The effects of simulated microgravity on avian embryonic development.

Based on the few reports available, microgravity (MG) can have adverse effects on the early development of vascularised extra-embryonic membranes in avian eggs. Whether gravity or oxygen availability is the stimulus for development of the blood vessels in the chorioallantoic membranes (CAM) remains unclear. Under gravity the blastoderm forms on top of the yolk sac, closest to the oxygen rich region beneath the shell membranes, and from there the CAM buds from an abdominal extension subsequently to form a close contact with shell membranes. Then as the embryo develops it spreads beneath the eggshell surface to maximise the surface area of the CAM vascular bed available for O2 uptake. To investigate how simulated MG influences development of the CAM and embryo we conducted experiments on chicken embryos during incubation in a 3D-clinostat (control or continuous MG treatment at 5 rpm). Further, to determine if CAM angiogenesis is directed towards regions of high O2 tension or gravity we investigated the effects of wax treatment (50% shell surface area) on development in MG. We found that clinostat MG caused embryonic failure between day 0-5 by preventing normal development of CAM-shell membrane complex. Thereafter acute MG promoted increases in CAM mass, but did not affect embryo mass. Preliminary findings suggest that combined acute MG and wax treatment did not significantly affect embryonic growth in either MG or control groups, but retarded CAM growth in control embryos only. Finally, we will present evidence to show that acute and prolonged exposure to MG does not prevent normal growth and hatching, but might have more subtle effects on hatchling physiology, including reduced heart mass.     

The effects of Scutellaria baicalensis extract on embryonic development in mice

BACKGROUND : Scutellaria baicalensis is a perennial herbaceous plant widely distributed in Oriental areas. Its roots, a commonly used medicinal source, reputedly calm fetuses in pregnant women; however, there is no sufficient evidence to date to assess its safety during pregnancy. This study aims to evaluate the effects of S. baicalensis aqueous extract on embryonic development in ICR mice. METHODS : Aqueous extract of S. baicalensis roots was prepared in accordance with clinical application. Pregnant mice were randomly divided into four groups, i.e., mice treated by gavage with water as negative control, with aqueous extract of 2 (1.8 times of human daily dose), 8 or 32 g/kg/day from gestation day (Gd) 6 to 15 as low-, middle-, and high-dose groups, respectively. The parameters of live and dead fetuses, resorptions, external and skeletal malformed fetuses, maternal body weight, maternal liver, kidneys, and heart weights were evaluated on Gd 18. RESULTS : There was no significant difference in fetal parameters among four groups. Maternal absolute liver and kidneys weights in the high-dose group were significantly higher than those in negative control (p<0.05). Relative liver and kidneys weights in this group were significantly higher than those in any other group (p<0.05). CONCLUSIONS : Oral administration of aqueous extract of S. baicalensis roots at or below 32 g/kg/day to ICR mice during organogenesis did not cause significant fetal external or skeletal malformations. However, 32 g/kg/day presented potential maternal toxicity. Birth Defects Res (Part B)86: 79-84, 2009. © 2009 Wiley-Liss, Inc.     

Combined effects of radiation and caffeine on embryonic development in mice

The combined effect of radiation and caffeine has been studied in mouse embryos. Radiation and/or caffeine were administered to ICR mice on Day 11 of gestation. Intrauterine death, gross malformation, and fetal body weight were selected as indicators of effects. Doses of whole-body gamma irradiation were 0.5 to 2.5 Gy and those of caffeine were 100 and 250 mg/kg maternal body wt. Intrauterine mortality increased with increasing radiation dose; this trend was more remarkable in combination with caffeine. Gross malformations such as cleft palate and defects of forelegs and hindlegs appeared frequently in the fetuses treated with both radiation and caffeine. Decreased fetal weight was observed even in mice treated with 0.5 Gy of radiation or 100 mg/kg caffeine. There was a linear relationship between dose and reduction of fetal weight. The fetal weight was a sensitive, precise, and easy-to-handle indicator for the effects of growth retardation. Intrauterine mortality and frequencies of cleft palate and defects of forelegs and hindlegs were higher than the sum of those induced by radiation and by caffeine separately. The results indicated that the combined action of radiation and caffeine on intrauterine death and malformations was synergistic.     

Inert gas enhancement of superoxide radical production.

Two free radical generating systems, xanthine oxidase/hypoxanthine or phenazine methosulfate/NADH, were exposed to air plus He, N2, or Ar at partial pressures ranging from 0.2 to 6.0 MPa, and the rates of production of superoxide, hydroxyl, singlet O2, and H2O2 were measured. All three inert gases acted similarly to enhance the production of superoxide radicals by facilitating interactions between iron and H2O2, or O2 and organic radicals. These reactions occurred at quite low gas partial pressures, only 0.28 MPa, and hydrostatic pressures of up to 6.0 MPa had no effect on radical reactions. Enhanced radical production may be the basis for the inhibition of cellular growth mediated by inert gases, and inert gas enhancement of O2 toxicity.     

