Studies of oogenesis in Urechis caupo. II. Accumulation during oogenesis of carbohydrate, RNA, microtubule protein, and soluble, mitochondrial, and lysosomal enzymes

In the echiuroid worm Urechis caupo, the oocytes develop independently as single cells in the coelomic fluid. The accumulation of oocyte constituents during oogenesis was determined in different size classes of coelomic oocytes, separated by fractionation on a Ficoll density gradient. Each size class was assayed for protein, carbohydrate, RNA, microtubule protein, glucose-6-phosphate dehydrogenase, acid phosphatase, and cytochrome c oxidase. All the constituents investigated accumulated continuously during oogenesis, and the rates of accumulation paralleled the volume increase of the oocytes. The electrophoretic pattern of soluble egg proteins supported this finding. These data suggest that the genes involved in the synthesis of all the oocyte constituents studied are activated early in oogenesis.     

Soluble microtubule proteins of the sea urchin embryo: partial characterization of the proteins and behavior of the pool in early development

A partial characterization of the soluble microtubule proteins of sea urchin eggs and embryos is presented. Vinblastine precipitation yielded a pellet with a high colchicine binding activity. This precipitate when electrophoresed on an alkaline SDS/urea gel system yields two protein bands which correspond to molecular weights of 57,000 ± 2000 and 52,000 ± 2000. These values are very close to our values and to the published values for axonemal microtubule proteins. Electrophoresis of the vinblastine precipitated proteins on a neutral SDS system without urea yielded only one band with an apparent molecular weight of 52,000 ± 2000. The amino acid composition of the vinblastine-precipitated microtubule protein was determined to be similar to that of axonemal protein.The pool of microtubule proteins was found to remain constant in size throughout early development in both control and actinomycin-treated embryos. Soluble microtubule proteins comprise about 0.37% of the total protein of the sea urchin (Arbacia) egg. Approximately 20% of the total microtubule protein in the egg appears to be particle bound.     

Cytochemical and Biochemical Analysis of Development of Urechis Oocytes

The echiuroid marine worm Urechis caupo is uniquely suited forthe study of oogenesis. A relatively large quantity of oocytesat various developmental stages can be obtained and subjectedto coordinated cytochemical and biochemical analysis Oocytesat the cluster, early diplotene, mid-diplotene, and diffusediplotene or lampbrush stages are active in the synthesis andaccumulation of ribosomal RNA, several proteins, carbohydrates,lipids, and also, perhaps, yolk constituents. Only corticalgranule formation, which occurs during later stages of oogenesis,appears to be stage specific. Ribosomal RNA genes are also transcribedin the nucleolus of the mature oocytes or unfertilized eggs.However, the rate of production in these eggs appears to beregulated at the level of maturation of rRNA precursor molecules.     

The polyspermy block in eggs of Urechis caupo: Evidence for a “rapid” block

Experiments have established the presence and time course of a polyspermy block in eggs of the echiuroid worm Urechis caupo. At various times after an initial insemination the eggs were reinseminated with concentrated sperm suspensions and the polyspermy determined. The block is rapid; partial protection against polyspermy is evident within a few seconds after insemination of the eggs. Although there are other rapid responses approximately correlated in time with the beginning of the block and possibly related to it, I also consider the possibility that a relatively ‘late’ event might account for the apparent ‘rapid’ block in these eggs.     

Release of acid and changes in light-scattering properties following fertilization of Urechis caupo eggs.

The release of a fertilization acid, monitored by measuring the pH of egg suspensions, begins within 10 sec of insemination of Urechis caupo eggs. This is 4 min before the vitelline layer begins to elevate and is apparently unrelated to that process. The eggs of two molluscs, Mytilus californianus and Acmaea incessa, do not form a fertilization acid. The acid of Urechis eggs is not accompanied by release of “fertilization” carbohydrate, sulfate, or a nonvolatile weak acid into the seawater. The light-scattering properties of Urechis eggs change during the first 10 min after insemination. A decrease in light scattering begins by 10 sec and is complete by 1 min (Phase I). This is followed by a further decrease (3–6 min, Phase II) and an increase (6–10 min, Phase III). In striking contrast to an overtly similar situation in sea urchin eggs (fertilization acid and coincident light-scattering decrease), the release of acid and the initial light-scattering change are not the result of cortical granule discharge, and the acid, at least, is not related to the changes in shape or surface area which the eggs undergo. The processes underlying these rapid events are not yet known.     

