ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

Some analyses of artificially incubated eggs and hatchlings of Green and Loggerhead sea turtles

Eggs of the Green sea turtle ( Chelonia mydas ) and the Loggerhead sea turtle ( Caretta caretta ) were incubated in coral sand or silica sand moistened with distilled water. The eggs and hatchlings were analysed for calcium, magnesium and phosphorus. There was less magnesium but more calcium in the hatchlings than in the original egg contents but this was not influenced by the type of sand used during incubation. The results are interpreted as indicating that the eggshell is the major source of calcium for the developing embryos. The Logger-head sea turtle shows a greater efficiency in the incorporation of yolk magnesium and phosphorus into the embryo than does the Green sea turtle.     

Activation of sea urchin eggs by inositol phosphates is independent of external calcium.

We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in sea urchin eggs. We show that: (a) inositol phosphates activate eggs of the sea urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes are independent of external calcium; (c) in calcium-free seawater, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free sea water are more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate sea urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports.     

MAGNESIUM ION-REQUIRING STEP IN FERTILIZATION OF SEA URCHINS*

The magnesium ion-requiring step in fertilization of sea urchins was investigated. When eggs were inseminated in Mg-free sea water, several spermatozoa were found to bind to each egg surface with their reacted acrosomes without elevation of fertilization membrane. The number of binding jelly-treated spermatozoa to an egg did not differ regardless of the presence or virtual absence of magnesium ions. Although fertilization did not occur in Ca, Mg-deficient sea water (CM-deficient SW) even when jelly-treated spermatozoa were employed, some eggs could be fertilized by the addition of magnesium to the CM-deficient SW 60 sec after insemination, when jelly-treated spermatozoa had completely lost their fertilizing capacity in the CM-deficient SW. The acrosomal process of jelly-treated spermatozoa appeared to penetrate the vitelline layer in the CM-deficient SW. DTT- or pancreatin-treated eggs could not be fertilized in the virtual absence of magnesium. Re-fertilization using the fertilized eggs deprived of fertilization membrane did not occur under conditions of magnesium deficiency. These results suggest that external magnesium ions are indispensable at least for the fertilization process following penetration of the vitelline layer by the spermatozoa, such as fusion of the plasma membrane between an egg and a reacted spermatozoon, or the subsequent step(s) such as sperm penetration into egg interior and egg activation which precedes the cortical reaction.     

Significance of an Increase of Intracellular Adenosine Concentration for Dormancy in Starfish Blastulae

Adenosine at concentrations greater than 6 μg/ml halted embryonic development of the starfish Asterina pectinifera specifically at the 256-cell stage which corresponds to the onset of blastulation. When a fertilized egg was cultured continuously in sea water containing adenosine from fertilization, a gradual increase in intracellular concentrations of free adenosine was observed before a cessation of development took place. On the other hand, intracellular concentrations of ATP, ADP and AMP in the embryo cultured in sea water containing adenosine were nearly the same as those of an embryo cultured in sea water without adenosine. By returning the development-arrested embryo to normal sea water the embyro developed normally to the bipinnaria stage accompanied by a gradual decrease in the intracellular cencentration of adenosine.
Treatment of fertilized eggs with 9-β-d-arabinofuranosyl-9H-purine-6-amine (25 μg/ml) or 2'-deoxyadenosine (10 μg/ml) halted development specifically before the onset of blastulation in an irreversible manner. Embryos treated with 3'-deoxyadenosine (50 μg/ml) shortly after fertilization developed to healthy blastulae but hatching never occurred. These results exclude the possibilities that the action of adenosine is mediated by the inhibition of DNA synthesis or RNA polyadenylylation.     