Pleiotropic effects of zebrafish protein-tyrosine phosphatase-1B on early embryonic development

Protein tyrosine phosphorylation is an important mechanism of eukaryotic cell signalling which is regulated by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we report the molecular cloning of the first zebrafish protein-tyrosine phosphatase, zf-PTP-1B, the homologue of human PTP-1B. Zf-PTP-1B was catalytically active and localised to the endoplasmic reticulum, like human PTP-1B. Zf-PTP-1B was maternally expressed in zebrafish embryos, and low ubiquitous expression was detected up to day 7 of development. Microinjection of zf-PTP-1B RNA induced pleiotropic, but reproducible developmental defects. Evaluation of the live embryos at 24 h post fertilisation indicated that zf-PTP-1B induced defects in somite formation. The phenotype was dependent on protein-tyrosine phosphatase activity of zf-PTP-1B, since embryos injected with catalytically inactive zf-PTP-1B-C213S developed normally. Co-injection of wild type and inactive zf-PTP-1B led to a rescue of the zf-PTP-1B-induced phenotype, suggesting that zf-PTP-1B-C213S had dominant negative activity. The zf-PTP-1B-induced phenotype suggests that proper tyrosine phosphorylation of key proteins is essential for early development, most notably somitogenesis.     

Temperature effects on embryonic development and the life cycle of Diphyllobothrium dendriticum

Diphyllobothrium dendriticum eggs collected from hamster faeces were incubated at 10 or 20 degrees C, or maintained at 4 degrees C for 11-30 months. On day 65, 20-50% of eggs failed to hatch at 10 degrees C and 42-51% did not hatch by day 21 at 20 degrees C. Our study indicates that eggs begin to hatch in mid-August and persist until October in many lakes within the natural range of D. dendriticum. Our results demonstrate that eggs stored at 4 degrees C will hatch, suggesting they can persist in the environment for long periods and contribute to the D. dendriticum life cycle in high Arctic lakes by hatching months or years after release.     

Stage-specific excitation of cannabinoid receptor exhibits differential effects on mouse embryonic development

Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. We have previously demonstrated that preimplantation mouse embryos express mRNA for these receptors and that the periimplantation uterus contains the highest level of anandamide yet discovered in a mammalian tissue. We further demonstrated that 2-cell mouse embryos exposed to low levels of anandamide (7 nM) or other known cannabinoid agonists in culture exhibit markedly compromised embryonic development to blastocysts and that this effect is mediated by CB1-R. In contrast, the present study demonstrates that blastocysts exposed in culture to the same low levels of cannabinoid agonists exhibited accelerated trophoblast differentiation with respect to fibronectin-binding activity and trophoblast outgrowth. Again, these effects resulted from activation of embryonic CB1-R. There was a differential concentration-dependent effect of cannabinoids on the trophoblast, with an observed inhibition of differentiation at higher doses. These results provide evidence for the first time that cannabinoid effects are differentially executed depending on the embryonic stage and cannabinoid levels in the environment. Since uterine anandamide levels are lowest at the sites of implantation and highest at the interimplantation sites, the new findings imply that site-specific levels of anandamide and/or other endogenous ligands in the uterus may regulate implantation spatially by promoting trophoblast differentiation at the sites of blastocyst implantation.     

Studies on effects of co-culturing on the development of embryonic and postnatal cerebellar cells

Experiments were conducted to compare the in vitro development of different cerebellar cells in primary surface cultures to that observed in their co-cultures. Single cultures of embryonic (E.19) and postnatal (P 7) cerebellar cells as well as their co-cultures were established. Changes in cell-size distribution and in high-affinity GABA-uptake were studied during the first three weeks in the different cultures. Morphological analyses showed that in single cultures of embryonic (E 19) cells a number of large and middle-size neurons survived on the top of confluent monolayer of large flattened astroglial cells. In cultures of postnatal (P 7) cerebellar cells small tetanus toxin positive non-GABAergic neurons proved to be the most abundant cell-constituents while no confluent feeder-layer of non-neuronal cells was formed. In co-cultures both embryonic and postnatal morphological features were observed but we could not observe any improvement in either survival or maturation of the neuronal types studied. A transient increase in glial GABA-uptake, however, was observed in both postnatal and co-cultures soon after plating of postnatal cerebellar cells.     

Temperature effects on embryonic abscisic acid levels during development of wheat grain dormancy

Abscisic acid (ABA) levels were determined in both the embryo and remaining grain remnant during development of wheat caryopses under temperature conditions which produced either high or low levels of dormancy in mature grain. Higher levels of grain dormancy were produced in grain from plants grown at 15°C as compared to 25°C. In grain grown at 15°C, embryonic ABA levels steadily increased during development, reaching a maximum at stage IV, just before grain desiccation. At 25°C, ABA levels were very high at the earliest stages of embryonic development, but dropped rapidly during maturation. Only small cultivar differences in ABA levels were observed during development at either temperature. In general, higher levels of dormancy in mature grain correlated with prolonged elevation of ABA levels during grain maturation.Contribution from USDA-ARS and the College of Agriculture and Home Economics Research Center, Washington State University, scientific paper no. 8901-13.Mention of a specific product name by the United States Department of Agriculture does not constitute an endorsement and does not imply a recommendation over other suitable products.     