Protein Compositional Analysis of the Eggs of Black Widow Spider (Latrodectus tredecimguttatus): Implications for the Understanding of Egg Toxicity

Previous work found that high‐molecular‐weight fractions in the egg extract of Latrodectus tredecimguttatus exhibited strong toxicities. For investigating the possible relationship of proteins in the eggs with the toxic effect, the protein composition of the eggs was analyzed using proteomic strategies and compared with that of the spider's venom. SDS‐PAGE showed that the proteins of eggs were primarily distributed in the molecular weight range of higher than 55 kDa as well as around 34 kDa, having high abundance proteins with molecular weights of about 60 kDa and 130 kDa. A total of 157 proteins were identified from the egg extract, which were involved in important cellular functions and processes including catalysis, transport, and metabolism regulation. Comparison indicated that the protein composition of eggs is more complex than that of venom, and there are few similarities between the protein composition of the two materials, demonstrating that the eggs have their own distinct toxic mechanism. © 2012Wiley Periodicals, Inc. J BiochemMol Toxicol 26:510‐515, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21460     

SYNTHESIS AND STORAGE OF MICROTUBULE PROTEINS BY SEA URCHIN EMBRYOS

Studies employing colchicine binding, precipitation with vinblastine sulfate, and acrylamide gel electrophoresis confirm earlier proposals that Arbacia punctulata and Lytechinus pictus eggs and embryos contain a store of microtubule proteins. Treatment of 150,000 g supernatants from sea urchin homogenates with vinblastine sulfate precipitates about 5% of the total soluble protein, and 75% of the colchicine-binding activity. Electrophoretic examination of the precipitate reveals two very prominent bands. These have migration rates identical to those of the A and B microtubule proteins of cilia. These proteins can be made radioactive at the 16 cell stage and at hatching by pulse labeling with tritiated amino acids. By labeling for 1 hr with leucine-³H in early cleavage, then culturing embryos in the presence of unlabeled leucine, removal of newly synthesized microtubule proteins from the soluble pool can be demonstrated. Incorporation of labeled amino acids into microtubule proteins is not affected by culturing embryos continuously in 20 µg/ml of actinomycin D. Microtubule proteins appear, therefore, to be synthesized on "maternal" messenger RNA. This provides the first protein encoded by stored or "masked" mRNA in sea urchin embryos to be identified.     

Purification, characterization, and assembly properties of tubulin from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus

Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by chromatography of an egg supernatant fraction on DEAE-Sephacel or DEAE-cellulose followed by cycles of temperature-dependent microtubule assembly and disassembly in vitro. After two assembly cycles, the microtubule protein consisted of the alpha- and beta-tubulins (greater than 98% of the protein) and trace quantities of seven proteins with molecular weights less than 55 000; no associated proteins with molecular weights greater than tubulin were observed. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on urea-polyacrylamide gradient gels, the alpha- and beta-tubulins did not precisely comigrate with their counterparts from bovine brain. Two-dimensional electrophoresis revealed that urchin egg tubulin contained two major alpha-tubulins and a single major beta species. No oligomeric structures were observed in tubulin preparations maintained at 0 degrees C. Purified egg tubulin assembled efficiently into microtubules when warmed to 37 degrees C in a glycerol-free polymerization buffer containing guanosine 5'-triphosphate. The critical concentration for assembly of once- or twice-cycled egg tubulin was 0.12-0.15 mg/mL. Morphologically normal microtubules were observed by electron microscopy, and these microtubules were depolymerized by exposure to low temperature or to podophyllotoxin. Chromatography of a twice-cycled egg tubulin preparation on phosphocellulose did not alter its protein composition and did not affect its subsequent assembly into microtubules. At concentrations above 0.5-0.6 mg/mL, a concentration-dependent "overshoot" in turbidity was observed during the assembly reaction. These results suggest that egg tubulin assembles into microtubules in the absence of the ring-shaped oligomers and microtubule-associated proteins that characterize microtubule protein from vertebrate brain.     

