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1.
Fertilization of sea urchin eggs results in the rapid recruitment of stored messages into polyribosomes. Whether translational control in sea urchin eggs is mediated by macromolecules associated with the stored messages remains unknown, since preparations of messenger ribonucleoprotein complexes (mRNPs) were active in protein synthesis in a rabbit reticulocyte lysate. To facilitate the study of mRNPs, chromatography on oligo(dT)-cellulose was used to purify poly(A)-containing mRNPs from eggs and embryos of the sea urchin Strongylocentrotus purpuratus. Nonpolyribosomal mRNPs purified from eggs had a similar sedimentation in sucrose to unpurified mRNPs, a peak buoyant density in metrizamide of 1.22 g/cm3, and peak buoyant densities in Cs2SO4 in 1.42 g/cm3 after fixation with glutaraldehyde and 1.46 g/cm3 without fixation. Nonpolyribosomal mRNPs from eggs and zygotes contained 5-10 major proteins on sodium dodecylsulfate (SDS) polyacrylamide gels, and numerous minor bands. UV-irradiation of living eggs of the sea urchin Arbacia punctulata produced cross-linked mRNPs which contained a similar pattern of polypeptides to noncross-linked mRNPs. The polypeptides associated with embryonic polyribosomal mRNPs were also qualitatively similar to those present in nonpolyribosomal mRNPs, although stoichiometric differences may exist.  相似文献   

2.
In sea urchin eggs, 10 μg/mL melittin was found to induce fertilization envelope formation without any increase in [Ca2+]i (the intracellular free Ca2+ level). On the other hand, 10 μmol/L Br-A23187 and 100 μg/mL SDS induced fertilization envelope formation associated with [Ca2+]i increase. If EGTA was injected into eggs to make an intracellular concentration of 2 mmol/L, [Ca2+]i became quite low and was not altered by melittin, or by Br-A23187 and SDS. In eggs containing EGTA, fertilization envelope formation was induced by melittin even in Ca2+-free artificial sea water, but not by Br-A23187 or SDS. Thus [Ca2+]i is essential for induction of a fertilization envelope in sea urchin eggs by Br-A23187 or SDS but not by melittin. Melittin probably activates some Ca2+-independent reaction downstream of Ca2+-dependent reactions in a sequential reaction system that finally results in fertilization envelope formation.  相似文献   

3.
Abstract Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens were isolated. Sporulating cells were lysed by sonication in the presence of protease inhibitors. IB were isolated by centrifugation in linear gradients of sucrose, sodium bromide or sodium diatrizoate and banded at buoyant densities of 1.33–1.36 g/cm3, 1.30–1.34 g/cm3 and 1.33 g/cm3, respectively. Isolated IB were treated with detergent to remove attached cell membrane. They ranged in size from 0.5–1.4 μm long and from 0.2–0.5 μm wide. They were found to be serologically related to purified enterotoxin.  相似文献   

4.
Ryanodine Activates Sea Urchin Eggs   总被引:3,自引:2,他引:1  
We have studied the effect on sea urchin eggs of ryanodine, a plant alkaloid that causes muscle contraction by opening calcium channels in the sarcoplasmic reticulum terminal cisternae. Ryanodine, although it is less effective that IP3, produces full or partial activation in 62% of injected sea urchin eggs. In addition ryanodine inhibits in a dose dependant manner 45Ca pumping in the isolated egg cortex or in eggs permeabilized with digitonin. Efflux experiments show that in fact ryanodine as IP3 stimulates the release of calcium sequestered intracellularly. We further show that these effects of ryanodine are inhibited by Mg++, ruthenium red and heparin. Our results suggest that ryanodine-sensitive intracellular calcium channels exist in the sea urchin egg.  相似文献   

5.
In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus , fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca2+ free- or Ca2+, Mg2+ free- artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4-dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO-treated eggs exhibited cyanide-insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.  相似文献   

6.
Immediately after fertilization sea urchin eggs undergo an increase in cytoplasmic pH from 6.8 to 7.2. This pH change occurs by activation of a Na+/H+ antiporter, and is a necessary signal for later steps in metabolic activation of development. Activators of protein kinase C such as phorbol myristate acetate (PMA) and diacylglycerol produce a similar pH increase in eggs. Phosphorylation of the antiporter or a regulatory protein may be a step in activating Na+/H+ exchange. Here we show that treatment of sea urchin eggs ( S. purpuratus ) with PMA results in increased phosphorylation of over a dozen proteins. Of these, three proteins of Mr=240, 92 and 80 kD are located in the egg cortex; under-representation of these bands in isolated cortical granules suggests that they are plasma membrane-associated. Phosphorylation of the 92 kD band is concentration-dependent over a range of 10 to 1000 nM PMA and occurs over a time-course of 1 to 3 min. Phosphoamino acid analysis indicates that phosphorylation is on serine residues. Phosphorylation appeares to be mediated by protein kinase C since the inactive PMA analogue, 4α-phorbol 12, 13-didecanoate, does not induce phosphorylation nor does experimental alkalinization of the egg cytoplasm.  相似文献   

