Influence of cosmic radiation and/or microgravity on development of Carausius morosus.

Eggs of Carausius morosus were exposed to spaceflight conditions in two spaceflight missions, the German 7 day Spacelab Mission D1 and the Soviet 12.56 day Biosatellite Mission "COSMOS 1887". During spaceflight the eggs continued their development. Eggs of five different ages representing different sensitivity to radiation and different capacity to regeneration were used to investigate the influence of cosmic radiation and/or microgravity on insect development. Using the Biostack concept--eggs in monolayers sandwiched between nuclear track detectors--and the 1 g reference centrifuge of BIORACK in D1 we were able to separate effects of heavy ions of the cosmic radiation from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, embryonic and larval growth kinetics and anomaly frequencies were determined. Microgravity leads to a reduced hatching rate of eggs exposed in the early stages of development. Hatching was normal in eggs which were exposed on the 1 g reference centrifuge. Hits by heavy ions caused body anomalies. The combined action of heavy ions and microgravity resulted in an unexpectedly high frequency of anomalies. These results obtained from the Spacelab Mission D1, were confirmed in an experiment onboard of COSMOS 1887. In addition to the previous analysis, embryonic development before hatching was followed which showed no major difference between flight and the ground control specimens. Since a reconfirmation of reduced hatching rates was observed in COSMOS 1887, too, the above results suggest some microgravity induced functional impairment of the hatching activity, rather than blockage in embryonic development.     

Xenopus laevis embryos can establish their spatial bilateral symmetrical body pattern without gravity.

One assumes that gravity cooperates with the sperm in the establishment of bilateral symmetry in the embryo, particularly in species with yolky eggs. However, only experiments under genuine microgravity can prove this. May 2nd 1988 on the TEXUS-17 Sounding Rocket, eggs of Xenopus laevis became the first vertebrate eggs ever successfully fertilized in Space. Fertilization was done in fully automated hardware; the experiment was successfully repeated and extended in 1989. Here we report a "Space First" from the IML-1 Space Shuttle mission (January 1992): In similar hardware and under microgravity, artificially fertilized Xenopus eggs started embryonic development. Histological fixation was pre-programmed at the time gastrulation would occur on Earth and indeed, gastrulae were fixed. Thus after fertilization in near weightlessness Xenopus embryos do develop bilaterally symmetrically, very probably cued by the sperm alone.     

Embryogenesis and organogenesis of Carausius morosus under spaceflight conditions

The influence of cosmic radiation and/or microgravity on insect development was studied during the 7 day German Spacelab Mission D1. Eggs of Carausius morosus of five stages differing in sensitivity to radiation and in capacity to regeneration were allowed to continue their development in the BIORACK 22°C incubator, either at microgravity conditions or on the 1 g reference centrifuge. Using the Biostack concept - eggs in monolayers were sandwiched between visual track detectors - and the 1 g reference centrifuge, we were able to separate radiation effects from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, growth kinetics and anomaly frequencies were determined in the different test samples. The early stages of development turned out to be highly sensitive to single hits of cosmic ray particles as well as to the temporary exposure to microgravity during their development. In some cases, the combined action of radiation and microgravity even amplified the effects exerted by the single parameters of space. Hits by single HZE particles caused early effects, such as body anomalies, as well as late effects, such as retarded growth after hatching. Microgravity exposure lead to a reduced hatching rate. A synergistic action of HZE particle hits and microgravity was established in the unexpectedly high frequency of anomal larvae. However, it cannot be excluded, that cosmic background radiation or low LET HZE particles are also causally involved in damage observed in the microgravity samples.     

Fertilization of sea urchin eggs in space and subsequent development under normal conditions.

Sea urchin eggs are generally considered as most suitable animal models for studying fertilization processes and embryonic development. In the present study, they are used for determining a possible role of gravity in fertilization and the establishment of egg polarity and the embryonic axis. For this purpose, eggs of the particularly well known and suitable species Paracentrotus lividus have been automatically fertilized under microgravity conditions during the Swedish sounding rocket flights MASER IV and MASER V. It turns out, that fertilization "in Space" occurs normally and that subsequent embryonic and larval development of such eggs, continued on the ground, is normal, leading to advanced pluteus stages.     