Stimulation of protein phosphorylation during fertilization-induced maturation of Urechis caupo oocytes

Protein phosphorylation was studied during fertilization of Urechis caupo oocytes both in vivo, by measuring [³²P]phosphate incorporation into ³²P preloaded oocytes and in vitro, by measuring endogenous protein kinase and phosphatase activities in homogenates. During fertilization (and maturation) the rate of protein phosphorylation is dramatically increased. No change in the [³²P]phosphate uptake, or the nucleotide levels was observed at fertilization, so the increase cannot be attributed to changes in substrate availability. In vitro enzyme assays showed changes in protein kinase activity which approximately mirrored the changes in the in vivo phosphorylation pattern. As there were no changes in protein phosphatase activity, these results suggest the phosphorylation change results from an increase in protein kinase activity. The pattern of change, investigated by SDS-polyacrylamide gel electrophoresis, shows that proteins that were phosphorylated in the unfertilized egg become phosphorylated to a greater degree after fertilization. One protein (48 kd) undergoes an increase followed by a decrease of its phosphorylation level.     

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.     

Potential use of seaweeds in the laying hen ration to improve the quality of n-3 fatty acid enriched eggs

An association between dietary fish oil and decreased yolk weight and reduced sensory quality of eggs has been reported when eggs are enriched with n-3 FA from fish oil. Seaweeds are an important source of compounds that seem to increase egg weight when included in the laying hen diet. The objectives of this study were to determine the influence of the dietary seaweeds Macrocystis pyrifera, Sargassum sinicola and Enteromorpha sp. on the physical quality, lipid composition and consumer acceptability of n-3 FA enriched eggs. One-hundred and forty-four 35-week-old Leghorn hens were randomly distributed in four treatments that consisted of the inclusion of 2% of sardine oil (SO) and 10% of each marine alga (MA) in laying hens' diets; a control diet (C) was also prepared. The study lasted 8 weeks and egg physical quality, egg lipids and sensory attributes were evaluated. The results showed that incorporation of 10% M. pyrifera in the diets is an effective way of increasing the n-3 FA content, the albumen height and yolk color, but not the egg weight, when these are enriched with n-3 FA from fish oil. The egg flavor was also not affected.     

Reproductive biology of brown trout, Salmo trutta macrostigma Dumeril 1858, in a tributary of the Ceyhan River which flows into the eastern Mediterranean Sea

The study describes some key elements of the reproductive biology, including spawning season, age at sexual maturity, fecundity and egg diameter of the native brown trout, Salmo trutta macrostigma, in a tributary of the Ceyhan River. A total of 197 brown trout (118 females and 79 males) were captured in 2000–2001 by electric fishing. In observations on monthly changes, the gonadosomatic index (GSI) and the monthly frequency distribution of egg diameter confirmed that spawning lasted from November to January. Some 27.7% of the females and 62.5% of the males attained sexual maturity in their second year. The smallest fork length (FL) of brown trout attaining sexual maturity was 17.4 cm for males and 17.8 cm for females. Mean fecundity in age groups II, III, IV and V were 360, 452, 693 and 1283 eggs per female, respectively. One 9‐year‐old female had a unique 3232 egg count. The mean fecundity of the sampled population was 554 eggs per fish, positively correlated with the FL (mm) (R = 0.8227 ) and body weight (R = 0.8130). The diameter of mature eggs in the spawning season ranged from 3.250 to 5.930 mm, with a 4.146 mm average. Mean egg diameter in age groups II, III, IV and V in the spawning season were 0.813, 3.799, 4.663 and 5.243 mm, respectively. Fecundity, egg weight and diameter were statistically different in all age groups.     

