Effects of incubation temperature on growth and development of embryos ofAlligator mississippiensis

Summary Eggs ofAlligator mississippiensis were incubated at 30 °C and 33 °C throughout incubation up to hatching. Every four days several eggs were opened and the albumen, yolk and extra-embryonic fluids removed and weighed. The embryo was removed and fixed prior to being staged, weighted and measured for various morphometric criteria. Development at 33 °C was accelerated compared with 30 °C in terms of yolk and albumen utilization and embryo growth. Significant losses in yolk mass did not occur until stage 22 at 33 °C but occurred at stage 18 at 30 °C. Different patterns in growth were observed in embryos at the two temperatures at similar morphological stages: between stages 18 and 22 embryos at 33 °C were smaller (in mass and length) compared with embryos at 30 °C despite being morphologically similar. The differences in growth and physiology between embryos at 30 °C (females) and 33 °C (males) were dependent on incubation temperature but not sex. Incubation at 33 °C accelerated both growth and development inAlligator; initially morphogenesis was accelerated by the higher temperature but later, growth rate was accelerated.     

Developmental expression of aquaporin-3 in zebrafish embryos (Danio rerio)

Fish embryos have never been successfully cryopreserved because of the low permeability of cryoprotectants into the yolk. Recently, we used aquaporin-3 fused with a green fluorescent protein (AQP3GFP) to modify the zebrafish embryo, and demonstrated that the pores functioned physiologically. This increased the water and cryoprotectant permeability of the membranes. We have continued our work on AQP3-modified embryos and here we report their developmental expression of AQP3, the success of various culture media on their survival and development, and their reproductive success. The AQP3GFP expression begins within 30 m after the mRNA AQP3GFP injection into the yolk of the 1- to 4-cell embryo. This expression is distributed in the membranes throughout the blastoderm and the yolk syncytial layer within 24 h. It diminishes after 96 h. We found no difference in the survival or normal development of embryos from AQP3GFP or wild-type adults. Additionally, zebrafish embryos did not require special culture medium to survive after AQP3GFP modification. In fact, they survived best in embryo medium (ca. 40 mOsm). Embryos reared entirely in embryo medium had a higher percent survival and a higher percent normal development than those exposed to a high osmolality sucrose culture medium (ca. 330 mOsm). The mechanism whereby these embryos can maintain their internal osmolality in a hypoosmotic solution with water channels in their membranes is unknown.     

Embryo aberrations in the amphipod Monoporeia affinis as indicators of toxic pollutants in sediments: A field evaluation

Reproduction in the amphipod Monoporeia affinis is sensitive to contaminant exposure, and embryo aberrations are used in the Swedish National Monitoring Program to indicate biological effects of contaminants. However, empirical support for a causal relationship between contaminant load and frequency of aberrations is largely based on experimental studies. A field evaluation of aberration frequency in relation to contaminant exposure is required, if we are to use reproductive disorders as indicators of contaminant effects in risk assessment. In this study, we evaluated occurrence of females carrying malformed embryos and frequency of specific embryo aberrations in broods of M. affinis in relation to concentrations of various pollutants in sediments. Gravid M. affinis and surface sediment were sampled at ten sites in the Gulf of Bothnia located at varying proximity to historical and current polluting point sources. At each site, concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and trace and heavy metals in surface sediments were determined. Amphipod fecundity and embryo development status were assayed with regard to morphologically recognizable aberrations (malformed embryos, membrane-damaged embryos, embryos with arrested development and females with dead brood). Our key finding is that different types of embryo aberrations were significantly associated with specific contaminant groups in the sediment. In particular, occurrence of females with embryo malformations was strongly related to elevated concentrations of Cd and PCBs, while females with membrane-damaged embryos occurred at high PAH concentrations. Also, frequency of embryos with arrested development was linked to elevated concentrations of PAHs and metals. Finally, frequency of females carrying dead broods, which was earlier considered to be a hypoxia-induced pathology, was significantly positively related to PAH concentrations. Thus, these reproductive aberrations in M. affinis can serve as contaminant-specific indicators of PCB, PAH and heavy metal exposure in biological effect monitoring.     