Cytoskeleton and gravity at work in the establishment of dorso-ventral polarity in the egg of Xenopus laevis

The establishment of polarities during early embryogenesis is essential for normal development. Amphibian eggs are appropriate models for studies on embryonic pattern formation. The animal-vegetal axis of the axially symmetrical amphibian egg originates during oogenesis and foreshadows the main body axis of the embryo. The dorso-ventral polarity is epigenetically established before first cleavage. Recent experiments strongly suggest that in the monospermic eggs of the anuran Xenopus laevis both the cytoskeleton and gravity act in the determination of the dorso-ventral polarity. In order to test the role of gravity in this process, eggs will be fertilized under microgravity conditions during the SL-D1 flight in 1985. In a fully automatic experiment container eggs will be kept under well-defined conditions and artificially fertilized as soon as microgravity is reached; eggs and embryos at different stages will then be fixed for later examination. Back on earth the material will be analysed and we will know whether fertilization under microgravity conditions is possible. If so, the relation of the dorso-ventral axis to the former sperm entry point will be determined on the whole embryos; in addition eggs and embryos will be analysed cytologically.     

Transient effects of microgravity on early embryos of Xenopus laevis.

In order to study the role of gravity on the early development of the clawed toad Xenopus laevis, we performed an experiment on the Maser-6 sounding rocket launched from Kiruna (Sweden) on 4 Nov 1993. The aim was to find out whether a short period of microgravity during fertilization and the first few minutes of development does indeed result in abnormal axis formation as was suggested by a pilot experiment on the Maser 3 in 1989. On the Maser 6 we used two new technical additions in the Fokker CIS unit, viz. a 1-g control centrifuge and a video recording unit which both worked successfully. The 1-g control centrifuge was used to discriminate between the influences of flight perturbations and microgravity. After fertilization shortly before launch, one of the first indications of successful egg activation, the cortical contraction, was registered in microgravity and on earth. Analysis of the video tapes revealed that the cortical contraction in microgravity starts earlier than at 1 g on earth. After recovery of the eggs fertilized in microgravity and culture of the embryos on earth, the morphology of the blastocoel has some consistent differences from blastulae from eggs fertilized in the 1-g centrifuge of the rocket. However from the gastrula stage onward, the microgravity embryos apparently recover and resume normal development: the XBra gene is normally expressed, and histological examination shows normal axis formation.     

Amphibian egg cytoplasm response to altered g-forces and gravity orientation

Elucidation of dorsal/ventral polarity and primary embryonic axis development in amphibian embryos requires an understanding of cytoplasmic rearrangements in fertile eggs at the biophysical, physiological, and biochemical levels. Evidence is presented that amphibian egg cytoplasmic components are compartmentalized. The effects of altered orientation to the gravitational vector (i.e., egg inversion) and alterations in gravity force ranging from hypergravity (centrifugation) to simulated microgravity (i.e., horizontal clinostat rotation) on cytoplasmic compartment rearrangements are reviewed. The behavior of yolk compartments as well as a newly defined (with monoclonal antibody) non-yolk cytoplasmic compartment, in inverted eggs and in eggs rotated on horizontal clinostats at their buoyant density, is discussed.     

A direct approach to the study of the effect of gravity on axis formation in birds.