Influence of honey and maternal age on egg load of lab-cultured <Emphasis Type="Italic">Cotesia marginiventris</Emphasis>

Laboratory experiments were conducted to determine the impact of feeding status and maternal age on egg load of Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae), a solitary, koinobiont endoparasitoid of noctuid pests. Egg load was defined as the number of mature (i.e., fully-chorionated) eggs found in the ovaries and oviducts. Significantly more mature eggs were stored in honey-fed than starved females. For honey-fed females, egg load increased within several days of isolation from hosts. This study suggests that C. marginiventris is weakly synovigenic because females emerge with a considerable number of mature eggs and are capable of maturing many more eggs. Feeding on a suitable source of carbohydrate should increase the egg load (i.e., potential fecundity) of this insect within 3–4 days in an in vivo rearing system.     

Reduced realised fecundity in the pine looper Bupalus piniarius caused by host plant defoliation

1. Whether the potential fecundity of herbivorous insects is realised or not may depend on female behaviour, which in turn may be influenced by host plant acceptability. Female Bupalus piniarius were observed to discriminate against needles growing out the year following defoliation (current‐year needles) of its host plant Pinus sylvestris. 2. It was hypothesised that the discriminatory behaviour was due to current‐year needles being less secure as a substrate. Field and laboratory experiments were designed to test this hypothesis and to estimate the discrepancy between potential and realised fecundity when females were offered defoliated branches. 3. In a laboratory oviposition experiment, B. piniarius females were exposed to branches bearing either current‐year needles only or both mature and current‐year needles. Daily oviposition rate, egg batch size, longevity, and mature eggs and fat retained at death were recorded for each female. In field experiments, the rate at which eggs dropped from expanding needles and the capacity of neonate larvae hatching from the dropped eggs to colonise a tree were assessed. 4. Significantly fewer eggs were laid when females were exposed to defoliated branches. 5. Twenty‐six and 16% of the eggs laid on current‐year needles dropped from the needles in 1998 and 1999 respectively, whereas no eggs dropped from mature needles in 1998 and only one egg (< 1%) dropped in 1999. 6. A very small proportion of larvae hatching on the forest floor (simulated egg drop) was able to recolonise host trees. 7. These results emphasise the importance of oviposition behaviour on realised fecundity when analysing insect population dynamics. In the case of B. piniarius, egg placement, although a minor detail during the normal course of events, became of key importance when defoliation deprived females of their preferred egg attachment site.     

Maximum realised lifetime parasitism and occurrence of time limitation in Gonatocerus ashmeadi (Hymenoptera: Mymaridae) foraging in citrus orchards

The relationship between body size (hind tibia length), <12 h egg load, wing wear and parasitoid age was used to estimate realised lifetime parasitism of recently dead Gonatocerus ashmeadi collected in a citrus orchard. Under prevailing field conditions and methodology assumptions, it was estimated that female G. ashmeadi lived on average for 183 ± 17 degree-days, parasitised a total of 87 ± 9 Homalodisca vitripennis eggs, and died with 34 ± 5 eggs remaining in the ovaries. Only 17% of dead G. ashmeadi died with no mature eggs suggesting that 83% of G. ashmeadi were not egg limited at time of death. Estimates of realised lifetime parasitism for female G. ashmeadi under prevailing field conditions in July and August in a southern California citrus orchard indicated that time of year had a significant effect on reproductive output. Additionally, live G. ashmeadi captured daily during June through August 2006 had body size, egg load and wing wear recorded to detect possible monthly changes in parasitoid age and egg load. Foraging G. ashmeadi captured alive in June were older and oviposited more eggs in the field compared with August. Only 0.5% of live G. ashmeadi were captured with no mature eggs in their ovaries indicating that the vast majority of live G. ashmeadi were not egg limited.     

Release of mouse eggs from metaphase arrest by protein synthesis inhibition in the absence of a calcium signal or microtubule assembly

Mouse egg activation, which includes release from meiotic metaphase II arrest, results from fertilization-induced increase in intracellular calcium concentration ([Ca²⁺]_i). However, during egg activation caused by exposure to the protein synthesis inhibitor, cycloheximide, [Ca²⁺]_i did not change. Although eggs fertilized in the presence of microtubule inhibitors remain arrested at metaphase, eggs treated for 32 hr with cycloheximide and the microtubule inhibitor, colcemid, formed nuclei. In untreated eggs aged in culture for 24 hr, the microtubule spindles became deformed. These eggs formed nuclei after exposure to cycloheximide, but not the calcium ionophore A23187. Our results indicate that eggs in which protein synthesis is inhibited are released from metaphase without an increase in [Ca²⁺]_i, and despite disruption of the Spindle. © 1995 Wiley-Liss, Inc.     