A marker of animal-vegetal polarity in the egg of the sea urchin Paracentrotus lividus. The pigment band

We have examined the subequatorial accumulation of pigment granules (the so-called 'pigment band') in the egg of the sea urchin Paracentrotus lividus, which constitutes an unambiguous marker of animal-vegetal polarity. Most of the reddish pigment granules are situated at the periphery of the egg. They exhibit occasional saltatory movements and can aggregate into large patches. Pigment granules are retained as a band in the isolated cortex when the egg surface complex is isolated by shearing eggs attached to polylysine-coated surfaces with calcium-free isotonic solutions. Pigment granules remain as the main vesicular component of fertilized egg cortices or of unfertilized egg cortices perfused with calcium to provoke cortical granule exocytosis. They may be anchored to the isolated cortex through associations with the plasma membrane and with an extensive subsurface network of rough endoplasmic reticulum (rough ER). Pigment granules contain antimonate-precipitable calcium and, in this respect and many others, resemble acidic vesicles recently identified in the cortex of unpigmented sea urchin eggs. We discuss the similarities observed between granules and acidic vesicles in various urchin egg species and their possible functions.     

银鲴性腺发育和胚胎发育的观察

生长在湖南江河、池塘、水库环境的银鲴,性成熟年龄一般是二冬龄(2+),雄性比雌性提前一年成熟。江河银鲴每到生殖季节,可以自行产卵、受精,繁衍后代;池养银鲴却不能自然产卵,其卵母细胞只能发育到初级卵母细胞阶段(Ⅳ时相),必须通过人工催情,才使卵母细咆由第Ⅳ时相发育到第Ⅴ时相。池养雄性银鲴,能够完成由精原细胞到精子的全部发育过程。冬季,达性成熟的银鲴,雌性卵巢为Ⅲ期,雄性精巢为Ⅳ期。每年的3月份,银鲴开始进入性成熟期,4月份为它的生殖盛期。银鲴是鲴亚科中产半浮性卵的鱼类。银鲴在胚胎发育早期行盘状卵裂。当水温21—23℃时,其胚胎发育历经26小时10分,胚胎发育总热量为550—602度时;银鲴从受精卵到仔鱼期结束(鱼苗下池),整个过程历时112小时,区分胚胎发育、仔鱼发育两个阶段。胚胎发育又划为5个时期。       

Effects of salinity on egg development and hatching in grey mullet Mugil cephalus L.

Grey mullet, Mugil cephalus , collected from ponds were induced to spawn with carp pituitary homogenate and human chorionic gonadotropin (HCG) at 30%_o salinity and 26° C. The spontaneously spawned and fertilized eggs were transferred to sea water of different salinities in the range of 5–70%_o, either at the 2-blastomere or the gastrula stage. Those eggs transferred at the gastrula stage were more tolerant to the salinity change than were those transferred at the 2-blastomere stage. The eggs did develop to the embryonic stage within the salinity range from 5 to 60%_o. Hatching occurred in all salinities between 10 and 55%_o. However, no larvae survived at 10 or at 55%_o. The optimal salinity range for eggs incubating at 22.0–25.5° C was from 30 to 40%_o salinity, with the peak at 35%_o.     

盐度对沙蜇有性繁殖阶段早期发育的影响

近几十年来,沙蜇的频繁暴发给东亚海域的海洋生态系统带来了广泛影响。在秋季,沙蜇成熟的雌雄水母体在沿岸水域聚集产卵,有性繁殖产生的受精卵发育成新的底栖螅状体,为螅状体种群数量进行补充。河口浅滩海域为沙蜇的繁育地,沿岸盐度较低,在秋季降雨期盐度多变,较低、多变的盐度可能对沙蜇有性繁殖阶段的早期发育产生重要作用,从而影响螅状体种群数量的补充。实验设置了4种不同盐度(15、20、25、30)试验组,在不同盐度下对沙蜇受精卵进行培养,探讨盐度对沙蜇早期发育过程中受精卵、浮浪幼虫发育以及早期螅状体生长及存活的影响。试验结果:沙蜇受精卵胚胎发育的适宜盐度为20,发育基本与盐度25、30同步,盐度15受精卵细胞发育迟缓,发育率显著降低;浮浪幼虫发育适宜盐度为20和25,两组浮浪幼虫附着变态率高于盐度15、30,盐度15时浮浪幼虫活力明显降低、发育迟缓,浮浪幼虫在盐度15时水中存活时间较长可达8 d,但附着时间集中在培养后的3、4天,与其他组相同;早期螅状体幼体适宜盐度为20、25、30,早期螅状体存活率、相对增长率及特定生长率均显著高于盐度15,三组间差异不显著。结果表明,盐度显著影响沙蜇有性繁殖阶段...     