A system has been developed to enable the normal development of aborted very early uterine avian embryos, outside the female's uterus. The shell-less aborted egg was put into a foster shell of a sister egg, previously laid by the same female. The empty space between the shell and aborted egg was filled with artificial uterine fluid. The reconstructed eggs were incubated at 42 degrees C for 30 hours in a vertical position. The atmosphere contained a high concentration of CO2 (8-10%). At the termination of the 30 h the eggs were transferred to incubation at 37 degrees C in normal atmospheric conditions. Normal development has been recorded for a certain percentage of eggs incubated up to 12 days. In other cases abnormalities, arrested development or development of extraembryonic membranes only, without a sign of an embryonic axis, have been observed. The three important conclusions from the above experiments were: 1. It is possible to develop a closed, self-contained system, disconnected from the female's body, that would support the development of early uterine embryos. 2. The incidence of embryo-less extraembryonic membranes in such a system, is correlated with the degree of detachment of the "yolk" from the outer envelopes. 3. Such a system can be further developed into an experiment suited for microgravity conditions which will be an alternative to an experiment with live birds. The experiment will be aimed at testing the importance of gravity in changing the radially symmetrical avian blastoderm into a bilaterally symmetrical blastoderm.     

Formation of otoconia in the Japanese red-bellied newt, Cynops pyrrhogaster.

Pre-mated adult female newts and fertilized eggs will be flown on the International Microgravity Laboratory-2 flight, in 1994. One objective of the flight will be to observe the influence of microgravity on the development of the gravity-sensing organs in the inner ear. These organs contain sensory hair cells covered by a layer of dense stones (otoconia). Gravity and linear acceleration exert forces on these masses, leading to excitation of the nerve fibers innervating the hair cells. If the production of the otoliths is regulated to reach an optimal weight, their development might be abnormal in microgravity. Ground-based control experiments are reported describing the developmental sequence in which both the otoliths and their associated sensory epithelium and the semicircular canals appear and develop. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos at stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. Reports of experiments in which fertilized frog eggs were flown on a Russian Cosmos mission conclude that the utricular otolith is increased in volume, whereas the saccular otolith maintains normal size, suggesting that at least in the utricle, the weight of the otolith might be regulated.     

Development of space-fertilized eggs and formation of primordial germ cells in the embryos of Medaka fish.

In the second International Microgravity Laboratory (IML-2) mission in 1994, four small Japanese killifish (Medaka, Oryzias latipes) made a space travel of 15 days aboard a space shuttle. These four adult Medaka fish successfully mated in space for the first time among vertebrate animals. Moreover, the eggs they laid developed normally, at least in their external appearance, hatching as fry (baby fish) in space. Fish mated and laid eggs every day during the first week. Near the end of the mission most of the eggs had a well-developed body with two pigmented eyes. In total, 43 eggs were laid (detected), out of which 8 fry hatched in space, as truly 'space-originated' babies. A further 30 fry hatched within 3 days after landing. This is the normal hatching rate, compared with the ground-based data. Among the 8 space-originated fry, four were killed for histological sections, and germ cells at the gonadal region were counted for each fry. Their numbers were in the range of the germ cells of the normal control fry (ground-kept samples). Thus, as embryos developed normally in their external appearance, inside the embryos the formation of primordial germ cells took place normally in space, and their migration to the genital ridges was not hindered by microgravity. The two of the remaining space-originated fry have grown up and been creating their offspring in the laboratory. This proved that the primordial germ cells formed in space were also normal from a functional point of view. The four space-travelled adult fish re-started mating and laying eggs on the 7th day after landing and continued to do so every day afterward. Fertilization rate and hatchability of these eggs were as high as the eggs laid by the laboratory-kept fish. This fact implies that in gametogenesis of adult fish, there are no specific stages of germ cells extremely susceptible to microgravity.     

Microgravity effects on "postural" muscle activity patterns.

Changes in neuromuscular activation patterns associated with movements made in microgravity can contribute to muscular atrophy. Using EMG to monitor "postural" muscles, it was found that free floating arm flexions made in microgravity were not always preceded by neuromuscular activation patterns normally observed during movements made in unit gravity. Additionally, manipulation of foot sensory input during microgravity arm flexion impacted upon anticipatory postural muscle activation.     

The pleurodele, an animal model for space biology studies.