Fertilization acid release in Urechis eggs: I. The nature of the acid and the dependence of acid release and egg activation on external pH

The amount of fertilization acid produced by eggs of Urechis caupo, monitored by automatically back-titrating egg suspensions with base, depends linearly on the pH of the seawater. Above pH 7.0, at which no acid is released (Paul, M., Dev. Biol.43, 299–312, 1975), acid release increased approximately 0.34 pmole/egg/0.1 pH unit. Activation (germinal vesicle breakdown) depended on the amount of acid release in natural seawater; it did not occur if eggs released <1.5 pmole acid/egg. When fertilization acid is released into HCO^?₃-free seawater and the pH permitted to decrease, the supernatant can be tested for the presence of a volatile acid, such as CO₂, by bubbling with N₂ and comparing the increase in pH as volatile acid is driven off with experiments in which HCl or CO₂ is substituted for fertilization acid. An increase in pH of <0.2 pH units occurred on N₂ bubbling when fertilization acid or HCl was used to acidify HCO^?₃-free seawater compared to an increase of >0.5 pH units when CO₂ was used. Therefore, most, if not all, of Urechis fertilization acid is not volatile, and since Paul (1975) showed that it is not a nonvolatile weak acid, it must be H⁺.     

Breeding biology aspects of spotted flapshell Turtle,Lissemys punctata (Lacepede 1788), in Bangladesh

This study was conducted to determine the breeding season, gonadal development, egg laying period, clutch size and other biological aspects of spotted flapshell turtle, Lissemys punctata, of Bangladesh between January 1997 and December 2001. The egg laying period of L. punctata, was found between August and March. The nesting sites were elevated fallow lands in secluded areas. The female turtle laid all the mature eggs at a time for each clutch at night. A gravid female turtle laid three clutches of eggs in each year and the mean clutch size was 13.0 ± 1.9 eggs and mean weight of each egg was 10.3 ± 1.3 g. The eggs are spherical in shape and whitish in color. The mean incubation period was 173 ± 34 days (range 119–225 days). The incubation period of first clutch was the longest than the second and third clutches. Hatching success was found 41%. Maximum hatching was observed in June. The present investigation was made to explore the possibility to raise turtle farming in captive condition. The findings would, hopefully, help to rear the species and to assess the commercial potentiality of turtle farming in captive condition, that is, in the eco-climatic condition of Bangladesh.     

Limitation of reproduction by feeding condition in a carabid beetle,Carabus yaconinus

1. Effects of the amount of food consumed on reproduction of the carabid beetle, Carabus yaconinus B., were studied in the laboratory by rearing beetles at different food levels, and the feeding and oviposition rates in the field were estimated on the basis of the relationships between the amount of food consumed, body weight and egg production obtained in the experiment.
2. The maximum amount of food consumed was 150 mg of minced beef per day. The number of eggs laid per day and the mean body weight increased with an increase in the amount of food consumed. High mortality occurred only when the beetles consumed less than 25 mg of minced beef per day.
3. The ratio of current body weight to the minimum one just before death by starvation, W/W_min, was used for the estimation of the rates of food consumption and egg production. The relationships between mean W/W_min ratio, the amount of food consumed and the number of eggs laid per day were clarified.
4. The relationships between ovary states (ovary weight and the number of mature eggs in the ovary) and W/W_min ratio were examined for the females caught in the field. Females with higher values of W/W_min ratio had more mature eggs.
5. The amount of food consumed by females in the field during the reproductive period was estimated to be 50–70% of the maximum value attained in the experiment and the estimated rate of oviposition was 45–59% of the maximum rate attained in the experiment.