Temperature related effects on embryonic development of the Mediterranean locust, Dociostaurus maroccanus

Laboratory studies were conducted to assess the effect of temperature on the development of the eggs of Dociostaurus maroccanus (Thunberg) (Orthoptera, Acrididae) during anatrepsis (stages I–XIV) and during catatrepsis (stages XV–XX). The developmental rates of anatrepsis were studied at five constant temperatures ranging from 10 to 30°C. Egg development occurred over the entire range but at 10°C the embryos were unable to complete anatrepsis. The relationship between temperature and developmental times for completing anatrepsis was analysed by the non‐linear Logan type III model. The optimal temperature estimated for the development of eggs during anatrepsis was 24.7°C; the lower and upper thermal thresholds were 9°C and 31°C, respectively. Once the embryos completed anatrepsis, only those incubated at 15°C continued morphogenesis beyond stage XIV (diapause stage) without a low‐temperature exposure period. The developmental rate of catatrepsis was studied at four constant temperatures ranging from 15°C to 30°C after exposure to low‐temperature, 10°C, for 30, 60 or 90 days. For catatrepsis, temperature and developmental time were linearly and inversely related. Linear regression was used to estimate the lower developmental threshold and the degree days requirements for catatrepsis. Both decreased with longer exposure to the low temperature; the former from 13.8°C to 10.5°C and the latter from 212.8 to 171.5 degree days, following 30 and 90 days at 10°C, respectively. Our results improve the ability of decision support systems for Mediterranean locust pest management by providing better forecasts to land managers and pest advisors.     

Temperature effects on embryonic abscisic acid levels during development of wheat grain dormancy

Abscisic acid (ABA) levels were determined in both the embryo and remaining grain remnant during development of wheat caryopses under temperature conditions which produced either high or low levels of dormancy in mature grain. Higher levels of grain dormancy were produced in grain from plants grown at 15°C as compared to 25°C. In grain grown at 15°C, embryonic ABA levels steadily increased during development, reaching a maximum at stage IV, just before grain desiccation. At 25°C, ABA levels were very high at the earliest stages of embryonic development, but dropped rapidly during maturation. Only small cultivar differences in ABA levels were observed during development at either temperature. In general, higher levels of dormancy in mature grain correlated with prolonged elevation of ABA levels during grain maturation.     

Inert gas diffusion in heterogeneous tissue I: Without perfusion

A mathematical model is proposed to describe transient gas diffusion into a block of heterogenous tissue placed on an impermeable base. The corresponding asymptotic sultion of mass uptake of the gas is derived on the assumption that the diffusion constant is very much smaller in the cellular phase. It is expected that this will be useful in evaluating the diffusion constant in cellular material, and the volume fraction of extracellular fluid, providing the partition coefficient is known. The phenomenon of mutual interaction and multiple feedback between cellular and extracellular fluid is clearly seen in the overall response of the tissue. In this regard it is shown that the extraction of the two least dominant time constants, by backward projection of the experimental data curve of gas uptake, is likely to confuse the numerical evaluation of the physical parameters of the system. In an appendix, the problem of diffusion straight through a tissue slice is solved at the asymptotic stage, before steady state is reached. The resulting expression predicts the by-passing of cells by the diffusing gas and shows how the parameters cannot reliably be, determined.     

Reconstructing embryonic development

Novel approaches to bio-imaging and automated computational image processing allow the design of truly quantitative studies in developmental biology. Cell behavior, cell fate decisions, cell interactions during tissue morphogenesis, and gene expression dynamics can be analyzed in vivo for entire complex organisms and throughout embryonic development. We review state-of-the-art technology for live imaging, focusing on fluorescence light microscopy techniques for system-level investigations of animal development, and discuss computational approaches to image segmentation, cell tracking, automated data annotation, and biophysical modeling. We argue that the substantial increase in data complexity and size requires sophisticated new strategies to data analysis to exploit the enormous potential of these new resources.     

Inert gas diffusion in heterogeneous tissue II: With perfusion

In this paper, a tractable mathematical model is proposed to describe transient inert gas diffusion in heterogeneous tissue with perfusion controlling gas input to the cellular region. The corresponding solution of overall mass uptake of the inert gas is derived exactly and should be useful in interpreting washout curves from particular tissue zones, whether there is any interaction with cellular diffusion or not. It is shown that the solution contains effectively nearly all models hitherto proposed to describe gas uptake in tissue. However some indication is given of a possible situation where perfusion, extra-cellular and cellular diffusion will need to be treated separately.     

On the possible effects of homeostatic shifts in human embryonic development

Possible etiological factors of congenital malformations as well as of human trisomies are considered in the framework of the repressor hypothesis. In this approach gene expression is envisaged from the point of view of the functional variations of the total activation energy for normal gene expression in homeostatic equilibrium. We restrict our attention to variations of the total activation energy under the effect of temperature gradients. We discuss the evidence that hyperthermia may be an etiological factor for trisomies in humans.Also at the Instituto Internacional de Estudios Avanzados Apartado 17606, Parque Central, Caracas 1015-A, Venezuela.