Water Content, Raffinose, and Dehydrins in the Induction of Desiccation Tolerance in Immature Wheat Embryos

Desiccation tolerance is initiated in wheat (Triticum aestivum L.) embryos in planta at 22 to 24 d after anthesis, at the time that the embryo water content has decreased from about 73% fresh weight (2.7 g water/g dry weight) to about 65% fresh weight (1.8 g water/g dry weight). To determine if desiccation tolerance is fully induced by the loss of a relatively small amount of water, detached wheat grains were treated to reduce the embryo water content by just a small amount to approximately 69% (2.2 g water/g dry weight). After 24 h of such incipient water loss, subsequently excised embryos were able to withstand severe desiccation, whereas those embryos that had not previously lost water could not. Therefore, a relatively small decrease in water content for only 24 h acts as the signal for the development of desiccation tolerance. Embryos that were induced into tolerance by a 24-h water loss had no detectable raffinose. The oligosaccharide accumulated at later times even in embryos of detached grains that had not become desiccation tolerant, although tolerant embryos (i.e. those that previously had lost some water) contained larger amounts of the carbohydrate. It is concluded that desiccation tolerance and the occurrence of raffinose are not correlated. Immunodetected dehydrins accumulated in embryos in planta as desiccation tolerance developed. Detachment of grains induced the appearance of dehydrins at an earlier age, even in embryos that had not been made desiccation tolerant by incipient drying. It is concluded that a small reduction in water content induces desiccation tolerance by initiating changes in which dehydrins might participate but not by their interaction with raffinose.     

三江源区高寒草甸退化对土壤水源涵养功能的影响

三江源区是我国重要的水源涵养区,研究草地退化对土壤水源涵养功能的影响,可为三江源区水源涵养功能的科学评估与合理监测提供科学依据。以实地采样与室内测试分析相结合的方法研究了三江源区内不同土壤类型高寒草甸生物量特征、土壤水文物理性质及土壤水源涵养量。结果表明:高寒草甸在重度退化阶段地上生物量、地下生物量、毛管孔隙度、总孔隙度、自然含水量、最大持水量、土壤水源涵养量显著低于未退化和中度退化阶段(P<0.05)。随着高寒草甸退化程度加剧,土壤容重逐渐增大,且非毛管孔隙度规律不显著。未退化、中度退化、重度退化草甸的土壤水源涵养量范围分别为1884.32—1897.44t/hm2、1360.04—1707.79t/hm2、1082.38—1550.10t/hm2。中度退化草甸土壤水源涵养量比未退化草甸低9.37%—10.35%,重度退化草甸低18.31%—27.82%。草甸退化进程中土壤总孔隙度与毛管孔隙度的降低是影响土壤水源涵养量下降的直接原因,而草甸退化进程中地上生物量与地下生物量的减少则是间接原因。度量三江源区高寒草甸土壤水源涵养功能时应着重考虑毛管孔隙度的蓄水作用。研究表明高寒草甸地上生物量与土壤水源涵养量之间存在显著的正相关关系(P<0.05),该结果能够推动水源涵养功能评估向空间化、精细化发展,为探索利用遥感技术监测三江源区水源涵养功能提供参考依据。     

Pathway for the movement of water and cryoprotectants in bovine oocytes and embryos

The permeability of cells is important for cryopreservation. Previously, we showed in mice that the permeability to water and cryoprotectants of oocytes and embryos at early cleavage stages (early embryos) is low because these molecules move across the plasma membrane predominantly by simple diffusion through the lipid bilayer, whereas permeability of morulae and blastocysts is high because of a water channel, aquaporin 3 (AQP3). In this study, we examined the pathways for the movement of water and cryoprotectants in bovine oocytes/embryos and the role of AQP3 in the movement by determining permeability, first in intact bovine oocytes/embryos, then in bovine morulae with suppressed AQP3 expression, and finally in mouse oocytes expressing bovine AQP3. Results suggest that water moves through bovine oocytes and early embryos slowly by simple diffusion, as is the case in mice, although channel processes are also involved in the movement. On the other hand, water appears to move through morulae and blastocysts predominantly by facilitated diffusion via channels, as in mice. Like water, cryoprotectants appear to move through bovine oocytes/early embryos mostly by simple diffusion, but channel processes could also be involved in the movement of glycerol and ethylene glycol, unlike that in mice. In bovine morulae, although glycerol and ethylene glycol would move predominantly by facilitated diffusion, mostly through AQP3, as in mice, dimethylsulfoxide appears to move predominantly by simple diffusion, unlike in mice. These results indicate that permeability-related properties of bovine oocytes/embryos are similar to those of mouse oocytes/embryos, but species-specific differences do exist.     