CHANGES IN ACTIVITIES OF THYMIDYLATE SYNTHASE AND DIHYDROFOLATE REDUCTASE IN SEA URCHIN EGGS AFTER FERTILIZATION

Thymidylate synthase activity in sea urchin eggs increases just after fertilization and decreases 30 min later. Then, cyclic variation in the activity occurs in association with the cleavage cycle. Dihydrofolate reductase activity in fertilized eggs is almost the same as in unfertilized eggs and shows no marked change within 3 hr after fertilization. Aminopterin, an analogue of dihydrofolate, inhibits dihydrofolate reductase, and arrests cleavage. On incubation in sea water containing aminopterin (20-100μM) from the time of fertilization, the development of Clypeaster and Pseudocentrotus eggs was arrested at the 32–64 cell stage, and that of Anthocidaris eggs was arrested at the morula stage. Dihydrofolate (100μM) counteracts the inhibitory effect of aminopterin on egg cleavage. Thymidine at concentrations above 10μM also prevents inhibition by aminopterin. Other deoxyribonucleosides at concentrations of 10μM to 100μM do not affect inhibition of egg cleavage by aminopterin. Deoxyadenosine at concentrations above 5 mM inhibits egg cleavage, but other deoxyribonucleosides have no effect.     

Calcium mobilization, water balance, and growth in embryos of the agamid lizard Amphibolurus barbatus

Embryos of the agamid lizard Amphibolurus barbatus are at developmental stage 29 of Dufaure and Hubert at the time of oviposition. Mobilization of calcium and other nutrients from the yolk proceeds slowly for the first half of incubation, during which time embryonic growth also proceeds slowly. During the second half of incubation, however, embryos withdraw calcium and other nutrients from the yolk very rapidly, and growth rates are correspondingly high. Approximately 60% of the calcium used by developing embryos is obtained from the yolk, but fully 40% of their requirement is met by calcium mobilized from the eggshell. Very little calcium remains in residual yolk of hatchlings, so this yolk must be used in maintenance metabolism rather than in growth of neonates. No dichotomy exists among oviparous, amniotic vertebrates with respect to sources of calcium used by developing embryos, but one does exist with respect to patterns of mobilization of this element. Whereas calcium is extracted from yolk of embryonic reptiles throughout incubation, it actually is deposited in yolk of embryonic birds after the midpoint in development.     

Mechanism for Increase in Intracellular Concentration of Free Calcium in Fertilized Sea Urchin Egg : A Method for Estimating Intracellular Concentration of Free Calcium

Intracellular free calcium concentration in the sea urchin egg was calculated to increase from 0.1 mM in an unfertilized egg to 1 mM in a fertilized egg 10 min after fertilization, based on measurement of the dissociation constant between free calcium and sea urchin egg homogenate. The dissociation constant between free calcium (dialyzable calcium) and homogenate of sea urchin eggs was measured by means of dialysis equilibrium. The dissociation constant of the unfertilized egg was about 10^–4 M and that of the fertilized egg was about 10–3 M in three species of sea urchin, Hemicentrotus pulcherrimus, Anthocidaris crassispina, and Pseudocentrotus depressus. An increase in the dissociation constant of the unfertilized egg homogenate was observed after the addition of calcium ion at a concentration above 0.3 mM, the dissociation constant becoming the same as that observed in the fertilized egg homogenate after the administration of CaCl₂ at a concentration above 1 mM. Sodium ion also caused a decrease in the calcium-binding ability of the unfertilized egg homogenate. Therefore, penetration of calcium ion or sodium ion upon fertilization might induce an increase in the dissociation constant and then intracellular concentration of free calcium would increase at fertilization. Almost all calcium-binding ability of the egg homogenate was found in the microsomal fraction, and the substance which bound calcium was thought to be protein in nature, since trypsin could decrease the level of calcium-binding substance in the homogenate of the eggs.     