Radioactive labeling of proteins in cultured postimplantation mouse embryos I. Influence of the embryo preparation method

Summary Conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations followed by fluorography. The aim was to obtain highly radioactive proteins under conditions as physiological as possible. Embryos at Days 10, 11, and 12 of gestation were prepared in different ways and incubated for 4 h in Tyrode’s solution containing [³H]amino acids (mixture) at a concentration of 27 μCi/ml medium. The preparations were: a) yolk sac opened, placenta and blood circulation intact; (b) yolk sac and amnion opened, placenta and blood circulation intact (Day 10 embryos only); c) placenta, yolk sac, and amnion removed (embryo “naked”); d) naked embryos cut randomly into pieces (Day 10 mebryos only). After incubation whole embryos or certain parts (tail, liver, rest body) were investigated by determining the radioactivity taken up by the protein. The results are given in dpm per mg protein per embryo. Radioactivity of proteins was about 3 times higher in naked mebryos than in embryos left in their yolk sacs. This was true for all three stages investigated. However, the degree of radioactivity in the various parts of naked embryos differed by a factor of 15, whereas radioactivity was evenly distributed in embryos incubated in their yolk sacs. Therefore, embryos prepared according to the first methods (see above) fulfilled the conditions required at the best. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic Analysis of Cell Proteins).     

Biomass Measurement of Methanogens in the Sediments of Tokyo Bay Using Archaeol Lipids

An archaeal ether-linked lipid, archaeol, was determined to be a biomass indicator for methanogens both in the laboratory enriched culture and in marine sediments. The archaeol measurement method described by Ohtsubo et al. in 1993 was modified and applied to marine sediments. We compared the amount of archaeol with the cell number of methanogens or methane concentration in laboratory enriched culture of methanogens from marine sediment. Good correlations were obtained as follows: (Methane, mmol) = 11.2 × (Archaeol, mg): r= .996 or (Cell number) = 1.13 × 10¹¹× (Archaeol, mg): r= .995, respectively. In the sediments of Tokyo Bay, archaeol was measured from approximately 46 to 561 ng/dry g sediment at the entrance to 267 to 4160 ng/dry g sediment at the innermost area. Using the coefficient from the laboratory experiment, these data corresponded to cell numbers of 5.2 × 10⁶ to 4.7 × 10⁸/dry g sediment. These values were 1 or 2 orders of magnitude higher than those obtained by culture methods in previous studies. Although dead or decomposed cells might be detected, archaeol measurement is useful for estimating the biomass of methanogens because of the good correlation between methane concentration and archaeol content in marine environments. In this study, we found a correlation of (Methane, mmol) = 0.012 × (Archaeol, mg): r= .932, n= 17 in marine sediments. Received December 21, 1998; accepted June 16, 1999     

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O₂ / 5% CO₂ in a rolling bottle culture apparatus at 37 °​C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.     

Recent advances in the use of meiofaunal polychaetes for ecotoxicological assessments

While whole sediment toxicity tests with macrofaunal polychaetes are well developed and standardized, they are oftentimes not very sensitive to environmental contaminants. Meiofaunal polychaetes, however, are sensitive to contaminants, easy to culture, and representative of the interstitial habitat. These are desirable attributes for ecotoxicological assessments of marine and estuarine sediments. The meiofaunal polychaete, Dinophilus gyrociliatus Schmidt, 1857, is a cosmopolitan species that has become a useful tool for ecotoxicological assessments, particularly for its use in toxicity testing with sediment pore waters. Due to its short life cycle it is suitable for sublethal toxicity tests, with egg production by the females as a sensitive endpoint, which can be assessed in a 7-day exposure period. Toxicity assessments of pore waters from an industrialized bay in Texas, U.S.A., and of pore waters from sediments spiked with nitroaromatic explosives, demonstrated that the D. gyrociliatus reproduction endpoint was consistently among the most sensitive, when compared to early-life stage tests with several other marine species, including macro-algae, sea urchins and fish. There was also excellent agreement among the results of porewater tests with D. gyrociliatus, nauplii of the meiofaunal copepod, Longipedia americana, and embryos of the sea urchin, Arbacia punctulata in a survey with pore waters extracted from sediments collected in the vicinity of an oil platform in the Gulf of Mexico, where metals were the primary contaminants of concern.     