广东白盆珠水库水源林土壤水源涵养能力研究

通过对广东省白盆珠水库水源林土壤类型调查及土壤水分物理性质的测定,结果表明:库区水源林水平地带性土壤属赤红壤,山地土壤垂直带谱明显,分布有赤红壤、山地红壤、山地黄壤和山顶灌丛草甸土4个类型。土壤容重约为1.338g·cm^-3,随海拔升高土壤砂粒含量增加。土壤总孔隙度在45%～50%,非毛管孔隙度在5%～9%之间,毛管孔隙度35%～50%。不同森林类型土壤的最大持水量在30%～50%,即50～60mm,变化不大;蓄水容量有较大区别,范围在500～850t·hm^-2,灌丛草甸土最大,针阔混交林次之,沟谷阔叶林最小;排水能力约在130～180t·hm^-2,并以灌丛草甸土为最大,次生阔叶林为最小。该库区水源林土壤的排水和蓄水容量分别为62.69万t、316.29万t,消洪补枯能力明显。但水源林土壤非毛管孔隙度较小,蓄水量小于广东各种有林地森林类型平均蓄水量,所以该库区的水源林还需加强保育,以提升土壤的水源涵养能力。     

Dermatan sulfate formation in gastrulae of the sea urchin Clypeaster japonicus

Gastrullation of sea urchin embryos is arrested in sulfate-free sea water. This developmental arrest has been considered to be due to lack of sulfation of glycosaminoglycans in the extracellular matrix of the embryos. In the present study, we characterized a dermatan sulfate type component formed in gastrula-stage embryos of the sea urchin Clypeaster japonicus and examined the effects of sulfate deprivation on the formation. Glycosamino-glycans were prepared from gastrula-stage embryos incubated with [3H]acetate in normal and sulfate-free sea water. Enzymatic analyses indicated that embryos formed a glycosaminoglycan of the dermatan sulfate type which contained an N-acetylgalactosamine-6-sulfate-containing disaccharide as a major unit, plus a minor unidentified component. Under sulfate-free conditions, embryos formed an under-sulfated chondroitin/dermatan sulfate copolymer which mainly consisted of non-sulfate, glucuronic acid-containing (chondroitin) disaccharide units. These results suggest that sulfate deprivation diminishes not only the degree of sulfation but also the formation of L-iduronic acid-containing (dermatan) disaccharide units in dermatan sulfate in sea urchin embryos.     

Effect of water hardness on oocyte quality and embryo development in the African clawed frog (Xenopus laevis)

Husbandry and health of the African clawed frog, Xenopus laevis, greatly influences the quality of oocytes produced. One factor affecting oocyte quality is the water conditions in which females are maintained. Dechlorination and adequate salt concentration are known to affect oocytes, but water hardness has not been considered an important factor in Xenopus husbandry by the research community. We found that, when females were kept in soft water or water with marine salts alone, even when it was cooled to 17 to 18 degrees C, the quality of oocytes decreased; only 20 to 25% of resulting embryos developed to tailbud stages. Survival and normal development of embryos increased significantly within one month of addition to the laboratory housing water of salts that mimic conditions in African Rift Valley lakes. These salts greatly increased water hardness; development of embryos to tailbud stages remained high (50 to 70% on average) for more than a year after their addition to the water housing females. Water from South African ponds where X. laevis are collected, and from wells used by the major suppliers of X. laevis, also was moderately to very hard. Our results suggest that X. laevis is naturally adapted to hard water, and indicate that increasing general hardness during laboratory housing is more important for oocyte quality and embryo development than is increasing carbonate hardness (alkalinity) in the water used to house females.     