Temperature‐mediated survival,development and hatching variation of Pacific cod Gadus macrocephalus eggs

Laboratory‐validated data on the survival, development and hatching responses of fertilized Pacific cod Gadus macrocephalus eggs from the northern Japan stock were determined through an incubation experiment. The optimum temperature for survival until hatching ranged from 4 to 8° C. No significant difference in development rates was found between the populations from Mutsu Bay, Japan, and western Canadian coastal waters even though the samples may belong to different G. macrocephalus stocks. Gadus macrocephalus larvae hatched asynchronously from egg batches despite incubation under the same environment during their development. Both incubation temperature and temperature‐mediated hatch rank affect size and yolk reserve. These data suggest that variations in water temperatures within an ecological range markedly influence the development rates, survival and hatching of the eggs, as well as the stage at hatch larvae of G. macrocephalus. Asynchronous hatching and the production of offspring with variable sizes and yolk reserves are considered evolutionary bet‐hedging strategies that enable the species to maximize their likelihood of survival in an environment with variable temperatures.     

Solubilization and Identification of Hen Eggshell Membrane Proteins During Different Times of Chicken Embryo Development Using the Proteomic Approach

A fertilized chicken egg is a unit of life. During hatching, transport of nutrients, including calcium, have been reported from the egg components to the developing embryo. Calcium is mobilized from the eggshell with the involvement of Ca²⁺-binding proteins. In addition, other unknown proteins may also play some important roles during embryo developing process. Therefore identification and prediction of biological functions of eggshell membrane (ESM) proteins during chick embryo development was conducted by proteome analysis. Comparison of different lysis solutions indicated that the highest ability to extract ESM proteins could be obtained with 1 % sodium dodecyl sulfate in 5 mM Tris–HCl buffer pH 8.8 containing 0.1 % 2-mercaptoethanol. In this study fertilized Cornish chicken eggs were incubated at 37 °C in humidified incubators for up to 21 days. At selected times (days 1, 9, 15 and 21), samples were taken and the ESMs were carefully separated by hand, washed with distilled water, and air-dried at room temperature. The ESM proteins were then solubilized and analyzed by proteome analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with high performance liquid chromatography and mass spectrometry revealed 62 proteins in the ESM; only keratin is known ESM protein, 8 of which are egg white proteins and related while 53 others have not previously been reported. Some differences in the types of proteins and their molecular functions were noted in ESM at different incubation times. One protein which was present only at days 15 and 21 of egg incubation was identified as a calcium binding protein i.e. EGF like repeats and discoidin I like domain 3 (EDIL3 homologous protein).     

Influence of calcium on Manduca sexta plasmatocyte spreading and network formation

Plasmatocytes are a class of insect hemocytes important in the cellular defense response. In some species, they are phagocytic, protecting the insect from smaller pathogens. In many insects, they work in concert with other hemocytes (particularly other plasmatocytes and granular cells) to form nodules and to encapsulate foreign material. To perform these functions, plasmatocytes attach to, spread on, and surround suitable targets. Because of their importance, because we had previously observed that prolonged incubation of hemocytes in solutions containing the divalent cation chelator ethylenediaminetetraacetic acid (EDTA) inhibited plasmatocyte spreading, and because of the importance of divalent cations in many immune-related functions, we investigated the effect of calcium and magnesium on spreading of plasmatocytes from fifth instar Manduca sexta larvae. On glass slides, plasmatocytes spread more quickly and elongated in Grace's medium containing 5 mM calcium, compared to calcium-free medium. In the presence of calcium, plasmatocyte adhesion, spreading, and network formation were not visibly different in magnesium-free and magnesium-containing Grace's medium. Using immunomicroscopy with a monoclonal antibody specific for plasmatocytes, we measured the length and width of plasmatocytes incubated with several different concentrations of calcium. Plasmatocyte length positively correlated with calcium concentration to 5 mM (maximum concentration tested and approximately the hemolymph concentration). Mean plasmatocyte width was less in 0 and 5 mM calcium than in 0.05 or 0.5 mM calcium. On plastic, hemocytes survived longer than on glass (they survived beyond 24 h) and, in 5 mM calcium, formed an extensive network readily visible by phase-contrast microscopy. This network was never as extensive in the absence of calcium. Network formation in the absence of magnesium, but presence of calcium, resembled network formation in standard Grace's medium.     