DIFFERENTIAL EFFECTS OF SEDIMENTS ON SURVIVAL AND GROWTH OF FUCUS SERRATUS EMBRYOS (FUCALES,PHAEOPHYCEAE)1

Recruitment of seaweeds through small reproductive stages is limited on sediment inundated rocky shores and largely unsuccessful in soft sediment environments. Burial in sediment has several potentially negative effects for seaweed propagules, and these effects were differentiated in a laboratory experiment. We investigated how light deprivation, sediment type (grain size, organic content, and origin), and sediment chemistry (oxygen presence and toxicity through hydrogen sulfide) affected survivorship and growth of Fucus serratus L. embryos. Presence of hydrogen sulfide had overriding negative impacts on both survivorship and growth of Fucus embryos, independently of sediment type and light availability. In contrast, simple anaerobiosis generally did not impair survival or growth of the embryos. Fine sediments, 3 mm thick, significantly reduced embryo survivorship, presumably through accumulation of metabolic waste products in the immediate vicinity of the embryos as a consequence of constrained diffusion. This effect was equally pronounced in the presence of a 1‐mm layer of organically rich biodeposits. Irradiance levels did not affect survival of embryos but influenced growth. Decreasing thickness and increasing coarseness of sediments together represented a gradient of enhanced light penetration and diffusion. Growth of embryos increased along this gradient. In nature, soft sediment environments with organically enriched muds (e.g. tidal flats and salt marshes) represent habitats least favorable for colonization through small reproductive stages of seaweeds.     

New Approaches for Studying the Permeability of Fish Embryos: Toward Successful Cryopreservation

This paper describes some new approaches for understanding the permeability of teleost embryos. The dechorionated zebrafish (Brachydanio rerio) was used as a model for basic studies of water and cryoprotectant permeability. These embryos are composed of two compartments, a large yolk (surrounded by the yolk syncytial layer) and differentiating blastoderm cells. Cellular water was distributed unequally in each compartment. Measurements indicated that the total water in the embryo was 74%, while the total water in the yolk was 42%, and total water in the blastoderm was 82%. The internal isosmotic value for the zebrafish embryo is unknown. However, for one-compartment modeling studies of membrane permeability, the mean Lp (±SEM) values were 0.022 ± 0.002 to 0.049 ± 0.008 μm × min⁻¹atm⁻¹at 40 mOsm (assuming this was one possible internal isosmotic value for the entire embryo) and 0.040 ± 0.004 to 0.1 ± 0.017 μm × min⁻¹atm⁻¹at 300 mOsm (assuming this was another possible internal isosmotic value for the entire embryo). When three- and six-somite embryos were placed in 1.5 and 2.0Mcryoprotectants (dimethyl sulfoxide and propylene glycol), osmometric measurements of volume changes indicated no cryoprotectant permeation. However, similar measurements with methanol revealed a small volume decrease (ca. 8%) and recovery (ca. 5%) for six-somite embryos in a 2.0Msolution. Magnetic resonance (MR) images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that only methanol permeated the entire embryo within 15 min. The other cryoprotectants exhibited little or no permeation into the yolk over 2.5 h. The results from MR spectroscopy and cryoprotectant microinjections into the yolk suggested that the yolk syncytial layer plays the critical limiting role for cryoprotectant permeation throughout the embryo.     

Comparative proteome analysis of the embryo proper and yolk sac membrane of day 11.5 cultured rat embryos

BACKGROUND: Proteomic analysis of cultured postimplantation rat embryos is expected to be useful for investigation into embryonic development. Here we analyzed protein expression in cultured postimplantation rat embryos by two-dimensional electrophoresis (2-DE) and mass-spectrometric protein identification. METHODS: Rat embryos were cultured from day 9.5 for 48 h or from day 10.5 for 24 h. Proteins of the embryo proper and yolk sac membrane were isolated by 2-DE and differentially analyzed with a 2-D analysis software. Selected protein spots in the 2-DE gels were identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometric analysis and protein database search. RESULTS: About 800 and 1,000 protein spots were matched through the replicate 2-DE gels each from one embryo in the embryo proper and yolk sac membrane, respectively, and virtually the same protein spots were observed irrespective to the length of culture period. From protein spots specific to the embryo proper (126 spots) and yolk sac membrane (304 spots), proteins involved in tissue-characteristic functions, such as morphogenesis and nutritional transfer, were identified: calponin, cellular retinoic acid binding protein, cofilin, myosin, and stathmin in the embryo proper, and Ash-m, dimerization cofactor of hepatocyte nuclear factor, ERM-binding phosphoprotein, cathepsin, and legumain in the yolk sac membrane. CONCLUSION: Proteomic analysis of cultured postimplantation rat embryos will be a new approach in developmental biology and toxicology at the protein level.     