Ventilatory mechanisms and the effect of hypoxia and temperature on the embryonic lesser spotted dogfish

Small, intermediate and large-sized embryos of the dogfish Scyliorhinus canicula utilize different ventilatory methods; small and intermediate embryos rely on body movement alone to stir either the jelly or sea water in the capsule, large embryos use conventional pharyngeal pumping to pump water through the case. The effects of environmental changes in O₂ tension (0.5–100% air saturation) and temperature (6–18°C) upon ventilatory mechanisms in the developing embryo in situ were studied using non-invasive ultrasonography. All three embryo classes increased ventilation rate with rising temperature: for small embryos, y=2.02x+3.295 ( P <0.01); for intermediate embryos, y=3.51x+0.395 ( P <0.01); and for large embryos, y=3.81x+9.39 ( P <0.01); where y=ventilatory frequency (tail beats min⁻¹ or pump cycles min⁻¹) and x=temperature (°C). Q ₁₀ (6–16°C)=5.0, 2.45, and 2.08 for small, intermediate and large embryos, respectively; corresponding Q ₁₀ (8–18°C) values were 2.09, 2.62, and 2.02. It is suggested that the extreme response of small embryos to 6°C is related to a different state of development in either chemoreceptors or muscle blocks. There was no significant change in ventilatory frequency induced by chronic (2 h) hypoxia. Dogfish embryos are oxyconformers at 8°C but oxyregulators at higher temperatures. Water flow through an eggcase occupied by a large embryo was studied also. Water enters the open eggcase of a large embryo, drawn in by the buccal/opercular pump of the respiring embryo, via holes at the posterior end of the eggcase. Expired water exits holes at the anterior end of the eggcase. The mean residence time for water in the case is 50 s at 8°C, giving a transit velocity of 1.36 mm s⁻¹.     

Critical factors affecting the permeabilization of Drosophila embryos by alkanes.

Because of waxes in the vitelline membrane, the Drosophila egg is effectively impermeable to liquid water and to aqueous solutes, and consequently it cannot be cryopreserved unless it can be permeabilized. The more successful of the few published permeabilization procedures involve the removal of the chorion mechanically or by hypochlorite solution, the removal of all surrounding water by air drying or alcohol, the exposure of eggs to pure alkanes like octane or hexane for some 30 s, the removal of the alkane and the transfer of the eggs to aqueous culture medium without their desiccation, and lastly incubation of the permeabilized embryos under mineral oil. In following these procedures we opted for a somewhat different approach to applying hypochlorite, water, alcohol, and alkane; namely, eggs were placed between two Nucleopore filters, and the fluids drawn sequentially through the filters by vacuum. Extensive initial attempts were mystifying and discouraging in that although permeabilization was good, survivals were poor, and modifications that increased the latter reduced the former. The explanation turned out to be that permeabilization and survival depended critically on the amount of carry-over alcohol that contaminated the alkane. To determine the effects of alcohol concentration in the alkane, it was essential first to effectively eliminate carry-over contamination and then re-add precise amounts of alcohol (isopropanol) to the alkane (n-hexane, heptane, or octane). When the alcohol concentration is less than or equal to 0.2%, permeabilization is poor; when it is greater than or equal to 0.5%, permeabilization is good but survival (hatching) is poor. There are strong interactions between alcohol concentration and exposure time to alkane/alcohol mixtures with respect to the fraction of embryos that become permeabilized and the percentage that survive. There are also significant but less critical effects from the type of alcohol and alkane. The best results for 12-h embryos (greater than or equal to 90% permeabilization and 70-80% hatching) were achieved with eggs exposed to 0.3 or 0.4% 1-butanol in n-heptane for 90 s. High survivals of permeabilized 12-h embryos did not require incubation under mineral oil. Permeabilized embryos are permeable to water, ethylene glycol, glycerol, and the stain rhodamine B (which was used to assess permeabilization). They are effectively impermeable to sucrose. Embryo age is important. Between 14 and 16 h the above permeabilization procedures become dramatically less effective.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in the biochemical composition of Euphausia superba Dana embryos during early development