Pleurodeles waltl, an Urodele amphibian is proposed as a model for space biology studies. Our laboratory is developing three types of experiments in space using this animal: 1) in vivo fertilization and development ("FERTILE" project); 2) influence of microgravity and space radiation on the organization and preservation of specialized structures in the neurons and muscle cells (in vitro; "CELIMENE" PROJECT); 3) influence of microgravity on tissue regeneration (muscle, bone, epidermis and spinal cord).     

Capillary movement of liquid in granular beds in microgravity.

A more complete understanding of the dynamics of capillary flow through an unsaturated porous medium would be useful for the development of an effective water and nutrient delivery system for the growth of plants in space. An experiment was conducted on the Mir Space Station that used an experimental cuvette called "Capillary Test Bed" to compare fluid migration under terrestrial laboratory conditions by positioning the cuvette such that the hydrostatic force is negated and on Mir under microgravity conditions. Differences in fluid migration in the cuvette were observed with migration being slower in microgravity compared with some ground control experiments.     

Fertilization of frog eggs on a Sounding Rocket in space.

During the TEXUS-17 flight (April/May 1988) eggs of a higher organism, the anuran amphibian Xenopus laevis, have for the first time been successfully fertilized under microgravity on a Sounding Rocket. This result also implies that Life Sciences Experiments of Short Duration can be carried out on Sounding Rockets. The latter can therefore function as additional carriers for such experiments. Histological sections of the experimental material demonstrated the penetration of sperm into eggs, while SEM analysis revealed the differentiation of characteristic egg surface structures. Our TEXUS-17 experiment convincingly shows that the modified automatic experiment container, originally designed for experimental BR 52NL on the D1-mission, now functions flawlessly. Eight containers were flown in an airtight, well-isolated box (TEM 06-15), and a similar set was activated on Earth, two hours later. The analysis of the biological material is in progress.     

Development of plant protoplasts during the IML-1 mission.

During the 8 day IML-1 mission, regeneration of cell walls and cell divisions in rapeseed protoplasts were studied using the Biorack microscope onboard the Space Shuttle "Discovery". Samples from microgravity and 1g protoplast cultures were loaded on microscope slides. Visual microscopic observations were reported by the payload specialist Roberta Bondar, by down-link video transmission and by use of a microscope camera. Protoplasts grown under microgravity conditions do regenerate cell walls but to a lesser extent than under 1g. Cell divisions are delayed under microgravity. Few cell aggregates with maximum 4-6 cells per aggregate are formed under microgravity conditions, indicating that microgravity may have a profound influence on plant cell differentiation.     

Retinal photoreceptor and related gene expression in normal and clinostat-treated fish embryos

Medaka fish had performed mating behavior successfully in space for the first time among vertebrate, and the eggs which were laid in space developed normally, and hatched during the space travel of 15 days aboard the space shuttle in the second International Microgravity Laboratory (IML-2) mission in 1994 (Ijiri 1994). But there has been few studies whether microgravity affects the development of rather complex tissues in this fish. Investigating this problem, we focused on the organogenetic events in the retina in developing Medaka under normal and simulated microgravity conditions (by a three-dimensional clinostat, 3D-clinostat). Our results showed that both normal and 3D-clinostat-treated Medaka embryos developed on almost equal time course. Moreover, we investigated the development of the retina in normal and 3D-clinostat-treated embryos, but there were no differences in organogenesis of their retina. Lamination of retina occurred almost at equal timing and the expressions of opsin genes in the 3D-clinostat-treated group also began almost at the same time as control. Our observations suggest that there were no definite effects of simulated microgravity on the organizations of a complex tissue such as retina in developing fish embryos.     

A step in embryonic axis specification in Xenopus laevis is simulated by cytoplasmic displacements elicited by gravity and centrifugal force.