Presence and distribution of E-cadherin in the 4-cell golden hamster embryo. Effect of maternal age and parity

Maternal age dependency of gestation time in hamster and in other mammals is a well demonstrated fact. We have recently shown that adult nulliparous and multiparous hamster females show significant asynchrony and retard on early embryo development (from two blastomeres to morula stages) when compared with nulliparous young females. The number of cell-cell adhesions between blastomeres in early embryo development has been reported to be a good indication of the ability of embryos to cleave and develop. In this work we studied, by indirect immunofluorescence, the presence and distribution of E-cadherin in 4-cell embryos obtained from nulliparous young (NYF), nulliparous adult (NAF) and multiparous adult (MAF) hamster females. Distribution and intensity of fluorescence was observed and registered using confocal microscopy. Staining intensities for E-cadherin were quantified by computed densitometry in the free membrane regions, in the cytoplasm region and in the cell-cell adhesion zones of each embryo. E-Cadherin in all the studied zones was significantly higher (p<0.01) in NYF. Cadherin concentration in the intercellular membranes was always statistically higher (p<0.05) than in the free membrane regions. An appreciable concentration of E-cadherin was found in the cytoplasm of the 4-cell embryos obtained from the three groups of females, but was significantly higher in NYF. No statistical differences were observed in any of the parameters studied between NAF and MAF. Our results seem to indicate that changes in the reproductive behavior related to age and/or multiparity may be correlated with changes in the processes related to intercellular adhesions during early cleavage.     

Testing generalizations about latitudinal variation in reproduction and recruitment patterns with sicyoniid and caridean shrimp species

Summary

Study of latitudinal variation in seasonality of reproduction and recruitment of benthic marine invertebrates is useful in generating and testing hypotheses about causal factors acting on reproduction such as temperature and larval food supply that might be altered by changes in world climate. Analysis of latitudinal variation in reproductive patterns might be made with comparisons (a) among species with a common phylogenetic history from different latitudes and habitats and (b) among phylogenetically different taxa from the same location. Hypotheses on variation of reproductive seasonality with latitude are tested here with results of a study on nine species of caridean and two species of sicyoniid shrimp sampled from a tropical seagrass meadow in Puerto Rico. Breeding condition was determined by the presence or absence of incubated embryos (carideans) and the state of ovarian development in both carideans and sicyoniids. Recruitment was estimated from the percentage of individuals of monthly population samples in the juvenile size classes. Comparison of reproductive patterns among tropical, subtropical, and cool temperate Sicyonia spp. supports the paradigm of continuous reproduction in the tropics with increased restriction of breeding season with an increase in latitude. A greater intensity of breeding effort appears to accompany the shorter breeding period associated with an increase in latitude. At the tropical site most females of all caridean species carried embryos during all months of the year. With the onset of sexual maturity, caridean females produced consecutive broods for the rest of their relatively short (< 6 month) life span. In both sicyoniid and caridean species, recruitment occurred throughout the year but was highly variable, i.e., episodic rather than truly continuous or seasonal. Patterns of recruitment were highly concordant among but not between sicyoniid and caridean species, indicating that different sets of environmental factors controlled recruitment in the two groups. It is suggested that simultaneous study of adult reproduction and larval ecology is necessary to understand patterns of reproduction and recruitment. Coordinated effort on a global scale in studying latitudinal variation in reproduction and recruitment is suggested in order to predict the consequences of climate change on commercially and ecologically important marine invertebrate species.     

The role of the yolk sac in craniofacial development of cultured rat embryos

To study the role of the yolk sac and amnion in craniofacial development, the effects of opening the yolk sac and amnion on facial formation of rat embryos were examined in vitro. Rat embryos were cultured for 72 hr from day 11.5 of gestation using an improved rotation apparatus. In experiments, the yolk sac and amnion were opened at the time of explantation (day 11.5) in one group (D11 open) and were opened 24 hr after the beginning of the culture (day 12.5) in another group (D12 open). Cleft lip developed in 100% of cultured embryos when the yolk sac and amnion were opened at day 11.5 (D11 open). In the D12 open group, however, cleft lip occurrence decreased to 3.0%. Protein content, wet weight, and somite number of cultured embryos were not significantly different in the two groups. The results of this study demonstrate that it is beneficial to open the yolk sac and amnion after 24 hr in culture for normal facial formation of rat embryo cultured from day 11.5 of gestation.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

Feeding, growth, and fecundity of Abarenicola pacifica in relation to sediment organic concentration