Summary The biochemical composition of developing Euphausia superba Dana embryos has been examined at three stages: fresh spawned, gastrula, and limb bud. Fresh spawn embryos had 31% lipid, 1.0% carbohydrate, and 57% protein on a gram dry weight basis. Throughout development lipids were utilized more slowly than in other crustacean embryos for a total utilization of 37.5%. Overall, 35.6% of the starting protein was utilized. On a weight basis, twice as much protein than lipid was used during development, and it appeared that, energetically, protein and lipid contributed equally to the energetics of the developing embryo. Carbohydrate was evidently a minor substrate in early development, although the level increased 38% during development. Average water content was 86% in fresh spawned embryos and 88% in the gastrula stage. The average dry weight of the embryos throughout development was 30 g. The features of planktonic embryos are contrasted with demersal embryos and the atypical metabolic pattern of krill embryos is discussed.MOA dedicates this paper to the memory of Dr. Mary Alice McWhinnie, for her encouragement during my years at DePaul University and for the opportunity to participate in her research     

Reduction of embryotoxicity by protein in embryo culture media.

Experiments tested the hypothesis that one role of protein in embryo culture media is protection of embryos against potentially embryotoxic substances in the media. Mouse embryos were cultured in modified Krebs-Ringer bicarbonate medium and in modified Tyrode's medium, aliquots of which were supplemented with 4 mg/ml of the protein bovine serum albumin (BSA), while other aliquots were left protein free. The media were prepared using water samples that differed in purity, as reflected by differences in conductivity, with tap water being least pure (and considered to have the greatest potential for being embryotoxic) and water that had been purified by reverse osmosis, Milli-Q filtration, and triple distillation being most pure. Embryos were placed in the media while in the two-cell stage of development and their development was assessed after 24, 48, and 72 hr of culture. Rate of embryo development in BSA-supplemented media was greater than that in protein-free media only when the media were prepared with the least purified water samples. Because these water samples would have contained substances not contained in media prepared with purer water, or would have contained the substances in higher concentration, the data supported the hypothesis that protein can protect embryos during culture by negating effects of embryotoxic substances in the media.     

Importation of in vitro-produced Bubalus bubalis embryos from Italy into the United States: a case report

On December 19, 2005, 14 in vitro-fertilized water buffalo (Bubalus bubalis) embryos, which had been cryopreserved by vitrification, were thawed and transferred into B. bubalis recipients in California. The embryos had been produced in Italy, following transvaginal oocyte pickup (TVOPU), with subsequent in vitro maturation, insemination, and culture. This case study relates our experience in meeting the regulatory criteria, established by the Animal Import/Export Office of the USDA-Animal and Plant Health Inspection Service (USDA-APHIS), in order to successfully import these embryos into the USA.     

The Effects of lithium and Zinc Ions on the Pattern of Acidic Glycans in the Sea Urchin (Hemicentrotus pulcherrimus) Embryo

Among several acidic glycan components found in Hemicentrotus embryos, the "F"- and "S"-components were specifically affected by treatment with Li⁺and Zn²⁺, respectively. The amount of the "F"-component in Li⁺-treated embryos was about 60% that in normal embryos. This fact was in accordance with the reduced alcian blue staining of the surfaces in Li⁺-treated embryos. Moreover, the "F"-component in Li⁺-treated embryos appeared to be composed of two subcomponents, while in normal and Zn²⁺-treated embryos it appeared to be single. The "S"-component in Zn²⁺-treated embryos was about 8% that in normal embryos. According to histochemistry with a lectin probe, it was found that UEA-I was much more strongly associated with a hyaline layer in Li⁺-treated than in normal and Zn²⁺-treated embryos. Li⁺-treated embryos developed into exogastrulas, which were divided by a constriction into two parts; an animal half which stained intensely with alcian blue, and a vegetal half which stained poorly. On the other hand, Zn²⁺-treated embryos remained as permanent blastulas. Considering the above, it is suggested that change in the acidic glycan pattern leads to alterations in the morphogenesis of sea urchin embryos.     