7.
Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca2+ dependence to M-phase onset. A potential target of this Ca2+ dependence may be CaM kinase-II (the multifunctional [type II] Ca2+/calmodulin [CaM]-dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK-II is activated after fertilization and whether it interacts with other known M-phase regulators, such as p34cdc2. We report that total CaMK-II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK-II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK-II phosphoproteins after fertilization. Finally, CaMK-II activity and only the 62 kDa phosphoprotein physically associate with p34cdc2, but again only after fertilization. These changes in CaMK-II activity and p34cdc2-association after fertilization may ensure that Ca2+ signals are targeted to the M-phase machinery at the appropriate developmental times.  相似文献   

8.
Spermatozoa of the sea urchin, Hemicentrotus pulcherrimus (108 cells/ml), preincubated with unfertilized eggs deprived of jelly coats (more than l05 cells/ml) at 20°C for 20min in Mg2+ free artificial sea water containing 1 mM Ca2+ (MFASW), exhibited very low respiration, which was enhanced by 2, 4 dinitrophenol (DNP). The fertilization rate in MFASW was usually less than 5% and was about 25% at most. Preincubation with fertilized eggs (with and without a fertilization membrane) in MFASW did not reduced the respiratory rate of spermatozoa. The rate of sperm respiration was lower in MFASW than in artificial sea water (ASW), but was higher than the respiratory rate of spermatozoa preincubated in MFASW with unfertilized eggs. Sperm respiration in MFASW or in ASW was not stimulated by 2, 4 dinitrophenol. Almost complete inhibition of sperm respiration was obtained with unfertilized eggs fixed with glutaraldehyde at concentrations of above 105 cells/ml in MFASW and of about l04 cells/ml in ASW. The respiratory rate of spermatozoa treated with fixed eggs was enhanced by DNP. It is concluded that the respiratory rate of the spermatozoa is reduced by their interaction with unfertilized eggs before their penetration into the eggs.  相似文献   

9.
Protein kinase C (PKC) has been shown to play a role in events involved in fertilization such as activation of the Na+/H+antiporter and an NADPH dependent oxidase. In addition, it is involved in cell fate programming later in development of the sea urchin embryo. In order to further address the role of PKC in sea urchin development, we have screened a Lytechinus pictus ovary tissue cDNA library and identified one clone for sea urchin protein kinase C (suPKC1). This clone encodes a deduced protein with a molecular mass of 72.4 kDa, which shows strong homology to invertebrate and mammalian protein kinase C (PKC) sequences. PKC has been partially purified from eggs of L. pictus. This kinase activity has been shown to be dependent upon phosphatidylserine, diacylglycerol and Ca2+. In agreement with this biochemical data, suPKC1 has a C2 or Ca2+-binding domain suggesting its activity would be Ca2+-dependent. Polyclonal antibodies raised against peptides of the suPKC1 sequence recognize an antigen of approximately 71 kDa in DE52 fractions that contain PKC activity; this reactivity is not observed in fractions that lack PKC activity. Using a ribonuclease protection assay, we have demonstrated the presence of suPKC1 message throughout developmental stages of the sea urchin embryo.  相似文献   

10.
Thapsigargin (Tg), an inhibitor of microsomal Ca2+ ATPase, is used as a tool to study the changes in Ca2+ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP-dependent Ca2+ sequestration in a dose-dependent and non-reversible manner, depending on the bulk of biological material used. IC5O values are 1 nmol/L and 1–10μmol/L, respectively, in the cortical Ca2+ stores (isolated cortices preparation) and in digitonin-permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca2+ release from 45Ca pre-loaded cortices but leads to a loss of 25% of the total Ca2+ content from the cortical area. Using microspectrofluorimetry of fura-2-loaded eggs, we found that 10 μmol/L Tg induced a moderate rise in cytosolic Ca2+ activity as compared with the fertilization-induced Ca2+ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10μmol/L Tg but with a delayed onset relative to sperm-induced effects. The present findings indicate that although it triggers most fertilization-related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca2+ sequestration.  相似文献   