For marine deposit-feeding invertebrates, the distribution of species with different life history strategies has long been known to be correlated with sediment organic concentration. Large populations of opportunistic species are found in sediments with enriched organic concentration, while equilibrium species populate low organic concentration sediments. Differences in both behavioral (e.g. feeding rate) and physiological (e.g. growth rate, reproductive output) adaptations determine the ability of species to establish populations in different environments. By systematically documenting differences in the way these factors vary as sediment organic concentration varies for both opportunistic and equilibrium species, we can better understand the mechanisms underlying this correlation between sediment organic concentration and species distributions. Here, we present the results of experiments examining the interactions among food concentration, feeding rate, growth rate, and reproductive output (measured as egg number and size) for the equilibrium species Abarenicola pacifica. A. pacifica is a large, long-lived, iteroparous, sub-surface deposit-feeding polychaete. Individual worms were reared throughout most of one generation in sediments differing only in the concentration of organic matter. Juveniles (<20 mg AFDW) had higher feeding rates and growth rates in sediments of higher organic concentration throughout the range tested. These results are consistent with the predictions from optimal foraging theory. As worms grew, however, these patterns changed. Once worms reached a mean body size of approximately 50 mg AFDW, feeding rate was greater on sediments of lower organic concentration (although it took worms in the sediments with lower organic concentration longer to reach this size). Differences in growth rates among treatments decreased as worms grew. For worms >100 mg AFDW, growth rates were uniformly low ( approximately 1%/day) on all sediments, but the early advantage obtained by worms in the high organic treatments resulted in much greater body sizes after 200 days. Worms had higher tissue triacylglyceride concentrations and produced more eggs (independent of worm size) as sediment organic concentration increased. We conclude that A. pacifica alters its feeding rate in response to variations in food resources in such a way as to maximize its energy intake and thereby maximize fitness. Future studies should investigate whether opportunistic species (as well as other equilibrium species) also have this ability.     

Effect of supplementation of different growth factors in embryo culture medium with a small number of bovine embryos on in vitro embryo development and quality

When embryos are cultured individually or in small groups, blastocyst yield efficiency and quality are usually reduced. The aim of this work was to investigate the effect of supplementation of the embryo culture medium (CM) with several growth factors (GFs) on embryo development and apoptosis rate when a reduced number of embryos were in vitro cultured. Two experimental studies (ES) were carried out. In ES 1, five treatments were tested to study the effect of GF on embryo development: Control (∼30 to 50 embryos cultured in 500 μl of CM); Control 5 (Five embryos cultured in 50 μl microdrops of CM), without addition of GF in either of the two control groups; epidermal GF (EGF); IGF-I; and transforming GF-α (TGF-α) (Five embryos were cultured in 50 μl microdrops of CM with 10 ng/ml EGF, 10 ng/ml IGF-I or 10 ng/ml TGF-α, respectively). In ES 2, following the results obtained in ES 1, four different treatments were tested to study their effect on embryo development and quality (number of cells per blastocyst and apoptotic rate): Control; Control 5; EGF, all three similar to ES 1; EGF + IGF-I group (five embryos cultured in 50 μl microdrops of CM with 10 ng/ml EGF and 10 ng/ml IGF-I). In both ESs, it was observed that a higher proportion of embryos cultured in larger groups achieved blastocyst stage than embryos cultured in reduced groups (22.6% v. 14.0%, 12.6% and 5.3% for Control v. Control 5, IGF-I, TGF-α groups in ES 1, and 24.9% v. 17.1% and 19.0% for Control v. Control 5 and EGF in ES 2, respectively; P < 0.05), with the exception of embryos cultured in medium supplemented with EGF (18.5%) or with EGF + IGF-I (23.5%), in ES 1 and ES 2, respectively. With regard to blastocyst quality, embryos cultured in reduced groups and supplemented with EGF, alone or combined with IGF-I, presented lower apoptosis rates than embryos cultured in reduced groups without GF supplementation (11.6% and 10.5% v. 21.9% for EGF, EGF + IGF-I and Control 5 groups, respectively; P < 0.05). The experimental group did not affect the total number of cells per blastocyst. In conclusion, this study showed that supplementation of the CM with EGF and IGF could partially avoid the deleterious effect of in vitro culture of small groups of bovine embryos, increasing blastocyst rates and decreasing apoptosis rates of these blastocysts.     