Enhancing frequency of regeneration of somatic embryos of avocado (Persea americana Mill.) using semi-permeable cellulose acetate membranes

Regeneration of avocado via somatic embryogenesis is difficult due to poor embryo maturation, resulting in low frequencies of germination. In this study, the influence of semi-permeable cellulose acetate membranes and culture media, containing high levels of sucrose along with coconut water, on maturation and germination of somatic embryos of avocado have been evaluated. The culture of embryogenic calli on top of cellulose acetate membranes significantly increased the number of mature, white-opaque embryos that were recovered after 5 weeks of culture. These embryos showed a much more normal appearance and better quality compared with the control embryos, although the embryo size was significantly reduced. To increase the embryo size and to complete maturation, several two-step maturation treatments were tested. The culture of white-opaque somatic embryos in a modified MS medium with B5 macronutrients gelled with 10 g L^?1 agar (B5m10A medium) over a 5-week period, followed by 5 additional weeks in B5m10A with 45 g L^?1 sucrose and 20 % coconut water, yielded the best results, reducing the percentage of necrotic embryos and the number of calli formed. The beneficial effects of this maturation treatment were enhanced when using embryos that were pre-matured on cellulose acetate membranes. Following this two-step maturation treatment, the germination rate of the control somatic embryos, which were not cultured on cellulose membranes, was lower than 10 %, but it significantly improved when the embryos had been pre-matured on cellulose acetate membranes for 5 weeks, reaching a germination rate close to 40 %. The water availability was significantly reduced when somatic embryos were cultured on cellulose membranes, and after this pre-maturation treatment, the white-opaque embryos showed lower water potential and ABA content compared with the control embryos. These results suggest that culturing over cellulose membranes causes a controlled embryo desiccation that enhances the recovery of plants.     

The Structure and Development of the Apical Ganglion in the Sea Urchin Pluteus Larvae of Strongylocentrotus droebachiensis and Mespilia globulus

The structure of the apical ganglion is described using transmission electron microscopy and ultrastructural immunohistochemical localization with anti-serotonin, and the development of these nerve cells in animalized and vegetalized embryos, and embryos treated with Ca²⁺-deficient sea water are demonstrated with immunofluorescence microscopy. The axons within the neuropile contain clear and dense-core vesicles, but all the vesicles appear anti-serotonin immunoreactive. The principal type of neuron within the apical ganglions is anti-serotonin immunoreactive. In the animalized embryos, serotonergic neuroblasts and neurons appear in the second quarter of animal-vegetal axis. Serotonergic cells were not detected in vegetalized exogestrulae. Mespilia globulus embryos treated with Ca²⁺-deficient sea water ruptured to form a cellular sheet on the substratum. In ruptured embryos serotonergic neurons formed a ring around the area corresponding to the apical plate of normal embryos. These findings indicated that the serotonergic preoral nerve cells of echinopluteus derive from cells on the periphery of the apical plate.     

Japanese flounder (Paralichthys olivaceus) embryos are difficult to cryopreserve by vitrification

The first successful cryopreservation of fish embryos was reported in the Japanese flounder by vitrification [Chen and Tian, Theriogenology, 63, 1207-1219, 2005]. Since very high concentrations of cryoprotectants are needed for vitrification and fish embryos have a large volume, Japanese flounder embryos must have low sensitivity to cryoprotectant toxicity and high permeability to water and cryoprotectants. So, we investigated the sensitivity and the permeability of Japanese flounder embryos. In addition, we assessed the survival of flounder embryos after vitrification with solutions containing methanol and propylene glycol, following Chen and Tian's report. The embryos were relatively insensitive to the toxicity of individual cryoprotectants at lower concentrations, especially methanol and propylene glycol as their report. Although their permeability to water and cryoprotectants could not be measured from volume changes in cryoprotectant solutions, the embryos appeared to be permeable to methanol but less permeable to DMSO, ethylene glycol, and propylene glycol. Although vitrification solutions containing methanol and propylene glycol, which were used in Chen and Tian's report, were toxic to embryos, a small proportion of embryos did survived. However, when vitrified with the vitrification solutions, no embryos survived after warming. The embryos became opaque during cooling with liquid nitrogen, indicating the formation of intracellular ice during cooling. When embryos had been kept in vitrification solutions for 60 min after being treated with the vitrification solution, some remained transparent during cooling, but became opaque during warming. This suggests that dehydration and/or permeation by cryoprotectants were insufficient for vitrification of the embryos even after they had been over-treated with the vitrification solutions. Thus, Chen and Tian's cryopreservation method lacks general application to Japanese flounder embryos.     