11.
We have purified and extensively characterized a 19-S particle from sea urchin eggs. This particle is the sea urchin homologue of the "prosome", a particle originally identified in duck erythroblasts. We now show that these sea urchin prosomes contain multiple proteolytic activities. As shown for analogous particles from other cells, these particles hydrolyze synthetic substrates containing neutral hydrophobic or basic amino acids at the carboxy terminus of the synthetic peptides. They contain 16-20 small proteins ranging in molecular weight from 20,000 to 32,000. Peptide mapping shows that most of the polypeptides are unique, however, three exist in two isoelectric forms. We have investigated the possible function of the sea urchin multicatalytic proteases (MCPs) by determining their subcellular distribution, their relationship to egg snRNPs, and their possible role in translational repression. There are almost as many MCPs (2 x 10(8] as ribosomes (6.6 x 10(8] or mRNPs (1.8 x 10(7] per egg. This suggests that like ribosomes, the MCPs are stored in the egg for use during later development. We find that a substantial proportion of egg MCPs move into nuclei by the late blastula stage. Using a specific antibody against one of the sea urchin MCP proteins and antibodies against U1-U6, La, and Ro RNPs, we show that the sea urchin particle is distinct from these RNPs, although the anti-U1-U6 RNP antibody cross-reacts with a single MCP protein. In addition, the sea urchin MCP appears to be associated with a large structure in the cytoplasm of unfertilized eggs and is released under the same conditions that activate egg mRNPs in vitro.  相似文献   

12.
The sulfating system in sea urchin embryos was examined, using the labeled precursor inorganic [35S]sulfate in vivo and [35S]3'-phosphoadenosine 5'-phosphosulfate ([35S]PAPS) in a cell-free system. In vivo incorporation of [35pS]sulfate into the trichloroacetic acid (TCA)-insolubte fraction increased gradually during sea urchin development, whereas radioactivity of [35S]sulfate contained in the TCA-soluble fraction showed a conspicuous peak at the late gastrula stage.
In a cell-free system, the particulate fraction showed marked incorporation of [35pS]JPAPS. This sulfating activity was highest at pH 6.4 to 7.2 and at 27°C, and it was strongly inhibited by Hg 2+and p-chloromercuribenzoic acid.
The sulfating activity was quite low in fertilized eggs, but then increased rapidly up to the swimming blastula stage. The activity in the particulate fraction precipitated at 10,000 xg increased gradually and that in the particulate fraction precipitated at 100,000 xg was almost constant from the swimming blastula stage to the pluteus stage.  相似文献   

13.
Eggs of the sea urchin, Hemicentrotus pulcherrimus , were stimulated by halothane, known to induce Ca2+ release from sarcosome, to cause fertilization membrane formation in normal and Ca2+ free artificial sea water. In the absence of external Ca2+, halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca2+ release from sarcosome, but was not blocked by nifedipine, a Ca2+ antagonist specific to Ca2+ channels in plasma membrane. Ca2+ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. 45Ca2+ influx was substantially stimulated by halothane in eggs exposed to 45CaCl2. Halothane-induced 45Ca2+ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca2+ release from intracellular organellae is blocked, Ca2+ transport through Ca2+ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca2+ level, resulting in egg activation.  相似文献   

14.
Fertilization of sea urchin eggs results in a large increase in the rate of protein synthesis which is mediated by the translation of stored maternal mRNA. The masked message hypothesis suggests that messenger ribonucleoprotein particles (mRNPs) from unfertilized eggs are translationally inactive and that fertilization results in alterations of the mRNPs such that they become translationally active. Previous workers have isolated egg mRNPs by sucrose gradient centrifugation and have assayed their translational activity in heterologous cell-free systems. The conflicting results they obtained are probably due to the sensitivity of mRNPs to artifactual activation and inactivation. Previously, we demonstrated that unfractionated mRNPs in a sea urchin cell-free translation system were translationally inactive. Now, using large-pore gel filtration chromatography, we partially purified egg mRNPs while retaining their translationally repressed state. Polysomal mRNPs from fertilized eggs isolated under the same conditions were translationally active. The changes in the pattern of proteins synthesized by fractionated unfertilized and fertilized mRNPs in vitro were similar to those changes observed in vivo. Treatment of egg mRNPs with buffers containing high salt and EDTA, followed by rechromatography, resulted in the activation of the mRNPs and the release of an inhibitor of translation from the mRNPs. Analysis of the inhibitory fraction on one-dimensional sodium dodecyl sulfate gels indicated that this fraction contains a complex set of proteins, several of which were released from high-salt-EDTA-activated mRNPs and not from inactive low-salt control mRNPs. One of the released proteins may be responsible for the repression of egg mRNPs in vitro and be involved in the unmasking of mRNPs at fertilization.  相似文献   