Determination of the body pattern in Xenopus embryos is known to involve at least six steps. One of these steps can be experimentally simulated by inclining the fertilized egg with respect to gravity or centrifugal force (10-30 g x 4 min, directed 90 degrees to the animal-vegetal axis). In these eggs, the dorsal structures of the body axis form from the side of the egg that was uppermost in the gravitational or centrifugal field. This topography is seen even if the sperm entry point side (the prospective ventral side in control eggs) was uppermost. In addition, conjoined twin embryos form at very high frequencies in response to certain conditions of single or double centrifugation. Cytological analysis shows that the dorsal structures invariably form from the side(s) of the egg away from which vegetal cytoplasm was displaced. This is similar to the situation in the unperturbed egg, where the subcortical cytoplasm of the vegetal hemisphere rotates some 30 degrees relative to the surface, and the dorsal structures form from the side of the egg away from which the subcortical cytoplasm moved. The displacements elicited by centrifugation probably substitute for the normal displacements brought about by the subcortical rotation. These and other data suggest that the subcortical rotation is a crucial step in the process of axis determination. The subcortical rotation is an autonomous activity of the activated egg, and can displace cytoplasm against gravity. I believe that the subcortical rotation will function normally at microgravity, and I expect that overall development and axis polarity at microgravity will be normal. This will be tested in spaceflight.     

Fertilization and development of eggs of the South African clawed toad, Xenopus laevis, on sounding rockets in space.

Egg rotation and centrifugation experiments strongly suggest a role for gravity in the determination of the spatial structure of amphibian embryos. Decisive experiments can only be made in Space. Eggs of Xenopus laevis, the South African clawed toad, were the first vertebrate eggs which were successfully fertilized on Sounding Rockets in Space. Unfixed, newly fertilized eggs survived reentry, and a reasonable number showed a seemingly normal gastrulation but died between gastrulation and neurulation. Only a few reached the larval stage, but these developed abnormally. In the future, we intend to test whether this abnormal morphogenesis is due to reentry perturbations, or due to a real microgravity effect, through perturbation of the reinitiation of meiosis and other processes, or started by later sperm penetration.     

Pigment composition and concentrations within the plant (Ceratophyllum demersum L.) component of the STS-89 C.E.B.A.S. Mini-Module spaceflight experiment.

The Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) Mini-Module, a Space Shuttle middeck locker payload which supports a variety of aquatic inhabitants (fish, snails, plants and bacteria) in an enclosed 8.6 L chamber, was tested for its biological stability in microgravity. The aquatic plant, Ceratophyllum demersum L., was critical for the vitality and functioning of this artificial mini-ecosystem. Its photosynthetic pigment concentrations were of interest due to their light harvesting and protective functions. "Post-flight" chlorophyll and carotenoid concentrations within Ceratophyllum apical segments were directly related to the quantities of light received in the experiments, with microgravity exposure (STS-89) failing to account for any significant deviation from ground control studies.     

Urodelean amphibians in studies on microgravity: effects upon organ and tissue regeneration.

Results obtained from nine experiments performed onboard Russian biosatellites have shown that microgravity promotes tissue regeneration in the newt, Pleurodeles waltl. The effect has been reproduced in all flights and on a clinostat as well for eye tissues (lens and retina), limbs and tail. The effect was demonstrated in 1.5- to 2-fold increase in cell proliferation in the early stages of regeneration in space flight. Animals "flown" intact and operated after flight regenerated faster than control ones and showed long-lasting micro-"g" effect. The most recent experiment flew aboard the Bion-11 biosatellite. This test was performed for study on microgravity effect on neural retina regeneration after optic nerve lesioning in the newt. Obtained results confirmed our previous information about intensification of regenerative processes in detached neural retina in urodela exposed to simulated weightlessness (Grigoryan et al., 1998). In particular, we found the increase and activation of cell populations participating in neural retina restoration and maintenance of retinal structure. Our findings suggest that promoting effect of microgravity upon regeneration could be influenced by several factors, largely influenced by a response of the whole organism to changed gravity vector. We hypothesized the synthesis of the specific range of stress proteins induced by micro-"g" and their regulative role in cell proliferation. Such a hypothesis for the existence of "altered gravity stress proteins" is discussed.