Advanced reproductive technology in the water buffalo

Embryo transfer techniques in water buffalo were derived from those in cattle. However, the success rate is much lower in buffaloes, due to their inherent lower fertility and poor superovulatory response. The buffalo ovary has a smaller population of recruitable follicles at any given time than the ovary of the cow (89% fewer at birth). In addition, estrus detection is problematic. Progress in the field of embryo transfer in water buffalo has been slow, and is primarily due to a poor response to superovulation. The average yield of transferable embryos is less than one per superovulated donor. In vitro embryo production could considerably improve the efficacy and logistics of embryo production. The technique of Ovum Pick Up is superior to superovulation; it can yield more transferable embryos per donor on a monthly basis (2.0 versus 0.6). The feasibility of intergeneric embryo transfer between buffalo and cattle has been investigated. No pregnancy resulted after transfer of 13 buffalo embryos to synchronized Holstein heifers. Preliminary successes with nucleus transfer of Bubalus bubalis fetal and adult somatic nuclei into enucleated bovine oocytes and subsequent development to the blastocyst stage have been reported.     

Nature and distribution of porosity and permeability in jurassic carbonate reservoirs of the Arabian Gulf basin

Summary The Middle-Upper Jurassic section in the Arabian Gulf basin forms one of the most prolific sequences in the world, in which an excellent combination of source, reservoir and seal rocks was developed within a major sedimentary cycle. The sequence consists of a) relatively quiet deep-water mudstone, wackestone and shale (source facies), b) shallow-water high enery grainstone and packstone (reservoir facies), and c) very shallow supratidal anhydrite (seal facies). The principal factors, which controlled the sedimentation of this sequence, are considered to have been eustatic sea-level change and epeirogenic movement of carbonate shelves. The Jurassic reservoirs of the major oil fields in this region show exceptionally high porosity up to 30% for their relatively old geologic age (some 150 million years old) and depths of burial in the range between 1,200 and more than 2,700 m. Porosity occurs most commonly as intergranular/remnant primary pore spaces, but its distribution is quite uneven and very complicated. To account for the existence of such high porosity (and permeability) in the Jurassic reservoirs, probable geological, physical and chemical factors for preserving and enhancing porosity (and permeability), such as acidic formation fluids, reduced fluid mobility, tectonic forces, ductility of intercalated beds (e.g. anhydrite), and dolomitization were examined. It has been observed in various fields in the region that oilsaturated portions of the Jurassic reservoirs tend to retain higher porosity than the surrounding water-saturated zones. Porosity preservation by hydrocarbons is possible primarily because of excess hydrocarbon pressure and of reduced mobility of water in such oil-saturated zones. To continue sediment diagenesis, a steady supply of minerals by formation water and the mobility of the water may have been essential. Because the entrapment of oil in the Jurassic reservoirs in the region is considered to have been as late as early Tertiary, some other (pre-migration) mechanisms which may have worked in the earlier geologic stages for preserving and creating porosity (and permeability) seem to be necessary.     