15.
The Mg2+ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg2+, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca2+, were used, the fertilization rate remained quite low in the absence of Mg2+. In Strongylo-centrotus intermedius , the lowest concentration of Mg2+ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca2+, whereas that of calcium was 3 mM in the presence of 49 mM Mg2+. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg2+ or Ca2+ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg2+ and/or Ca2+. These results indicate that Mg2+ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.  相似文献   

16.
Sea urchin eggs kept in artificial sea water (ASW) containing 0.01–0.3 M NaSCN in place of NaCI from within 2 min after insemination formed thin, enlarged fertilization envelopes, which were broken on mild agitation of egg suspensions more easily than those formed in Ca2+-free ASW. The blastomeres of almost all embryos derived from eggs treated with 0.2M SCN for 1 hr dissociated spontaneously, and did not reassociate with other blastomeres appreciably. Thus SCN probably denaturated some compound(s) participating in blastomere binding and hardening of the fertilization envelope. Abnormal arrangements of blastomeres, probably due to incomplete blastomere dissociation, were observed in embryos derived from eggs treated with 0.1 M SCN for 1 hr. Treatment of fertilized or unfertilized eggs with 0.05–0.1 M SCN for a short period caused concentration-dependent block of morphogenic processes such as formation of the archenteron and pluteus arms in the post-hatching period. The effects of SCN on morphogenesis were not inhibited by furosemide or 4,4'-diisothiocyano 2,2'-disulfonic stilbene. Presumably, the denaturation of several compounds in the egg surface by SCN causes abnormal morphogenesis of embryos. The inhibitory effects of SCN on hardening of the fertilization envelope, blastomere binding and morphogenesis were greater in the absence of Ca2+.  相似文献   

17.
Analysis of the literature on pelagic fish eggs enabled generalizations to be made of their energy densities, because the property of being buoyant in sea water appears to constrain the proximate composition of the eggs and thus to minimize interspecific variation. An energy density of 1.34 J μl−1 of total egg volume is derived for most species spawning eggs without visible oil globules. The energy density of eggs with oil globules is predicted by     x (J μ11) where x is the fractional volume of the oil globule.  相似文献   

18.
Two rapid methods, direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) on swab resuspension fluids, were compared with the traditional total viable count (TVC) on swab resuspension fluids for their ability to enumerate surface populations of attached bacteria. The degree of error in estimating surface populations was shown to be significantly less with DEM than DEFT followed by TVC. DEM estimated populations in the range 3 times 103 to 5 times 107 colonies/cm2 whilst DEFT enumerated populations above 3 times 104 colonies/cm2 and TVC above 3 times 105 colonies/cm2 (as measured by DEM). Swabbing was shown to remove a constant proportion of organisms from the surface populations tested, although below 3 times 105 colonies/cm2 most of the organisms remained in the cotton matrix and were difficult to resuspend. DEFT was more able to enumerate swab resuspension fluids obtained from surface populations below 3 times 105 colonies/cm2 than was TVC.  相似文献   

19.
Eriophorum vaginatum tillers were collected at Eagle Creek, Alaska and cultivated in aerated solutions under controlled environmental conditions. The nutrient solutions contained traces, 1.05 and 21 mg l−1 N (nitrate) and traces, 0.15 and 3.10 mg l−1 P (phosphate), pH was maintained at 5.5. The high N, 21 mg l−1, and P, 3.18 mg l−1, nutrient solution produced significant biomass increases. Functional leaf areas were significantly enhanced by high N and P doses in the solutions. Root surface areas varied considerably between treatments; however, significant differences were not found. The mean root surface area of a tiller reached 126 cm2 (range 35–290 cm2), whereas the functional leaf area was 6.8 cm2 (range 3.3–20.3 cm2). Tillers growing in the highest N + P solutions produced twice the number of daughter tillers as tillers growing in solutions with trace amounts of N and P.  相似文献   

20.
ABSTRACT. Pentrich ciliates attached to small stones from the beds of two streams, one large with hard water, the other small with soft water, were enumerated throughout an annual cycle. Throughout the year, Platycola was the dominant peritrich in both streams, except for a brief period during the spring when Vorticella and Carchesium predominated. Vorticella reached peak levels of 89 ciliates cm2 of stone surface, and up to 102 Platycola per cm2 of stone surface were found. Mean volumes of samples of the main species were calculated, and used to estimate the standing stock biomasses. using a standard value of dry weight per unit volume. Published values of the growth rates of representatives of the main genera were used to estimate production values, which totalled about 6.5 g dry weight of peritrich cytoplasm/m2 of stream bed per annum in the large stream (mean annual density = 8.3 peritrichs/cm2 of stone surface), and 33 g dry weight/m2 of stream bed per annum in the small stream (mean annual density = 47 peritrichs/cm2 of stone surface). Food supply, temperature and predation were the primary factors determining peritrich abundance  相似文献   

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