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

Growth and uptake of mineral by embryos of the direct-developing frog Eleutherodactylus coqui

Embryos of the direct-developing frog Eleutherodactylus coqui take up small quantities of yolk and yolk mineral early in incubation but increase their uptake of yolk reserves at later stages of development. Growth and accumulation of calcium and magnesium by embryos also occur slowly at first and at a higher rate later. Accumulation of calcium and magnesium by embryos is largely a function of variation in size of embryos, but uptake of phosphorus is unrelated to size. Althrough patterns of growth and uptake of mineral by embryonic coquis resemble those for embryos of oviparous amniotes, embryonic coquis do not deplete the yolk of its nutrients to the same degree. Thus, residual yolk of coqui hatchlings contains a high percentage of the nutrient reserves originally present in the egg. This difference between embryonic coquis and embryos of oviparous amniotes may indicate that transfer of nutrients from yolk to embryo becomes limiting during the grwoth phase. Alternatively, some aspects of the neurologic system are so poorly developed at hatching that coqui may not be able to find prey effectively. A large nutrient reserve could sustain hatchling while the neurologic system continues to mature.     

Calcium-dependent exocytosis in an in vitro secretory granule plasma membrane preparation from sea urchin eggs and the effects of some inhibitors of cytoskeletal function

Egg cortical granules remain attached to the egg plasma membrane when the egg is ruptured. We present evidence that demonstrates that, when the cytoplasmic face of the egg plasma membrane is exposed to micromolar calcium concentrations, an exocytosis of the cortical granules occurs which corresponds to the cortical granule exocytosis seen when the egg is fertilized. The calcium sensitivity of the preparation is decreased by an increase in magnesium concentration and increased by a decrease in magnesium concentration. Exocytosis is inhibited by trifluoperazine (half inhibition at 6 microM), a drug that inhibits the action of the calcium-dependent regulatory protein calmodulin. Colchicine, vinblastine, nocodazole, cytochalasin B, phalloidin, N-ethylmaleimide-modified myosin subfragment 1, and antibody to actin are without effect on this in vitro exocytosis at concentrations that far exceed those required to disrupt microtubules and microfilaments. Conditions are such that penetration to the exocytotic site is optimal. It is unlikely, therefore, that either actin or tubulin participate intimately in exocytosis. Our data also exclude on quantitative grounds several other mechanisms postulated to account for the fusion of the secretory granule with the plasma membrane.     

金线蛙早期胚胎发育的初步观察

报道了金线蛙的早期胚胎发育。自受精卵期至鳃盖完成期共分为 2 6个时期 ,其发育历程及各时期胚胎外形特征与黑斑蛙基本相似 ,但孵化期比黑斑蛙推迟三个胚期而早于泽蛙 ,在水温 ( 2 0± 0 5)℃及常温 ( 2 1 5～ 2 4℃ )条件下 ,其胚胎发育的全时程分别为 2 1 2 94小时和 1 70 95小时。     

牛蒡抗虫活性及抗虫成分的初步研究

在水温(23±0.5)℃、pH 7.0～8.0、溶解氧＞5 mg· L-1的条件下,研究孵化过程中不同盐度对拟目乌贼胚胎发育和孵化率的影响.结果表明,拟目乌贼卵透明略带奶油色,近椭圆形,短径为0.596～1.758 cm,长径为1.452～3.136 cm,刚产出的卵重约2g;孵化完成时,卵膜内的重量达到4.5g.在盐度为28‰时,经过28～30 d孵化,卵的短径和长径平均分别增加了0.601、0.615 cm,孵化率为57.0％;盐度为23‰时,卵的长径和短径也相应增加,增幅分别为0.567、1.263 cm,孵化率为13.5％;盐度为33‰时,经过28～30 d孵化,卵短径平均增长0.150 cm,而长径却缩短了0.141 cm,孵化率为9.5％;在盐度为13‰、18‰、38‰和43‰条件下,胚胎停止发育,孵化率为0.结果表明,拟目乌贼属于狭盐性种类,盐度变化对拟目乌贼胚胎发育和孵化有直接影响.