Osmoregulation, growth and sucrose accumulation in germinated Trigonella foenum-graecum (fenugreek) seeds treated with polyethylene glycol

Germinated seeds of Trigonella foenum-graecum L. (fenugreek) were grown in water or in polyethylene glycol (PEG) solutions. After endosperm removal, the water relations, growth, dry weight, sucrose and reducing sugar content of the embryo were determined. Under water sstress conditions, water content and osmotic potential (π₀) at saturation, growth and dry weight were lower than in non-stressed controls. The reduction in dry weight indicated a lower uptake of solutes from the endosperm and the decrease in π₀ was not accompanied by an increase in the amount of the accumulated solutes. It is suggested that embryos of stressed fenugreek seeds control osmotic potential by reduction of water uptake and that this results in reduction of growth. Embryos isolated from germinated seeds ("naked" embryos) were grown in water or in PEG solutions, with or without galactose (as an external solute source substituting for the endosperm). The results indicate that a decrease in the external solute did not account for growth reduction under conditions of water stress, and that decreased solute transport to the embryo may be important. The sucrose contents of "naked" embryos and of embryos from whole seeds were higher after PEG treatment, while reducing sugar contents were lower compared to non-stressed controls. The increased sucrose accumulation may be due to decreased sucrose hydrolysis.     

Organic,inorganic, and caloric composition of eggs,pentaculae, and adults of the brooding sea cucumber Cucumaria curata cowles (Echinodermata: Holothuroidea)

Levels (percentage composition) of water, ash, carbohydrate, lipid, protein, and calories were determined for eggs, pentaculae, and adults of the sea cucumber Cucumaria curata Cowles. Component contents (μg/individual) were calculated for eggs and pentaculae. During the 28 days of development to hatching, the large yolky eggs gain water and ash, the total dry weight increasing from 169 to 190 μg/egg during embryogenesis. There were no statistically significant changes in lipid, protein, and caloric contents during embryogenesis, but carbohydrate decreased by 0.82 μg/egg.The decrease in carbohydrate is sufficient to account for estimated embryonic energy requirements. Based on the utilization of carbohydrate, embryos of C. curata show a nutritional pattern similar to that of the planktonic embryos of sea urchins and different from that of embryos developing from terrestrial eggs, freshwater eggs, and planktonic and demersal marine eggs.Although broods varied widely in egg number and mean egg dry weight, C. curata gives eggs which contain a constant proportion of organic components.Levels of ash, water, and protein in the adults exceeded those in the pentacula, and lipid comprises a much smaller proportion of the adult body than it did of the pentacula.     

Pipetting causes shear stress and elevation of phosphorylated stress-activated protein kinase/jun kinase in preimplantation embryos

Shear stress at 1.2 dynes/cm(2) induces stress-activated protein kinase/jun kinase phosphorylation that precedes and causes apoptosis in embryos (Xie et al., 2006b, Biol Reprod). Pipetting embryos is necessary for many protocols, from in vitro fertilization to collecting embryos prior to analyzing gene expression by microarrays. We sought to determine if pipetting upregulates phosphorylated MAPK8/9 (formerly known as stress-activated protein kinase/jun kinase/SAPK/JNK1, 2). We found that phosphorylated MAPK8/9, a marker of MAPK8/9 activation, is upregulated in a dose-dependent manner by pipetting. Whereas embryos with the zona pellucida removed were more sensitive to stress-induced lethality mediated by 1.2 dynes/cm(2) shear force, phosphorylated MAPK8/9 was induced at lower numbers of pipet triturations in hatched embryos at E4.5. E4.5 embryos were more sensitive to induction of MAPK8/9 than unhatched embryos at E2.5 or E3.5. E3.5 embryos also showed a pipetting dose-dependent induction of FOS protein (formerly known as c-fos), a marker of shear stress in many cell types. Phosphorylated MAPK8/9 measured in ex vivo embryos from E1.5 to E4.5 were expressed at low levels. Embryos that had been pipetted sufficiently to induce phosphorylated MAPK8/9 and FOS had the same number of cells as untreated embryos 24 hr later. This suggests that rapid phosphorylation of MAPK8/9 due to transient shear stress does not mediate long-term negative biological outcomes. But, it is possible that techniques requiring multiple handling events would induce MAPK8/9 and cause biological outcomes or that other biological outcomes are affected by low amounts of transient shear stress. This study suggests that embryo handling prior to experimental measurement of signal transduction phosphoproteins, proteins and mRNA should be performed with care. Indeed, it is likely that shear stress may cause rapid transient changes in hundreds of proteins and mRNA.