 相似文献
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Induction of cyclic AMP phosphodiesterase in Blastocladiella emersonii and its relation to cyclic AMP metabolism. 总被引:2,自引:2,他引:0
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Extracts of vegetative cells of Blastocladiella emersonii contain 5% or less of the cyclic AMP phosphodiesterase activity in zoospore extracts. This difference in activity could be accounted for entirely by an increase in the differential rate of phosphodiesterase synthesis during sporulation, beginning after a lag period of about 60 min and extending for at least an additional 90 min into the 4-h sporulation process. To examine the relation between enzyme synthesis and cyclic nucleotide metabolicm, we determined the substrate specificity of phosphodiesterase synthesized during sporulation and partially purified from zoospores. Zoospore extracts contain two components, separable by gel filtration chromatography, with cyclic AMP phosphodiesterase activity. The larger component accounts for 20% of the total activity and the smaller component for 80%. Both components show essentially an absolute substrate specificity for cyclic AMP among several cyclic purine and cyclic pyrimidine nucleotides tested. Nevertheless, we found no change in the total cyclic AMP content of sporulating cells before, during, or after enzyme activity increased. We speculate that some other component of cyclic AMP metabolism or function limits the rate of cyclic AMP hydrolysis in sporulating cells. 相似文献