Extracellular matrix of sea urchin and other marine invertebrate embryos

The extracellular matrix surrounding the sea urchin embryo (outer ECM) contains fibers and granules of various sizes which are organized in recognizable patterns as shown by ultrastructural studies, particularly stereoimaging techniques. The use of the ruthenium red method for retaining and staining the ECM, with modifications of the Luft (Anatomical Record 171:347-368, 1971) method for invertebrate embryos, allows for the clarification of certain structures, particularly fiber compaction in the interzonal region, and microvillus-associated bodies. The inner ECM in the sea urchin embryo includes the basal lamina and blastocoel matrix. Stereoimages show that the fibers which are loosely distributed in the blastocoel matrix become compacted around the periphery of the blastocoel to form the basal lamina. The ruthenium red method was also used on a number of marine invertebrate embryos and larvae, representing different phyla, to facilitate comparisons between their surface coats. The similarities observed in the specimens shown suggest that ECMs are widely found on marine invertebrate eggs, embryos, and larvae, and that they resemble vertebrate ECMs and may, therefore, have similar functions.     

Early development of johnny darter,Etheostoma nigrum,and fantail darter,E. flabellare,with a discussion of its ecological and evolutionary aspects

Synopsis Fantail darters,Etheostoma flabellare, have larger eggs with greater yolk volume than johnny darters,E. nigrum, and have a longer embryo period. This longer embryo period is associated with accelerated finfold differentiation, producing a larger, better formed individual at the onset of exogenous feeding. Developmental differences among these two species and the rainbow darter,Etheostoma caeruleum, primarily in timing of ontogenetic events, are described in detail. These differences in timing are considered largely early developmental phenomena as they affect embryos, larvae, and aievins but apparently not juveniles or adults. Demographic aspects of early life history were assessed using data from studies of Ontario streams. Differences in early survival among species appear to play an important role in recruitment of mature females, but cannot be attributed to parental care or egg size differences.     

Proteomic analysis of selenium embryotoxicity in cultured postimplantation rat embryos

BACKGROUND: Developmental toxicity of selenium (Se) is a nutritional, environmental and medicinal concern. Here, we investigated Se embryotoxicity by proteomic analysis of cultured rat embryos. METHODS: Rat embryos at day 9.5 or 10.5 of gestation were cultured for 48 or 24 h, respectively, in the presence of sodium selenate (100 or 150 µM) or sodium selenite (20 or 30 µM). Proteins from the embryo proper and yolk sac membrane were analyzed by two‐dimensional electrophoresis for quantitative changes from those in control embryos. Proteins with quantitative changes were identified by mass spectrometric analysis. RESULTS: Growth inhibition and morphological abnormalities of cultured embryos were observed in all the Se treatment groups. By the analysis of the embryo proper, actin‐binding proteins were identified as proteins with quantitative changes by selenate: increased phosphorylated‐cofilin 1, increased phosphorylated‐destrin, decreased drebrin E, and decreased myosin light polypeptide 3. Many proteins showed similar changes between selenate and selenite, including increased ATP‐synthase, decreased acidic ribosomal phosphoprotein P0, and decreased pyrroline‐5‐carboxylate reductase‐like. In the yolk sac membrane, antioxidant proteins were identified for protein spots with quantitative changes by selenite: increased peroxiredoxin 1 and increased glutathione S‐transferase. CONCLUSION: The identified proteins with quantitative changes by selenate or selenite were considered to be candidate proteins involved in Se embryotoxicity: the actin‐binding proteins for selenate embryotoxicity, proteins with the similar changes for the common Se embryotoxicity and antioxidant proteins for modification of Se embryotoxicity by redox‐related treatments. These proteins may also be used as biomarkers in developmental toxicity studies. Birth Defects Res (Part B), 2008. © 2008 Wiley‐Liss, Inc.     

EMBRYO AND ORGAN CULTURES OF THE HORSESHOE CRAB,TACHYPLEUS TRIDENTATUS*

This paper deals with some preliminary technical experiments on cultures of the embryos and organs of Tachypleus tridentatus. In embryo culture, using the embryos at various stages of development, embryonic areas with hardly any yolk remaining in them were cultured in various media and by various methods (Series A); hemispheres of embryos containing the embryonic area (Series B) and whole embryos without egg membranes (Series C) were cultured in sterilized sea water. The suitability of the culture media and methods is described; the medium containing calf serum or 199, and the hanging-drop method were found to be the most appropriate of those used in respective series of experiments. The changes in external and internal structures of the explants from embryos at various developmental stages cultured in various media and by various methods are described; dorsal closure, secretion of inner egg membrane, thickening of the cell layer and the elongation of limb buds were all observed in the Series A experiments. In Series B and C, coelom formation and also normal development of the embryos cultured after the 2nd embryonic moulting were observed. In organ culture, fragments of the embryonic heart were cultured in various media and by various methods. The hanging-drop method resulted in longer survival than the method using agar media.